DISS. ETH No. 20973
Polyphenol content and profile in apples and its potential
relevance to human health
A dissertation submitted to
ETH Zurich
for the degree of
Doctor of Science
presented by
MARIA CEYMANN
Lebensmittelchemikerin, Bayerische Julius-Maximilians-Universität Würzburg,
Germany
born 26.05.1982
Krumbach (Schwaben), Germany
accepted on the recommendation of
Prof. Dr. Richard F. Hurrell, examiner
Prof. Dr. Laura Nyström, co-examiner
Eva Arrigoni, co-examiner
2013
ACKNOWLEDGEMENTS
I would gratefully acknowledge Prof. Richard F. Hurrell for giving me the opportunity to join
the Human Nutrition Laboratory as an external PhD student. You introduced me to the field
of human nutrition and all the discussions and feedback helped to accomplish my work.
***
I am very grateful to Prof. Laura Nyström for offering her expertise as co-examiner.
***
I would like to thank Eva Arrigoni for providing me the research topic and for supervising my
thesis. You supported me with all the discussion and additionally with practical work during
my pregnancy and maternity leave. Thank you so much.
***
I would like to thank the research station Agroscope Changins-Wädenswil in Wädenswil,
Switzerland, for financing my PhD.
***
Many thanks to present and former members of the „Gruppe LM“ for the enjoyable working
environment and the cheerful coffee breaks: Dr. Anna Bozzi Nising, Constance Reif, Claudia
Good, Eva Arrigoni, Dr. Daniel Baumgartner, Hans Schärer, Thomas Eppler, Sonia PetignatKeller, Martin Heiri, Christine Brugger, Dr. Franz Gasser, Brigitte Kamm and Werner
Naunheim.
***
My sincere thanks go to Daniel and Hans for all your help in the lab, your support with the
UPLC-MS and in doing statistics.
***
Very special thanks go to Conny, Melanie, Pina and Claudia for being such great colleagues.
Your support and especially friendship was very important to me.
***
I would like to thank all apple providers: Reto Leumann, Claudia Good, Andreas Naef, Danilo
Christen, Otto Läubli, Michael Gölles, Martin Kockerols, Werner Naunheim and Isabel
Mühlenz. Thanks are also given to Dr. Markus Kellerhals and Simon Egger for scientific
discussion. You all introduced me in the world of apple production and I enjoyed working
with you very much.
***
Many thanks go to Melanie Erzinger, Jennifer Kläui, Martin Heiri and Dominic Ritler for
their support during sample preparation and analysis.
***
I would like to thank my parents Resi and Hans for supporting me during my studies and for
all your practical help during the last month.
***
Last but not least, I would like to thank you, Andi, for your great support and patience.
Additionally, very special thanks go to my children, Sara and Konstantin, who slept quiet in
the afternoon and bore with me spending a lot of time with the computer.
TABLE OF CONTENTS
ABBREVIATIONS ......................................................................................................................... 1
SUMMARY................................................................................................................................... 3
ZUSAMMENFASSUNG .................................................................................................................. 7
INTRODUCTION ........................................................................................................................ 13
LITERATURE REVIEW .............................................................................................................. 15
1
APPLES AND CONSUMERS .............................................................................................. 17
2
POLYPHENOLS IN APPLES............................................................................................... 19
2.1
Flavan-3-ols ......................................................................................................... 20
2.2
Phenolic acids ...................................................................................................... 22
2.3
Dihydrochalcones ................................................................................................. 23
2.4
Flavonols .............................................................................................................. 23
2.5
Polyphenol content of apples and apple juices .................................................... 25
2.5.1 Apples............................................................................................................... 25
2.5.2 Apple juices ...................................................................................................... 27
2.6
Pre-harvest factors influencing the polyphenol content and profile of apples .... 28
3
BIOACCESSIBILITY OF APPLE POLYPHENOLS .................................................................. 32
4
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY ..................................................... 37
5
6
4.1
Absorption and metabolism of flavan-3-ols ......................................................... 38
4.2
Absorption and metabolism of phenolic acids ..................................................... 43
4.3
Absorption and metabolism of dihydrochalcones ................................................ 47
4.4
Absorption and metabolism of flavonols .............................................................. 51
4.5
Comparison of the fate of apple polyphenols after ingestion............................... 54
APPLE POLYPHENOLS AS ANTIOXIDANTS ....................................................................... 57
5.1
Analytical methods of the antioxidant potential ................................................... 57
5.2
Antioxidant action in humans ............................................................................... 58
HEALTH EFFECTS OF APPLE POLYPHENOLS .................................................................... 61
6.1
Health effects of flavan-3-ols ............................................................................... 62
6.1.1 Flavan-3-ols in the prevention of cardiovascular diseases ............................... 63
6.1.2 Flavan-3-ols in the prevention of other Western diseases ................................ 81
6.2
Health effects of phenolic acids............................................................................ 84
6.3
Health effects of dihydrochalcones ...................................................................... 87
6.4
Health effects of flavonols .................................................................................... 88
6.5
Conclusion: potential health effects of polyphenols in apples ............................. 90
REFERENCES ......................................................................................................................... 91
PAPER 1 .................................................................................................................................. 109
PAPER 2 .................................................................................................................................. 125
PAPER 3 .................................................................................................................................. 145
PAPER 4 .................................................................................................................................. 169
CONCLUSIONS ........................................................................................................................ 185
CURRICULUM VITAE.............................................................................................................. 189
ABBREVIATIONS
ABTS
2,2’-azonobis(3-ethylbenzothiazoline-6-sulphonate)
ADP
adenosine diphosphate
BSA
bovine serum albumin
C
catechin
CA
chlorogenic acid
CAD
cardioartery diseases
CE
catechin equivalents
CF
caffeic acid
CI
confidence interval
COMT
catechol-O-methyl transferase
CQA
p-coumaroylquinic acid
cv.
cultivar
CVD
cardiovascular diseases
DAD
diode array detection
DHC
dihydrocaffeic acid
DPPH
1,1-diphenyl-2-picrylhydrazyl radical
EC
epicatechin
EST
esterase
FC
flow cytometry
FFQ
food frequency questionnaires
FM
edible fresh matter
FMD
flow mediated dilation
FRAP
ferric reducing antioxidant power
HDL
high density lipoprotein
LDL
low density lipoprotein
LPH
lactase-phloridzin-hydrolase
MM
microbial metabolites
NO
nitric oxide
ORAC
oxygen radical absorbance capacity
PC B1
procyanidin B1
PC B2
procyanidin B2
PFA
platelet functional analyser
1
Phl
phloretin
Phz
phloridzin
PP
polyphenols
PXG
phloretin-xyloglucoside
Q
quercetin
QA
quinic acid
QG
quercetin-glucoside
QGal
quercetin-galactoside
QR
quercetin-rhamnoside
R
rutin
RR
relative risk
SULT
sulphotransferase
TC
total cholesterol
TEAC
Trolox® equivalent antioxidant potential
TPC
total polyphenol content
TUV
tuneable UV
UGT
uridindiphosphate-glucuronosyltransferase
UHPLC-MS
ultra high pressure liquid chromatography mass spectrometry
UHPLC-UV
ultra high pressure liquid chromatography ultra violet detection
WHS
Women’s Health Study
ß-gly
ß-glucosidase
2
SUMMARY
Background Polyphenols are a diverse group of secondary plant metabolites which are
present in all plant foods. Apple polyphenols belong to the groups of flavan-3-ols, phenolic
acids, dihydrochalcones and flavonols. Their content and profile in apples vary widely
depending on the cultivar. In addition, pre-harvest-factors such as light, rootstock, crop load,
production method and growing season are reported to have an influence on the polyphenol
content. During digestion, polyphenols are released from the apple matrix and become
bioaccessible for absorption into the mucosal cells, where before the passage into the plasma,
they are mainly metabolised to glucuronides, methylates and sulphates. Polyphenol
compounds are bioactive, including their antioxidant properties. They are reported to be
protective against chronic diseases such as cardiovascular diseases and various cancers, and
they may modulate glucose absorption in the human body.
Aims of this thesis The overall aim of this thesis was to evaluate the potential of apples to
protect against chronic diseases by quantifying those polyphenols with reported health
benefits in common and regional apple cultivars, and investigating the pre-harvest factors that
could influence their levels in the fruits. The specific aims were to develop an analytical
method to identify and quantify the 12 main apple polyphenols (PAPER 1); to screen the
polyphenol content and profile of a broad range of apple cultivars (PAPER 2); to quantify and
assess the influence of pre-harvest factors on polyphenol content and profile (PAPER 3), and to
investigate the in vitro accessibility of polyphenols in apples (PAPER 4).
Papers These aims were addressed by developing a method adapted to UHPLC-MS, by
comparing polyphenol contents of apples grown under different conditions over a three year
period, and by mimicking the digestion of fresh apples with an in vitro method simulating
gastric and intestinal digestion. The UHPLC-MS method was used for quantifications of
individual polyphenols in all studies.
PAPER 1 For the development of the analytical method and measurement of the major
polyphenols in four apple cultivars, apple slices were frozen in liquid nitrogen, ground to a
fine powder and extracted with methanol containing formic acid (1 % v/v). After dilution and
filtration, the filtrate was measured directly by UHPLC-MS and the total polyphenol content
(TPC) by Folin-Ciocalteu was quantified. The analytical method developed was simple with a
rapid extraction procedure and a single chromatographic run. The sample preparation was
consumer adapted as it analysed polyphenols in apples with the skin but without the core area.
3
The 12 most important apple polyphenols were quantified. These were catechin, epicatechin,
procyanidin B1 and B2, chlorogenic acid, coumaroylquinic acid, phloridzin, phloretinxyloglucoside, quercetin-galactoside/-glucoside, rutin, and quercetin rhamnoside. The TPC
was analysed in the same extracts and showed 3-4 times higher polyphenol concentrations
than the sum of individual polyphenols determined by UHPLC-MS. This difference was
explained by dilution and filtration losses of oligomeric polyphenols during sample
preparation for UHPLC-MS analysis. The oligomeric polyphenols are not expected to have
major health benefits so that the losses appear negligible from a nutritional point of view. The
individual polyphenols quantified in the cultivars Braeburn, Fuji, Gala Galaxy and Golden
Reinders were comparable to published values.
PAPER 2 For the screening of polyphenols in a wide range of apples, the UHPLC-MS method
was used to quantify polyphenols in 104 European cultivars, mainly harvested in Switzerland.
TPC by Folin-Ciocalteu and the antioxidant potential by Trolox® equivalent antioxidant
potential (TEAC) as well as ferric reducing antioxidant power (FRAP) were measured for
comparison. For individual polyphenols, the amount of the different polyphenols in the 104
cultivars varied from below the limit of detection (<lod) to 70 mg/100 g FM. The highest
concentrations were found for epicatechin, procyanidin B2 and chlorogenic acid and the
individual apple polyphenols varied strongly between the different apple cultivars. Highest
variability was obtained for phenolic acid values ranging from 1-70 mg/100 g for chlorogenic
acid and from <lod to 18 mg/100 g edible fresh matter (FM) for coumaroylquinic acid.
Flavan-3-ols showed a slightly smaller range (catechin: <lod-10 mg, epicatechin: 0.5-34 mg,
procyanidin B1: <lod-11 mg, procyanidin B2: 0.6-32 mg/100 g FM). Dihydrochalcones
(phloridzin: 0.4-13 mg, phloretin xyloglucoside: 0.7-20 mg/100 g FM) and flavonols
(quercetin-galactoside/glucoside: 0.4-4 mg, rutin: <lod-1 mg, quercetin-rhamnoside: 0.45 mg/100 g FM) had more constant values with a smaller range. Based on their polyphenol
profile, apple cultivars could be divided into flavan-3-ol dominated or phenolic acid
dominated cultivars. The latter possess a flavan-3-ol to phenolic acid ratio ≤ 0.9, whereas the
flavan-3-ol dominated cultivars show a ratio ≥ 1.1. In one serving size (150 g), a flavan-3-ol
dominated cultivar such as cv. Topaz can contain an equal amount of epicatechin as a serving
size of dark chocolate (20 g) and a phenolic acid dominated cultivar, for example. cv.
Jonagold can reach the chlorogenic acid level of a cup of coffee (150 mL). Such quantities in
cocoa and coffee are reported to have beneficial health effects in humans.
When the 104 apple cultivars were screened for antioxidant potential, the TPC varied from 52
to 379 mg catechin equivalents/100 g FM. They were considerably higher than the sum of
4
individual polyphenols analysed by means of UHPLC-MS, but a reasonable correlation
between the two measures was found. Moreover, similar correlations were observed for
TEAC and FRAP. Therefore, these simple photometric methods can be considered as a good
predictor for the polyphenol content in apples, but provide no information about the profile or
potential effects on health.
PAPER 3 The investigation of pre-harvest factors revealed that the main factor influencing the
polyphenol content was the cultivar, which was also the only pre-harvest factor having an
effect on the polyphenol profile. The mutant variety and growing year additionally had a
small influence on the polyphenol content of apples. The influence of the rootstock, growing
environment, crop load and production methods resulted in small but non-significant
differences between treatments. If the apple grower wants to grow apples high in the
polyphenols such as epicatechin or chlorogenic acid, which are reported to have health
benefits, the best advice is to select the cultivar high in these specific polyphenols and to
optimise the pre-harvest factors if possible.
PAPER 4 For investigating the bioaccessibility of apple polyphenols, fresh apples and apple
juices were digested with an in vitro method simulating gastric and intestinal digestion. Pure
apple flavan-3-ols, six different apple cultivars and four apple juices were incubated with
artificial gastric juice (1 h) and afterwards with artificial duodenal juice (2 h) at 37° C. The
supernatants of the digested samples were freeze-dried and redissolved in methanolcontaining 1 % formic acid (v/v) for the determination of catechin, epicatechin, procyanidin
B1 and B2 as well as chlorogenic acid by UHPLC-UV. The in vitro digestion of fresh apples
indicated a relatively high bioaccessibility of apple polyphenols with nearly 50 % recovered
after simulated gastric digestion and simulated duodenal digestion. The same values were
found for apple juices. As the polyphenols of apple juice are expected to be 100 % accessible,
the recovery values reflect mainly the destruction of the polyphenols during the simulated in
vitro digestion but also include formation of monomers from oligomers and epimerisation of
monomers as detected for isolated pure monomeric (60-70 % recovery) and dimeric flavan-3ols (traces of recovery). As polyphenols from other foods are reported to arrive intact in the
small intestine, the current in vitro method needs improvement to better predict
bioaccessibility in humans.
Conclusion The apple cultivar appeared to be the main factor for increasing the polyphenol
content followed by some pre-harvest factors. Moreover, the polyphenol profile can only be
modified by choosing a specific cultivar. Therefore, the choice of the apple cultivar appeared
5
to be the most important decision for the consumer but also for the producer. A flavan-3-ol
dominated cultivar may have a beneficial effect on cardiovascular health mainly assigned to
epicatechin. Two flavan-3-ol dominated apples would provide a level of epicatechin that has
been shown to improve blood flow when consumed with cocoa. In contrast, the level of
chlorogenic acid in the phenolic acid dominated apples is much lower than the levels used in
intervention studies which were reported to improve the glucose metabolism and possibly be
beneficial to weight control. Another health aspect could be obtained by one apple of a
cultivar which contains sufficient dihydrochalcones to help prevent type 2 diabetes through
the inhibition of glucose uptake. Furthermore, a reduced cancer risk could be associated with
eating daily two apples with red skin through the inhibitory effect on cancer cell proliferation
of quercetin-glycosides. Overall, the polyphenol profile of the apples commonly consumed in
Switzerland would indicate that two or more apples per day would be necessary to help
reduce the risk of common chronic diseases. The old saying should be adapted to “Two apples
a day keep the doctor away”. In this context, an increase in apple consumption should be
recommended to the Swiss population, even though the postulated health effects of apple
polyphenols should be investigated in further human studies.
6
ZUSAMMENFASSUNG
Hintergrund Polyphenole sind eine vielfältige Gruppe von sekundären Pflanzenstoffen, die
in allen pflanzlichen Lebensmitteln vorkommen. Polyphenole in Äpfeln gehören zu den
Gruppen der Flavan-3-ole, phenolischen Säuren, Dihydrochalcone und Flavonole. Ihr Gehalt
ist stark von der Sorte abhängig. Als weitere Einflussfaktoren auf den Polyphenolgehalt gelten
Vorerntefaktoren wie Licht, Unterlage, Behang, Produktionsmethode und Produktionsjahr. Im
Verdauungsprozess werden Polyphenole aus der Apfelmatrix freigesetzt. Dadurch werden sie
für die Aufnahme in die Mukosazellen, wo sie hauptsächlich zu Glucuroniden, Methylaten
und Sulfaten verstoffwechselt werden, biozugänglich. Polyphenole sind bioaktive Substanzen
mit antioxidativen Eigenschaften. Ihnen wird einen schützender Effekt gegen chronische
Erkrankungen, wie beispielsweise die Prävention kardiovaskulärer Erkrankungen und
verschiedener Krebsarten, zugeschrieben. Auch bei der Regulierung der Glucoseaufnahme im
menschlichen Körper entfalten sie möglicherweise eine positive Wirkung.
Ziele der Arbeit Das generelle Ziel dieser Arbeit war es das Potential von Äpfeln zum Schutz
vor chronischen Krankheiten zu ermitteln. In gängigen Apfelsorten wurden dazu Polyphenole
mit
einem
bekannten
positiven
Einfluss
auf
die
Gesundheit
quantifiziert
und
Vorerntefaktoren, die diese Gehalte beeinflussen können, untersucht. Die spezifischen Ziele
dieser Arbeit waren die Entwicklung einer analytischen Methode zur Identifizierung und
Quantifizierung der 12 wichtigsten Apfelpolyphenole (PAPER 1); die Untersuchung des
Polyphenolgehalts und -profils eines breiten Sortiments an Apfelsorten (PAPER 2); die
Bestimmung und Bewertung des Einflusses von Vorerntefaktoren auf Polyphenolgehalt
und -profil (PAPER 3) sowie die Analyse der in vitro Zugänglichkeit von Polyphenolen in
Äpfeln (PAPER 4).
Paper Um die oben erwähnten Fragestellungen zu beantworten, wurde eine UHPLC-MS
Methode entwickelt, welche es ermöglicht Polyphenolgehalt und -profil von Äpfeln, die im
Zeitraum von drei Jahren unter verschiedenen Bedingungen angebaut wurden, zu
untersuchen. Des Weiteren wurde die Zugänglichkeit von Polyphenolen aus frischen Äpfeln
mit einer in vitro Verdauungsmethode ermittelt. Die UHPLC-MS Methode wurde in allen
weiteren Analysen für die Quantifizierung einzelner Polyphenole verwendet.
PAPER 1 Für die Entwicklung der analytischen Methode und Messung der wichtigsten
Polyphenole in vier Apfelsorten wurden Apfelschnitze in flüssigem Sticksoff gefroren, zu
feinen Pulver gemahlen und mit Ameisensäure-haltigem Methanol (1 % v/v) extrahiert. Nach
7
der Verdünnung und Filtrierung der Extrakte, wurden diese direkt mittels UHPLC-MS
analysiert und zusätzlich der Gesamtpolyphenolgehalt (TPC) mittels Folin-Ciocalteu
gemessen. Die entwickelte analytische Methode war auf Grund eines schnellen
Extraktionsprotokolls sowie eines einzigen chromatographischen Laufs einfach durchführbar.
Die Probenvorbereitung war an das Konsumentenverhalten angepasst, indem die Polyphenole
im Apfel mit der Schale, aber ohne Kerngehäuse analysiert wurden. Die 12 wichtigsten
Apfelpolyphenole wurden quantifiziert. Dies waren Catechin, Epicatechin, Procyanidin B1
und
B2,
Chlorogensäure,
Cumaroylchinasäure,
Phloridzin,
Phloretinxyloglucosid,
Quercetingalactosid/-glucosid, Rutin und Quercetinrhamnosid. Der in den gleichen Extrakten
analysierte TPC zeigte drei- bis viermal höhere Polyphenolkonzentrationen als die Summe der
einzelnen Polyphenole, die mittels UHPLC-MS gemessen wurden. Dieser Unterschied wurde
mit Verdünnungs- und Filtrationsverlusten von oligomeren Polyphenolen während der
Probenvorbereitung für die UHPLC-MS-Analysen erklärt. Da von den oligomeren
Polyphenolen bislang keine wichtigen Gesundheitseffekte bekannt sind, erschienen die
Verluste aus ernährungswissenschaftlicher Sicht vernachlässigbar. Die Quantifizierung der
einzelnen Polyphenole in den Sorten Braeburn, Fuji, Gala Galaxy und Golden Reinders war
mit publizierten Werten vergleichbar.
PAPER 2 Für das Screening von Polyphenolen in Äpfeln wurden in 104 europäischen,
hauptsächlich in der Schweiz geernteten Apfelsorten, die einzelnen Polyphenole mit UHPLCMS quantifiziert. Zum Vergleich wurden TPC mittels Folin-Ciocalteu und das antioxidative
Potential mittels Trolox® equivalent antioxidative potential (TEAC) und ferric reducing
antioxidant power (FRAP) bestimmt. Die verschiedenen Polyphenolgehalte der 104
Apfelsorten schwankten von unterhalb der Detektionsgrenze (<lod) bis zu 70 mg/100 g
essbare Frischmasse (FM). Die höchsten Konzentrationen wurden für Epicatechin,
Procyanidin
B2
und
Chlorogensäure
detektiert.
Dabei
variierten
die
einzelnen
Apfelpolyphenole stark zwischen den einzelnen Sorten. Die grössten Unterschiede wurden in
den Gehalten von phenolischen Säuren gefunden, die zwischen 1-70 mg/100 g FM für
Chlorogensäure
und
unterhalb
des
Detektionslimits
und
18 mg/100g
FM
für
Cumaroylchinasäure lagen. Flavon-3-ole zeigten etwas kleinere Unterschiede (Catechin:
<lod-10 mg, Epicatechin: 0.5-34 mg, Procyanidin B1: <lod-11 mg, Procyanidin B2: 0.632 mg/100 g FM). Die Konzentrationen der Dihydrochalcone (Phloridzin: 0.4-12 mg,
Phloretinxyloglucosid: 0.7-20 mg/100 g FM) und Flavonole (Quercetingalactosid/-glucosid:
0.4-4 mg, Rutin: <lod-1 mg, Quercetinrhamnosid: 0.4-5 mg/100 g FM) waren konstanter bei
8
kleinerer Variabilität. Auf Grund ihres Polyphenolprofils konnten die Apfelsorten in Flavan3-ol-dominierte und in phenolische Säuren-dominierte Sorten unterteilt werden. Letztere
besitzen ein Verhältnis von Flavan-3-olen zu phenolischen Säuren von < 0.9, wohingegen die
Flavan-3-ol-dominierten Sorten ein Verhältnis von > 1.1 aufweisen. Eine Portion (150 g)
einer Flavan-3-ol-dominierten Apfelsorte, wie beispielsweise Topaz, verfügt über
Epicatechingehalte vergleichbar mit einer Portion Schokolade (20 g). Eine Portion einer
phenolische
Säuren-dominierten
Sorte
wie
Jonagold
hingegen
kann
die
Chlorogensäuregehalte einer Tasse Kaffee (150 ml) erreichen. Diesen Gehalten in Kakao und
Kaffee werden gesundheitsfördernde Effekte beim Menschen nachgesagt.
Das Screening des antioxidativen Potentials der 104 Apfelsorten zeigte TPC im Bereich von
52-379 mg Catechinäquivalente/100 g FM. Diese lagen massgeblich höher als die ermittelte
Summe der einzelnen Polyphenole, die mittels UPLC-MS analysiert wurden. Allerdings
konnte eine ausreichende Korrelation zwischen beiden Messungen aufgedeckt werden.
Zusätzlich konnten ähnlich ausgeprägte Zusammenhänge für die TEAC- und FRAPMessungen gezeigt werden. In diesem Kontext können die einfachen photometrischen
Messungen als gute Methoden zur Abschätzung der Polyphenolgehalte in Äpfeln angesehen
werden, wobei keine Aussagen über das Polyphenolprofil möglich sind. Auch die postulierten
Gesundheitseffekte der Einzelsubstanzen können somit nicht abgeschätzt werden.
PAPER 3 Die Untersuchung der Vorerntefaktoren zeigte, dass der wichtigste Einflussfaktor auf
den Polyphenolgehalt die Sorte war. Diese stellte auch den einzigen Vorerntefaktor dar, der
einen Einfluss auf das Polyphenolprofil hatte. Zusätzlich hatten die Mutante und das
Produktionsjahr einen marginalen Einfluss auf den Polyphenolgehalt von Äpfeln. Der
Einfluss der Unterlage, des Standortes, des Behangs und der Produktionsmethode resultierte
in kleinen, aber nicht signifikanten Unterschieden zwischen den Behandlungen. Wenn
Apfelproduzenten Äpfel mit einem hohen Gehalt an Polyphenolen wie beispielsweise
potentiell gesundheitsförderndem Epicatechin oder Chlorogensäure anbauen wollen, kann die
entsprechende Wahl einer Apfelsorte mit hohen Gehalten dieser spezifischen Polyphenole
sowie eine Optimierung der Vorerntefaktoren empfohlen werden.
PAPER 4 Um die Biozugänglichkeit von Apfelpolyphenolen zu untersuchen, wurden frische
Äpfel und Apfelsäfte mit einer in vitro Methode verdaut. Sechs verschiedene Apfelsorten und
vier Apfelsäfte wurden mit künstlichem Magensaft (1 h) und anschliessend mit künstlichem
Duodenalsaft (2 h) bei 37° C inkubiert. Die Überstände der verdauten Proben wurden
9
gefriergetrocknet und in Ameisensäure-haltigem Methanol (1 % v/v) gelöst, um die
Konzentration an Catechin, Epicatechin, Procyanidin B1 und B2 sowie Chlorogensäure
mittels UHPLC-UV zu bestimmen. Nach der in vitro Verdauung der Apfelproben zeigte sich
eine relativ hohe Biozugänglichkeit der Apfelpolyphenole mit etwa 50 % Wiederfindung nach
Versetzen mit Magensaft und genauso nach zusätzlichem Versetzen mit Duodenalsaft. Die
gleichen Werte wurden für Apfelsäfte ermittelt. Da für Polyphenole aus Apfelsaft eine
Zugänglichkeit von 100% erwartet wurde, zeigten die ermittelten Wiederfindungsraten einen
Polyphenolabbau während der in vitro Verdauung. Zusätzlich beinhaltet diese Wiederfindung
auch die Bildung von Monomeren aus Oligomeren sowie die Epimerisierung von
Monomeren, wie es für isolierte Reinsubstanzen von monomeren (60-70% Wiederfindung)
und dimeren Flavan-3-olen (Spuren in der Wiederfindung) analysiert wurde. Da gezeigt
wurde, dass Polyphenole von anderen Lebensmitteln intakt den Dünndarm erreichen, ist eine
Verbesserung der aktuellen in vitro Methode notwendig, um die Biozugänglichkeit im
Menschen besser vorhersagen zu können.
Schlussfolgerungen Es stellte sich heraus, dass die Sorte der Haupteinflussfaktor für den
Polyphenolgehalt in Äpfeln ist, gefolgt von einzelnen Vorerntefaktoren. Ausserdem kann das
Polyphenolprofil nur durch die Auswahl einer spezifischen Sorte modifiziert werden.
Deswegen ist die Wahl der Apfelsorte die wichtigste Entscheidung für Konsumenten und
Produzenten. Eine Flavan-3-ol-dominierte Sorte kann sich schützend auf die kardiovaskuläre
Gesundheit auswirken, wobei diese Wirkung hauptsächlich Epicatechin zugesprochen wird.
Zwei Flavan-3-ol-dominierte Äpfel können einen Epicatechingehalt liefern, von welchem
gezeigt wurde, dass er verzehrt in Kakao den Blutfluss erhöhen kann. Im Gegensatz dazu sind
die Chlorogensäuregehalte in phenolischen Säuren-dominierten Sorten niedriger als die
Konzentration, die in Interventionsstudien eine Verbesserung im Glucosestoffwechsel und
einen postulierten positiven Effekt bei der Gewichtskontrolle zeigten. Ein weiterer
gesundheitsfördernder Aspekt könnte durch einen Apfel einer Sorte mit hohen
Dihydrochalcongehalten erreicht werden, da dieser Polyphenolgruppe ein präventiver Effekt
gegen Typ 2 Diabetes durch Hemmung der Glucoseaufnahme zugesprochen wird. Des
Weiteren könnte der tägliche Verzehr von zwei rotschaligen Äpfeln mit einem geringeren
Krebsrisiko assoziiert werden, da Quercetinglycoside die Krebszellenproliferation hemmen.
Allgemein betrachtet weisen die Polyphenolprofile in gängig verzehrten Schweizer
Apfelsorten darauf hin, dass täglich zwei oder mehr Äpfel konsumiert werden sollten, um das
Risiko von chronischen Erkrankungen zu reduzieren. Das alte Sprichwort sollte diesbezüglich
angepasst werden: „Two apples a day keep the doctor away“. In diesem Zusammenhang sollte
10
der Schweizer Bevölkerung ein gesteigerter Apfelkonsum empfohlen werden, wobei die
Gesundheitseffekte der einzelnen Apfelpolyphenole in weiteren Humanstudien untersucht
werden müsste.
11
12
INTRODUCTION
Polyphenols are secondary plant metabolites with an aromatic structure containing two or
more hydroxyl groups. They are prevalent in fruits, vegetables, cocoa and beverages such as
tea, coffee and wine (HAN et al. 2007). Secondary plant metabolites have little or no role in
photosynthesis, respiration, development or growth of plants (CROZIER et al. 2009).
Polyphenols are produced to protect the plant from microbial infections and from herbivores.
When a plant is wounded, polyphenols and polyphenol oxidase come in contact initiating
oxidation and the defence mechanism is activated. They also protect the plant from UV
radiation and are responsible for colouration of flowers and fruits, for example apples and
grapes.
In apples, the four predominating polyphenol groups are phenolic acids with a C6-C3 carbon
skeleton as well as flavan-3-ols, dihydrochalcones and flavonols with a C6-C3-C6 carbon
skeleton, the latter also called flavonoids (CROZIER et al. 2009). The amount of the different
polyphenols depends on pre-harvest factors, which have been reviewed by TREUTTER et al.
(2010). Variations of crop load, rootstock, environment, plant nutrition, light, plant protection
strategy, growing year and cultivar might have more or less impact on polyphenol content in
the fruit.
Eating apples contributes to the intake of antioxidants, as polyphenols are the most abundant
antioxidants in our diet (D’ARCHIVIO et al. 2007). However, polyphenols are non-nutritive for
humans and therefore they are treated as “xenobiotics” in the human body and metabolised so
as to eliminate them efficiently (CROZIER et al. 2009). Consequently, the absorption rates for
the individual polyphenols are estimated to be rather low and the rapid metabolism of the
polyphenols complicates the investigation of polyphenol absorption. Nevertheless,
polyphenols have been reported to have beneficial health effects. They have been thought to
be
cardio-protective,
neuro-protective,
immune-protective,
antidiabetic,
antiallergic,
antioxidant and to improve endothelial function (HAN et al. 2007). Moreover, dietary
polyphenols showed positive effects on gastrointestinal health and chronic obstructive
pulmonary diseases. Additionally, they may have antiinflammatory, antitumor and hormone
modulation effects (HAN et al. 2007). By ingesting at least 5 servings of fruits and vegetables
per day, as recommended by nutrition and health authorities in several countries, dietary
polyphenols may contribute to these postulated health effects. However, polyphenol contents
and profiles of fruits and vegetables differ considerably and an estimation of their potentially
13
beneficial health effect needs the knowledge of the factors influencing their content and
profile. Apples are a very popular fruit worldwide and in the United States of America, they
were estimated to contribute to 20-25 % of the per capita fruit polyphenol consumption
(BIEDRZYCKA et al. 2008).
Due to the frequent apple consumption and their varying polyphenol content and profile,
modulating polyphenols in apples may be a possible nutritional tool to support the prevention
of degenerative diseases. To provide the relevant data for estimating potential health effects of
apples, the following thesis deals with polyphenols in apples. In the literature review, apples
and apple polyphenols, their contents and factors influencing them are introduced (Chapter 1
and 2). Bioaccessibility of apple polyphenols and their fate in the human body are discussed
in Chapter 3 and 4. In addition, the function of polyphenols as antioxidants (Chapter 5) and
their postulated health effects (Chapter 6) complete the literature review. The 4 papers deal
with the analytical method to determine polyphenols in apples (Paper 1), the variability of
polyphenol content and profile in different apple cultivars (Paper 2), the pre-harvest factors
influencing polyphenol content and profile (Paper 3) and finally an evaluation of an in vitro
digestion approach to estimate bioaccessibility and stability of apple polyphenols during
digestion (Paper 4).
Biedrzycka, E. and R. Amarowicz (2008). "Diet and health: Apple polyphenols as antioxidants." Food
Reviews International 24(2): 235 - 251.
Crozier, A., I. B. Jaganath, et al. (2009). "Dietary phenolics: chemistry, bioavailability and effects on
health." Natural Product Reports 26(8): 1001-1043.
D’Archivio, M., C. Filesi, et al. (2007). "Polyphenols, dietary sources and bioavailability." Annali
dell'Istituto Superiore di Sanita 43(4): 348-361.
Han, X., T. Shen, et al. (2007). "Dietary polyphenols and their biological significance." International
Journal of Molecular Sciences 8: 950-988.
Treutter, D. (2010). "Managing phenol contents in crop plants by phytochemical farming and
breeding-visions and constraints." International Journal of Molecular Sciences 11(3): 807857.
14
LITERATURE REVIEW
15
16
LITERATURE REVIEW
1
APPLES AND CONSUMERS
Apples and consumers
Apples (Malus domestica) belong to the family of Rosaceae which also includes other edible
plants such as apricots, cherries and strawberries. They were originally found in Western Asia
and cultivated from small, sour and bitter fruits to the present edible cultivars. Their dietary
usage has been described over many thousands of years (WIKIPEDIA 2012a). In history, this
fruit has been linked to much symbolism. In the bible, the consumption of the apple was
forbidden to Adam and Eve. In the Middle Ages, the symbol for the Holy Roman Emperor
was the orb, in German called “Reichsapfel” (“apple of the empire”). Later in history, Isaac
Newton was inspired to formulate his theory of gravitation by watching the fall of an apple
from a tree and in the Swiss history William Tell is famous for his apple-shooting.
Today, apples are also important as a fruit available around the whole year. Apples contain
mainly water (85 %) and carbohydrates (13.8 %), which are composed of 11.6 g/100 g sugar,
2.1 g/100 g dietary fibres and 0.1 g/100 g starch (SWISSFIR 2012). Fats and proteins account
for 5 % and 2 % of the total energy of 51 kcal/100 g edible part, respectively. Moreover,
apples also provide the consumer with a balanced intake of various vitamins and minerals.
However, the content of the apple components can vary between the different cultivars and
apples are available with green, yellow and red skin colour and the combination of them. The
diversity of apple cultivars, more than 7500 are reported worldwide (WIKIPEDIA 2012b),
provides the consumer a broad range of sugar to acid ratios, aromas and sizes of the fruit.
Therefore, nearly every taste can be suited. This explains the relatively high consumption with
a global mean of 26 g/capita/d (FAOSTAT 2012c) in 2009, although the average mean
consumption varies between the countries. In Europe, most apples were consumed in
Montenegro, Slovenia and Austria with 149, 119 and 117 g/capita/d, respectively, with the
amount consumed in Montenegro representing the average weight of an apple. In Switzerland,
the average consumption was 62 g/capita/d, which is higher than the European mean
(55 g/capita/d). In the United States of America higher amounts were consumed in 2009
(66 g/capita/d), and consumption in Oceania reached 70 g/capita/d (FAOSTAT 2012c).
Concerning the apple production, the global apple production increased between 2000 and
2010 from 62 Mio t up to 70 Mio t with Asia representing the main apple production area
with the increasing yields. In Europe and the United States of America the apple production
over the same time period declined slightly, but still reached 14 Mio t and 4 Mio t,
respectively. The main apple producers in Europe are Italy (16 % of the European
production), Poland (13 %) and France (12 %). Switzerland (1.4 % of the European
17
LITERATURE REVIEW
APPLES AND CONSUMERS
production) followed at position 18 in the descending order of the European countries in 2010
(FAOSTAT 2012b). In Switzerland, the 85 most popular apples cultivars are cultivated on
3900 hectares with cultivar (cv.) Golden Delicious (29 % of the Swiss production), Gala
(28 %) and Braeburn (12 %) being the most important (FÜGLISTER 2012). The production of
cv. Golden Delicious decreased by one third between the year 2000 and 2010, whereas the
production of cv. Gala increased 2.5-fold and production of cv. Braeburn in 2010 was 7-fold
higher than in 2000. This decline of the cv. Golden Delicious represents a change in consumer
taste, and there is increasing evidence that apples also provide beneficial effects to humans’
health. As reviewed by HYSON (2011), there are reports that apple consumption may lead to
reduced risks of chronic diseases such as cancer, cardiovascular disease, asthma and
Alzheimer’s disease. Moreover, apples have been reported to have a positive effect on
cognitive decline, diabetes, weight management, bone health, pulmonary function and
gastrointestinal protection (HYSON 2011).
18
LITERATURE REVIEW
2
POLYPHENOLS IN APPLES
Polyphenols in apples
Polyphenols are secondary plant metabolites and contain one or more aromatic rings and at
least two hydroxyl groups. Several thousand different polyphenols are known (SIES 2010).
The two main classes of polyphenols in foods are phenolic acids and flavonoids. Flavonoids
are further divided into flavones, flavonols, flavan-3-ols, flavanones, isoflavones and
anthocyanidins (SCALBERT et al. 2005). The five polyphenol classes found in apples are
flavan-3-ols, phenolic acids, dihydrochalcones, flavonols and anthocyanidins, the lastmentioned being responsible for the red colour of the skin (VAN DER SLUIS et al. 2001). Due to
their low amounts in apples, anthocyanidins are not considered in this thesis.
The average daily intake of total polyphenols is estimated to be as high as 1 g/d with phenolic
acids account for one third and flavonoids for the remaining two thirds (SCALBERT et al.
2000). The range of the intake is reported to be from less than 100 mg/d up to 2 g/d depending
on consumption habits (RAWEL et al. 2007). However, the exact intake is difficult to estimate,
due to the lack of comprehensive data of polyphenol contents of foods (SCALBERT et al.
2000). Additionally, polyphenol intake is dependent on the consumption of different food
groups, for example, phenolic acids are mainly consumed via the ingesting of coffee (HOELZL
et al. 2010) and flavan-3-ols by tea or cocoa consumption (NEVEU et al. 2010), whereas the
main source for flavonols are onions, kale, broccoli and apples with decreasing amounts of
the mentioned sources (HOLLMAN et al. 2000). Estimating the intake of polyphenols via
biomarkers in urine was proposed by MENNEN et al. (2007), but this method needs more
human intervention studies.
All polyphenols are produced in plants via the shikimic acid pathway (ARTS et al. 2005)
during their secondary metabolism (FRAGA 2007;
VAN DER SLUIS
et al. 2001). CROZIER at al.
(2009) described the plant biosynthesis of polyphenols, whereas TREUTTER et al (2001)
focused on the polyphenol biosynthesis in apples. As summarised in Figure 1, the precursor of
all apple polyphenol groups is phenylalanine. This amino acid is metabolised to cinnamic acid
which represents a direct precursor of coumaric acid. The hydroxycinnamic acids, the
phenolic acids of apples, are derived from coumaric acid, whereas for the biosynthesis of
dihydrochalcones first the synthesis of chalcones occurs. Flavan-3-ols, flavonols and
anthocyanidins are produced by further metabolism of the chalcones. The complete
biosynthesis of polyphenols in plants still needs further investigation but it has been
recognised, that biosynthesis and accumulation of polyphenols are tissue specific (TREUTTER
2001). For example in apples, biosynthesis occurs mostly during fruit development, i.e. until
19
LITERATURE REVIEW
POLYPHENOLS IN APPLES
cell proliferation stops. Afterwards, mainly cell enlargement takes place which leads to a
dilution of polyphenols (GUYOT et al. 2003; RENARD et al. 2007). In the following subchapters, distribution and prevalence of the main apple polyphenol classes are discussed.
Figure 1:
2.1
Biosynthetic pathway of polyphenols in apple, PAL phenylalanine ammonia lyase,
CHS chalcone synthase, CHI chalcone flavanone isomerise, DFR dihydroflavonol
reductase, ANS anthocyanidin synthase, GT glycosyl transferase, (TREUTTER 2001)
Flavan-3-ols
The flavan-3-ols are the most abundant group of polyphenols in apples. They can be divided
into monomeric, oligomeric and polymeric flavan-3-ols. The only two monomeric flavan-3ols in apples are catechin (C, Figure 2 a) and epicatechin (EC, Figure 2 b), whereby the
amount of epicatechin is higher in average.
20
LITERATURE REVIEW
Figure 2:
POLYPHENOLS IN APPLES
Chemical structure of a catechin, b epicatechin, c procyanidin B1 and d procyanidin B2
The condensation of this two monomers results in oligomeric flavan-3-ols, which are called
procyanidins (ARON et al. 2008). The most important dimeric procyanidins in apples are
procyanidin B1 (PC B1, condensation of EC and C, Figure 2 c) and B2 (PC B2, condensation
of EC and EC, Figure 2 d). The online database Phenol Explorer, an internet database
summing up various food polyphenol contents, reports a range of 0-3.4 mg/100 g FM for C
and 1.8-19.2 mg/100 g FM for EC in whole dessert apples (NEVEU et al. 2010). Treutter at al.
(2001) reported a range of 1-8.4 mg/100 g FM for PC B1 and 1.5-23 mg/100 g FM for PC B2
in fresh apple fruits. The content in the apple skin ranged from 10-100 mg/100 g FM for PC
B2. Additionally, higher oligomeric procyanidins with an average degree of polymerisation of
5.7-7.1 are found in apples and they seem to be the most abundant polyphenols in apples
(GUYOT et al. 2002). However, the analysis of higher oligomeric flavan-3-ols is difficult due
to their high molecular weight (MANACH et al. 2004) and additionally their limited solubility
and resulting extractability. Moreover, the different extraction methods affect the
polymerisation degree and thus leads to different results (VRHOVSEK et al. 2004). Until now,
oligomeric procyanidins are determined by thiolysis-HPLC (GUYOT et al. 2001), but a simple
method for direct analysis is still lacking.
Monomeric and oligomeric flavan-3-ols are present in both flesh and skin, but higher amounts
are quantified in the skin (TSAO et al. 2003). Polymeric flavan-3-ols are responsible for the
astringent taste of apples or other fruits and beverages (RENARD et al. 2007). The
21
LITERATURE REVIEW
POLYPHENOLS IN APPLES
procyanidins form complexes with salivary proteins, which are responsible for this
organoleptic sensation (MANACH et al. 2004). Moreover, this sensation seems to be dependent
on polymerisation degree; the higher the polymerisation degree, the more astringent the
sensation (VIDAL et al. 2003). The smaller procyanidins are reported to be predominantly
bitter and are responsible for the bitterness of for example dark chocolate (MANACH et al.
2004; RENARD et al. 2007). Another characteristic of flavan-3-ols is their contribution to
enzymatic browning. In intact plant tissues, the flavan-3-ols and the polyphenoloxidase are
separated. By disruption of the cell structure flavan-3-ols are oxidised by the
polyphenoloxidase and they polymerise to brown pigments, which reduced the flavan-3-ol
level but is requested at black tea fermentation (HOLLMAN et al. 2000).
2.2
Phenolic acids
Phenolic acids are phenols that possess one carboxylic acid group and belong to organic acids.
The two subclasses are hydroxycinnamic and hydroxybenzoic acids. The carbon skeleton is
either cinnamic acid or benzoic acid and due to hydroxylation at the aromatic ring, the
different phenolic acids are built (ROBBINS 2003). For example, p-coumaric acid contains
only one hydroxyl group, whereas caffeic acid posses an additional one at the neighbouring C
atom in the aromatic ring. In apples, only the hydroxycinnamic acids are relevant. The main
phenolic acid in apples is chlorogenic acid (CA, see Figure 3), the ester of caffeic acid (CF)
and quinic acid (QA). p-Coumaroylquinic acid (CQA) is the other phenolic acid in apples,
and isomers of both CA and CQA occur only in low amounts (TREUTTER 2001). The
concentration ranges reported in whole apple fruits are from 3-43 mg/100 g FM for CA and
from 0.3-3.5 mg/100 g FM for CQA. These values are comparable to the amounts measured
in the apple skin: 0.1-44 mg/100 g FM for CA and 0.54-3.9 mg/100 g FM for CQA
(TREUTTER 2001).
Figure 3:
Chemical structure of chlorogenic acid
Peeling of an apple consequently does not reduce the phenolic acid concentration per unit of
weight. Moreover, the main polyphenol in apple juice is CA independent of the polyphenol
22
LITERATURE REVIEW
POLYPHENOLS IN APPLES
profile (TREUTTER 2001) due to its good water solubility. As reported for flavan-3-ols, CA is
also a substrate of the polyphenoloxidase (TREUTTER 2001).
2.3
Dihydrochalcones
Dihydrochalcones are exclusively found in apples and apple products. As derivates of
phloretin (Phl, see Figure 4) their chemical structure includes an open ring (CROZIER et al.
2009). Major dihydrochalcones contained in apples are phloridzin (Phz, substituted with
glucose at the superior hydroxyl group in Figure 4) and phloretin-xyloglucoside (PXG,
xyloglucose at the superior hydroxyl group in Figure 4) (VRHOVSEK et al. 2004). Their
amount in apple fruits range from 0.3-16 mg/100 g FM for Phz and 1-23 mg/100 g FM for
PXG, whereas the levels in apple skin are reported to be higher with 0.6-65.4 mg/100 g FM
and 7-27 mg/100 g FM, respectively (TREUTTER 2001)
Figure 4:
Chemical structure of phloretin
Additionally, dihydrochalcones represent up to 60 % of the polyphenols in the seeds (TOMÁSBARBERÁN et al. 2000). Therefore, removing of the skin and the core area reduces the amount
of dihydrochalcones ingested with an apple. Moreover, bruising the fruit causes browning by
the action of polyphenoloxidase which leads to a reduction between 20 and 40 % in the
content of dihydrochalcones (TOMÁS-BARBERÁN et al. 2000).
2.4
Flavonols
Flavonols are a polyphenol group of minor importance in apples. They are glycosylated
quercetin (Q, see Figure 5) derivatives, whereby the glycosylation occurs in direct
neighbourhood of the ketone group. The major sugar attached is galactose but glucose,
xylose, arabinose, rhamnose and rhamnoglucose derivatives are also reported (TREUTTER
2001). The amounts in fresh apple fruits range from 1-92 mg/100 g FM for quercetingalactoside (QGal), from traces to 2 mg/100 g FM for quercetin-glucoside (QG), from 166 mg/100 g FM for quercetin-rhamnoside (QR) and from 1-19 mg/100 g FM for quercetinrhamnoglucoside, otherwise known as rutin (R). The values of the skin are for QGal 795 mg/100 g FM, QG traces to 20 mg/100 g FM, QR 6-66 mg/100 g FM and R 0.519 mg/100 g FM (TREUTTER 2001).
23
LITERATURE REVIEW
Figure 5:
POLYPHENOLS IN APPLES
Chemical structure of quercetin
Flavonol-glycosides are mainly located in the apple skin (TSAO et al. 2003), where their
biosynthesis is induced by light (D’ARCHIVIO et al. 2007). Consequently, the amounts found
in individual fruits of the same tree and also within one fruit can vary significantly depending
on sun exposure (MANACH et al. 2004). By peeling an apple, most of the quercetin-glycosides
are removed and therefore the intake of apple flavonols depends on the way of consumption.
Due to the glycosylation at the described position, no oxidation by polyphenoloxidase occurs
because of steric hindrance (VAN
DER
SLUIS et al. 2002). Consequently, only removing the
skin leads to losses of flavonols in apples.
24
LITERATURE REVIEW
2.5
POLYPHENOLS IN APPLES
Polyphenol content of apples and apple juices
2.5.1
Apples
As mentioned above, a whole apple contains the individual polyphenols in different
concentrations. Table 1 summarises their mean values obtained in 5 experiments on dessert
apples and compares them to the data given by the Phenol Explorer, (NEVEU et al. 2010).
Table 1:
reference
number of
cultivars
flavan-3-ols
Mean values and ranges (in brackets) of individual polyphenol contents of apples as
reported in literature and the Phenol Explorer (NEVEU et al. 2010) [mg/100 g FM]
(NEVEU et
al. 2010)
between 1036
(VALAVANIDIS
et al. 2009)
(WOJDYLO
et al. 2008)
(VRHOVSEK
et al. 2004)
(LEE et al.
2003)
(PODSEDEK
et al. 2000)
4
67
5
6
10
C
1.2 (0-3)1
1.8 (1-3)
11.7 (1-72)
1.0 (0-2)
1.0 (0-2)
EC 8.3 (2-19)2
9.3 (7-15)
58.5 (7-276)
6.6 (5-11)
8.7 (2-19)
6.0 (2-9)
PC B1
2.3 (0-6)
14.3 (2-61)
PC B2 14.6 (1-38)3
7.4 (6-10)
60.4 (7-200)
6.5 (6-8)
9.4 (3-22)
2.3 (1-5)
phenolic acids
CA 13.3 (2-43)2
7.1 (4-11)
46.9 (1-296)
8.4 (4-12)
9.0 (4-14)
17.3 (3-43)
CQA
2.3 (0-7)4
5.1 (0.4-26)
1.7 (1-2)
dihydrochalcone
s
Phz
2.8 (1-9)5
3.8 (1-10)
10.1 (0-135)
1.6 (1-3)
5.6 (1-3)
PXG
2.6 (1-8)6
1.0 (1-2)
flavonols
QGal
2.4 (1-5)7
2.7 (1-4)
17.1 (1-54)
2.7 (1-4)
QG
0.6 (0-2)7
4.6 (0.2-21)
0.6 (0-1)
QR
1.3 (1-5)7
12.0 (2-69)
0.8 (0-1)
R
0.2 (0-1)8
0.9 (0-9)
0.2 (0-1)
1
number of cultivars: 30, 2number of cultivars: 36, 3number of cultivars: 24, 4number of cultivars: 10, 5number of
cultivars: 20, 6number of cultivars: 15, 7number of cultivars: 18, 8number of cultivars: 13
As indicated by Table 1, the highest amounts of individual polyphenols in apples are reached
by EC, PC B2 and CA. PC B2 shows comparable levels to EC and they seem to be related in
apples during polyphenol synthesis. A comparable relationship could be found for C and PC
B1, both with lower values than the other flavan-3-ols. The PC B2 value of PODSEDEK et al.
(2000) is an exception as it is lower compared to the other levels reported in Table 1. A reason
for this difference could be the lower water solubility of PC B2 compared to the other
polyphenols investigated, because these authors used concentrated aqueous solutions for
analysis, whereas all other authors used at least 50 % organic solvents. In Table 1, the mean
value of the three most important polyphenols, EC, PC B2 and CA vary considerably
compared to the other individual polyphenols. The mean values of WOJDYLO et al. (2008) in
Table 1 are high, due to the inclusion of dessert and cider apples and old and new cultivars
included in this investigation. Old cultivars, which are mostly cultivated for more than 75
years, have been reported to have a higher polyphenol content and cider apples showed
mainly higher polyphenol contents (WOJDYLO et al. 2008). For example, 5 French cider
25
LITERATURE REVIEW
POLYPHENOLS IN APPLES
cultivars reached mean values of 8.6 mg/100 g of C, 40.3 mg/100 g of EC, 28.0 mg/100 g of
PC B2, 64.4 mg/100 g of CA, 10.1 mg/100 g of CQA, 3.0 mg/100 g of Phz and 3.6 mg/100 g
of PXG (GUYOT et al. 2003). These values are higher than the values of Table 1 with
exception of the data of WOJDYLo et al. (2008) and are responsible for the specific
organoleptic characteristic of cider apples and render them inappropriate for the usage as
dessert apples.
During the last 13 years, a large number of studies reporting polyphenol concentrations of
apples has been published (ALONSO-SALCES et al. 2004a; ALONSO-SALCES et al. 2004b;
ALONSO-SALCES et al. 2005; ALONSO-SALCES et al. 2006; ARTS et al. 2000a; AWAD et al.
2000; GUYOT et al. 2002; GUYOT et al. 2003; HECKE et al. 2006; HYSON 2011; KAHLE et al.
2005a; KHANIZADEH et al. 2008; KUMAZAWA et al. 2007; LAMPERI et al. 2008; LEE et al.
2003; MANGAS et al. 1999; MARI et al. 2010; MCGHIE et al. 2005; NAPOLITANO et al. 2004;
OSZMIANSKI et al. 2007; PODSEDEK et al. 2000; RENARD et al. 2007; SANONER et al. 1999;
TSAO et al. 2003; VALAVANIDIS et al. 2009;
VAN DER SLUIS
et al. 2001;
VAN DER SLUIS
et al.
2004; VEBERIC et al. 2005; VRHOVSEK et al. 2004; WOJDYLO et al. 2008; WU et al. 2007).
However, a comparison of the studies remains difficult due to the different methods used. For
example, some authors investigated apple skin and apple flesh separately and other reported
data of freeze dried samples. Additionally, the cultivars investigated were common dessert
apple cultivars or regionally cultivated ones. Clearly, many studies concerning polyphenol
contents in apples have already been made, however more investigations are needed which
compare the different cultivars with the same analytical technique. The Phenol Explorer
(NEVEU et al. 2010) is a useful tool to get an overview of potential polyphenol contents and
ranges, however, the analysis of individual cultivars remains necessary particulary if health
recommendations relevant to individual polyphenols are to be given.
Concerning peeled apples, flavan-3-ols, dihydrochalcones and flavonols are located in the
apple skin and were removed in the peeled apples as reported in Table 2. Also the individual
polyphenol contents of cvs. Golden, Granny, Braeburn and Jonagold indicated that by peeling
an apple, a main part of the polyphenols is removed. However, in the flesh of cider apples
also higher polyphenols contents are reported than in dessert apples. This knowledge could be
important for the evaluation of consumption data. The contribution of peeled apples to the
daily polyphenol intake is much less in comparison to whole apples.
26
LITERATURE REVIEW
Table 2:
POLYPHENOLS IN APPLES
Mean values of the Phenol Explorer and individual polyphenol contents of 4 dessert
apples divided in flesh and skin [mg/100 g FM]
cv. Braeburn2
cv. Jonagold3
skin
flesh
skin
flesh
skin
C
1.6
5.6
0.5
0.8
2.3
4.0
EC
6.7
28.7
5.9
12.4
9.6
17.0
PC B1
5.7
6.4
PC B2
10.0
19.6
7.2
15.0
13.4
24.1
phenolic acids
CA
18.2
49.3
8.4
5.5
3.5
0.9
CQA
1.4
3.0
1.4
0.9
0.3
0.3
dihydrochalcones
Phz
1.5
2.5
1.1
4.0
0.6
1.3
PXG
0.5
3.3
1.1
4.2
1.3
4.1
flavonols
QGal
0.2
0.01
QG
0.04
0.06
QR
0.2
0.22
1
(NEVEU et al. 2010), 2(GUYOT et al. 2002), 3(VAN DER SLUIS et al. 2001)
0.8
12.4
0.8
13.2
6.7
24.5
7.1
2.0
4.3
0.9
1.4
14.8
0.7
0.9
3.4
4.2
17.0
6.6
0
0
0.7
12.6
2.0
14.5
dessert
apples1
flesh
cider
apples1
flesh
cv. Golden2
flesh
skin
cv. Granny2
flesh
flavan-3-ols
2.5.2
Apple juices
In the literature, data are available concerning polyphenol content in apple juices (ALONSOSALCES et al. 2004a; ALONSO-SALCES et al. 2004b; GUYOT et al. 2003; KAHLE et al. 2005a;
OSZMIANSKI et al. 2007; SANONER et al. 1999;
VAN DER SLUIS
et al. 2001) and on the factors
influencing them during apple juice production (VAN DER SLUIS et al. 2004; VAN DER SLUIS et
al. 2002; VAN DER SLUIS et al. 2005).
The polyphenol content of apple juices differs from that of whole apples. The flavonol levels
are very low (Table 3 compared to Table 1), due to their location in the apple skin and due to
their low water solubility. On the other hand, phenolic acids are present in relatively high
amounts due to their location in the apple flesh and their good water solubility. Table 3 also
compares the differences in individual polyphenols in juices made from dessert or cider
apples. KAHLE et al. (2005a) reported higher amounts of all individual polyphenols in cider
apple juice, whereas the Phenol Explorer (NEVEU et al. 2010) reported similar or lower
amounts of PC B1, PC B2, Phz and the flavonols in cider apple juice. For most apple juices
the major polyphenol is CA (Table 3). However, this seems to be cultivar specific and there
are exceptions. EC and PC B2 were the predominant apple polyphenols in the apple cultivar
Champion (OSZMIANSKI et al. 2007) and also in the dessert apple juices reported in the Phenol
Explorer (NEVEU et al. 2010).
27
LITERATURE REVIEW
Table 3:
number of
cultivars
POLYPHENOLS IN APPLES
Mean values of individual polyphenol contents of apple juices as reported in literature
[mg/100 mL]
(NEVEU et al.
2010)
dessert
apple,
between 9-27
(NEVEU et
al. 2010)
cider apples,
between 4137
(GUYOT et
al. 2003)
5 cider
apples
(KAHLE et
al. 2005a)
4 dessert
apples
(KAHLE et
al. 2005a)
7 cider
apples
(OSZMIANSKI
et al. 2007)
1, cloudy
juice
flavan-3-ols
C
2.01
4.68
8.6
0.4
1.7
14.7
1
EC
7.8
9.09
23.6
3.7
8.1
118.8
PC B1
0.92
0.710
0.4
0.7
29.3
1
PC B2
7.9
6.611
19.6
3.8
6.3
149.6
phenolic acids
CA
7.03
21.512
61.4
4.5
27.3
83.3
4
CQA
0.8
2.613
12.0
1.4
5.8
16.0
dihydrochalcones
Phz
2.45
1.512
3.3
0.7
3.5
16.0
6
PXG
1.5
3.311
5.4
1.1
5.7
17.3
flavonols
QGal
0.27
0.214
traces
0.4
2.5
7
QG
0.08
0.0914
traces
0.1
1.6
QR
0.72
0.214
0.1
0.3
3.8
1
number of cultivars: 11, 2number of cultivars: 9, 3number of cultivars: 22, 4number of cultivars: 16, 5number of
cultivars: 27, 6number of cultivars: 25, 7number of cultivars: 20, 8number of cultivars: 49, 9number of cultivars:
137, 10number of cultivars: 7, 11number of cultivars: 124, 12number of cultivars: 132, 13number of cultivars: 4,
14
number of cultivars: 36
2.6
Pre-harvest factors influencing the polyphenol content and profile of apples
The polyphenol content of apples depends on their biosynthesis, which can be influenced by
light, nutrition of the plant, rootstock (TREUTTER 2010), crop load, production method,
growing season, growing region, cultivar and mutant variety. However, at the present time,
the influence of pre-harvest factors on polyphenol profiles has only been investigated in
relation to growing season, and the impact of the other pre-harvest factors on polyphenol
profile remains unknown.
Beside the cultivar, the influence of the mutant variety has been investigated, but the authors
failed to show significant differences (AWAD et al. 2000; SILVEIRA et al. 2007). Therefore, the
potential of mutation to increase the polyphenol content needs further investigations.
The influence of light on apple polyphenols was investigated in cv. Jonagold apples.
Measurements of light levels at the top, the outside of the canopy and the canopy interior
showed a direct correlation to the quercetin glycoside levels in the apples. C, CA and Phz
contents were not influenced by the position on the tree and consequently by the light (AWAD
et al. 2001b). These results are in accordance with the findings of DʼARCHIVIO et al. (2007)
who reported that the biosynthesis of quercetin glycosides is induced by light (see 2.4).
28
LITERATURE REVIEW
POLYPHENOLS IN APPLES
Additionally, the elevated light level provides more energy for carbon assimilation and thus
more carbon resources for biosynthesis of polyphenols (TREUTTER 2010).
Experiments with cv. Elstar apples indicated an influence of nutrition of the tree. The
concentration of N and Mg were negatively correlated with total flavonoid concentrations.
The greatest influence was detected for N concentrations on flavonols, catechins and to a
lesser extent on Phz, whereas CA remains unaffected (AWAD et al. 2002). The explanation of
the influence of plant nutrition seems to be the influence of certain nutrients on biosynthetic
enzymes (TREUTTER 2010).
For optimising fertility, fruit yield and for high fruit quality, specific scion/rootstock
combinations have been developed over centuries of fruit growing (TREUTTER 2010). MAINLA
et al (2011) reported differences between different rootstocks for 6 Estonian apple cultivars.
An explanation for this effect could be the variation of nutrient supply to the fruit, changes in
the hormonal balance and potentially elevated metabolic stress for the tree (TREUTTER 2010).
Generally, elevated metabolic stress results in increased polyphenol content (TREUTTER
2005).
The influence of crop load on polyphenol content was tested in the skin of the cvs. Jonagold
and Red Elstar. The fruit weight inversely correlated with the crop load, but the polyphenol
content in the skin of the apples failed to show significant differences (AWAD et al. 2001a).
This result could be dependent on the analytical method and the evaluation of the skin only,
because STOPAR et al. (2002) reported significant differences in the cortex with skin of cv.
Jonagold. High crop load resulted in low polyphenol contents and vice versa. This could be
due to fruits with higher fruit weight in the low crop load that leads to a dilution of
polyphenols during ripening of the fruit (RENARD et al. 2007).
The influence of the production method has been repeatedly investigated. The elevated
metabolic stress in organically produced fruits was described as mechanism behind this factor.
This is due to the fact that a reduced plant protection strategy forces the plant to protect itself
by increasing the polyphenol content (VEBERIC et al. 2005). HECKE et al. (2006) and VEBERIC
et al. (2005) compared organically produced apples with conventionally produced apples of
different cultivars. Both groups reported higher polyphenol contents in organically produced
cultivars. In contrast, authors comparing the same cultivars produced with both methods
detected no difference in polyphenol contents (LAMPERI et al. 2008; VALAVANIDIS et al.
2009). In one study, significantly higher polyphenol concentrations in organically produced
29
LITERATURE REVIEW
POLYPHENOLS IN APPLES
compared to conventionally grown fruits were reported for cv. Golden Delicious but only in
one of the three growing years (STRACKE et al. 2009). Another group reported significantly
higher amounts of polyphenols in apples produced with integrated production (economical
production of high quality with priority to ecologically safe methods) compared to organically
grown fruits (CHINNICI et al. 2004a). In summary, the production method appears to have only
a small impact on the polyphenol content. Moreover, metabolic stress seems also to be
relevant for conventionally produced fruits. For a general conclusion concerning production
method, further investigations are needed.
Another influencing factor for the polyphenol content is the growing season. Various authors
detected significant differences between the polyphenol content of the different growing
seasons (KEVERS et al. 2011; LATA et al. 2005; MAINLA et al. 2011; STRACKE et al. 2009).
LATA et al (2007) and
VAN DER
SLUIS et al. (2001) reported a cultivar dependent influence.
The 19 cultivars investigated by LATA et al. (2007) showed higher or lower polyphenol
contents in 2005 compared to 2004, only 6 cultivars produced similar amounts in both years.
In the trial of VAN DER SLUIS et al. (2001), the polyphenol content of cv. Cox’s Orange varied
more between three years compared to cvs. Jonagold, Golden Delicious and Elstar. However,
according to GUYOT et al. (2003) the polyphenol profile remains constant between the
different growing seasons. The reason for variation in polyphenol content appear to be the
weather and consequently the influence of light, water and nutrient supply. As mentioned
above, light induces the biosynthesis of flavonols and mineral nutrition plays a role for the
biosynthetic enzymes. Sufficient water supply may result in fruits with higher fruit weight; in
arid years the fruit weight may be reduced including a lower dilution of the polyphenols in
fruits, in contrast to a high fruit weight with a greater dilution.
In addition to the growing season, the growing region has an impact on the polyphenol
content in apples. As determined in different regions in New Zealand (MCGHIE et al. 2005),
Italy (LAMPERI et al. 2008) and Chile (YURI et al. 2009), the growing region has an influence
on the polyphenol content in apple fruits. However, this influence was cultivar dependent
(MCGHIE et al. 2005) or detectable only in the apple skin (LAMPERI et al. 2008). In Chile, this
effect appears to be latitude dependent, due to different light conditions (YURI et al. 2009).
Therefore, metabolic stress seems to be also responsible. Nevertheless, it seems possible that
each cultivar needs specific growing conditions in order to develop the maximum amounts of
polyphenols.
30
LITERATURE REVIEW
POLYPHENOLS IN APPLES
In summary, for predicting polyphenol contents in apples, it is necessary to know the cultivar
and additionally the other pre-harvest factors, although their influence on polyphenol content
is smaller than for the cultivar. However, based on the trials published so far, it would seem
necessary that production conditions should be controlled if apples are to be used for human
intervention studies investigating health effects and that polyphenol content should be directly
measured.
31
LITERATURE REVIEW
3
BIOACCESSIBILITY OF APPLE POLYPHENOLS
Bioaccessibility of apple polyphenols
Polyphenols in apples are stored in the vacuole within the cell structure (TREUTTER 2010), so
they have to be released before being absorbed, metabolised and possibly showing any
beneficial health effect. Therefore, the term bioaccessibility is defined as the amount of an
ingested nutrient that reaches the enterocytes in a form suitable for absorption (PALAFOXCARLOS et al. 2011; SCHOLZ et al. 2007). In clear apple juice, all polyphenols are in solution
and should therefore be potentially absorbable or 100 % bioaccessible (PALAFOX-CARLOS et
al. 2011). With fresh apples, a release of polyphenols from the matrix is necessary and this
would be expected to occur during mastication of the fruit as the chewing process should
break down cell structures and the polyphenols should be released from the vacuole.
However, the extent to which they are released by mastication and further by carbohydrate
and protein digestion in the mouth, stomach and small intestine needs further investigations.
In the following section, the in vitro bioaccessible fraction, i.e. the amount solubilised in
simulated gastrointestinal media, is discussed in relation to its ability to predict
bioaccessibility in humans. The influence of different polyphenol containing food matrices
and the effect of other food components being simultaneously ingested with polyphenol
containing food on polyphenol solubility and bioaccessibility is also discussed.
In Table 4, current literature using in vitro digestion methods as a tool for estimating the
bioaccessibility is summarised. All authors used pepsin and a pH of 2 for 1 or 2 h for
simulating the gastric phase and pancreatin and bile salts (exceptions indicated in Table 4) at
neutral pH for simulating conditions in the small intestine. Moreover, the duration of the
intestinal digestion varied between 2 and 6 h. Only BERMUDEZ-SOTO et al. (2007) digested the
sample under absence of light and oxygen. In these in vitro experiments, bioaccessibility was
defined as the amount being released from the matrix and quantified as the amount of
polyphenols in solution in the supernatants either globally by photometric methods (BOUAYED
et al. 2011; FOGLIANO et al. 2011) or by HPLC-diode array detection (DAD) (BERMÚDEZSOTO et al. 2007; TARKO et al. 2009). In contrast, HERVERT-HERNANDEZ et al. (2010) and
RODRIGUEZ-MATEOS et al. (2012) analysed the amount of insoluble polyphenols in the
digestion residue photometrically to calculate the release and therefore provided no
information about the intact polyphenols in the supernatant.
Overall, bioaccessibility values analysed by Folin-Ciocalteu were rather high with three
quarters released from dried pepper and cactus pear pulp (Table 4), whereas FOGLIANO et al.
(2011) recovered less than half of the amount of polyphenols present in water-insoluble
32
LITERATURE REVIEW
BIOACCESSIBILITY OF APPLE POLYPHENOLS
cocoa. A reason for this difference could be the shorter in vitro digestion time (3 h versus
7.5 h). However, these studies offer no information about the composition of the recoveries,
for example the amount being released, the amount being degraded and epimerised. HPLCDAD provides more detailed data concerning the fate of individual polyphenols or polyphenol
groups. As expected, polyphenols of chokeberry juice were completely recovered after
simulated gastric digestion (Table 4), but after intestinal digestion the recoveries decreased for
some polyphenol groups (BERMÚDEZ-SOTO et al. 2007) presumably due to degradation. No
changes were found for phenolic acids, very likely due to their good water solubility and
stability in neutral pH. The recovery of flavan-3-ols was greater than for flavonols perhaps
because the flavan-3-ols recovered in the simulated small intestine were a combination of
released flavan-3-ols and degradation products of oligomeric procyanidins. The low
degradation of flavonols (26 %) in this study could be explained by the absence of light and
oxygen.
In Table 4, the two in vitro studies with apples used a digestion system which simulated the
pancreatic phase with the aid of a dialysis bag to adjust the pH and the estimated
bioaccessibility (BOUAYED et al. 2011; TARKO et al. 2009). The bioaccessible fraction
determined by photometric methods were higher (mean 65 %) after simulation of gastric
digestion than after simulated gastric followed by small intestine conditions (mean 55 %)
(BOUAYED et al. 2011), indicating some degradation or modification of the substances
reacting photometrically as polyphenols. However, TARKO et al. (2009) reported a
considerably lower mean recovery (27 %) and a high variability in the recovery between the
single polyphenols by HPLC-diode array detection. The recoveries reported in Table 4
showed high differences but it was estimated that overall approximately 50 % of the
polyphenols are bioaccessible. This is in accordance with LAPARRA et al. (2008), who
reported a mean bioaccessibility of 54 % of mainly flavonols in beans and with SAURACALIXTO et al. (2007), who investigated the bioaccessibility of polyphenols in a whole diet.
The latter concluded that 48 % of all dietary polyphenols from solid vegetable foods are
bioaccessible in the small intestine and a further 42 % become accessible in the large
intestine.
33
LITERATURE REVIEW
Table 4:
BIOACCESSIBILITY OF APPLE POLYPHENOLS
In vitro digestion studies investigating the bioaccessibility of polyphenols in different
food sources
Source
duration
analytical method
quantified in:
bioaccessibility
hot dried pepper1
7.5 ha
Folin-Ciocalteu
digestion residue
75 %
cactus pear pulp2
7.5 ha
Folin-Ciocalteu
digestion residue
76 %
water-insoluble cocoa
fraction3
3 ha, d
Folin-Ciocalteu
supernatant
38.6 %
chokeberry juice4
2 hb, e
HPLC-DAD
supernatant
~100 %
c, e
2h
flavan-3-ols: 81 %
phenolic acids: 100 %
flavonols: 74 %
apple5
1 hb
Folin-Ciocalteu
supernatant
colourimetric assay
with AlCl3
2 h 45 mind, f
Folin-Ciocalteu
total flavonoids: ~65 %
dialysate
colourimetric assay
with AlCl3
apple cv. Malinowka6
6 ha, d, f
HPLC-DAD
~65 %
55 %
total flavonoids: 44 %
dialysate
C: 90 %
EC: 9 %
PC B1: 70 %
PC B2: 20 %
CA: 0 %
CQA: 14 %
Phz: 30 %
PXG: 28 %
QGal: 39 %
QG: 0 %
QR: 0 %
(HERVERT-HERNANDEZ et al. 2010), 2(RAMIREZ-MORENO et al. 2011), 3(FOGLIANO et al. 2011), 4(BERMÚDEZSOTO et al. 2007), 5(BOUAYED et al. 2011), 6(TARKO et al. 2009)
a
gastric followed by pancreatic phase, b gastric phase, c pancreatic phase, d pancreatic phase without bile salts, e
absence of light and O2, f with dialysis
1
Nevertheless, details what exactly occurs to apple polyphenols during the passage of the
stomach and the small intestine remain unclear. Most authors measured only the total amount
of polyphenol compounds reacting photometrically or the polyphenol content at the end of the
simulated digestion. SPENCER at al. (2000) and ZHU et al. (2002) reported a degradation of
cocoa procyanidins to monomers in a simulated stomach digestion and an additional
epimerization of flavan-3-ols. In an in vivo study (RIOS et al. 2002), however, the cocoa
34
LITERATURE REVIEW
BIOACCESSIBILITY OF APPLE POLYPHENOLS
flavan-3-ols were stable during the passage of the stomach, which could be due to the absence
of light and oxygen under in vivo conditions. Therefore, the utility of the in vitro data to
predict in vivo bioaccessibility is difficult to evaluate with the studies made so far. However,
optimizing the in vitro digestion methods, for example the exclusion of light and oxygen, may
help to mimic the in vivo conditions and may better approximate in vivo bioaccessibility.
So far, studies investigating the influence of processing on bioaccessibility of apple
polyphenols are not available. However, OGHBAEI et al. (2012) examined the influence of
sieving of finger millet flour on polyphenol bioaccessibility. During this process, the fibre
content is reduced. Polyphenol and flavonoid bioaccessibility were increased compared to
whole flour, although the absolute amount of polyphenols was reduced by sieving. This
demonstrates that processing may have an important influence on bioaccessibility and the
further bioavailability.
In chocolate, the production process supports the release of polyphenols from the cocoa bean
matrix and cocoa polyphenols seem to be completely released from the matrix. However, the
concurrent administration of fat or carbohydrates is reported to have no influence on the
solubility of polyphenols and consequently on their bioaccessibility (ORTEGA et al. 2009). In
an in vitro digestion of cocoa liquor, the basic product for producing cocoa powder (≈ 50 %
fat), and partially defatted cocoa powder (≈ 15 % fat) showed no influence on
bioaccessibility. In contrast, variability in bioaccessibility of the different polyphenol groups
was observed, very likely related to differences in their hydrophilic nature. For phenolic acids,
bioaccessibility was higher than for procyanidins and the more lipophilic flavone aglycones
had a low bioaccessibility due to their higher retention in the pellet and lower water solubility
(ORTEGA et al. 2009).
SCHOLZ et al. (2007) reviewed the factors affecting bioaccessibility and bioavailability of
polyphenols in vivo. They concluded that bioaccessibility is mostly influenced by solubility
and dependent on the sugar moieties attached to the polyphenol. However, the authors
reviewed the different polyphenol groups, but further informative details concerning the fate
of monomeric and dimeric flavan-3-ols were not discussed. Additionally, ethanol was
reported to enhance solubility in Q containing drinks. The ethanol containing white wine led
to an increased bioavailability compared to grape juice and vegetable juice and the authors
related this effect to a better solubility of Q in ethanol and consequently a higher
bioaccessibility (GOLDBERG et al. 2003). However, for flavan-3-ols, phenolic acids and
35
LITERATURE REVIEW
BIOACCESSIBILITY OF APPLE POLYPHENOLS
dihydrochalcones, the influence of ethanol may be less significant due to their good water
solubility.
In summary, bioaccessibility represents the amount of a compound reaching the enterocytes
(SCHOLZ et al. 2007) and being available for absorption. The polyphenols have to be released
from the matrix, i.e. plant cell structures have to be loosened or even destroyed to render them
solubilised in the digestion fluids. Due to the differences in water solubility of the polyphenol
groups and the varying characteristics of food matrices, the estimation of the bioaccessibility
of apple polyphenols needs further examination.
36
LITERATURE REVIEW
4
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
Fate of apple polyphenols in the human body
Bioavailability related to polyphenols is a term often used in literature to define the amount of
polyphenols potentially available in the human body to evoke beneficial health effects. The
US Food and Drug Administration defined bioavailability of drugs as “the rate and extent to
which the active ingredient or active moiety is absorbed from a drug product and becomes
available at the site of action” (FDA 2003). Bioavailability is the amount of substance found
in urine according to PRASAIN et al. (2007). However, the problem of this definition is the
underestimation of polyphenols being excreted via bile back to the intestinal lumen. This
fraction and also unabsorbed polyphenols reach the colon, where they are degraded by colonic
microflora and the resulting metabolites are absorbed and excreted via urine (NEILSON et al.
2011). Microbial metabolites, however, are often phenolic acids and difficult to differentiate
from other break down products of non phenolic food components excreted in urine.
Therefore, urinary excretion of intact polyphenols or polyphenols conjugated with
glucuronides, methylates or sulphates appear to be a reliable method to determine polyphenol
bioavailability, but this method underestimates the amount of polyphenols leaving the
digestive tract. Furthermore, beside urine, intact and conjugated polyphenols are also excreted
via faecal water (GILL et al. 2010; JENNER et al. 2005; MATEOS-MARTIN et al. 2012), but to a
very low extend (<5 % of the intake)(JENNER et al. 2005; WALLE et al. 2001). Due to the
described issues for estimating bioavailability, in this chapter, the term “bioavailability”
includes all forms of polyphenols leaving the digestive tract as original polyphenols or as
metabolites. Consequently, the amount entering cells and tissues are described as “fractional
absorption”.
The following subchapters cover only the bioavailability of apple polyphenols. Due to rare
data about polyphenol absorption from apples, studies in rats and humans dealing with tea,
coffee, cocoa and other foods are included. Figure 6 to Figure 9 and Table 5 to Table 7
represent a summary of metabolism and absorption data extracted from various published
articles. Metabolites found in the small intestine are summarised from data of in situ perfusion
models and studies with ileostomy volunteers. Moreover, the polyphenols are discussed
group-wise, but the amount of published studies strongly varies between the groups. The fate
of the polyphenols during passage along the digestive tract is described. Additionally,
subchapter 4.5 compares the absorption and metabolism of the different classes of apple
polyphenol groups.
37
LITERATURE REVIEW
4.1
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
Absorption and metabolism of flavan-3-ols
The fate of C, EC, PC B1 and B2 in the digestive tract is summarised in Figure 6 and Table 5
based on literature. Ingested C and EC are absorbed by passive diffusion (DONOVAN et al.
2001; DONOVAN et al. 2006) into enterocytes, where they are metabolised and transported into
the blood circulation or back into the intestinal lumen. Via blood, C and EC and their
metabolites reach the different tissues, liver (URPI-SARDA et al. 2010) and kidney. In all
organs further metabolism may occur. Excretion of a single ingested dose is completed in
24 h (MANACH et al. 1999; ROURA et al. 2007) by urinary excretion or flux back into the
intestinal lumen via bile (ZHANG et al. 2007). These monomeric flavan-3-ols reach the colon
and are metabolised by colonic microbiota or excreted in the stool (MATEOS-MARTIN et al.
2012). The fate of PC B1 and B2 is rather unclear. They may be absorbed and metabolised in
the similar way as monomeric flavan-3-ols, based on the corresponding analysis of these
substances in body fluids. However, cleavage of the procyanidins into the monomers also
seems possible. Nevertheless, most of the procyanidins reach the colon and are further
metabolised by colonic microflora by ring fission and degradation to phenolic acids that can
be detected in urine or stool (STOUPI et al. 2010; YANG et al. 2008).
Different opinions concerning the stability of procyanidins in gastric milieu exist in
literature. SPENCER et al. (2000) and ZHU et al. (2002) reported a degradation of the dimers
into monomers in simulated gastric juice. In contrast, RIOS et al. (2002) investigated the fate
of cocoa procyanidins in humans with a nasogastric tube and collected the stomach content
every 10 min. Ingested with a cocoa matrix, the procyanidins remained stable during the
passage through the stomach. Moreover, the absence of oxygen in vivo may have an influence
on the higher recoveries. Consequently, extrapolation of in vitro to in vivo results appears
difficult and the influence of the matrix needs further investigations. However, it could be
possible that the matrix has a buffering effect on procyanidins which allows procyanidins to
reach the small intestine in an intact form.
38
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
ENTEROCYTE
INTESTINAL
LUMEN
C
BLOOD
TISSUE
E
C
C
E
E
SULT
E
COMT
C
E
E
LIVER
UGT
E
E
C
E
C
C
C
E
C
E
E
E
C
E
E
C
E
C
URINE
KIDNEY
C
E
E
MM
COLON
E
E
C
E
MICROFLORA
E
C
E
C
C
E
E
E
MM
E
C
C
E
E
E
COMT
SULT
UGT
epicatechin
catechin
procyanidin B1
procyanidin B2
catechol-O-methyl-transferase
sulfotransferase
UDP-glucuronosyltransferase
Figure 6:
C
E
C
E
E
C
C
MM
epicatechin/catechin metabolites in intestine
epicatechin/catechin metabolites in plasma
procyanidin B1/B2 metabolites in plasma
epicatechin/catechin metabolites in urine
microbial metabolites (γ-valerolactones,
phenylproprionic, phenylacetic ,
phenylvaleric, benzoic, hippuric acids,
valerolactone-glucuronides, -sulfates, methyl-sulfates)
microflora
potential cleavage
Metabolism and absorption of flavan-3-ols. Symbols represent flavan-3-ols and their
metabolites (for details see Table 5) found in the different body fluids based on
literature (BABA et al. 2000; BORGES et al. 2010; CRESPY et al. 2003; DEL RIO et al.
2010; DONOVAN et al. 1999; DONOVAN et al. 2001; DUPONT et al. 2002; HOLT et al.
2002; KUHNLE et al. 2000; KWIK-URIBE et al. 2008; MANACH et al. 1999; MATEOSMARTIN et al. 2012; MONAGAS et al. 2010; NEILSON et al. 2011; OTTAVIANI et al.
2012; OTTAVIANI et al. 2011; RIOS et al. 2002; SANO et al. 2003; SERRA et al. 2010;
STALMACH et al. 2009; STALMACH et al. 2010b; STOUPI et al. 2010; URPI-SARDA et al.
2009; URPI-SARDA et al. 2010; WILLIAMSON et al. 2010; WILLIAMSON et al. 2011; ZHU
et al. 2002). Dotted lines show possible metabolic routes and metabolites.
Once absorbed into the enterocytes of the small intestine, C and EC are metabolised by
enterocyte enzymes. The metabolites can be transported to the blood circulation or back to the
luminal side, which was shown by analysis of ileostomy effluent after ingestion of 300 ml of
green tea (STALMACH et al. 2010b). Metabolites are mainly glucuronides or methylates
(KUHNLE et al. 2000; MONAGAS et al. 2010) due to the presence of phase II metabolism in the
enterocytes of the small intestine. Sulphated metabolites of C and EC (see Table 5) were also
reported in ileal effluents (STALMACH et al. 2010b) and may be derived from further
metabolism in the liver and transport back by bile into the small intestinal lumen (DONOVAN
39
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
et al. 2001; MONAGAS et al. 2010). Therefore, the recovery of C and EC and their metabolites
in ileal effluents represents a combination of unabsorbed, absorbed and re-excreted via the
enterocytes, as well as absorbed and re-excreted via the bile and is thus not an adequate tool
for the calculation of absorption rates. However, STALMACH et al. (2010a) reported that 70 %
of the ingested green tea flavan-3-ols were found in human ileostomy effluent, whereof 33 %
were parent compounds and 37 % metabolites. The values obtained from ileostomy patients
after ingesting 1 L of a cloudy apple juice resulted in no recovery for C, PC B1 and B2, but
17.4 % EC (KAHLE et al. 2005b). Regarding these two studies, it appears possible, that
monomeric and dimeric flavan-3-ols are absorbed in the small intestine, whereas the higher
oligomeric procyanidins found in green tea are scarcely absorbed or re-circulated back into
the intestinal lumen. The high levels of EC in ileostomy effluent could be derived from
breakdown products of oligomeric procyanidins (see Chapter 3) or from low absorption levels
of EC, although this contrasts with the high plasma levels of EC reported below. NEILSON et
al. (2011) reviewed that the absorption of flavan-3-ols from tea and cocoa were between 35 %
and 80 % of the ingested compounds and that 11-50 % of the absorbed polyphenols were recirculated back to intestine.
For the absorption of oligomeric flavan-3-ols, the Lipinski's rule of five (LIPINSKI et al. 2001)
seems appropriate. This rule describes the properties a molecule needs for absorption and
includes a molecular weight below 500 u for substances being absorbable in the small
intestine. Therefore, only procyanidin dimers and trimers could be directly absorbed (YANG et
al. 2008). Additionally, the cleavage of the interflavan bond of procyanidins is discussed as a
possibility. SPENCER et al. (2001) reported 96 % EC and only small amounts of the dimers on
the serosal side after rats’ small intestine perfusion of PC B2 and procyanidin B5. These
findings have not been confirmed by any other author until now.
BORGES et al. (2010) found no procyanidins in blood plasma after the intake of a polyphenol
rich juice drink in humans. In contrast, other authors have reported low levels of procyanidin
dimers in blood plasma in rats (GARCÍA-RAMÍREZ et al. 2006; SERRA et al. 2010) or in humans
after cocoa intake (HOLT et al. 2002) or after grape seed extract intake (SANO et al. 2003). A
recently published study (OTTAVIANI et al. 2012) investigated the plasma levels of monomeric
and dimeric/oligomeric (two to ten monomers) flavan-3-ols after ingestion of three different
test drinks in humans. The first drink contained only monomeric flavan-3-ols and resulted in
relatively high plasma levels (880 nmol/L) whereas the second drink contained only
dimeric/oligomeric procyanidins and led to approximately ten-fold lower plasma levels. The
40
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
third drink with monomeric and dimeric/oligomeric flavan-3-ols resulted in similar levels as
the first drink. For this drink, the highest PC B2 plasma level was given with approximately
4 nmol/L. The authors concluded that the absorption of oligomeric procyanidins occurs only
at low levels in humans, whereas the absorption of C and EC in the human body is
considerably higher. This finding concerning monomeric flavan-3-ols was also confirmed by
other authors (BABA et al. 2000; BORGES et al. 2010; DONOVAN et al. 1999; OTTAVIANI et al.
2011; SAHA et al. 2012; STALMACH et al. 2010a). As shown in Table 5, various metabolites of
C and EC have been identified. SERRA et al. (2010) suggested that with high doses of flavan3-ols, the metabolism mainly occurs in the liver, whereas at low doses the enterocytes were
responsible for the modification of polyphenols (SCALBERT et al. 2000). The two sites of
metabolism are indicated by the identification of metabolites of C and EC which have
undergone two or three different modifications (for example methyl-sulpha-glucuronides).
Peak plasma levels were reported to be between 0.8 and 2.2 h postprandial (STALMACH et al.
2010a).
Unabsorbed flavan-3-ols or flavan-3-ols re-excreted into the small intestine reach the colon
and are subject to microbial degradation. According to OLTHOF et al. (2003), green tea
catechins in humans are cleaved into γ-valerolactones by colonic microorganisms,
subsequently the γ-valerolactones are degraded to phenylproprionic acids and further
metabolised to benzoic acids and hippuric acid, that could be detected in urine. Monagas
(2010) reviewed the in vitro and in vivo studies conducted in this field and discussed the
metabolic pathway. Moreover, procyanidin dimers and EC have been reported to be excreted
with the stool of rats ingesting procyanidins in cinnamon (MATEOS-MARTIN et al. 2012). Also
a few microbial metabolites of procyanidins were found in the faeces, whereas in urine more
different metabolites were analysed. Therefore, the main excretion way of flavan-3-ols
appears to be the urine, which was quantified by STOUPI et al. (2010) to be 62 % of the
ingested dose of
14
C PC B2 in rats. In faeces, 41 % of the ingested radioactivity was
recovered, including intact PC B2 and microbial metabolites.
The microbial metabolites, mainly γ-valerolactones, phenylproprionic acid, phenylacetic acid,
phenylvaleric acid, benzoic acid and hippuric acids, can be absorbed in the colon, transported
via blood circulation and excreted via urine (URPI-SARDA et al. 2009). Additionally, γvalerolactone-glucuronides, -sulphates and -methyl-sulphates were reported in urine
(MONAGAS et al. 2010). In a rat experiment, 63 % of the oral 14C PC B2 dose was excreted in
urine by detecting radioactivity without examination of substances and metabolites (STOUPI et
41
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
al. 2010). C, EC and their metabolites were also discovered in urine (Table 5). The pattern of
metabolites is comparable to that of blood plasma with methylates, sulphates and
glucuronides and their combinations (BABA et al. 2000; OTTAVIANI et al. 2012; ROURA et al.
2007; SAHA et al. 2012; STALMACH et al. 2010a). PC B2 was found in low amounts in the
urine of humans after ingestion of cocoa or a test drink (OTTAVIANI et al. 2012; URPI-SARDA
et al. 2009). Consequently, the pattern of flavan-3-ol metabolites in urine is composed of
metabolites either directly absorbed, metabolised, and excreted in the urine or derived from
microbial degradation.
Table 5:
Summary of flavan-3-ol metabolites found in different body fluids; + represents
detected metabolite.
metabolite
in intestine
catechin
epicatechin
glucuronides
methylates
sulphates
methyl-glucuronides
methyl-sulphates
+
+
+
+
+
+
+
+
+
+
glucuronides
methylates
sulphates
methyl-glucuronides
methyl-sulphates
methyl-sulpho-glucuronides
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
procyanidin B1
procyanidin B2
in plasma
+
+
in urine
glucuronides
methylates
sulphates
methyl-glucuronides
methyl-sulphates
sulpho-glucuronides
methyl-sulpho-glucuronides
+
+
+
+
+
+
Although several studies have tried to estimate fractional absorption in humans, this is
complicated due to rapid metabolism in the enterocyte and other tissues, re-extraction into the
intestinal lumen via the enterocyte or bile, and colonic degradation. In a perfusion experiment
with isolated jejunum and ileum in rats, the net transfer of C across the brush border was 35 %
(CRESPY et al. 2003). Other authors have reported lower rates of passage between 17 and
26 % depending on perfusion concentration (DONOVAN et al. 2006). In humans, the fractional
absorption of the combination of C and EC was reported to be 25.2 % as measured in urine
after the intake of a polyphenol rich juice drink (BORGES et al. 2010). However, as the authors
42
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
state, the urinary calculation of absorption neglects other excretion routes such as via bile and
stool. BABA et al. (2000) reported similar absorption values from a human study after cocoa
ingestion, as did STALMACH et al. (2010a), and summarised that the fractional absorption of C
and EC as measured by appearance in the urine would appear to be between 25 % and 30 %.
It seems that up to 80 % of the monomeric flavan-3-ols can pass into the enterocytes but a
large fraction is excreted directly back into the lumen without passing into the blood
(NEILSON et al. 2011).
Additionally, the stereochemistry of C and EC is reported to be important for absorption
values (OTTAVIANI et al. 2011). Humans ingested a low-flavan-3-ol cocoa dairy drink
containing either (-)EC (contained in apples), (+)EC, (+)C (contained in apples) or (-)C. The
highest flavan-3-ol amounts were absorbed in the test drink only containing (-)EC, followed
by (+)EC and (+)C and the lowest absorption level was for (-)C. Comparable results were
reported with (-)C and (+)C after in situ perfusion of rats jejunum plus ileum (DONOVAN et al.
2006).
4.2
Absorption and metabolism of phenolic acids
The fate of CA, the main apple phenolic acid (see 2.2), and CQA in the digestive tract is
summarised in Figure 7 and Table 6 based on literature. Data dealing with CQA are rare and
only one study detected this compound after enzymatic treatment with glucuronidase and
sulphatase in plasma (0.17 % of the ingested dose) and urine (0.33 % of the ingested dose in
24 h) of humans ingesting apple juice (KAHLE et al. 2011). CA reaching the small intestine is
partly absorbed (about 30 %) by enterocytes, nearly completely metabolised within the
enterocyte and recovered in the intestinal lumen, blood plasma and urine as glucuronides and
sulphates (KAHLE et al. 2011; OLTHOF et al. 2003; RENOUF et al. 2010; STALMACH et al.
2010b; WILLIAMSON et al. 2011). The hydrolysis of CA in the small intestine results in CF (<
1 %) which reaches blood plasma after 0.5 h postprandial (LAFAY et al. 2006; MONTEIRO et
al. 2007), but the hydrolysis of CA by intestinal esterase (EST) is controversially discussed.
About two third of the ingested CA reaches the colon, where the gut microflora degrades and
metabolises the phenolic acids (GONTHIER et al. 2003; WILLIAMSON et al. 2011). Their
metabolites are also present in blood plasma at later time points, in urine and in the stool. The
sites of metabolism other than the microflora are still unknown. Possible sites are enterocytes,
liver, muscle tissue and kidney.
43
LITERATURE REVIEW
INTESTINAL
LUMEN
CQA
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
ENTEROCYTE
BLOOD
CA
TISSUE
CQA
CA
SULT
CF
LIVER
COMT
CA
CA
DHC
EST
CF
QA
UGT
MM
CF
DHC
CF
KIDNEY
URINE
CA
DHC
CF
CF
DHC
CQA
COLON
CA
CF
DHC
EST
MICROFLORA
COMT
QA
SULT
MM
UGT
CA
CF
QA
CQA
DHC
Figure 7:
MM
chlorogenic acid
caffeic acid
quinic acid
coumaroylquinic acid
dihydrocaffeic acid
CA
C
C
DHC
C
DHC
MM
Esterase
catechol-O-methyl-transferase
sulphotransferase
UDP-glucuronosyltransferase
chlorogenic/caffeic acid metabolites in intestine
caffeic/dihydrocaffeic acid metabolites in plasma
caffeic/dihydrocaffeic acid metabolites in urine
microbial metabolites (coumaric acids,
phenylproprionic acids, phenylacetic
acids, benzoic acids, hippuric acids)
microflora
Metabolism and absorption of phenolic acids. Symbols represent phenolic acids and
their metabolites found in the different body fluids based on results of literature
(GONTHIER et al. 2003; KAHLE et al. 2011; OLTHOF et al. 2003; RENOUF et al. 2010;
STALMACH et al. 2009; STALMACH et al. 2010b; WILLIAMSON et al. 2011). Dotted lines
show possible metabolic routes and metabolites.
The stomach is reported to be the earliest site of absorption of intact CA. The infusion of
buffered CA in rats’ stomach showed a fractional absorption of 16 % (LAFAY et al. 2006).
The transport via the bilitranslocase, which is involved in anthocyanidin transport, other
unidentified organic anion transporters, or the passive transport were suggested as possible
absorption mechanisms.
CA seems to be predominantly stable during passage through both stomach and small
intestine, because of the supposed absence of esterase activity (AZUMA et al. 2000; OLTHOF
et al. 2001). Accordingly, no activity to hydrolyse CA into CF and QA was reported by
ANDREASEN et al. (2001), although esterases which are able to hydrolyse hydroxycinnamates
have been detected along the intestine of other mammals (Figure 7).On the other hand, small
amounts of CF were found in the intestinal lumen (LAFAY et al. 2006; NARDINI et al. 2002;
STALMACH et al. 2010b) leading to the assumption that mechanisms for the hydrolysis of CA
44
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
exist in the intestinal lumen or directly in the enterocytes. This however would appear to be of
minor importance. Further metabolism of CA seems to occur in the enterocytes, since
metabolites of CA and CF have been reported in the intestinal lumen (Table 6) and based on
the sulphates, glucuronides and methyl-glucuronides detected in the intestinal lumen, it would
appear that the small intestine enterocytes contain sulphotransferase (SULT), UDPglucuronosyltransferase (UGT) and catechol-O-methyl-transferase (COMT) (STALMACH et al.
2010b), also described in Table 6. Additionally, a study with everted sacs of jejunum of rats
and Caco-2 cells reported comparable metabolites of dihydrocaffeic acid (DHC), when DHC
was gives as phenolic acid (POQUET et al. 2008b) confirming the presence of SULT, UGT and
COMT in the enterocytes and their ability to metabolise phenolic acids.
One third of the ingested CA is absorbed directly in the small intestine (OLTHOF et al. 2001;
STALMACH et al. 2010b; WILLIAMSON et al. 2011), whereas the remaining two thirds of the
ingested CA remain in the digestive tract and reach the colon (OLTHOF et al. 2001; STALMACH
et al. 2010b). There, colonic microflora first hydrolyses CA into CF and Q. CF is further
metabolised into DHC. All three metabolites, i.e. CF, QA and DHC are absorbed and further
metabolised by colonic epithelial cells, transported via blood circulation into the liver, kidney
and other tissues where further metabolism occurs (WILLIAMSON et al. 2011). According to
GONTHIER et al. (2003), the fraction of these metabolites in urine represents only 0.48 % of
the ingested CA. However, half of the CA (57 % of the intake) is reported to be metabolised
by the microflora and excreted in urine as coumaric acid, derivates of phenylproprionic,
phenylacetic, benzoic and hippuric acid (GONTHIER et al. 2003) (Figure 7). Furthermore, a
part of the microbial metabolites seem to be excreted via stool, because they have been
reported in the faeces of humans ingesting their normal diet (JENNER et al. 2005).
In plasma, most of the microbial metabolites cannot be assigned uniquely to phenolic acid
metabolism because of their ubiquity after food consumption. Polyphenol metabolites in the
plasma have often been identified after treatment with glucuronidase and sulphatase so as to
identify metabolites as free polyphenols. RENOUF et al. (2010) fed CA in form of instant
soluble coffee to human volunteers and determined plasma metabolites after treatment with
glucuronidase and sulphatase. This study and a similar experiment by WITTEMER et al. (2005)
with artichoke leaf extracts ingested by humans reported peak plasma levels for CF and
methylated CF after 1-2 h and an additional peak for DHC and methylated DHC at 6-8 h.
FUMEAUX et al. (2010) detected sulphates and methylated sulphates of CF and DHC directly
in the plasma of human volunteers after the ingestion of 200 mL instant coffee. Traces of
45
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
methyl glucuronides of CF and DHC were detected by STALMACH et al. (2009). Based on
these reports, it would appear that methylation and sulphation occurs mainly in small intestine
enterocytes, various tissues and the liver and that small amounts of glucuronides may be
produced in the kidney, resulting in a more rapid clearance from the blood circulation
(FUMEAUX et al. 2010). However, the exact site of metabolism is difficult to identify, because
of the fast metabolism of CA and its derivatives, the differences between human and rats and
the complexity of metabolites and their analysis. After coffee consumption, intact CA (<5 %
of the ingested dose) has also been reported to be present in plasma in addition to CF and
DHC metabolites (FARAH et al. 2008; MONTEIRO et al. 2007; STALMACH et al. 2009;
WILLIAMSON et al. 2011).
After CA consumption, urine has been reported to contain sulphates, methyl-glucuronides
and methyl-sulphates metabolites of CF and glucuronides, methylates, sulphates, methylsulphates and methyl-glucuronides of DHC (FUMEAUX et al. 2010; STALMACH et al. 2009)
(see Table 6). In addition, intact CA and CF were also detected in urine after coffee
consumption in humans (GONTHIER et al. 2003; ITO et al. 2005; KEMPF et al. 2010; MENNEN
et al. 2007; MONTEIRO et al. 2007) (Figure 7). The fact that only small amounts (<10 % of
ingested CA) of these metabolites were recovered indicates that renal excretion is not a major
pathway in humans for the excretion of CA derivates from the metabolism of absorbed CA
(CHOUDHURY et al. 1999; MONTEIRO et al. 2007). This contrasts to microbial degradation
products, which were excreted in urine at levels estimated to represent 57 % of the intake
(GONTHIER et al. 2003).
The estimated fractional absorption values of ingested CA reported in literature vary
widely, although there seems to be a consensus that intestinal absorption in humans is around
30 %. STALMACH et al. (2009) reported a urinary excretion of CA and its metabolites from
coffee which represents 29.1 % of the ingested dose in healthy humans, whereas GONTHIER et
al. (2003) calculated 57 % of pure substance intake, detected as microbial metabolites in urine
of rats, were excreted by this route. Comparable results (46 % of the intake) were obtained by
BORGES et al. (2010) after the consumption of a polyphenol rich juice drink by humans.
However, in ileostomy subjects, recovery of CA from apple juice in the small intestine was
only 10 % suggesting an absorption of 90 % (KAHLE et al. 2005b). In contrast, the fractional
absorption of pure CA in ileostomy subjects was lower with 33 % of the ingested dose
(OLTHOF et al. 2001). The effect of food matrix on the fractional absorption values was
investigated with milk in coffee. Although an interaction of milk proteins and CA has been
46
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
reported, the absorption values were not changed in the plasma of rats fed with coffee with or
without milk (DUPAS et al. 2006).
Table 6:
Summary of phenolic acid metabolites found in different body fluids; + represents
detected metabolite.
metabolite
in intestine
chlorogenic acid
glucuronides
sulphates
methyl-glucuronides
caffeic acid
+
+
dihydrocaffeic acid
+
+
in plasma
methylates
sulphates
methyl-glucuronides
methyl-sulphates
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
in urine
glucuronides
methylates
sulphates
methyl-glucuronides
methyl-sulphates
These large differences in estimated absorption may depend on the different food matrices,
amounts ingested, study designs and analytical methods. For example, different authors used
different models, which includes humans (STALMACH et al. 2010b) or rats (GONTHIER et al.
2003). Other authors used pure substances (OLTHOF et al. 2001) in contrast to apple juice
(KAHLE et al. 2005b), that contains more polyphenol groups than only phenolic acids.
Therefore the exact value of the CA bioavailability is difficult to estimate. As mentioned
before, WILLIAMSON et al. (2011) estimated the intestinal absorption rate of phenolic acids
from coffee to be about one third. The remaining two thirds are thought to be degraded by
colonic microflora and these metabolites can be also absorbed to some extent. The fractional
absorption of CA from apples has not been reported.
4.3
Absorption and metabolism of dihydrochalcones
The fate of apple dihydrochalcones in the small intestine is illustrated in Figure 8 and Table 7
based on literature. Data concerning metabolism or absorption in the stomach are not
available. Ingested Phz is hydrolysed by lactase-phloridzin-hydrolase (LPH) in the brush
border of the small intestine and the resulting Phl enters the enterocytes. After metabolism of
Phl to sulphates and glucuronides, non metabolised Phl and its metabolites have been
recovered in the intestinal lumen, blood plasma and urine. Un-absorbed and re-excreted Phl
reaches the colon and no information on metabolism or absorption of metabolites is available.
47
LITERATURE REVIEW
INTESTINAL
LUMEN
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
ENTEROCYTE
BLOOD
TISSUE
PXG
Phz
LPH
Phz
UGT
Phl
LIVER
Phl
SULT
ß-gly
Phl
Phz
PXG
Phl
KIDNEY
URINE
Phl
Phl
Phl
Phl
Phz
PXG
LPH
UGT
ß-gly
SULT
phloretin
phloridzin
phloretin-xyloglucoside
Lactase-phloridzin-hydrolase
UDP-glucuronosyltransferase
ß-glycosidase
sulphotransferase
Figure 8:
Phl
Phl
Phl
phloretin metabolites in intestine
phloretin metabolites in plasma
phloretin metabolites in urine
Metabolism and absorption of dihydrochalcones. Symbols represent dihydrochalcones
and their metabolites found in the different body fluids based on results of literature
(CRESPY et al. 2002a; CRESPY et al. 2001; DUPONT et al. 2002; MARKS et al. 2009)
Phl is partly absorbed in the small intestine. The perfusion of rats’ jejunum and ileum with a
Phl containing buffer solution showed 42 % of the perfused Phl in its intact form and 33 % as
glucuronides and sulphates at the end of perfusion, including a Phl net absorption of 25 % in
rats (CRESPY et al. 2001). For the perfusion with Phz, 19 % of the perfused dose was
recovered in its native form and 47 % as aglycone at the end of perfusion. For Phl, the net
absorption was higher (34 % of the perfused dose) (CRESPY et al. 2001). CRESPY et al. (2001)
reported LPH being responsible for the hydrolysis of Phz. This enzyme showed the ability to
hydrolyse all Phz ingested in humans, because no Phz was recovered in the effluent of
ileostomy subjects after the consumption of 500 mL of apple cider (MARKS et al. 2009) or 1 L
of a polyphenol-rich apple juice (KAHLE et al. 2005b). For phloretin-xyloglucoside (PXG), the
hydrolysis was not completely, as only 22 % of the intake from apple cider and 20 % of the
intake from apple juice were found in the ileostomy effluent. In addition, MARKS et al. (2009)
identified Phl glucuronides, glucuronides-sulphates and sulphates in ileostomy effluent after
ingestion of 500 mL of apple cider in humans (Table 7). UGT and SULT appear to be
responsible for metabolism of Phl. Overall, 38.6 % of ingested dihydrochalcones and their
metabolites were recovered in ileal effluent after apple cider intake (MARKS et al. 2009).
48
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
What occurs to the remaining part of the ingested Phz, Phl and PXG needs to be examined in
further experiments.
In plasma, Phl and Phl glucuronides or sulphates have been reported in rats after the
administration of either Phz or Phl (CRESPY et al. 2002a). Four hours postprandial, the major
part (95 %) was represented by the Phl metabolites, whereas 24 h postprandial, the Phl
concentration increased up to 14 %, due to an accumulation of unmetabolised Phl, which is
more complicated to excrete in comparison to Phl metabolites. In addition, the Phl and Phl
metabolite concentrations in the plasma depend on the ingested form of Phz or Phl. After
feeding of Phl, Phl appeared earlier in the plasma, due to direct absorption (CRESPY et al.
2002a). In humans, only Phl glucuronides (see Table 7) have been reported in plasma after the
ingestion of 500 mL apple cider. This compound mainly appeared half an hour after the
ingestion (MARKS et al. 2009). In addition to those metabolites, KAHLE et al. (2011) reported
approximately 10 % of free Phl in human blood serum after the intake of 1 L cloudy apple
juices. This difference could be explained by the higher Phz intake with apple juice (81 μmol)
than with apple cider (46 μmol), which leads to an enzymatic saturation in the enterocytes
occurs.
In urine, the excretion rates of Phz and Phl metabolites were similar in rats and reached a
value of ≈10 % of the ingested dose (CRESPY et al. 2002a). These authors analysed the
metabolites after treatment with glucuronidase and sulphatase. No details of the metabolite
composition were provided. In humans, however, Phl glucuronides have been reported in
plasma after the intake of a polyphenol-rich fruit juice (BORGES et al. 2010) and Phl
glucuronide sulphates in urine (see Table 7) after consumption of apple cider (MARKS et al.
2009). The overall urinary excretion (4.9 %) mainly occurred within the first 5 h indicating
that the Phl absorption takes place in the small intestine (BORGES et al. 2010). Phlglucuronides-sulphates seem to be rapidly excreted, based on their absence in blood plasma
(MARKS et al. 2009).
The fractional absorption value of apple dihydrochalcones estimated based on urinary
excretion in rats has been reported to be 10 % after ingestion of a single meal of either Phl or
Phz (CRESPY et al. 2002a). In humans, the fractional absorption based on urinary excretion
values has been described to be very low with only ≈5 % of the ingested dihydrochalcones
(BORGES et al. 2010; MARKS et al. 2009). A further study with humans ingesting 1.1 L of an
alcoholic cider beverage resulted in an urinary recovery of 21 % of the dose of Phz after
treatment with glucuronidase and sulphatase (DUPONT et al. 2002). A reason for this
49
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
difference could be the treatment with glucuronidase and sulphatase compared to the direct
measurement of the metabolites by MARKS et al. (2009) and BORGES et al. (2010). However,
the amount being absorbed in the small intestine by enterocytes and subsequently distributed
in the human body appears to be higher than 5 %, due to observations in the ileostomy
effluent, where only 39 % of the ingested dihydrochalcones were recovered (MARKS et al.
2009). From the 61 % Phl disappearing in the small intestine, which are apparently absorbed,
only a small amount (5 %) is recovered as Phl and its metabolites in urine. According to
BORGES et al. (2010) the urinary excretion rate underestimates the absorption in small
intestine. Due to the detection of Phl in the faecal water of human volunteers (JENNER et al.
2005), it appears possible that small amounts of Phl are excreted via stool. Furthermore, to
date no microbial degradation products of Phl have been reported, but in accordance to the
other polyphenol classes, this metabolism route seems to be possible. The dihydrochalcones
bioavailability from apples, defined as the amount being available in different body tissues to
benefit human health, needs further investigations.
Table 7:
Summary of dihydrochalcone and flavonol metabolites found in different body fluids;
+ represents detected metabolite.
metabolite
in intestine
phloretin
quercetin
glucuronides
methylates
sulphates
methyl-glucuronides
methyl-sulphates
sulpho-glucuronides
+
+
+
+
+
+
glucuronides
sulphates
methyl-glucuronides
sulpho-glucuronides
methyl-sulpho-glucuronides
+
+
+
+
+
+
glucuronides
methylates
methyl-glucuronides
sulpho-glucuronides
+
+
+
+
+
+
+
in plasma
in urine
+
50
LITERATURE REVIEW
4.4
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
Absorption and metabolism of flavonols
The fate of apple flavonols in the gastrointestinal tract is illustrated in Figure 9 and Table 7
based on literature. QG is completely hydrolysed by the LPH located at the brush border of
the small intestine and the aglycone Q enters the enterocytes by passive diffusion (CERMAK et
al. 2003). Enzymes located in the enterocytes metabolise Q to methylates, glucuronides,
sulphates and different combinations of these derivatives (Table 7), which are found in
plasma and in the intestinal lumen. In the liver, further metabolism occurs and Q metabolites
are returned to the small intestine via biliary excretion. These metabolites as well as
metabolites from enterocyte metabolism, Q, QR and R reach the colon (Figure 9), where
microbial hydrolysis and further degradation occur. The metabolites can be detected at later
time points in plasma, urine (Table 7) and small amounts in stool.
ENTEROCYTE
INTESTINAL
LUMEN
BLOOD
TISSUE
Q
SULT
QG
UGT
LPH
R
COMT
Q
R
LIVER
ß-gly
Q
Q
Q
Q
KIDNEY
QR
URINE
Q
MM
COLON
Q
R
MICROFLORA
MM
Q
COMT
ß-gly
SULT
QG
Q
R
QR
LPH
UGT
Figure 9:
quercetin glucoside
quercetin
rutin
quercetin-rhamnoside
lactase-phloridzin-hydrolase
UDP-glucuronosyltransferase
Q
Q
Q
MM
catechol-O-methyl-transferase
ß-glycosidase
sulphotransferase
quercetin metabolites in intestine
quercetin metabolites in plasma
quercetin metabolites in urine
microbial metabolites (phenylacetic acids,
phloroglucinol, benzoic acids)
microflora
Metabolism and absorption of flavonols. Symbols represent flavonols and their
metabolites found in the different body fluids based on results of literature
(CHOUDHURY et al. 1999; CRESPY et al. 2003; CRESPY et al. 2001; CRESPY et al. 2002b;
CRESPY et al. 1999; MORAND et al. 2000a; MULLEN et al. 2006; OLTHOF et al. 2003;
WILLIAMSON et al. 2010). Dotted lines show possible metabolites.
51
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
As illustrated in Figure 9, Q, Q metabolites and R are the apple flavonols reported in the
intestinal lumen. After the hydrolysis of QG by LPH, Q is reported to enter the enterocytes
by passive diffusion (CERMAK et al. 2003). According to these authors, the concentration
gradient of Q at the enterocyte membrane between the intestinal lumen and enterocytes results
in a 2-fold higher absorption rate of QG compared to the Q aglycone. This observation was
also reported by CRESPY et al (2001), who investigated the absorption of Q and QG in an in
situ perfusion experiment of rats’ ileum and jejunum, by MORAND et al.(2000a; 2000b), who
examined the absorption rates of different Q glycosides in rats, and by HOLLMAN et al.
(1995), who studied the difference between Q glycosides as contained in fried onions
compared to pure Q in capsules in healthy ileostomy subjects. CRESPY et al. (2001; 1999)
identified the Q metabolites found after in situ perfusion in rats’ ileum and jejunum as
glucuronides, methylates and sulphates (Table 7). As described for the other categories of
polyphenol compounds, these metabolites are produced by SULT, COMT and UGT in the
enterocytes (see Table 7). However, LPH appears to completely hydrolyse QG and QGal due
to the absence of these glycosides in the ileostomy effluent after the ingestion of 1 L of a
cloudy apple juice (KAHLE et al. 2005b). As indicated in Figure 9, and reviewed by ZHANG et
al. (2007), a part (35 %) of intact Q is excreted via bile back to the small intestine (ZHANG et
al. 2007).
KNAUP et al. (2007) incubated various Q glycosides with ileostomy effluent and detected no
hydrolysis of QR. This indicates that the microflora of the small intestine is not able to cleave
QR, in contrast to colonic microbiota, which hydrolyses QR at high rates. The fate of the main
apple flavonol QGal is not well known, as it has been only investigated in one study with
ileostomy subjects ingesting 1 L of a cloudy apple juice, which reported the disappearance of
QGal in ileostomy effluent (KAHLE et al. 2005b). Considering this hydrolysis and the
comparable chemical structure of QGal to QG, it seems to be possible that QGal behaves like
QG during absorption and metabolism.
The main part (83 %) of QR and R reach the colon in humans, where they are hydrolysed by
the colonic microflora (OLTHOF et al. 2003), and the resulting Q can be absorbed by the colon
cells and further be metabolised. OLTHOF et al. (2003) compared the urinary metabolites of
humans ingesting QR with either a intact colon or without a colon. The authors mainly
identified phenylacetic acids (summarised in Figure 9) as the metabolite, transported via
blood circulation and excreted in urine, but found only traces of these microbial metabolites in
the urine of humans without a colon. Colonic microflora appears to be responsible for this
conversion of QR supported by the late (≥3 h postprandial) appearance of these metabolites in
52
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
the plasma (CERMAK et al. 2003; WILLIAMSON et al. 2010). Small amounts of intact Q were
detected in faecal water of humans ingesting their normal diet (JENNER et al. 2005). In
addition, humans ingesting 14C labelled Q excreted <5 % of the ingested dose in the stool, but
up to 80 % of the ingested dose was expired as 14CO2 (WALLE et al. 2001). This indicates, that
Q has to been metabolised by colonic microflora before the human metabolism seems to be
able to use it for further metabolism.
Various Q metabolites have been reported in the plasma and this is summarised in Table 7.
Sulphates and glucuronides appeared first between 0.6-0.8 h after intake of lightly fried
onions in humans (MULLEN et al. 2006). Methylates have also been detected in humans, but to
a lesser amount than sulphates and glucuronides in volunteers ingesting pure Q for two weeks
(EGERT et al. 2008). This contrasts to the extent of methylation rates in rats fed with Q, which
can be as high as 60 % (MORAND et al. 2000b). A second peak of Q metabolites has been
reported in the plasma 5 h after ingestion of Q-glycosides which was attributed to the
hydrolysis of QR and R by colonic microflora and a subsequent colonic absorption as
reviewed by DEL RIO et al. (2010). According to these authors, these latter Q metabolites
lacked sulphates indicating that sulphation takes place exclusively in the small intestine.
Compared to C, plasma levels of Q were higher (4.5 μmol versus 50 μmol) 24 h postprandial
in rats. A reason for this could be that Q metabolites have a higher affinity to albumin so that
they accumulate in the blood plasma (MANACH et al. 1999).
Microbial metabolites, as well as Q metabolites from intestinal cells, have been detected in
urine (see Figure 9 and Table 7). After intake of lightly fried onions in humans, glucuronides,
methyl-glucuronides and sulpho-glucuronides of Q in the urine represented 4.7 % of the
ingested Q dose (MULLEN et al. 2006). However, the profile of urinary metabolites differed
from the profile of plasma metabolites due to further metabolism in liver and kidney (DEL RIO
et al. 2010). After ingestion of a single 200 mg dose of Q, humans showed a urinary excretion
of 7.6 % (2009), as reviewed by the Phenol-Explorer (NEVEU et al. 2010). The higher
recovery of LOKE et al. (2009) compared to MULLEN et al. (2006) could be derived from
different ingestion doses and the analysis after glucuronidase and sulphatase treatment (LOKE
et al. 2009) compared to direct analysis by HPLC-tandem mass spectrometry (MULLEN et al.
2006). Because of the re-excretion via bile and loss in the stool, the urinary excretion is not
the major excretion way of Q metabolites and therefore, measurements of urinary Q
metabolites underestimates absorption rates of Q and Q-glycosides.
53
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
Consequently, as with all polyphenols, the calculation of fractional absorption remains
difficult. Based on urinary excretion of Q metabolites, DEL RIO et al. (2010) estimated in their
review that only 4 % of the Q intake appears in the urine, whereas in in situ perfusion models
in rats the uptake in the intestine wall was reported as 15 % for Q and 25 % for QG (CRESPY
et al. 2003; CRESPY et al. 2001; CRESPY et al. 1999). HOLLMAN et al. (1995) reported
absorption rates of 52 % for QG, 17 % for R and 24 % for Q in healthy ileostomy subjects
ingesting fried onions. Healthy humans ingesting 14C-Q orally also showed a mean absorption
rate of 45 % and half of the ingested Q was expired as 14CO2 produced in intestine (WALLE et
al. 2001). WILLIAMSON et al. (2010) concluded that the absorption of QG, one of the major
apple flavonols, was 50 % of the ingested dose in ileostomy volunteers. Summing up, urinary
excretion of metabolites seems to greatly underestimate the bioavailability of Q and
presumably other apple polyphenols. This is perhaps expected as Q metabolites can be
extensively degraded by the colonic microflora before re-absorption and only the major
metabolites are quantified in urine not the multitude of minor metabolites. In addition, small
amounts are excreted via the stool, but the major part seems to be expired as CO2.
4.5
Apple
Comparison of the fate of apple polyphenols after ingestion
polyphenols
from
all
four
categories,
i.e.
flavan-3-ols,
phenolic
acids,
dihydrochalcones and flavonols, are partly absorbed in the small intestine by active and
passive mechanisms (Table 8). After absorption into enterocytes, the polyphenols are
metabolised and transformed to varying extents primarily into glucuronides, sulphates and
methylates. These metabolites and the non-metabolised intact polyphenols are transferred to
blood plasma or re-extracted into the intestinal lumen. Un-metabolised intact polyphenols in
blood plasma seem to appear only when high doses of individual polyphenols are provided,
either in the isolated form, as concentrated extracts, or in foods. The polyphenols and their
metabolites pass via blood circulation and reach the liver and kidney, where further
metabolism occurs. The major part of the ingested flavan-3-ols and flavonols are transferred
via bile back to the intestinal lumen, perhaps due to their relative high molecular mass. The
part remaining in blood circulation and the absorbed phenolic acids and dihydrochalcones are
excreted via kidney and urine, where polyphenol glucuronides, sulphates and methylates
(exception dihydrochalcones) and combinations of these derivatives have been detected.
UGT, COMT and SULT are reported to be responsible for the formation of these metabolites.
The only exception is a lack of Phl methylates and it appears that COMT is unable to
metabolise dihydrochalcones. Microbial metabolites resulting from the breakdown in the
colon of intact polyphenols and their primary metabolites have also been detected in the urine,
54
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
indicating some colonic absorption and renal excretion. Until now, no microbial metabolites
have been reported for dihydrochalcones.
Peak plasma levels of intact polyphenols and their primary metabolites are reached between
30 min and 1.5 h after ingestion of all apple polyphenols due to the fast absorption in small
intestine. Microbial decomposition products show peak plasma levels between 5 h and 7 h
after ingestion of the polyphenol (Table 8).
Bioavailability has been defined as the amount of unchanged polyphenols plus metabolites
found in urine (PRASAIN et al. 2007). According to this definition, the bioavailability of
flavan-3-ols and phenolic acids is about one third of the intake, whereas the bioavailability of
dihydrochalcones and flavonols is low due to low urinary excretion (Table 8). However, as
mentioned above, urinary excretion underestimates the amount being absorbed in the small
intestine due to reflux of metabolised polyphenols into the small intestine from enterocytes or
via bile. A better method to estimate bioavailability is to quantify the amount being absorbed
in small intestine by analysing ileostomy effluent. This results in amounts comparable to
urinary excretion for flavan-3-ols and phenolic acids, but a ten-fold higher amount for
dihydrochalcones and flavonols as summarised in Table 8. The higher bioavailability values
for dihydrochalcones and flavonols could be explained by the hydrolysis of their glycosides
by LPH, which leads to a higher concentration of the corresponding aglycons directly at the
enterocyte membrane and enhance passive diffusion. Regarding postulated health effects of
apple polyphenols, the last-mentioned bioavailability values, calculated by the difference
between ingested dose and recovery in ileostomy effluent, appear to be more realistic.
Table 8:
location of
absorption
Comparison of metabolism and absorption values of apple polyphenols
flavan-3-ols
phenolic acids
dihydrochalcones
flavonols

 small intestine
 colon
 stomach
 small intestine
 colon

 small intestine
 colon

 small intestine
 colon
glucuronides
 glucuronides
 glucuronides
 glucuronides
methylates


 methylates
sulphates
 sulphates
 sulphates
 sulphates
microbial
 microbial
 microbial
metabolites
metabolites
metabolites
a
Tmax plasma levels
1.5 h
1 h/7 h
1h
<1 h/5 ha
b
recovery in urine
~27 %
~30 %
~5 %
~4 %
c
bioavailability
~30 %
~30%
~60 %
~50 %
a
First plasma peak level/second plasma peak level derived from microbial degradation; b parent compounds and
metabolites; c calculated as difference of ingestion and recovery in ileostomy effluent
metabolites




To attribute possible beneficial health effects to single polyphenols, their concentration at the
site of possible action is necessary to know. This is however difficult to analyse due to the
55
LITERATURE REVIEW
FATE OF APPLE POLYPHENOLS IN THE HUMAN BODY
different body tissues where polyphenols can exert a physiological action. The amount
reaching the target tissue can be estimated by examining blood plasma levels of polyphenols
as blood is the transporter of intact and metabolised polyphenols. The Phenol-Explorer
(NEVEU et al. 2010) provides a summary of available literature dealing with kinetic data in
plasma of humans and rats. In humans ingesting 1 L of a cloudy apple juice maximal plasma
concentrations were observed between 1.75 and 2.33 h. The concentrations in plasma after
treatment with glucuronidase and sulphatase were of 0.05 μmol/L for EC, 0.73 μmol/L for
CA, 0.09 μmol/L for CQA, 0.17 μmol/L for Phl, and 0.25 μmol/L for Q (KAHLE et al. 2011).
These values represent <0.5 % of the amounts ingested with apple juice with the exception of
Q (3.5 % of amount ingested). BORGES et al. (2010) reported slightly higher plasma levels
(1.3 μmol/L C+EC+metabolites, 0.2 μmol/L Phl metabolites) in humans having ingested
350 mL of a polyphenol-rich apple juice. The review of 97 bioavailability studies of pure
substances and foods in humans (MANACH et al. 2005) summarised the range of the maximal
plasma levels for C and EC (0.09-1.10 μmol/L), procyanidin dimers (0.008-0.03 μmol/L), CA
(0.26 μmol/L, range not indicated) and quercetin glycosides (0.51-3.80 μmol/L). Overall,
plasma concentrations stay mostly below 1 μmol/L (MANACH et al. 2004). Reasons for these
low plasma levels could be the fast excretion of polyphenol metabolites from plasma and the
lack of a complete detection of all metabolites in plasma, indicating the need of further
investigations in this research area.
56
LITERATURE REVIEW
5
APPLE POLYPHENOLS AS ANTIOXIDANTS
Apple polyphenols as antioxidants
Antioxidants are substances which are able to quench potentially damaging radicals or to
retard or prevent oxidisable substances from oxidation (BECKER et al. 2004). The chemical
structure of polyphenols is optimised to act as free-radical scavengers by hydrogen or electron
donation by the aromatic hydroxyl groups (FERNANDEZ-PANCHON et al. 2008). The resulting
polyphenol derived radical is stabilised by delocalization of an unpaired electron and by the
reaction with other antioxidants (BIEDRZYCKA et al. 2008). In apples, all polyphenol groups
demonstrate an antioxidant potential, but the flavan-3-ols showed the highest values among
the polyphenol groups in apples. Moreover, EC and PC B2 were the major contributors to the
antioxidant potential of an apple and also the phenolic acids have a significant role among
apple antioxidants (TSAO et al. 2005). LEE et al. (2003) specified the apple polyphenols with
decreasing antioxidant potential. The highest value was obtained for Q, followed by EC, PC
B2, Vitamin C, Phl and CA.
5.1
Analytical methods of the antioxidant potential
For measuring the antioxidant potential in foods, several in vitro methods were developed and
various fruits and vegetables have been investigated. The main methods used are the TEAC
(Trolox® equivalent antioxidant potential), FRAP (ferric reducing antioxidant power), DPPH
(1,1-diphenyl-2-picrylhydrazyl radical) and ORAC (oxygen radical absorbance capacity)
assays. These assays measure directly the electron transfer (TEAC, FRAP, DPPH) or
hydrogen atom donation (ORAC) from the antioxidant to free radicals in lipid free systems
(BECKER et al. 2004). In the TEAC assay, the coloured cation radical (measured at 734 nm) of
2,2’-azonobis(3-ethylbenzothiazoline-6-sulphonate) (ABTS), a non-biological radical, is
reduced by the antioxidant. The decolouration of the cation represents the antioxidant capacity
and is quantified in Trolox® equivalents (APAK et al. 2007; RE et al. 1999). The mechanism
behind this test is an electron transfer, the same as for the FRAP assay. In this assay, a ferric
ion is reduced by the antioxidant to a blue (measured at 595 nm) ferrous 2,4,6-tripyridyl-striazine complex (APAK et al. 2007; TSAO et al. 2005). In the DPPH assay, working also with
electron transfer, the stable DPPH radical is scavenged by the antioxidants and the decrease of
colouration in quantified spectrophotometrically at 516 nm (APAK et al. 2007; BECKER et al.
2004). The most relevant assay for human biology is the ORAC test, due to the short-lived
radicals. Oxidation induced by peroxyl radicals is inhibited by the antioxidant via classical
chain breaking antioxidant activity by hydrogen atom transfer which is measured as reduction
of fluorescence (PRIOR et al. 2005) with fluorescein as probe (BECKER et al. 2004). The
57
LITERATURE REVIEW
APPLE POLYPHENOLS AS ANTIOXIDANTS
different assays aree often calibrated with catechin, vitamin C, gallic acid or Trolox®, but
whereby the standard substance should be chosen by adaption to the investigated samples.
However, due to inter-laboratory variance and different methods used these values are
inefficient to quantify polyphenols.
5.2
Antioxidant action in humans
In humans, ingested polyphenols are metabolised (see Chapter 4) and therefore their
antioxidant potential is usually changed. Some authors reported that the metabolism of
polyphenols in the human body decreases their antioxidant potential (HOLLMAN et al. 2011).
For example, methylation of C or Q decreased their antioxidant potential due to blocked
hydroxyl groups (MANACH et al. 1999). On the other hand, a recent research note showed that
a metabolite of CA, dehydrocaffeic acid displayed higher antioxidant activities than the intact
functional CA in vitro (ISHIMOTO et al. 2012). Microbial metabolites contain free hydroxylgroups and show a similar antioxidant potential than their precursors (FERNANDEZ-PANCHON
et al. 2008). Consequently, the human polyphenol metabolism complicates the prediction of
the antioxidant potential of the ingested food.
However, in humans, antioxidants are reported to prevent radical reactions by reducing the
stress induced by reactive oxygen species, which are involved in plaque formation and DNA
damage, and may therefore help in the prevention of atherosclerosis and cancer (DRAGSTED
2003; FERNANDEZ-PANCHON et al. 2008). To be active and of potential health benefit, the
ingested antioxidants should be absorbed and maintain their antioxidant potential after
metabolism so as to increase the antioxidant potential in human tissues and improve several
biomarkers and symptoms of oxidative damage (DRAGSTED 2003). Praticò (2005) reviewed
the effect of dietary antioxidants on endothelium protection. According to this author,
endothelial functions are sensitive to oxidative stress induced by reactive oxygen species and
exogenous antioxidants are thought to antagonise these reactive species. Later work shows
that the effect of polyphenols on endothelial function is due to their action on the nitric oxide
system and not due to the antioxidant action. The antioxidant action would include an effect
for all polyphenols due to their antioxidant potential, but this has not been reported until now.
Only flavan-3-ols, mainly EC, have been reported to activate the nitric oxide system, which
induces the vasodilation (see Chapter 6.1.1).
In relation to in vivo studies evaluating the antioxidant potential of polyphenols, JALIL et al.
(2008a) reported that the circulating plasma free fatty acids and 8-isoprostane were reduced
after cocoa intake in obese rats and that cocoa flavan-3-ols may enhance the antioxidant
58
LITERATURE REVIEW
APPLE POLYPHENOLS AS ANTIOXIDANTS
defence system. Free fatty acids are elevated in obesity, cause insulin resistance and are
thought to be the major link between obesity and the development of metabolic syndrome
(BODEN 2008). In addition, 8-isoprostane is a biomarker of in vivo peroxidation of
arachidonic acid, which is independent from cyclooxygenase activity (ENGLER et al. 2004b).
In contrast, a human intervention study measuring the oxidation of endogenous urate, αtocopherol and lipids ex vivo failed to show any resistance to oxidation after high amounts of
apple polyphenol intake (LOTITO et al. 2004). Therefore, the authors concluded that apple
polyphenols may not result in equivalent in vivo antioxidant effects due to low plasma
concentration. According to GALLEANO et al. (2010), who reviewed the thermodynamics of
antioxidant action, the plasma concentrations are too low to meet the kinetic requirements
necessary to reach physiologically relevant reaction rates. Other authors also found no
evidence to support an antioxidant effect as the reason for reported beneficial health effects of
polyphenols (HOLLMAN et al. 2011). HOLST et al. (2008) similarly rejected the direct
antioxidant hypothesis due to the in vivo redox status being under a strict homeostatic control
in the body. This means the body specific antioxidant compounds and enzymes are able to
maintain the normal redox status in the body, as polyphenols are non-nutritive and not
essential for humans. Consequently, the redox status in the body is well controlled and the
influence of nutritional antioxidants on the body redox status is questionable.
Thus, although an antioxidant effect was the original hypothesis to explain the health benefits
of polyphenols, most subsequent investigations in humans failed to show that the observed
health effects of polyphenols are derived directly from the antioxidant potential of dietary
antioxidants after absorption and distribution into the target tissues. One exception may be the
antioxidant action of dietary polyphenols in the digestive tract, which is not directly related to
free radical scavenging activity. Fraga (2007) proposed the polyphenol-lipid and polyphenolprotein interactions as relevant mechanisms for plant polyphenols to show antioxidant effects
in the gastrointestinal tract as location of action. This means, polyphenols may protect the
gastrointestinal tract against food-borne reactive species or radicals generated within the
stomach and intestine (HALLIWELL 2007; HALLIWELL et al. 2005), by the reason of being
present in high concentrations at this location. There, they may scavenge reactive nitrogen,
chlorine and oxygen species and perhaps inhibit cyclooxygenase and lipoxygenase activity
(HALLIWELL et al. 2005). HOLLMAN et al. (2011) recently sustained the hypothesis that
polyphenols might reduce the burden of prooxidant intake. They mentioned that the
knowledge of these indirect antioxidant actions, the antioxidant action in the food prior to
consumption, needs further investigation.
59
LITERATURE REVIEW
APPLE POLYPHENOLS AS ANTIOXIDANTS
In summary, the antioxidant potential of polyphenol appears to be unlikely after absorption in
the human body, due to their low concentration and other regulatory mechanisms concerning
redox status. Therefore, the quantified in vitro antioxidant potential values hardly represent
the antioxidant action of polyphenols in vivo. However, polyphenols could protect the
gastrointestinal tract via the antioxidant mechanism by reducing the amount of reactive
species there, but much remains uncertain until now. Nevertheless, the different polyphenol
classes are reported to have beneficial health effects assigned to other mechanisms, which are
discussed in the following chapter.
60
LITERATURE REVIEW
6
HEALTH EFFECTS OF APPLE POLYPHENOLS
Health effects of apple polyphenols
Apples are generally considered “healthy” with the existing saying “An apple a day keeps the
doctor away”. Epidemiological studies investigated these effects by correlating the incidence
of cancer, cardiovascular diseases (CVD), lung function and diabetes to the dietary apple
intake. These studies were reviewed in 2004 by BOYER et al. (2004) and updated in 2011 by
HYSON (2011).
For cancer prevention, GALLUS et al. (2005) reported an inverse association between the
consumption of ≥ 1apple/d and various types of cancer (oral cavity and pharynx, oesophagus,
larynx, colorectum, breast, ovary and prostate) in a case-control meta-analysis in Italy. The
strongest chemopreventive effects of apples were found for lung cancer (ARTS et al. 2001a;
FESKANICH et al. 2000; LE MARCHAND et al. 2000; LINSEISEN et al. 2007) and colorectal
cancer (GALLUS et al. 2005; MICHELS et al. 2006; THEODORATOU et al. 2007). GERHAUSER
(2008) also reviewed the potential chemopreventive effects of apples from published in vitro,
in vivo and epidemiological studies and concluded, that at least one apple a day may have
preventive effect against cancer. The proposed mechanisms for this effect include
antimutagenic activity, modulation of carcinogen metabolism and signal transduction
pathways, anti-inflammation, antiproliferation and apoptosis-inducing activity.
The effect of apples on cardiovascular diseases was investigated over 6.9 years in the
Women’s Health Study (WHS) in 38445 women using food frequency questionnaires (FFQ).
In this study, a non-significant reduction of CVD risk was observed with increasing apple
consumption (SESSO et al. 2003). Moreover, the Finnish Mobile Clinic Health Examination
Survey reported that a lower intake of flavonoids, of which apples were the main provider,
increased the risk of coronary diseases (KNEKT et al. 2000; KNEKT et al. 1996). In addition, in
the Zutphen Elderly Study, an inverse association between apple consumption and mortality
from coronary heart diseases was found (ARTS et al. 2001b; HERTOG et al. 1993), whereas tea
catechins, for example gallates, failed to show an association. However, the authors were not
able to exclude a lifestyle effect, so it is possible that people consuming apples may have a
healthier lifestyle which may help prevent cardiovascular diseases.
BUTLAND et al. (2000) investigated lung function, as measured by a high maximum forced
expiratory volume in one second, in middle aged men (45-59 years) over 4 years in relation to
apple consumption per week. They reported that an increase in lung function was strongly
positive associated with a consumption of ≥ 5 apples/week. Since that study, there are reports
61
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
of an inverse association between apple consumption and chronic obstructive pulmonary
disease (TABAK et al. 2001) and asthma (ROMIEU et al. 2006; SHAHEEN et al. 2001). An apple
intake of > 4 apples/week by women during pregnancy is also reported to have a weak but
significant protective effect on asthma in their children of the age of 5 years (WILLERS et al.
2007). On the other hand, OKOKO et al. (2007) found no association between fresh apple
consumption and wheezing in primary school children, and GARCIA et al. (2005) failed to
detect an association between intake of flavan-3-ols, flavonols and flavones on asthma,
asthma severity and chronic sputum production in adults. One reason for these contradictory
results could be the limited information on the polyphenol composition in the databases, the
strong variability of polyphenol profile depending diet composition, differences in study
design, and differences in the physiological status of the different population groups studied.
In the WHS study, the effect of apple intake on type 2 diabetes was also investigated in
38018 women over 8.8 years of follow-up. The risk of developing type 2 diabetes was
significantly reduced by 28 % when ingesting ≥ 1 apple/d compared to no apple consumption
(SONG et al. 2005), but the bioactive compounds responsible for this effect were not
identified.
In most cohort and case-controlled epidemiological studies, apples showed mainly a positive
effect on health when consumption was > 1 apple/d. Therefore, the saying should be changed
to “Two apples a day keep the doctor away” with the cultivars available today, until the
bioactive compounds, and apples which are rich in these compounds, are identified. However,
despite of the reported beneficial effects of apples, the compounds and the mechanism behind
these effects remain largely unknown. The following sub-chapters discuss separately the
beneficial health effects of flavan-3-ols, phenolic acids, dihydrochalcones and flavonols based
on the published intervention studies. Most studies have been made with flavan-3-ols,
whereas intervention studies investigating the health effects of the phenolic acids,
dihydrochalcones and flavonols are limited. As there are relatively few studies with apples,
intervention studies with other foods containing the same polyphenols as present in apples,
are also reviewed.
6.1
Health effects of flavan-3-ols
Health effects of flavan-3-ols have mainly been investigated in relation to cocoa, cocoa
products and tea. However, as apple flavan-3-ols are similar to cocoa flavan-3-ols, both
consisting mainly of C, EC and procyanidins, this chapter reviews mainly the intervention
studies conducted with cocoa and chocolate. Flavan-3-ols have been reported to have a
62
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
beneficial impact on cardiovascular diseases, brain function, skin health, glucose tolerance,
obesity and cancer, all of which are discussed in the following sub-chapters.
6.1.1
Flavan-3-ols in the prevention of cardiovascular diseases
Cardiovascular diseases are the leading cause of death world-wide (DING et al. 2006) and the
risk of cardiovascular diseases can be influenced by diet and lifestyle factors. Diet
components can both increase and decrease the risk, with soluble dietary fibre, phytosterols,
long chain polyunsaturated fatty acids and flavan-3-ols being reported to have a protective
effect. Thus, by increasing cocoa or chocolate intake, the risk of cardiovascular events may be
decreased (BUITRAGO-LOPEZ et al. 2011). In a meta-analysis, these authors estimated a
reduction of 37 % in CVD (relative risk [RR] 0.63, 95 % confidence interval [CI]: 0.44; 0.90)
and a reduction of 29 % in strokes (RR: 0.71, 95 % CI: 0.52; 0.98) for the highest chocolate
consumption (between ≥ once a week and daily) as compared to the lowest (never). Coronary
heart disease and stroke are the main CVD outcomes and the main physiological risk factors
for these diseases are endothelial dysfunction, platelet dysfunction, an imbalance in blood
lipids and high blood pressure. The influence of flavan-3-ols on these risk factors is discussed
in the following paragraphs.
The endothelium of blood vessels is responsible for maintaining normal vascular tone and
blood flow (HEISS et al. 2006). Endothelial dysfunction is associated with cardiovascular
diseases and is therefore a predictor of future coronary events (CORTI et al. 2009). The
endothelium is the continuous, smooth nonthrombogenic surface of all blood vessels which
synthesises the nitric oxide (NO) via endothelial NO synthase in times of shear stress. NO
leads to vasodilation and induces a relaxation of vascular smooth muscle cells. Consequently,
endothelial dysfunction is associated with reduced endothelial NO synthase expression and
NO bioavailability (CORTI et al. 2009; JIMENEZ et al. 2012; KATZ et al. 2011). This effect can
be non-invasively measured by flow mediated dilation (FMD). A pneumatic tourniquet is
placed around the forearm and inflated to a supersystolic blood pressure. Rapid deflation
stimulates an increase in blood flow with an increased flow through the upstream brachial
artery, which can be measured by ultrasound technology (STONER et al. 2012) and FMD is
expressed as increase in artery diameter above the baseline. Table 9 summarises intervention
studies which have investigated the short-term or long-term effects of cocoa and chocolate
intake on endothelial function as measured by FMD.
In Table 9, the values represent the difference in FMD before and after treatment with flavan3-ols. Two main time points for measuring FMD were used, either 2 h after the last ingestion
63
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
of flavan-3-ols, which corresponds to the peak level of flavan-3-ol metabolites in plasma (see
4.1) or after an overnight fast. The latter was used to measure long-term effects of flavan-3ols. The trials measuring FMD after an overnight fast reported no (FAROUQUE et al. 2006) or
only small improvements in FMD (GRASSI et al. 2005b; WANG-POLAGRUTO et al. 2006). The
study of HEISS et al. (2010) was the only study reported in Table 9 which showed an
improvement in the low and high flavan-3-ol intervention groups. All five studies listed in
Table 9 investigated the effect of a single dose and showed a significant effect (< 7 %
improvement in FMD) compared to baseline 2 h after the intake (BALZER et al. 2008; BERRY
et al. 2010; FARIDI et al. 2008; HEISS et al. 2005; HERMANN et al. 2006). The ingested flavan3-ol single doses that resulted in improved blood flow ranged from 100 mg/dose to 1 g/dose.
The four chronic investigations (7 days to 6 weeks) measuring FMD 2 h after the last dose
reported a maximal 3 % significant improvement in FMD after the administration of
160 mg/d to 1 g/d cocoa flavan-3-ols (BALZER et al. 2008; ENGLER et al. 2004b; HEISS et al.
2007; NJIKE et al. 2011). In cocoa, more than half of the reported flavan-3-ols are monomeric
(56 %) with EC representing the major part (33 % of all flavan-3-ols). In dark chocolate, EC
is the major flavan-3-ol (46 %) followed PC B2 (24 %) (NEVEU et al. 2010). Due to the lower
absorption of dimeric and oligomeric procyanidins (see Chapter 4.1) and the high content of
EC, EC and not C is considered the cocoa flavan-3-ol with the main impact on FMD.
A dose-dependent improvement of FMD with cocoa flavan-3-ols has been reported by HEISS
et al. (2007). The half-maximal FMD response was achieved after the acute ingestion of
616 mg flavan-3-ols, which is somewhat higher than normal dietary intake. In addition to the
improvement in FMD caused by the cocoa beverage, NJIKE et al. (2011) reported a further
non-significant increase of 0.9 % in FMD in overweight men when they replaced the sugarsweetened cocoa beverage by a sugar-free un-sweetened cocoa beverage. Therefore, sugared
cocoa products may reduce the effect of the flavan-3-ols in relation to FMD. The studies
listened in Table 9 lead to the assumption, that a single 113 mg dose of flavan-3-ols (22 mg
EC) or a daily intake of 160 mg cocoa flavan-3-ols (48 mg EC) is sufficient to improve FMD.
64
dark
chocolate bar
7
cocoa drink
6
cocoa
beverage
5
cocoa drink
4
chocolate
3
liquid cocoa
2
chocolate
bar
2
cocoa
beverage
1
food
Table 9:
963 mg/dose
75 mg/d
963 mg/d
high flavan-3-ols
low flavan-3-ols
high flavan-3-ols
traces
259 mg/d
46 g low flavan-3-ols
46 g high flavan-3-ols
393 mg/d
371 mg/dose
medium flavan-3-ols
3 times per day
75 mg/dose
181 mg/dose
100 ml high flavan-3-ols
low flavan-3-ols
< 11 mg/dose
nr
40 g dark chocolate
100 ml low flavan-3-ols
nr
40 g white chocolate
161.3 mg/dose
22 g sugar-free cocoa
0
placebo
161.3 mg/dose
113.3 mg/dose
22 g cocoa powder bar
22 g sugared cocoa
0
701 mg/doseg
high flavan-3-ols
cocoa-free bar
22 mg/dose
flavan-3-ol
content
low flavan-3-ols
intervention
46 mg/d
177 mg/d
203 mg/d
17 mg/d
203 mg/dose
79 mg/dose
17 mg/dose
72 mg/dose
nr
nr
48 mg/dose
48 mg/dose
22 mg/dose
139 mg/dose
0
EC content
r, db,
pc
r,
doublemarked
r, db,
co
r
r, pc,
sb, co
r, db,
co
design
2w
2w
7d
30 d
30 d
single
dose
single
dose
single
dose
single
dose
single
dose
duration
healthy humans
smokers with endothelial
dysfunction
medicated diabetic
patients
diabetic patients
smokers
male smokers
overweight average BMI
30 kg/m2
overweight average BMI
30.6 kg/m2
subjects
11
10
6
41
41
10
10
10
11
10
10
44
21
n
2h
2h
2h
2h
2h
2h
2h
2h
2h
2h
measured
after
2h
FMD
1.3 % ↑#
no effect
2.9 % ↑*
1 % ↑**
no effect
1.7 % ↑**
0.9 % ↑**
no effect
2.4 % ↑*
no effect
2.6 % ↑*
no effect
5.7 % ↑##
2.0 % ↑##
1.5 % ↓
4.3 % ↑##
1.8 % ↓
6.1 % ↑#
Δ to
baseline a
3.4 % ↑
Intervention studies investigating the effect of flavan-3-ols on endothelial function, measured as changes in FMD compared to baseline
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
65
100 g/d
88 mg/d
0
446 mg/d
high flavan-3-ols
90 g/d
43 mg/d
444 mg/d
chocolate bar +
cocoa beverage
low flavan-3-ols
19.6 mg/d
236 mg/d
high flavan-3-ols
placebo
10 mg/d
161 mg/d
22 g/d cocoa powder
low flavan-3-ols
161 mg/d
22 g/d cocoa powder
66 mg/d
nr
nr
107 mg/d
4.7 mg/d
118 mg/d
2 mg/d
48 mg/d
48 mg/d
EC content
r, co
r, db,
parallelarm
r, db, pc
r, db, co,
controlled
r, co,
controlled,
design
15 d
15 d
6w
6w
6w
30 d
6w
6w
6w
duration
patients with essential
hypertension
postmenopausal
hypercho-lesteromic
CAD patients
CAD patients
overweight men (BMI
25-35 kg/m2)
subjects
10
9
8
40
16
37
na
≥ 12 h
≥ 12 h
after
overnight fast
overnight fast
overnight fast
measured
after
2h
FMD
1.5 % ↑**
no effect
2%↑
1.5 % ↓
no effect
no effect
3.8 % ↑**
1.3 % ↑**
2.4 % ↑##
1.5 % ↑#
Δ to
baselineb
↓
40 g lyophilised,
172 mg/d
16 mg/d
r, db, co
4w
hypercholesteromic
30
after
no effect
apple low PP
men
overnight fast
40 g lyophilised
1152 mg/d
104 mg/d
4w
no effect
apple, high PP
1
(BERRY et al. 2010), 2(FARIDI et al. 2008), 3(HERMANN et al. 2006), 4(HEISS et al. 2005), 5(BALZER et al. 2008), 6(HEISS et al. 2007), 7(ENGLER et al. 2004b), 8(NJIKE et al.
2011), 9(HEISS et al. 2010), 10(FAROUQUE et al. 2006), 11(WANG-POLAGRUTO et al. 2006), 12(GRASSI et al. 2005b), 13(AUCLAIR et al. 2010); a number of subjects, nr not
reported, r randomised, pc placebo-controlled, db double-blind, c controlled, pd parallel design, co cross-over, sb single blind, bchanges to baseline measurement;* p<0.05; **
p<0.001; # p<0.05 compared to placebo,;## p<0.001 compared to placebo
apple
13
dark chocolate
white chocolate
12
12
cocoa beverage
11
chocolate and
cocoa
10
cocoa beverage
9
0
flavan-3-ol
content
0 cocoa powder
intervention
to be continued
sugar-sweetened
cocoa-free placebo
8
sugar-sweetened
cocoa beverage
8
sugar-free cocoa
beverage
8
food
Table 9:
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
66
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
Only one study has investigated the effect of apple polyphenols on blood flow and that study
failed to show any effect on FMD (AUCLAIR et al. 2010). These authors fed two lyophilised
apple cultivars to adult subjects for four weeks. The cultivars were cv. Golden Delicious, with
low polyphenol content (0.21 g/d polyphenols, 172 mg/d flavan-3-ols, 16 mg/d EC) and cv.
Marie Ménard with high polyphenol content (1.43 g/d polyphenols, 1152 mg/d flavan-3-ols,
104 mg/d EC) but both with comparable polyphenol profile. The failure to find an effect on
FMD was possibly due to the measurement of FMD after an overnight fast which has failed to
show an effect in other studies. Moreover, although the reported flavan-3-ol dose in the high
polyphenol apple is high, the value included mainly the highly polymerised flavan-3-ols that
are reported to reach the colon without being absorbed (see 4.1) and would not be expected to
influence endothelial function.
A meta-analysis published by HOOPER et al. (2008) reported a chronic (≥ 2 weeks; typically
used doses: 50-100 g chocolate/d correspondent to 77-153 mg/d flavan-3-ols) beneficial effect
of chocolate and cocoa on FMD of 1.45 % (95 % CI: 0.6 %; 2.28 %) and an acute (typically
used doses: 50-100 g chocolate/d correspondent to 77-153 mg/d flavan-3-ols) effect of 3.99 %
(95 % CI: 2.86 %; 5.12 %), which may reduce the risk of CHD. For the acute studies, the
peak effect was 2 h after ingestion of the cocoa product as described above. A follow-up
meta-analysis by the same research group (HOOPER et al. 2012) resulted in comparable
outcomes. Chronic chocolate or cocoa consumption increased FMD by 1.34 % (95 % CI:
1.00 %; 1.68 %) and after acute ingestion an improvement in FMD of 3.19 % (95 % CI:
2.04 %; 4.33 %) was observed. Therefore, cocoa or chocolate flavan-3-ols increase FMD, but
because only a limited number of studies investigated the dose-dependence on FMD, no
conclusion could be made on this issue. The doses of cocoa or chocolate used in the published
trials were relatively high and represented ~10 % of the daily energy intake (HOOPER et al.
2012; HOOPER et al. 2008) which may not be a wise addition to a balanced diet. Nevertheless,
both two meta-analyses and the overview of Table 9, indicate that flavan-3-ols may improve
FMD and thus endothelial function. In addition, the European Food Safety Authority
reviewed the effect of cocoa flavan-3-ols on maintenance of endothelium-dependent
vasodilation and restricted a correspondent health claim to a minimum of 200 mg flavan-3-ols
(EFSA 2012) contained in the claimed food, even though this value is somewhat higher than
the minimum levels reported to improve FMD.
Over the last 8 years, various authors have reviewed the mechanism and effect of flavan-3-ols
on endothelial function (COOPER et al. 2008; ENGLER et al. 2004a; ENGLER et al. 2006;
67
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
JIMENEZ et al. 2012; STOCLET et al. 2004). The postulated mechanism behind this effect is an
induction of vasodilation via activation of the nitric oxide system. This was first shown by
FISHER et al. (2003), who reversed the effect of cocoa flavan-3-ols in humans by intravenous
administration of NG-nitro-L-arginine methyl ester, a specific nitric oxide synthase inhibitor.
Also, the nitric oxide inhibitor L-NG-monomethyl-arginine resulted in a reversion of the cocoa
flavan-3-ol induced increase in FMD (HEISS et al. 2005). Additionally, SCHEWE et al. (2008)
proposed that flavan-3-ols reduce the NADPH oxidase activity. NADPH oxidase isoforms
generate superoxide radicals that scavenge NO and therefore reduce the cellular level of NO
(KATZ et al. 2011). CORTI et al. (2009) related the inhibitory effect of flavan-3ols on NADPH
oxidase to short-term effects, whereas they suggested that the increased generation of NO was
involved in long-term effects.
In summary, intervention studies with apple flavan-3-ols are scarce but due to the positive
effect of cocoa flavan-3-ols on endothelial function, it seems likely that apple flavan-3-ols
may evoke comparable effects. However, the daily dose of apple polyphenols needed to
improve blood flow is unknown, because unlike cocoa, which has mainly monomeric flavan3-ols, apples have similar concentrations of monomeric and dimeric flavan-3-ols (see Table
1). This could be investigated by the administration of apple cultivars with different
polyphenol contents and profiles, for example two cultivars with similar total polyphenol
content, but high and low in monomeric flavan-3-ol levels comparable to cocoa flavan-3-ol
contents.
The beneficial effect of flavan-3-ols on blood pressure was reported the first time in Kuna
Indians living on islands near Panama. These Indians displayed a lower prevalence of agerelated hypertension compared to similar Indians living in Panama City. In relation to their
dietary patterns, their cocoa flavan-3-ol intake attracted the attention of researchers
(HOLLENBERG 2006). Further cohort studies have since confirmed that flavan-3-ols may
reduce blood pressure. For example in the Zutphen Elderly Study, the highest tertile (4.18 g
cocoa/d) of cocoa intake resulted in a 3.7 mmHg decrease in systolic blood pressure compared
to the lowest tertile (0 g cocoa/d) (BUIJSSE et al. 2006). Also in the Potsdam arm of the
European Prospective Investigation into Cancer and Nutrition, the mean systolic blood
pressure was decreased by 1 mmHg (95 % CI: -1.6 mmHg; -0.4 mmHg) and the mean
diastolic blood pressure by 0.9 mmHg (95 % CI: -1.3 mmHg; -0.5 mmHg) in the highest
quartile (7.5 g chocolate/d) compared to the lowest quartile (1.7 g/d) of chocolate intake
(BUIJSSE et al. 2010). However, regarding intervention studies, the effect of flavan-3-ols on
68
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
blood pressure remains unclear. Table 10 lists human intervention studies which have
investigated the effect of chocolate or cocoa beverages administration on blood pressure. In
overweight humans, a single dose of either cocoa beverage or a chocolate bar providing
> 100 mg flavan-3-ols/dose resulted in significant decreases in both systolic and diastolic
blood pressure (BERRY et al. 2010; FARIDI et al. 2008). However, sugared cocoa failed to
show any effect on blood pressure in contrast to its effect on FMD (Table 9). Two of the four
chronic studies (≥ 2 weeks) investigating blood pressure reported partly significant decreases
in blood pressure in healthy humans but independent of the dose (FRAGA et al. 2005; GRASSI
et al. 2005a). In hypertensive humans, subjects consuming the lower flavan-3-ol dose
(88 mg/d) showed significant decreases in both systolic and diastolic blood pressure (GRASSI
et al. 2005b) and cardioartery disease (CAD) patients also showed a response on systolic
blood pressure after ingestion of a cocoa beverage for 30 days. It appears possible therefore
that flavan-3-ols may decrease blood pressure especially in humans with elevated blood
pressure, but more intervention studies are needed with both healthy and hypertensive
humans. It seems likely, that EC is the major contributor to this effect, because it is absorbed
to a greater extent than dimeric flavan-3-ols and more than half of the cocoa flavan-3-ols
(NEVEU et al. 2010) are monomeric ones (40 % C, 60 % EC).
A meta-analysis of five randomised controlled trials with an average intervention duration of
two weeks reported a decrease of the pooled mean systolic blood pressure of -4.7 mmHg
(95 % CI: -7.6 mmHg; -1.8 mmHg; P=0.002) and of -2.8 mmHg (95 % CI: -4.8 mmHg;
-0.8 mmHg; P=0.006) for diastolic blood pressure in healthy and hypertensive humans after a
mean intake of 401.4 mg flavan-3-ols/d from chocolate (90.2 g/d mean chocolate intake). The
authors concluded, that the consumption of cocoa and cocoa products may reduce the blood
pressure (TAUBERT et al. 2007b). Another meta-analysis evaluating the effect of different
flavonoid subclasses in different food sources on CVD and risk factors reported a decrease of
systolic blood pressure of -5.88 mmHg (95 % CI: -9.55 mmHg; -2.21 mmHg) and of
-3.30 mmHg (95 % CI: -5.77 mmHg; -0.83 mmHg) of diastolic blood pressure without
indicating flavan-3-ol levels (HOOPER et al. 2008). These two meta-analyses reported a nearly
identical effect of cocoa on blood pressure, whereas a recent meta-analysis evaluating the
effects of chocolate, cocoa and flavan-3-ols on the major CVD risk factors failed to show a
significant effect on systolic blood pressure. The diastolic blood pressure was reduced by
-1.60 mmHg (95 % CI: -2.77 mmHg; -0.43 mmHg) (HOOPER et al. 2012). However,
subgrouping by EC dose, the authors suggested a reduction of both systolic and diastolic
69
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
blood pressure when the consumption of EC exceeds 50 mg/d that is comparable to two up to
five apples per day (NEVEU et al. 2010) depending on cultivar.
Various authors have reviewed the influence of cocoa flavan-3-ols on blood pressure. The
main conclusion was that flavan-3-ols of cocoa may reduce blood pressure, especially in
hypertensive patients (GRASSI et al. 2010; KHAWAJA et al. 2011). JIMENEZ et al. (2012)
indicated that each mmHg reduction in blood pressure is expected to lead to a risk reduction
of 2-3 % of cardiovascular events. The mechanism behind the blood pressure lowering effect
is suggested to be related to the ability of flavan-3-ols to modulate NO production and thus
the reduction of blood flow and their ability to inhibit the angiotensin I converting enzyme
(CORTI et al. 2009; ENGLER et al. 2004a; ENGLER et al. 2006).
In summary, cocoa derived flavan-3-ols have been reported to have a positive effect on blood
pressure especially in prehypertensive or high blood pressure patients. Although the effect of
apple flavan-3-ols on blood pressure has not been investigated, it seems likely that the
reported levels of flavan-3-ols and specifically EC in some cultivars would show a similar
effect.
70
white chocolate
dark chocolate
white chocolate
milk chocolate
white chocolate
dark chocolate
white chocolate
dark chocolate
90 g/d
100 g/d
90 g/d
100 g/d
105 g/d
105 g/d
300 mL/d
200 mL/d
35 g/d
45 g/d
46 g/d
46 g/d
cocoa-free bar
22 g cocoa powder bar
placebo
22 g sugared cocoa
22 g sugar-free cocoa
0
≈500 mg PP/d
0
88 mg/d
< 5 mg/d
168 mg/d
≈28 mg/d
≈900 mg/d
0
550 mg/d
traces
259 mg/d
0
113.3 mg/dose
0
161.3 mg/dose
161.3 mg/dose
22 mg/dose
701 mg/dose
flavan-3-ol
content
66 mg/d
66 mg/d
not reported
39 mg/d
1 mg/d
87 mg/d
not reported
46 mg/d
0
22 mg/dose
0
48 mg/dose
48 mg/dose
0
139 mg/dose
EC content
r, co
r, co
r
r, pc, db, co
r, sb
r, pc, db
r, pc, sb, co
r, db, co
design
15 d
15 d
15 d
15 d
14 d
14 d
2w
2w
2w
2w
2w
2w
single
dose
single
dose
duration
healthy
humans
hypertensive
humans
young male
soccer players
hypertensive
humans
healthy men
healthy
humans
humans BMI
30 kg/m2
humans BMI
31.6 kg/m2
subjects
15
10
14
14
20
19
20
10
11
44
21
na
control
↓#
no effect
↓**
no effect
↓
no effect
no effect
no effect
no effect
no effect
no effect
control
##
↓
control
no effect
##
↓
not
reported
control
↓
no effect
↓**
no effect
↓*
no effect
no effect
no effect
no effect
no effect
no effect
control
after
↓*
exercise
control
##
↓
control
no effect
↓#
effect on blood pressure
systolic
diastolic
low flavan-3-ols
10 mg/d
2 mg/d
r, db, co, c
30 d
CAD patients
16
no effect
no effect
high flavan-3-ols
236 mg/d
118 mg/d
30 d
no effect
↓*
1
(BERRY et al. 2010), 2(FARIDI et al. 2008), 3(ENGLER et al. 2004b), 4(SHIINA et al. 2009), 5(MUNIYAPPA et al. 2008), 6(FRAGA et al. 2005), 7(GRASSI et al. 2005b), 8(GRASSI
et al. 2005a), 9(HEISS et al. 2010); a number of subjects,b low flavan-3ol content, c high flavan-3-ol content, r randomised, pc placebo-controlled, db double-blind, c
controlled, co cross-over, sb single blind * p < 0.05, ** p < 0.001, # p < 0.05 compared to control, ## p < 0.001 compared to control
cocoa beverage
9
8
8
7
7
6
6
ocoa drinkb
5
cocoa drink
5c
4
4
dark chocolateb
3
dark chocolatec
3
liquid cocoa
2
chocolate bar
2
low flavan-3-ols
high flavan-3-ols
intervention
Intervention studies investigating the effect of flavan-3-ols on blood pressure
cocoa beverage
1
food
Table 10:
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
71
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
Platelet aggregation is, in addition to endothelial function, also involved in the development
of CVD, due to the ability of platelets to bind to collagen and therefore contribute to plaque
formation in blood vessels (OSTERTAG et al. 2010). The measurement of platelet aggregation
is complex and several different ex vivo methods have been developed. One frequently used
method is the measurement of shear stress-induced platelet aggregation by a Platelet
Functional Analyser (PFA). This instrument detects the closure time platelets need to
aggregate with collagen-epinephrine or collagen-adenosin diphosphate (ADP) coated
membranes (OSTERTAG et al. 2010). The four studies which have used this technique to
investigate the influence of polyphenols on platelet aggregation are summarised in Table 11.
All of them reported an increase of the closure time, which represents a decrease in platelet
aggregation and a decrease risk of CVD (BORDEAUX et al. 2007; PEARSON et al. 2002; REIN et
al. 2000; WANG-POLAGRUTO et al. 2006). According to OSTERTAG et al. (2010), this method
represents a good simulation of the conditions in small arteries within the human body. In
contrast, flow cytometry (FC), another method to analyse platelet aggregation, simulates only
low shear stress conditions and may be less suitable to mimic these processes. Two studies
listed in Table 11 (HAMED et al. 2008; MURPHY et al. 2003) used this method which is based
on platelet aggregation induced by arachidonic acid, ADP or collagen. Both studies reported a
significant decrease of platelet aggregation by administering flavan-3-ols, in form of
chocolate or flavan-3-ol containing tablets, respectively, which would also be expected to
decrease the risk of CVD. CARNEVALE et al. (2012) investigated platelet aggregation by
analysing the platelet activation of an oxidase producing reactive oxygen species and reported
a significant decrease of platelet aggregation only in smokers ingesting dark chocolate.
Similarly, the measurement of shear stress dependent platelet aggregation resulted in a
significant decrease after dark chocolate consumption in contrast to no effect after white
chocolate ingestion (HERMANN et al. 2006). Consequently, the studies in Table 11 show all a
tendency towards a decrease in platelet aggregation, but decreases were only found with
relatively high doses (≤ 200 mg/d or dose).
72
cocoa drink
caffeine drink
6
6
aspirin + cocoa
cocoa beverage
5
5
aspirin
5
cocoa drink
4
chocolate
3
no chocolate
3
dark chocolate
18.75 mg cocoa/dose
17 mg caffeine/dose
81 mg aspirin+18.8 g cocoa
18.75 g cocoa
897 mg/dose
0
897 mg/dose
897 mg/dose
0
446 mg/d
high flavan-3-ols
81 mg aspirin
43 mg/d
nr
nr
flavan-3-ol
content
nr
low flavan-3-ols
5.9 g cocoa
0
40 g/dose
40 g/dose
40 g/dose
40 g/dose
intervention
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
nr
EC
content
nr
co
r, db
24 h dietary
recall
r
r, co, sb
design
single
dose
single
dose
6w
6w
24 h
single
dose
single
dose
duration
Intervention studies investigating the effect of flavan-3-ols on platelet aggregation
white chocolate
2
2
milk chocolate
1
dark chocolate
1
food
Table 11:
humans
humans
postmenopausal
hypercholestero
mic women
healthy humans
10
10
16
9
8
141
1394
10
10
20
smokers
male smokers
20
20
smokers
healthy
20
na
healthy
subjects
PFA, epinephrine
induced
PFA, epinephrine
induced
PFA, ADP induced
PFA, epinephrine
induced
shear stress dependent
platelet function
platelet oxidase
activation
method
↓*
no effect
↓**
↓**
↓**
↓
control
↓
#
control
↓*
no effect
no effect
no effect
↓**
no effect
effect
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
73
3
234 mg/d
flavan-3-ol tablets
2
<6 mg/d
flavan-3-ol
content
700 mg/d
placebo
100 g/d
intervention
to be continued
nr
nr
4
EC
content
nr
blinded,
paralleldesign
design
28 d
28 d
1w
duration
5
humans
humans
subjects
13
15
28
na
↓*
↓*
FC, ADP induced
FC, AA induced
7
no effect
FC, AA induced
6
no effect
↓*
effect
FC, ADP induced
FC, AA induced
method
(CARNEVALE et al. 2012), (HERMANN et al. 2006), (BORDEAUX et al. 2007), (WANG-POLAGRUTO et al. 2006), (PEARSON et al. 2002), (REIN et al. 2000), (HAMED et al.
2008), 8(MURPHY et al. 2003); a number of subjects, nr not reported, r randomised, db double-blind, c0 cross-over, sb single blind, FC: flow cytometry; PFA: platelet
function analyser, AA arachidonic acid; * p < 0.05, ** p < 0.001, # p < 0.05 compared to control
1
tablets
8
dark chocolate
7
food
Table 11:
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
74
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
OSTERTAG et al. (2010) reviewed the published studies and concluded that flavan-3-ols may
reduce platelet aggregation, but due to the limited number of available studies and the
different methods used, a clear conclusion was not possible. One hundred g dark chocolate
with 70 % cocoa solids (≈33 g flavan-3-ols) was estimated to have a similar acute effect as
81 mg aspirin (OSTERTAG et al. 2010; PEARSON et al. 2002). Previous reviews (HEPTINSTALL
et al. 2006; HOLT et al. 2006; PEARSON et al. 2005) had also concluded that platelet
aggregation decreased after flavan-3-ol rich cocoa consumption and should thus lead to an
improvement of cardiovascular health. The postulated mechanisms of action for this effect are
that flavan-3-ols might induce changes in membrane fluidity, ligand-receptor affinity and
intracellular pathways. Moreover, it appears also possible that flavan-3-ols interact with the
lipid bilayer. However, these mechanisms have only been investigated in vitro and the effects
of flavan-3-ols after absorption depends on their bioactivity, the bioactivity of their
metabolites, and on their distribution in the human body. In summary, flavan-3-ols derived
from cocoa are able to positively influence platelet aggregation. Apple flavan-3-ols would be
expected to have similar effects but their bioactivity in humans in relation to platelet
aggregation remains to be investigated.
Intervention studies in human subjects examining the effect of flavan-3-ols on blood lipids,
especially total cholesterol (TC), low density lipoproteins (LDL) and high density lipoproteins
(HDL) are summarised in Table 12. Four of these trials used cocoa powder dissolved in water
(BABA et al. 2007a; BABA et al. 2007b; BALZER et al. 2008; TAUBERT et al. 2007a), two added
cocoa to a normal diet or to a chocolate intervention (CREWS et al. 2008; WAN et al. 2001)
whereas all other authors gave their participants chocolate with different amounts of flavan-3ols (traces to 1000 mg/d). The chocolate doses ranged from 5.6 g/d to 105 g/d, representing
high caloric loads per serving size.
Most studies of Table 12 reported a non significant effect or no effect compared to baseline
on TC plasma levels. Only FRAGA et al. (2005) showed a significant decrease in TC in young
male soccer players after they ingested milk chocolate compared to white chocolate low in
flavan-3-ols for two weeks. Additionally, the LDL levels in blood plasma decreased
significant by 15 % in this study. Three other interventions reported significant decreases in
LDL levels compared to baseline (GRASSI et al. 2005b), two of them with high (963 mg and
700 mg/d) flavan-3-ol intake (BALZER et al. 2008; HAMED et al. 2008). Concerning the effect
on HDL levels, three studies reported in a significant increase (≤ 10 %) in HDL levels
compared to baseline and three compared to control group, with the highest increase in HDL
75
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
of 23 %. The administered doses leading to significant increases in HDL range from 16 mg to
700 mg/d flavan-3-ols (Table 12), whereby the lowest and the highest doses resulted in the
same HDL level increase (9 %). This could be due to different subjects or intervention time.
However, the individual studies listed in Table 12 indicated that interventions with flavan-3ols might improve total cholesterol, LDL and HDL levels compared to baseline.
However, the meta-analysis (TOKEDE et al. 2011) including ten clinical trials also listed in
Table 12 (ALMOOSAWI et al. 2010; BALZER et al. 2008; CREWS et al. 2008; DAVISON et al.
2008; ENGLER et al. 2004b; FRAGA et al. 2005; GRASSI et al. 2005a; GRASSI et al. 2005b;
MUNIYAPPA et al. 2008; WAN et al. 2001) failed to show a statistical significant increase in
HDL levels after intervention periods between two and twelve weeks compared to untreated
control. The ingested foods were cocoa beverages and chocolate with flavan-3-ol contents
ranging from 88 mg to 1000 mg. The TC and LDL levels, however, showed significant
decreases. The authors noted that flavan-3-ols in doses lower than 500 mg/d evoke a higher
reduction in LDL concentration, and that patients with documented cardiovascular disease
risk factors may obtain higher reductions in LDL and TC levels by flavan-3-ol treatment. The
mechanisms behind these LDL lowering effects seem to be based on the ability of flavan-3-ol
to inhibit cholesterol absorption (TOKEDE et al. 2011) and/or on their ability to increase the
expression of LDL receptors in the tissues so that LDL together with its cholesterol can be
absorbed by the cells and thus removed from blood (MATSUI et al. 2005). However, the shortterm intervention studies (2 weeks) reviewed by TOKEDE et al. (2011) often lack an adequate
blinding. As a conclusion, the authors stated that chocolate seems to be the more effective
matrix for flavan-3-ols compared to cocoa beverages.
Another meta-analysis comparing intervention to placebo (JIA et al. 2010) of short-term
intervention trials (6 weeks) (BABA et al. 2007b; BALZER et al. 2008; FRAGA et al. 2005;
GRASSI et al. 2005a; GRASSI et al. 2005b; MUNIYAPPA et al. 2008; TAUBERT et al. 2007a;
WAN et al. 2001) found similar results to TOKEDE et al. (2011): significant lowering of LDL,
less reduction in TC and no changes on HDL. According to these authors, each mg/dL
reduction in LDL concentration can reduce the coronary artery disease risk by 1 %.
Table 12 lists individual studies and their outcomes after intervention mainly compared to
baseline. Nearly half of the studies reported no significant effects on one of the three
measured blood lipid parameters (HDL, LDL TC), indicating a strong heterogeneity between
the results of the individual studies. A conclusion can be deduced from the meta-analyses
76
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
described above (JIA et al. 2010; TOKEDE et al. 2011). In summary, LDL levels seem to be
improved by flavan-3-ols whereas total cholesterol and HDL levels remain unaffected.
Flavan-3-ols also have a potential antioxidant effect (see Chapter 5) and the oxidised LDL
level in the blood appears to be an even better biomarker for cardio-artery diseases than TC,
LDL and HDL levels (KATZ et al. 2011). Such a modified LDL is not absorbed by the LDL
receptors. According to BABA et al. (BABA et al. 2007a; BABA et al. 2007b), the level of
oxidised LDL was significantly reduced in the blood after cocoa ingestion. Another oxidation
biomarker for cardio-artery diseases is the level of plasma F2-isoprostanes, an indicator of in
vivo lipid peroxidation (WISWEDEL et al. 2004), as isoprostanes could oxidise cholesterol or
LDL proteins. These authors found a constant F2-isoprostane level in volunteers ingesting a
high flavan-3-ol cocoa drink containing 187 mg/100 mL, whereas in the low flavan-3-ol
group (14 mg/100 mL) the level of F2-isoprostanes increased postprandial. CORTI et al. (2009)
attributed this effect to the antioxidant activity of flavan-3-ols. Additionally, although cocoa
butter is a saturated fat which contains on average 33 % stearic acid, this fatty acid, unlike
other saturated fatty acids, has no influence on TC, LDL or HDL levels. Consequently, the
measured effects of cocoa on cholesterol levels should be derived from the flavan-3-ol content
in chocolate used as the intervention (CORTI et al. 2009).
In summary, flavan-3-ols may have a positive effect on blood lipid levels by decreasing
cholesterol absorption from the gastrointestinal tract, improving cellular cholesterol uptake
from blood by the tissues due to increased LDL receptors and additionally by protecting LDL
from oxidation due to their antioxidant activity. These effects could also be expected with the
ingestion of apple polyphenols.
77
diet+cocoa+chocolate
American diet
9
9
enriche chocolate
8
dark chocolate
white chocolate
8
8
dark chocolate
white chocolate
7
7
milk chocolate
white chocolate
6
6
chocolate
placebo
5
5
sugar+cocoa
sugar
4
4
cocoa
3
dark chocolate
2
dark chocolate
22 g+16 g/d
75 g/d
75 g/d
75 g/d
100 g/d
90 g/d
105 g/d
105 g/d
41 g/d
12 g+26 g/d
466 mg
procyanidins
< detection limit
475 mg
285 mg
0
88 mg/d
0
168 mg/d
< 5 mg/d
not reported
200 mg/d
111 mg/d
170 mg/d
114 mg/d
66.0 mg/d
39 mg/d
98 mg/d
266 mg/d
26 g/d
0
129 mg/d
200 mg/d
19.5 g/d
12 g/d
96.7 mg/d
133 mg/d
13 g/d
64.5 mg/d
46 mg/d
traces
EC content
traces
700 mg/d
259 mg/d
traces
flavan-3-ol
content
0
100 g/d
46 g/d
46 g/d
intervention
r, co, 2periods
not r
r, co
co
c, pd
r
db,
comparative
not reported
r, pc, db
design
Intervention studies investigating the effect of flavan-3-ols on blood lipids
low PP dark chocolate
1
1
food
Table 12:
4w
4w
3w
15 d
15 d
14 d
14 d
6w
12 w
12 w
4w
1w
2w
2w
duration
healthy humans
healthy humans
humans
young male
soccer players
healthy women
humans
humans
humans
humans
subjects
23
15
15
15
15
14
12
12
25
160
28
11
10
na
↓
↓
↓
↓
no effect
control
no effect
no effect
no effect
no effect
no effect
11 % ↓*
↑
↑
↓
↓
no effect
control
no effect
no effect
no effect
no effect
no effect
15 % ↓*
↓
↑
↑
↓
↓
↓
↓
↓
↓
6% ↓*
no effect
no effect
LDL
↓
no effect
no effect
TC
effect
4 % ↑#
control
14 % ↑##
11 % ↑##
no effect
no effect
no effect
↓
↑
↑
↓
23% ↑#
↑
10 % ↑*
8 % ↑*
6 % ↑*
3 % ↑*
9 % ↑*
no effect
no effect
HDL
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
78
chocolate
2
100 g/d
90 g/d
31 g/d
6.3 g/d
5.6 g/d
20 g/d
3
88 mg/d
0
900 mg/d
28 mg/d
28.3 mg/d
0
1000 mg/d
500 mg/d
963 mg/d
high flavan-3-ols
20 g/d
75 mg/d
8.3 g/d
low flavan-3-ols
45 g/d
45 g/d
37g+237mL/d
0.45 g/d
flavan-3-ol
content
4
66.0 mg/d
87 mg/d
1mg/d
5.1 mg/d
0
38 mg/d
19 mg/d
203 mg/d
17 mg/d
< 2 mg EC/d
16.6 mg EC/d
EC content
r, co
5
r, pc, co
r, db, pc
r, sb, co
r, doublemarked
r, pc, db
r, pc, pd
design
15 d
15 d
2w
18 w
2w
30 d
30 d
8w
8w
6w
6w
duration
6
hypertensive
humans
hypertensive
humans
prehypertensive
humans
overweight,
obese
type 2 diabetic
humans
type 2 diabetic
humans
humans
subjects
10
10
20
22
22
14
31
41
12
45
45
na
↓
12 % ↓*
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
↓*
no effect
no effect
no effect
9 % ↑*
no effect
no effect
HDL
no effect
no effect
no effect
LDL
7(GRASSI et al. 2005a) 8
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
no effect
TC
effect
(ENGLER et al. 2004b), (HAMED et al. 2008), (BABA et al. 2007a), (BABA et al. 2007b), (KURLANDSKY et al. 2006), (FRAGA et al. 2005),
, (MURSU et al.
2004), 9(WAN et al. 2001), 10(CREWS et al. 2008),11(MELLOR et al. 2010), 12(BALZER et al. 2008), 13(ALMOOSAWI et al. 2010), 14(TAUBERT et al. 2007a), 15(MUNIYAPPA et al.
2008), 16(GRASSI et al. 2005b), a number of subjects, r randomised, pc placebo-controlled, db double-blind, c controlled, pd parallel design, co cross-over, sb single blind, * p
< 0.05, # p < 0.05 compared to control, ## p < 0.001 compared to control
1
dark chocolate
white chocolate
16
16
cocoa beverage
placebo
15
15
dark chocolate
white chocolate
14
14
dark chocolate
dark chocolate
13
13
coca beverage
12
low PP chocolate
11
11
e
37g+237mL/d
intervention
to be continued
chocolate+beverag
placebo
10
10
food
Table 12:
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
79
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
The effect of flavan-3-ols on endothelial function may also evoke positive effects on brain
function. According to Spencer (2008), flavan-3-ols may enhance cortical blood flow that
further reduces the risk for dementia and stroke and enhances cognition in the elderly (FISHER
et al. 2006). The vascular cognitive decline in the elderly appears to be slowed down by
flavan-3-ols and the author concluded that increasing cerebral blood flow may help to support
the treatment of dementia and stroke.
In additional to the improvement in cerebral blood flow, ingested flavan-3-ols may also
increase dermal blood flow and therefore may be beneficial to skin health. In a cross-over
trial with ten healthy women ingesting either a low (6.6 mg EC) or a high (66 mg EC) cocoa
flavan-3-ol drink, dermal blood flow was significantly increased (1.7-fold compared to
baseline) two hours after high flavan-3-ol cocoa ingestion (NEUKAM et al. 2007). Due to
increased plasma concentrations measured 2 hours after administration, the authors concluded
that EC was responsible for this effect. The mechanism behind this improvement in dermal
blood flow seems to be similar to the improvement in endothelial function and thus blood
flow as described above. Moreover, this increase in cutaneous and subcutaneous blood flow is
also thought to increase the skin density and skin hydration. Another potential protective
effect of flavan-3-ols on skin health is the protection against UV light which may lead to
reduced erythema. HEINRICH et al. (2006) administered either a low (27 mg/d) or a high
(326 mg/d) flavan-3-ol cocoa drink to women for 12 weeks. The UV-induced erythema was
significantly decreased by 25 % in the high flavan-3-ol group compared to baseline, whereas
the low flavan-3-ol group showed no changes. WILLIAMS et al. (2009) confirmed this finding.
Thirty healthy subjects ingested 20 g of either high or low flavan-3-ol chocolate per day for
12 weeks. The minimal erythema dose, which is the lowest UV dose resulting in visible
erythema of the skin, doubled after the 12 weeks intervention compared to baseline in the
high flavan-3-ol chocolate group. This indicated a reduced sunburn risk and might also reduce
the risk for UV-induced skin damage. Consequently, flavan-3-ols seem to have a similar
effect in the human skin as in plants by acting as photo-protective components and therefore
might protect the human skin from sunburn, skin cancer and aging.
80
LITERATURE REVIEW
6.1.2
HEALTH EFFECTS OF APPLE POLYPHENOLS
Flavan-3-ols in the prevention of other Western diseases
A further potential beneficial health effect of flavan-3-ols is their influence on glucose
tolerance which has been investigated by measuring the fasting blood glucose level and by
performing an oral glucose tolerance test (Table 13). The blood glucose concentration is
strictly hormonally controlled and a failure of this control leads to metabolic syndrome, a
multi-symptom disorder causing impaired glucose tolerance, obesity, hyperglycemia,
hypertension, dyslipidaemia and type 2 diabetes (HANHINEVA et al. 2010).
The fasting blood glucose level in human subjects was significantly reduced after ingesting
dark chocolates with at least 66 mg flavan-3-ols/d over 2 weeks. Moreover, the response to
oral glucose tolerance test was improved, for example the plasma glucose level after glucose
intake was significantly reduced, in response to 15 days dark chocolate intake with 88 mg to
500 mg flavan-3-ols/d. No differences were observed between healthy, obese or hypertensive
humans or those with impaired glucose tolerance. Additionally, it seems possible that low
doses of flavan-3-ols may also provoke an improvement in glucose tolerance.
Searching for a mechanism, animal and cell line studies have shown that flavan-3-ols have no
influence on long-term glucose control, but could augment postprandial glucose metabolism
(JALIL et al. 2008b) by inhibiting active glucose transport (JOHNSTON et al. 2005). Moreover,
PINENT et al. (2006) have suggested that EC prevents hyperglycemia by inducing β-cell
regeneration. In support of a positive effect of flavan-3-ols on diabetes control, KATZ et al.
(2011) have reported that flavan-3-ols improve insulin resistance in patients with metabolic
syndrome thus slowing the progression of type 2 diabetes.
81
2
100 g/d
90 g/d
100 g/d
90 g/d
100 g/d
100 g/d
88 mg/d
0
nr
3
147.02 mg C+EC/d
0.04 mg C/d
1000 mg/d
20 g/d
66 mg/d
0
nr
111 mg/d
38.0 mg/d
19.0 mg/d
EC content
4
r, co
r, co
co
r, sb,
co
design
15 d
15 d
15 d
15 d
15 d
15 d
2w
2w
duration
grade one patients with
essential hypertension
healthy humans
humans with hypertension
and impaired glucose
tolerance
overweight and obese
humans
subjects
20
15
19
14
na
↓*
oral glucose tolerance test
a
↓**
no effect
oral glucose tolerance test
fasting blood glucose level
no effect
↓*
no effect
↓**
no effect
↓**
↓**
effect
fasting blood glucose level
oral glucose tolerance test
oral glucose tolerance test
fasting blood glucose level
method
(ALMOOSAWI et al. 2010), (GRASSI et al. 2008), (GRASSI et al. 2005a), (GRASSI et al. 2005b), nr not reported, r randomised, sb single blind, co cross-over, number of
subjects, * p < 0.05, ** p < 0.001
1
dark chocolate
4
white chocolate
4
dark chocolate
white chocolate
3
3
dark chocolate
white chocolate
2
2
500 mg/d
flavan-3-ol content
20 g/d
intervention
Intervention studies investigating the effect of flavan-3-ols on glucose tolerance
flavan-3-ol rich
dark chocolate
1
food
Table 13:
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
82
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
Flavan-3-ols may also be beneficial in preventing overweight and obesity. In some
intervention studies, high doses of chocolate were administrated to the subjects, however, in
spite of the high calorie load, no increase in body weight was observed (HAMED et al. 2008;
KURLANDSKY et al. 2006). In these studies, the subjects were instructed to make no changes
to their diet and to omit additional physical activity so as to compensate the extra energy
intake (528 kcal and 364 kcal). The chocolate intake ranged from 41 g/d to 100 g/d. Over a
period between seven days and six weeks, no significant weight changes were observed
compared to the weight assessment before the intervention. It may be possible therefore that
flavan-3-ols have a positive effect on obesity. PINENT et al. (2006) related this effect to a
direct influence of flavan-3-ols on the adipose tissue. Flavan-3-ols may modulate processes in
adipocytes such as lipolysis, lipid and glycogen synthesis, glucose uptake and differentiation.
MATSUI et al. (2005) reported results from a rat study, indicating that flavan-3-ols suppress
fatty acid synthesis in the liver and fatty acid transport systems with concurrent activation of
thermogenesis preventing triglyceride accumulation. However, further controlled studies are
needed to confirm or refute these effects of flavan-3-ols on weight control.
Finally, flavan-3-ols, due to their antioxidant capacity (as discussed in Chapter 5 and above in
relation to CVD) are suggested to have a preventive effect against cancer (LEE et al. 2006).
Antioxidants such as flavan-3-ols can quench free radicals and thus potentially protect against
DNA damage, which is associated with cancer development. However, only few
observational studies have reported a weak relationship between flavan-3-ol intake and a
reduced cancer risk (MASKARINEC 2009). Cell line studies, however, suggest flavan-3-ols may
protect from cancer. For example, PIERINI et al. (2008) reported that oligomers and polymers
of apple flavan-3-ols reduce the proliferation of oesophageal adenocarcinoma cells in vitro,
whereas monomers and dimers showed no activity. Although oligomeric and polymeric
flavan-3-ols are not absorbed in vivo, they are in contact with the epithelium of the
gastrointestinal tract and would be in contact with the oesophagus during swallowing. Other
authors reported a possible in vitro cancer chemopreventive potential against proinflammatory cytokine-mediated skin cancer (KIM et al. 2010). SHARMA et al. (2010)
investigated the effect of grape seeds procyanidins on lung cancer cells and showed a
significant inhibitory effect of the flavan-3-ols on the overexpression of cylcooxigenase-2 and
prostaglandins, which are associated, with cancer cell growth inhibition and induction of
apoptosis. In humans, ingesting either white chocolate (5 mg polyphenols, no EC) or dark
chocolate (860 mg polyphenols, 58 mg EC) DNA damage in the mononuclear blood cells was
significantly reduced two hours after the acute dose of dark chocolate intake compared to
83
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
white chocolate ingestion (SPADAFRANCA et al. 2010). The total antioxidant activity in blood
plasma was unaffected after both interventions, which leads to the assumption that EC may
improve DNA resistance to oxidative stress by another mechanism than the antioxidant
potential. However, this mechanism needs further investigations. To date, no human study has
reported positive effects in relation to cancer prevention and the in vivo mechanisms also need
further investigation. KATZ et al. (2011) suggested that flavan-3-ols may be able to protect
against cancer by increasing serum antioxidant status and by enhancing apoptosis of cancer
cells in vitro.
In summary, the main beneficial health effects of flavan-3-ols are those influencing CVD. The
potential mechanisms behind these CVD effects are the modulation of the nitric oxide system
and membrane fluidity, the inhibition of cholesterol absorption and active glucose absorption,
and reduction of cholesterol oxidation. These mechanisms, and other beneficial physiological
changes such as an improvement in endothelium function, platelet aggregation, blood
pressure, blood lipids profile, reduced DNA damages, might also improve brain function, skin
health, and glucose tolerance, and thus help prevent diabetes, obesity and cancer. Most studies
have investigated cocoa flavan-3-ols, but due to the high structural similarity to apple flavan3-ols, it seems to be possible that apple flavan-3-ols, especially EC, may have comparable
effects.
6.2
Health effects of phenolic acids
The health effects of phenolic acids and chlorogenic acid in particular, have been mostly
investigated with coffee. As coffee and apples both contain many different substances as well
as having different matrices (liquid and solid), a direct extrapolation of the health effects of
coffee to apples is not possible. However, health effects specifically attributed to the phenolic
acids in coffee may also have comparable effects to apples, because cider apples and coffee
can provide similar amounts of chlorogenic acid (NEVEU et al. 2010). MONTAGNANA et al.
(2012) reported the positive influence of chlorogenic acids in coffee on reducing blood
pressure, inflammation, type 2 diabetes and platelet aggregation.
In relation to cardiovascular diseases, CA may have a beneficial effect on blood flow as
described above for flavan-3-ols, but the high caffeine content of coffee is a complicating
factor. BUSCEMI et al. (2010) investigated the effect of phenolic acids in coffee on endothelial
function by measuring FMD in healthy subjects received either a cup of caffeinated or
decaffeinated coffee. Caffeinated coffee significantly decreased FMD due to the caffeine
contained, whereas after decaffeinated coffee ingestion FMD increased, but not significantly
84
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
(p=0.12). It is therefore possible that CA prevent the negative effect of caffeine on endothelial
function, however, this effect is not clear and further investigations using caffeine and CA as
pure substances are necessary.
Concerning blood pressure, WATANABE et al. (2006) reported a significant reduction in mildly
hypertensive patients after ingesting of 140 mg/d CA extracted from green coffee beans over
12 weeks compared to the placebo group. According to the authors, the improved NO
bioavailability induced by CA is responsible for this effect. BONITA et al. (2007) reviewed the
effects of phenolic acids in coffee related to cardiovascular diseases and concluded that CA
counteract the negative effects of caffeine.
The main beneficial effect attributed to phenolic acids however, and CA in particular, is their
ability to influence glucose metabolism. CA increased glucose tolerance measured by the
glucose tolerance test in mice (ONG et al. 2012) and stimulated glucose transport through
translocation of a glucose transporter in skeletal muscle. CA may therefore be beneficial for
patients with type 2 diabetes. A comparable trial in isolated rat skeletal muscle showed that
caffeic acid (CF) but not CA stimulates the insulin independent glucose transport and resulted
in a reduced intracellular energy status (TSUDA et al. 2012). These authors noted that CF is the
active compound and suggested that CA may lead to similar effects, when metabolised to CF
(see Chapter 4.2). Human intervention studies with coffee have reported comparable results.
In healthy volunteers consuming 25 g glucose in either 400 mL caffeinated or 400 mL
decaffeinated coffee, the glucose and insulin concentration tend to be lower in decaffeinated
coffee in the first 30 min. Thus, CA might have an antagonistic effect on glucose transport in
contrast to caffeine that seems to be responsible for the impaired glucose tolerance after
coffee consumption (JOHNSTON et al. 2003). In an oral glucose tolerance test with overweight
men, 1 g CA significantly reduced both glucose and insulin levels in the first 15 min after
administration compared to placebo, but not the glucose or insulin area under the curve values
over 2 h (VAN DIJK et al. 2009). Potential mechanisms behind this findings are explained by
BIDEL et al. (2008). CA might be able to retard the action of α-glucosidase or/and inhibit
glucose transporters both at the intestinal stage. Additionally, the inhibition of glucose-6phosphatase at the hepatic stage could result in reduced plasma glucose levels. Therefore, CA
may have a positive effect on hyperglycemia and hyperinsulinemia and may even retard the
development of type 2 diabetes.
In overweight subjects, the ingestion of high CA doses (1050 mg/d, 700 mg/d) extracted from
green coffee over 6 weeks resulted in significant reductions in body weight (VINSON et al.
85
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
2012). In addition to the influence of CA on glucose metabolism, the authors proposed that
CA and its metabolite CF inhibit α-amylase and therefore glucose cleavage from starch.
MONTAGNANA et al. (2012) reviewed the physiological effects of CA in coffee and concluded
that CA has the ability to reduce glucose uptake by adipocytes and to retard their
proliferation. It is possible therefore that apple phenolic acids may help to prevent type 2
diabetes and help reduce body weight.
As reported for flavan-3-ols, dihydrocaffeic acid (DHC), a major metabolite of CA has been
reported to protect keratinocytes cells from UV irradiation (POQUET et al. 2008a) by direct
radical scavenging of the reactive oxygen species. DHC also increase the antioxidant potential
of the cell itself and interfere directly with the pathways involved in cytokine stimulation.
However, this effect was investigated in vitro and the dose needed for an effect in humans is
unknown. It is also unknown whether DHC reach the target skin cells in vivo but nevertheless,
this study showed a potential effect of DHC on skin health.
Moreover, a pilot study with healthy older participants investigated the effect of high amounts
of CA (177 mg CA) in decaffeinated coffee on mood and cognition (CROPLEY et al. 2012).
This intervention was compared to decaffeinated coffee with regular CA level (76 mg CA),
caffeinated coffee (84 mg CA) and placebo. The decaffeinated coffee with high amounts of
CA was reported to improve some mood and behavioural measures, but to a lesser extent than
caffeinated coffee with regular CA content. Therefore, it appears possible that CA may
compensate the negative effects of decaffeination on mood and cognition but its activity on
neuro-cognitive functions needs further investigations.
Overall, phenolic acids have been reported to improve cardiovascular health, to reduce body
weight, to possibly protect from UV irradiation, and to positively influence mood and
cognition, whereas best evidence for a positive effect of phenolic acids on human’s health
seems to be related to their influence on glucose metabolism. Most investigations of the
beneficial effects of CA were conducted with coffee, which contains, in addition to CA, the
bioactive substance caffeine. Decaffeinated coffee was widely used to distinguish between the
effects of these two substances. It seems possible, however, that the beneficial effects related
to phenolic acids in coffee might also occur after consumption of apples, but further
investigations are clearly necessary.
86
LITERATURE REVIEW
6.3
HEALTH EFFECTS OF APPLE POLYPHENOLS
Health effects of dihydrochalcones
Phz has been used for many years as an anti-malaria agent (EHRENKRANZ et al. 2005). The
mechanism behind the antagonistic effect of Phz on malaria was first described by KUTNER et
al. (1987) as Phz irreversibly inhibiting the intraerythrocytic malaria growth.
However, during the investigations of Phz as a malaria treatment, an effect of Phz on glucose
metabolism was observed. EHRENKRANZ et al. (2005) reviewed the development in this field.
Phz and Phl are reported to have an inhibitory effect on the glucose transporters in the human
body. In the small intestine, glucose absorption occurs by a sodium-dependent glucose
transporter which can be inhibited by Phz (EHRENKRANZ et al. 2005). JOHNSTON et al. (2002)
investigated the effect of Phz (29 mg/L) on glucose plasma levels in humans ingesting
400 mL of clear and cloudy apple juice or water with the same glucose load. The glucose
response with apple juices was delayed compared to water, indicating that Phz might be
useful as a drug for diabetes treatment. Several studies have investigated the effect of Phz in
rats in which diabetes had been induced by alloxan (FREITAS et al. 2008), by streptozotocin
(BLONDEL et al. 1990; MASUMOTO et al. 2009) or by a partial removal of the pancreas
(ROSSETTI et al. 1987a; ROSSETTI et al. 1987b). All studies reported a decrease in plasma
glucose levels, but no influence on plasma insulin levels. Another sodium-dependent glucosetransporter is located in the kidney and is responsible for the reabsorption of filtered glucose.
The inhibitory effect of Phz on this transporter resulted in an increase in urinary glucose
excretion and a loss of glucose from the body. According to MATHER et al. (2010), the
inhibition of this glucose transporter in the kidney by Phz would be a good possibility for
diabetes treatment despite the side effect of diarrhoea. However, this seems unlikely as Phz is
metabolised to Phl before entering the blood circulation (see Chapter 4.3) and Phl has no
influence on this sodium-dependent glucose transporter, although it does inhibit the
facilitative glucose transporter located in essentially all cells on the basolateral side. In the
small intestine, the inhibition of this glucose transporter leads to a reduced release of glucose
into the blood circulation and also reduced blood glucose levels. This effect of Phl has mainly
been investigated in inverted sacs of rats’ small intestine, where the glucose was accumulated
in the tissue of the intestine (CRESPY et al. 2001; GROMOVA 2006; SCOW et al. 2011).
Nevertheless, Phz inhibits the glucose uptake in the small intestine and the reabsorption in the
kidney, whereas Phl inhibits the release of glucose from cell into the neighbouring tissues.
This helps explain why KOBORI et al. (2012) found 0.5 % of Phz in the diet of mice to be
sufficient to reduce their blood glucose levels. It would appear possible therefore to reduce the
87
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
development of type 2 diabetes in humans with Phz ingestion, but this needs further
investigation (HANHINEVA et al. 2010).
In addition to the effect of dihydrochalcones on glucose metabolism, Phl has been reported to
improve cancer treatment. Due to its ability to inhibit a glucose transporter, the transport of
glucose into liver cancer cells was inhibited. The inhibition of glucose transport into the liver
is the basis for a liver cancer treatment. The combined administration of Phl and paclitaxel, a
cancer drug, enhanced the effect of paclitaxel 4-fold compared to paclitaxel alone in a mouse
model (YANG et al. 2009). Also for melanoma cells (KOBORI et al. 1997) and leukaemia cells
(KOBORI et al. 1999), both tested in in vitro models, the potential of Phl to inhibit the transport
of glucose into cells is reported to be the main mechanism that led to Phl induced apoptosis.
KIM et al. (2009) and SZLISZKA et al. (2010) showed Phl induced apoptosis in human breast
and prostate cancer cells in vitro, respectively, and therefore highlighted the chemopreventive
potential of Phl. Overall, Phl seems to improve cancer treatment.
Another potential beneficial health effect of Phz is related to its structural analogy to
oestrogen and its possible effect in reducing bone loss and protecting against osteoporosis.
WANG et al. (2010) reported an oestrogenic and anti-oestrogenic effect of Phz in cell studies.
During oestrogen deficiency, Phz can act as a phyto-oestrogen and react with the
corresponding receptor. In the presence of high levels of oestrogen, Phz binds as a
competitive inhibitor to the receptor and shows anti-oestrogenic effects. In an investigation
with mice receiving Phz orally for 7 days, Phz induced a slight but not significant increase in
uterine weight and serum oestrogen contents (WANG et al. 2010). In an experiment with
ovariectomised rats with induced inflammation, an animal model for post-menopausal
osteoporosis, the animals received a diet with 55 mg Phz/day for 80 days (PUEL et al. 2005),
which corresponds to ten apples of 200 g FM per day (NEVEU et al. 2010). Phz was able to
prevent inflammation induced bone loss and generally improved bone mineral density. The
mechanism of action can be linked to the phyto-oestrogenic effect of Phz, which includes a
reduction of inflammation markers as an improvement in bone resorption (PUEL et al. 2005).
Consequently, Phz seems to elicit protective effects on bone loss but this effect has yet to be
demonstrated in humans.
6.4
Health effects of flavonols
Apple flavonols, mainly QG and QGal, have been little investigated in relation to beneficial
health effects. There are studies, however, with Q, and due to deglycosilation of QG and QGal
to Q in the small intestine and colon, it would seem likely that the proposed health effects of
88
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
Q would be similar for QG and QGal. Therefore, the positive effects of Q are reported in the
following paragraphs. However, human studies are scarce and mainly animal and cell studies
were reviewed.
Q and its glycosides are reported to have preventive effects on atherosclerosis. As reviewed
by ISHIZAWA et al. (2011), the metabolite of Q in the human body, quercetin glucuronide
inhibits the platelet-derived growth factor-induced cell migration and proliferation in vascular
smooth muscle cells in vitro. An in vivo study investigating the antioxidant effect of Q in
healthy humans ingesting a high or a low flavonol diet with onions and black tea as source of
polyphenols failed to show any significant differences between the two interventions by
measuring F2-isoprostanes and antibodies to malondialdehyde-modified LDL (O’REILLY et al.
2001). A reason for this outcome could be that the absorption of Q is too low to improve the
antioxidant status in humans. According to these authors and the review reported above, the
mechanism behind the described anti-atherosclerotic effect seems to be the cell proliferation.
Rat data indicates that flavonols might be able to attenuate symptoms of the metabolic
syndrome (PANCHAL et al. 2012). Dietary induced abdominal fat deposition in rats was
reduced with Q supplementation. Additionally, the systolic blood pressure was lowered but no
effect on body weight or dyslipidaemia was reported. The authors suggested that Q is able to
cause lipids trafficking away from the abdomen and therefore reduce the symptoms of the
metabolic syndrome.
The major health effects of flavonols, however, may be their ability to prevent and treat
cancer, although most of this evidence has been achieved so far by investigating the influence
of Q on cancer cell lines. Q is able to inhibit cell proliferation in colon carcinoma cells and
mammary adenocarcinoma cell lines. Moreover, RAMOS (2007) reported that Q induces
apoptosis of leukaemia, breast cancer, ovarian cancer, lung cancer, hepatoma, oral cancer and
colon cancer in cancer cell cultures. Another review covered the in vitro anti-invasive and in
vivo anti-metastatic activities of different flavonoids (WENG et al. 2012). The authors reported
that Q blocked the invasive activity in breast and prostate cancer. Furthermore in a cervical
cancer cell line, Q repressed the cell-matrix adhesion, migration and invasion, reduced the
ability of invasion of epidermal cancer cells and inhibited the motility and invasion of murine
melanoma cells. Moreover, NÖTHLINGS (2007) reported anti-cancer effects of flavonols in
humans. In a cohort study concerning flavonols and pancreatic cancer risk, Q intake showed
an inverse associated risk for pancreatic cancer during 8 years of follow up. A statistically
significant reduction of the relative risk was only obtained for the highest Q intake quintile
89
LITERATURE REVIEW
HEALTH EFFECTS OF APPLE POLYPHENOLS
(5 mg/1000 kcal per day) in current smokers with RR 0.55 (95 % CI: 0.30; 0.99; p trend
0.008). Regarding cell studies and this cohort study, it appears possible to prevent cancer by
increasing dietary Q intake.
In summary, the knowledge of postulated health effects of apple flavonols is predominantly
derived from in vitro cell studies and their effects in vivo remain uncertain. However, the
effect of Q on cancer, atherosclerosis and metabolic syndrome remains relevant for human
health, but would depend on the amount of flavonols ingested with apples.
6.5
Conclusion: potential health effects of polyphenols in apples
Summing up, the main beneficial health effects of flavan-3-ols, phenolic acids,
dihydrochalcones and flavonols of apples would be expected to be the prevention of CVD.
This effect can be mainly attributed to flavan-3-ols, and specifically EC, due to their
potentially high levels in apples and their widely accepted protective effect on CVD. Phenolic
acids and flavonols may support this effect to some extent. Dihydrochalcones, which are
unique to apples, influence glucose transport and may help prevent diabetes. Phenolic acids
may help to reduce weight and prevent obesity and diabetes by improving glucose
metabolism, and dihydrochalcones and flavonols may help prevent various cancers. The
advantage of apples therefore is the multiplicity of polyphenols they contain with a whole
range of bioactivities which help protect against all the major chronic diseases common in
industrialised countries. Apples appear to be an optimal snack and contribute to a healthy diet.
90
LITERATURE REVIEW
REFERENCES
REFERENCES
Almoosawi, S., L. Fyfe, et al. (2010). "The effect of polyphenol-rich dark chocolate on fasting
capillary whole blood glucose, total cholesterol, blood pressure and glucocorticoids in healthy
overweight and obese subjects." British Journal of Nutrition 103(6): 842-850.
Alonso-Salces, R. M., A. Barranco, et al. (2004a). "Polyphenolic profiles of Basque cider apple
cultivars and their technological properties." Journal of Agricultural and Food Chemistry 52:
2938-2952.
Alonso-Salces, R. M., C. Herrero, et al. (2004b). "Technological classification of Basque cider apple
cultivars according to their polyphenolic profiles by pattern recognition analysis." Journal of
Agricultural and Food Chemistry 52(26): 8006-8016.
Alonso-Salces, R. M., C. Herrero, et al. (2005). "Classification of apple fruits according to their
maturity state by the pattern recognition analysis of their polyphenolic compositions." Food
Chemistry 93(1): 113-123.
Alonso-Salces, R. M., C. Herrero, et al. (2006). "Polyphenolic compositions of Basque natural ciders:
A chemometric study." Food Chemistry 97(3): 438-446.
Andreasen, M. F., P. A. Kroon, et al. (2001). "Esterase activity able to hydrolyze dietary antioxidant
hydroxycinnamates is distributed along the intestine of mammals." Journal of Agricultural
and Food Chemistry 49(11): 5679-5684.
Apak, R., K. Güçlü, et al. (2007). "Comparative evaluation of various total antioxidant capacity assays
applied to phenolic compounds with the CUPRAC assay." Molecules 12: 1496-1547.
Aron, P. M. and J. A. Kennedy (2008). "Flavan-3-ols: Nature, occurrence and biological activity."
Molecular Nutrition and Food Research 52: 79 – 104.
Arts, I. C. and P. C. Hollman (2005). "Polyphenols and disease risk in epidemiologic studies."
American Journal of Clinical Nutrition 81(suppl): 317S-325S.
Arts, I. C., P. C. Hollman, et al. (2001a). "Dietary catechins and epithelial cancer incidence: the
Zutphen elderly study." International Journal of Cancer 92(2): 298-302.
Arts, I. C., D. R. Jacobs, Jr., et al. (2001b). "Dietary catechins in relation to coronary heart disease
death among postmenopausal women." Epidemiology 12(6): 668-675.
Arts, I. C. W., B. van de Putte, et al. (2000a). "Catechin contents of foods commonly consumed in The
Netherlands. 1. Fruits, vegetables, staple foods, and processed foods." Journal of Agricultural
and Food Chemistry 48(5): 1746-1751.
Auclair, S., G. Chironi, et al. (2010). "The regular consumption of a polyphenol-rich apple does not
influence endothelial function: a randomised double-blind trial in hypercholesterolemic
adults." European Journal of Clinical Nutrition 64(10): 1158-1165.
Awad, M. A. and A. de Jager (2002). "Relationships between fruit nutrients and concentrations of
flavonoids and chlorogenic acid in ‘Elstar’ apple skin." Scientia Horticulturae 92(3–4): 265276.
Awad, M. A., A. De Jager, et al. (2001a). "Formation of flavonoids and chlorogenic acid in apples as
affected by crop load." Scientia Horticulturae 91(3-4): 227-237.
Awad, M. A., A. Jager de, et al. (2000). "Flavanoid and chlorogenic acid levels in apple fruit:
characterisation of variation." Scientia Horticulturae 83: 249-263.
Awad, M. A., P. S. Wagenmakers, et al. (2001b). "Effects of light on flavonoid and chlorogenic acid
levels in the skin of 'Jonagold' apples." Scientia Horticulturae 88(4): 289-298.
Azuma, K., K. Ippoushi, et al. (2000). "Absorption of chlorogenic acid and caffeic acid in rats after
oral administration." Journal of Agricultural and Food Chemistry 48: 5496-5500.
Baba, S., M. Natsume, et al. (2007a). "Plasma LDL and HDL cholesterol and oxidized LDL
concentrations are altered in normo- and hypercholesterolemic humans after intake of different
levels of cocoa powder." Journal of Nutrition 137(6): 1436-1441.
Baba, S., N. Osakabe, et al. (2007b). "Continuous intake of polyphenolic compounds containing cocoa
powder reduces LDL oxidative susceptibility and has beneficial effects on plasma HDLcholesterol concentrations in humans." American Journal of Clinical Nutrition 85(3): 709-717.
Baba, S., N. Osakabe, et al. (2000). "Bioavailability of (-)-epicatechin upon intake of chocolate and
cocoa in human volunteers." Free Radical Research 33(5): 635-641.
91
LITERATURE REVIEW
REFERENCES
Balzer, J., T. Rassaf, et al. (2008). "Sustained benefits in vascular function through flavanol-containing
cocoa in medicated diabetic patients a double-masked, randomized, controlled trial." Journal
of the American College of Cardiology 51(22): 2141-2149.
Becker, E. M., L. R. Nissen, et al. (2004). "Antioxidant evaluation protocols: Food quality or health
effects." European Food Research and Technology 219: 561-571.
Bermúdez-Soto, M. J., F. A. Tomás-Barberán, et al. (2007). "Stability of polyphenols in chokeberry
(Aronia melanocarpa) subjected to in vitro gastric and pancreatic digestion." Food Chemistry
102(3): 865-874.
Berry, N. M., K. Davison, et al. (2010). "Impact of cocoa flavanol consumption on blood pressure
responsiveness to exercise." British Journal of Nutrition 103(10): 1480-1484.
Bidel, S., G. Hu, et al. (2008). "Coffee consumption and type 2 diabetes - An extensive review."
Central European Journal of Medicine 3(1): 9-19.
Biedrzycka, E. and R. Amarowicz (2008). "Diet and health: Apple polyphenols as antioxidants." Food
Reviews International 24(2): 235 - 251.
Blondel, O., D. Bailbe, et al. (1990). "Insulin resistance in rats with non-insulin-dependent diabetes
induced by neonatal (5 days) streptozotocin: evidence for reversal following phlorizin
treatment." Metabolism 39(8): 787-793.
Boden, G. (2008). "Obesity and free fatty acids." Endocrinol Metab Clin North Am 37(3): 635-646,
viii-ix.
Bonita, J. S., M. Mandarano, et al. (2007). "Coffee and cardiovascular disease: In vitro, cellular,
animal, and human studies." Pharmacological Research 55: 187-198.
Bordeaux, B., L. R. Yanek, et al. (2007). "Casual chocolate consumption and inhibition of platelet
function." Preventive cardiology 10(4): 175-180.
Borges, G., W. Mullen, et al. (2010). "Bioavailability of multiple components following acute
ingestion of a polyphenol-rich juice drink." Molecular Nutrition and Food Research 54:
S268–S277.
Bouayed, J., L. Hoffmann, et al. (2011). "Total phenolics, flavonoids, anthocyanins and antioxidant
activity following simulated gastro-intestinal digestion and dialysis of apple varieties:
Bioaccessibility and potential uptake." Food Chemistry 128(1): 14-21.
Boyer, J. and H. L. Rui (2004). "Apple phytochemicals and their health benefits." Nutrition
Journal(3): 5.
Buijsse, B., E. J. Feskens, et al. (2006). "Cocoa intake, blood pressure, and cardiovascular mortality:
the Zutphen Elderly Study." Advances in Internal Medicine 166(4): 411-417.
Buijsse, B., C. Weikert, et al. (2010). "Chocolate consumption in relation to blood pressure and risk of
cardiovascular disease in German adults." European Heart Journal 31(13): 1616-1623.
Buitrago-Lopez, A., J. Sanderson, et al. (2011). "Chocolate consumption and cardiometabolic
disorders: systematic review and meta-analysis." British Medical Journal 343: d4488.
Buscemi, S., S. Verga, et al. (2010). "Acute effects of coffee on endothelial function in healthy
subjects." European Journal of Clinical Nutrition 64(5): 483-489.
Butland, B. K., A. M. Fehily, et al. (2000). "Diet, lung function, and lung function decline in a cohort
of 2512 middle aged men." Thorax 55(2): 102-108.
Carnevale, R., L. Loffredo, et al. (2012). "Dark chocolate inhibits platelet isoprostanes via NOX2
down-regulation in smokers." Journal of Thrombosis and Haemostasis 10(1): 125-132.
Cermak, R., S. Landgraf, et al. (2003). "The bioavailability of quercetin in pigs depends on the
glycoside moiety and on dietary factors." Journal of Nutrition 133: 2802-2807.
Chinnici, F., A. Bendini, et al. (2004a). "Radical scavenging activities of peels and pulps from cv.
golden delicious apples as related to their phenolic composition." Journal of Agricultural and
Food Chemistry 52(15): 4684-4689.
Choudhury, R., S. K. Srai, et al. (1999). "Urinary excretion of hydroxycinnamates and flavonoids after
oral and intravenous administration." Free Radical Biology and Medicine 27(3-4): 278-286.
Cooper, K. A., J. L. Donovan, et al. (2008). "Cocoa and health: a decade of research." British Journal
of Nutrition 99(01): 1-11.
Corti, R., A. J. Flammer, et al. (2009). "Cocoa and cardiovascular health." Circulation 119(10): 14331441.
92
LITERATURE REVIEW
REFERENCES
Crespy, V., O. Aprikian, et al. (2002a). "Bioavailability of phloretin and phloridzin in rats." Journal of
Nutrition 132: 3227–3230.
Crespy, V., C. Morand, et al. (2003). "The splanchnic metabolism of flavonoids highly differed
according to the nature of the compound." American Journal of Physiology - Gastrointestinal
and Liver Physiology 284(6): G980-988.
Crespy, V., C. Morand, et al. (2001). "Comparison of the intestinal absorption of quercetin, phloretin
and their glucosides in rats." Journal of Nutrition 131(8): 2109-2114.
Crespy, V., C. Morand, et al. (2002b). "Quercetin, but not its glycosides, is absorbed from the rat
stomach." Journal of Agricultural and Food Chemistry 50(3): 618-621.
Crespy, V., C. Morand, et al. (1999). "Part of quercetin absorbed in the small intestine is conjugated
and further secreted in the intestinal lumen." American Journal of Physiology 277(1 Pt 1):
G120-126.
Crews, W. D., Jr., D. W. Harrison, et al. (2008). "A double-blind, placebo-controlled, randomized trial
of the effects of dark chocolate and cocoa on variables associated with neuropsychological
functioning and cardiovascular health: clinical findings from a sample of healthy, cognitively
intact older adults." American Journal of Clinical Nutrition 87(4): 872-880.
Cropley, V., R. Croft, et al. (2012). "Does coffee enriched with chlorogenic acids improve mood and
cognition after acute administration in healthy elderly? A pilot study." Psychopharmacology
219(3): 737-749.
Crozier, A., I. B. Jaganath, et al. (2009). "Dietary phenolics: chemistry, bioavailability and effects on
health." Natural Product Reports 26(8): 1001-1043.
D’Archivio, M., C. Filesi, et al. (2007). "Polyphenols, dietary sources and bioavailability." Annali
dell'Istituto Superiore di Sanita 43(4): 348-361.
Davison, K., A. M. Coates, et al. (2008). "Effect of cocoa flavanols and exercise on cardiometabolic
risk factors in overweight and obese subjects." International Journal of Obesity 32(8): 12891296.
Del Rio, D., G. Borges, et al. (2010). "Berry flavonoids and phenolics: bioavailability and evidence of
protective effects." British Journal of Nutrition 104: S67-S90.
Ding, E. L., S. M. Hutfless, et al. (2006). "Chocolate and prevention of cardiovascular disease: a
systematic review." Nutrition and Metabolism 3: 2.
Donovan, J. L., J. R. Bell, et al. (1999). "Catechin is present as metabolites in human plasma after
consumption of red wine." Journal of Nutrition 129(9): 1662-1668.
Donovan, J. L., V. Crespy, et al. (2001). "Catechin is metabolized by both the small intestine and liver
of rats." Journal of Nutrition 131(6): 1753-1757.
Donovan, J. L., V. Crespy, et al. (2006). "(+)-Catechin is more bioavailable than (-)-catechin:
relevance to the bioavailability of catechin from cocoa." Free Radical Research 40(10): 10291034.
Dragsted, L. (2003). "Antioxidant actions of polyphenols in humans." International Journal for
Vitamin and Nutrition Research 73(2): 112-119.
Dupas, C., A. M. Baglieri, et al. (2006). "Chlorogenic acid is poorly absorbed, independently of the
food matrix: A Caco-2 cells and rat chronic absorption study." Molecular Nutrition and Food
Research 50(11): 1053-1060.
DuPont, M. S., R. N. Bennett, et al. (2002). "Polyphenols from alcoholic apple cider are absorbed,
metabolized and excreted by humans." Journal of Nutrition 132(2): 172-175.
EFSA, P. o. D. P., Nutrition and Allergies (NDA) (2012). "Scientific Opinion on the substantiation of
a health claim related to cocoa flavanols and maintenance of normal endothelium-dependent
vasodilation pursuant to Article 13(5) of Regulation (EC) No 1924/2006." EFSA Journal
10(7): 2809.
Egert, S., S. Wolffram, et al. (2008). "Daily quercetin supplementation dose-dependently increases
plasma quercetin concentrations in healthy humans." Journal of Nutrition 138(9): 1615-1621.
Ehrenkranz, J. R. L., N. G. Lewis, et al. (2005). "Phlorizin: a review." Diabetes/Metabolism Research
and Reviews 21(1): 31-38.
Engler, M. B. and M. M. Engler (2004a). "The vasculoprotective effects of flavonoid-rich cocoa and
chocolate." Nutrition Research 24(9): 695-706.
93
LITERATURE REVIEW
REFERENCES
Engler, M. B. and M. M. Engler (2006). "The emerging role of flavonoid-rich cocoa and chocolate in
cardiovascular health and disease." Nutrition Reviews 64(3): 109-118.
Engler, M. B., M. M. Engler, et al. (2004b). "Flavonoid-rich dark chocolate improves endothelial
function and increases plasma epicatechin concentrations in healthy adults." Journal of the
American College of Nutrition 23(3): 197-204.
FAOSTAT. (2012b, 01.10.2012). "Food production data." Retrieved 01.10.2012, from
http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor.
FAOSTAT. (2012c, 01.10.2012). "Food supply data." Retrieved 01.10.2012, from
http://faostat.fao.org/site/609/DesktopDefault.aspx?PageID=609#ancor.
Farah, A., M. Monteiro, et al. (2008). "Chlorogenic acids from green coffee extract are highly
bioavailable in humans." Journal of Nutrition 138(12): 2309-2315.
Faridi, Z., V. Y. Njike, et al. (2008). "Acute dark chocolate and cocoa ingestion and endothelial
function: a randomized controlled crossover trial." American Journal of Clinical Nutrition
88(1): 58-63.
Farouque, H. M., M. Leung, et al. (2006). "Acute and chronic effects of flavanol-rich cocoa on
vascular function in subjects with coronary artery disease: a randomized double-blind placebocontrolled study." Clinical Science 111(1): 71-80.
FDA. (2003). "Guidance for industry: bioavailability and bioequivalence studies for orally
administered drug products — general considerations." Retrieved 02 May 2012, from
http://www.fda.gov/cder/guidance/index.htm.
Fernandez-Panchon, M. S., D. Villano, et al. (2008). "Antioxidant activity of phenolic compounds:
From in vitro results to in vivo evidence." Critical Reviews in Food Science and Nutrition
48(7): 649 - 671.
Feskanich, D., R. G. Ziegler, et al. (2000). "Prospective study of fruit and vegetable consumption and
risk of lung cancer among men and women." Journal of the National Cancer Institute 92(22):
1812-1823.
Fisher, N. D., M. Hughes, et al. (2003). "Flavanol-rich cocoa induces nitric-oxide-dependent
vasodilation in healthy humans." Journal of Hypertension 21(12): 2281-2286.
Fisher, N. D., F. A. Sorond, et al. (2006). "Cocoa flavanols and brain perfusion." Journal of
Cardiovascular Pharmacology 47 Suppl 2: S210-214.
Fogliano, V., M. L. Corollaro, et al. (2011). "In vitro bioaccessibility and gut biotransformation of
polyphenols present in the water-insoluble cocoa fraction." Molecular Nutrition and Food
Research 55 Suppl 1: S44-55.
Fraga, C. G. (2007). "Plant polyphenols: How to translate their in vitro antioxidant actions to in vivo
conditions." IUBMB Life 59(4-5): 308-315.
Fraga, C. G., L. Actis-Goretta, et al. (2005). "Regular consumption of a flavanol-rich chocolate can
improve oxidant stress in young soccer players." Clinical and Developmental Immunology
12(1): 11-17.
Freitas, H. S., G. F. Anhe, et al. (2008). "Na(+) -glucose transporter-2 messenger ribonucleic acid
expression in kidney of diabetic rats correlates with glycemic levels: involvement of
hepatocyte nuclear factor-1alpha expression and activity." Endocrinology 149(2): 717-724.
Füglister. (2012, 28.10.2012). "Apfelsorten-Lagerbestand in Tonnen per 31. Oktober in der Schweiz."
Retrieved
28.10.2012,
from
http://www.apfel.ch/produkte/lagerbestand_liste.aspx?LandID=1&Land=Schweiz&FruchtID=
1&Land_kurz=CH&Fruchtname2=Apfelsorten&PM=SwissGAP%20/%20GlobalGAP.
Fumeaux, R., C. Menozzi-Smarrito, et al. (2010). "First synthesis, characterization, and evidence for
the presence of hydroxycinnamic acid sulfate and glucuronide conjugates in human biological
fluids as a result of coffee consumption." Organic and Biomolecular Chemistry 8(22): 51995211.
Galleano, M., S. V. Verstraeten, et al. (2010). "Antioxidant actions of flavonoids: Thermodynamic and
kinetic analysis." Archives of Biochemistry and Biophysics 501(1): 23-30.
Gallus, S., R. Talamini, et al. (2005). "Does an apple a day keep the oncologist away?" Annals of
Oncology 16(11): 1841-1844.
94
LITERATURE REVIEW
REFERENCES
García-Ramírez, B., J. Fernández-Larrea, et al. (2006). "Tetramethylated dimeric procyanidins are
detected in rat plasma and liver early after oral administration of synthetic oligomeric
procyanidins." Journal of Agricultural and Food Chemistry 54(7): 2543-2551.
Garcia, V., I. C. W. Arts, et al. (2005). "Dietary intake of flavonoids and asthma in adults." European
Respiratory Journal 26(3): 449-452.
Gerhauser, C. (2008). "Cancer chemopreventive potential of apples, apple juice, and apple
components." Planta Medica 74(13): 1608-1624.
Gill, C. I., G. J. McDougall, et al. (2010). "Profiling of phenols in human fecal water after raspberry
supplementation." Journal of Agricultural and Food Chemistry 58(19): 10389-10395.
Goldberg, D. M., J. Yan, et al. (2003). "Absorption of three wine-related polyphenols in three different
matrices by healthy subjects." Clinical Biochemistry 36(1): 79-87.
Gonthier, M. P., M. A. Verny, et al. (2003). "Chlorogenic acid bioavailability largely depends on its
metabolism by the gut microflora in rats." Journal of Nutrition 133(6): 1853-1859.
Grassi, D., G. Desideri, et al. (2010). "Blood pressure and cardiovascular risk: what about cocoa and
chocolate?" Archives of Biochemistry and Biophysics 501(1): 112-115.
Grassi, D., G. Desideri, et al. (2008). "Blood pressure is reduced and insulin sensitivity increased in
glucose-intolerant, hypertensive subjects after 15 days of consuming high-polyphenol dark
chocolate." Journal of Nutrition 138(9): 1671-1676.
Grassi, D., C. Lippi, et al. (2005a). "Short-term administration of dark chocolate is followed by a
significant increase in insulin sensitivity and a decrease in blood pressure in healthy persons."
American Journal of Clinical Nutrition 81(3): 611-614.
Grassi, D., S. Necozione, et al. (2005b). "Cocoa reduces blood pressure and insulin resistance and
improves endothelium-dependent vasodilation in hypertensives." Hypertension 46(2): 398405.
Gromova, L. V. (2006). "Effect of phloretin and phloridzin on properties of digestion and absorption
in the rat small intestine." Journal of Evolutionary Biochemistry and Physiology 42(4): 454460.
Guyot, S., C. L. Bourvellec, et al. (2002). "Procyanidins are the most abundant polyphenols in dessert
apples at maturity." LWT- Food Science and Technology 35: 289-291.
Guyot, S., N. Marnet, et al. (2001). "Thiolysis-HPLC characterization of apple procyanidins covering
a large range of polymerization states." Journal of Agricultural and Food Chemistry 49: 1420.
Guyot, S., N. Marnet, et al. (2003). "Variability of the polyphenolic composition of cider apple (Malus
domestica) fruits and juices. (vol 51, pg 6240, 2003)." Journal of Agricultural and Food
Chemistry 51: 7522-7522.
Halliwell, B. (2007). "Dietary polyphenols: Good, bad, or indifferent for your health?" Cardiovascular
Research 73: 341-347.
Halliwell, B., J. Rafter, et al. (2005). "Health promotion by flavonoids, tocopherols, tocotrienols, and
other phenols: direct or indirect effects? Antioxidant or not?" American Journal of Clinical
Nutrition 81 (suppl): 268S-276S.
Hamed, M. S., S. Gambert, et al. (2008). "Dark chocolate effect on platelet activity, C-reactive protein
and lipid profile: a pilot study." Southern Medical Journal 101(12): 1203-1208.
Hanhineva, K., R. Torronen, et al. (2010). "Impact of dietary polyphenols on carbohydrate
metabolism." International Journal of Molecular Sciences 11(4): 1365-1402.
Hecke, K., K. Herbinger, et al. (2006). "Sugar-, acid- and phenol contents in apple cultivars from
organic and integrated fruit cultivation." European Journal of Clinical Nutrition 60: 11361140.
Heinrich, U., K. Neukam, et al. (2006). "Long-term ingestion of high flavanol cocoa provides
photoprotection against UV-induced erythema and improves skin condition in women."
Journal of Nutrition 136(6): 1565-1569.
Heiss, C., D. Finis, et al. (2007). "Sustained increase in flow-mediated dilation after daily intake of
high-flavanol cocoa drink over 1 week." Journal of Cardiovascular Pharmacology 49(2): 7480.
95
LITERATURE REVIEW
REFERENCES
Heiss, C., S. Jahn, et al. (2010). "Improvement of endothelial function with dietary flavanols is
associated with mobilization of circulating angiogenic cells in patients with coronary artery
disease." Journal of the American College of Cardiology 56(3): 218-224.
Heiss, C., P. Kleinbongard, et al. (2005). "Acute consumption of flavanol-rich cocoa and the reversal
of endothelial dysfunction in smokers." Journal of the American College of Cardiology 46(7):
1276-1283.
Heiss, C., H. Schroeter, et al. (2006). "Endothelial function, nitric oxide, and cocoa flavanols." Journal
of Cardiovascular Pharmacology 47 Suppl 2: S128-135; discussion S172-126.
Heptinstall, S., J. May, et al. (2006). "Cocoa flavanols and platelet and leukocyte function: recent in
vitro and ex vivo studies in healthy adults." Journal of Cardiovascular Pharmacology 47
Suppl 2: S197-205; discussion S206-199.
Hermann, F., L. E. Spieker, et al. (2006). "Dark chocolate improves endothelial and platelet function."
Heart 92(1): 119-120.
Hertog, M. G., E. J. Feskens, et al. (1993). "Dietary antioxidant flavonoids and risk of coronary heart
disease: the Zutphen Elderly Study." Lancet 342(8878): 1007-1011.
Hervert-Hernandez, D., S. G. Sayago-Ayerdi, et al. (2010). "Bioactive compounds of four hot pepper
varieties (Capsicum annuum L.), antioxidant capacity, and intestinal bioaccessibility." Journal
of Agricultural and Food Chemistry 58(6): 3399-3406.
Hoelzl, C., S. Knasmuller, et al. (2010). "Instant coffee with high chlorogenic acid levels protects
humans against oxidative damage of macromolecules." Molecular Nutrition and Food
Research 54(12): 1722-1733.
Hollenberg, N. K. (2006). "Vascular action of cocoa flavanols in humans: The roots of the story."
Journal of Cardiovascular Pharmacology 47: S99-S102.
Hollman, P. C. and I. C. Arts (2000). "Flavonols, flavones and flavanols – nature, occurrence and
dietary burden." Journal of the Science of Food and Agriculture 80: 1081-1093.
Hollman, P. C., A. Cassidy, et al. (2011). "The biological relevance of direct antioxidant effects of
polyphenols for cardiovascular health in humans is not established." Journal of Nutrition
141(5): 989S-1009S.
Hollman, P. C., J. H. de Vries, et al. (1995). "Absorption of dietary quercetin glycosides and quercetin
in healthy ileostomy volunteers." American Journal of Clinical Nutrition 62(6): 1276-1282.
Holst, B. and G. Williamson (2008). "Nutrients and phytochemicals: from bioavailability to
bioefficacy beyond antioxidants." Current Opinion in Biotechnology 19(2): 73-82.
Holt, R. R., L. Actis-Goretta, et al. (2006). "Dietary flavanols and platelet reactivity." Journal of
Cardiovascular Pharmacology 47 Suppl 2: S187-196; discussion S206-189.
Holt, R. R., S. A. Lazarus, et al. (2002). "Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] in
human plasma after the consumption of a flavanol-rich cocoa." American Journal of Clinical
Nutrition 76(4): 798-804.
Hooper, L., C. Kay, et al. (2012). "Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular
health: a systematic review and meta-analysis of randomized trials." American Journal of
Clinical Nutrition 95(3): 740-751.
Hooper, L., P. A. Kroon, et al. (2008). "Flavonoids, flavonoid-rich foods, and cardiovascular risk: a
meta-analysis of randomized controlled trials." American Journal of Clinical Nutrition 88(1):
38-50.
Hyson, D. A. (2011). "A comprehensive review of apples and apple components and their relationship
to human health." Advances in Nutrition 2(5): 408-420.
Ishimoto, H., A. Tai, et al. (2012). "Antioxidative properties of functional polyphenols and their
metabolites assessed by an ORAC assay." Bioscience, Biotechnology, and Biochemistry 76(2):
395-399.
Ishizawa, K., M. Yoshizumi, et al. (2011). "Pharmacology in health food: metabolism of quercetin in
vivo and its protective effect against arteriosclerosis." Journal of Pharmacological Sciences
115(4): 466-470.
Ito, H., M. Gonthier, et al. (2005). "Polyphenol levels in human urine after intake of six different
polyphenol-rich beverages." British Journal of Nutrition 94(4): 500-509.
Jalil, A. and A. Ismail (2008a). "Polyphenols in cocoa and cocoa products: Is there a link between
antioxidant properties and health?" Molecules 13(9): 2190-2219.
96
LITERATURE REVIEW
REFERENCES
Jalil, A. M., A. Ismail, et al. (2008b). "Effects of cocoa extract on glucometabolism, oxidative stress,
and antioxidant enzymes in obese-diabetic (Ob-db) rats." Journal of Agricultural and Food
Chemistry 56(17): 7877-7884.
Jenner, A. M., J. Rafter, et al. (2005). "Human fecal water content of phenolics: the extent of colonic
exposure to aromatic compounds." Free Radic Biol Med 38(6): 763-772.
Jia, L., X. Liu, et al. (2010). "Short-term effect of cocoa product consumption on lipid profile: a metaanalysis of randomized controlled trials." American Journal of Clinical Nutrition 92(1): 218225.
Jimenez, R., J. Duarte, et al. (2012). "Epicatechin: endothelial function and blood pressure." Journal of
Agricultural and Food Chemistry.
Johnston, K., P. Sharp, et al. (2005). "Dietary polyphenols decrease glucose uptake by human
intestinal Caco-2 cells." FEBS Letters 579(7): 1653-1657.
Johnston, K. L., M. N. Clifford, et al. (2002). "Possible role for apple juice phenolic compounds in the
acute modification of glucose tolerance and gastrointestinal hormone secretion in humans."
Journal of the Science of Food and Agriculture 82: 1800-1805.
Johnston, K. L., M. N. Clifford, et al. (2003). "Coffee acutely modifies gastrointestinal hormone
secretion and glucose tolerance in humans: glycemic effects of chlorogenic acid and caffeine."
American Journal of Clinical Nutrition 78(4): 728-733.
Kahle, K., M. Kempf, et al. (2011). "Intestinal transit and systemic metabolism of apple polyphenols."
European Journal of Nutrition 50(7): 507-522.
Kahle, K., M. Kraus, et al. (2005a). "Polyphenol profiles of apple juices." Molecular Nutrition and
Food Research 49: 797 – 806.
Kahle, K., M. Kraus, et al. (2005b). "Colonic availability of apple polyphenols – A study in ileostomy
subjects." Molecular Nutrition and Food Research 49: 1143-1150.
Katz, D. L., K. Doughty, et al. (2011). "Cocoa and chocolate in human health and disease."
Antioxidants and Redox Signaling.
Kempf, K., C. Herder, et al. (2010). "Effects of coffee consumption on subclinical inflammation and
other risk factors for type 2 diabetes: a clinical trial." American Journal of Clinical Nutrition
91(4): 950-957.
Kevers, C., J. Pincemail, et al. (2011). "Influence of cultivar, harvest time, storage conditions, and
peeling on the antioxidant capacity and phenolic and ascorbic acid contents of apples and
pears." Journal of Agricultural and Food Chemistry 59(11): 6165-6171.
Khanizadeh, S., R. Tsao, et al. (2008). "Polyphenol composition and total antioxidant capacity of
selected apple genotypes for processing." Journal of Food Composition and Analysis 21: 396401.
Khawaja, O., J. M. Gaziano, et al. (2011). "Chocolate and coronary heart disease: a systematic
review." Current Atherosclerosis Reports 13(6): 447-452.
Kim, J. E., J. E. Son, et al. (2010). "Cocoa polyphenols suppress TNF-alpha-induced vascular
endothelial growth factor expression by inhibiting phosphoinositide 3-kinase (PI3K) and
mitogen-activated protein kinase kinase-1 (MEK1) activities in mouse epidermal cells."
British Journal of Nutrition 104(7): 957-964.
Kim, M. S., J. Y. Kwon, et al. (2009). "Phloretin induces apoptosis in H-Ras MCF10A human breast
tumor cells through the activation of p53 via JNK and p38 mitogen-activated protein kinase
signaling." Annals of the New York Academy of Sciences 1171: 479-483.
Knaup, B., K. Kahle, et al. (2007). "Human intestinal hydrolysis of phenol glycosides – a study with
quercetin and p-nitrophenol glycosides using ileostomy fluid." Molecular Nutrition and Food
Research 51: 1423-1429.
Knekt, P., S. Isotupa, et al. (2000). "Quercetin intake and the incidence of cerebrovascular disease."
European Journal of Clinical Nutrition 54(5): 415-417.
Knekt, P., R. Jarvinen, et al. (1996). "Flavonoid intake and coronary mortality in Finland: a cohort
study." British Medical Journal 312(7029): 478-481.
Kobori, M., K. Iwashita, et al. (1999). "Phloretin-induced apoptosis in B16 melanoma 4A5 cells and
HL60 human leukemia cells." Bioscience, Biotechnology, and Biochemistry 63(4): 719-725.
Kobori, M., S. Masumoto, et al. (2012). "Phloridzin reduces blood glucose levels and alters hepatic
gene expression in normal BALB/c mice." Food and Chemical Toxicology.
97
LITERATURE REVIEW
REFERENCES
Kobori, M., H. Shinmoto, et al. (1997). "Phloretin-induced apoptosis in B16 melanoma 4A5 cells by
inhibition of glucose transmembrane transport." Cancer Letters 119(2): 207-212.
Kuhnle, G., J. P. E. Spencer, et al. (2000). "Epicatechin and catechin are O-methylated and
glucuronidated in the small intestine." Biochemical and Biophysical Research
Communications 277(2): 507-512.
Kumazawa, S., M. Ikenaga, et al. (2007). "Comprehensive analysis of polyphenols in fruits consumed
in Japan." Food Science and Technology Research 13: 404-413.
Kurlandsky, S. B. and K. S. Stote (2006). "Cardioprotective effects of chocolate and almond
consumption in healthy women." Nutrition Research 26(10): 509-516.
Kutner, S., W. V. Breuer, et al. (1987). "On the mode of action of phlorizin as an antimalarial agent in
in vitro cultures of Plasmodium falciparum." Biochemical Pharmacology 36(1): 123-129.
Kwik-Uribe, C. and R. M. Bektash (2008). "Cocoa flavanols: Measurement, bioavailability and
bioactivity." Asia Pacific Journal of Clinical Nutrition 17: 280-283.
Lafay, S., A. Gil-Izquierdo, et al. (2006). "Chlorogenic acid is absorbed in its intact form in the
stomach of rats." Journal of Nutrition 136(5): 1192-1197.
Lamperi, L., U. Chiuminatto, et al. (2008). "Polyphenol levels and free radical scavenging activities of
four apple cultivars from integrated and organic farming in different Italian areas." Journal of
Agricultural and Food Chemistry 56(15): 6536-6546.
Laparra, J. M., R. P. Glahn, et al. (2008). "Bioaccessibility of phenols in common beans (Phaseolus
vulgaris L.) and iron (Fe) availability to Caco-2 cells." Journal of Agricultural and Food
Chemistry 56: 10999-11005.
Lata, B., M. Przeradzka, et al. (2005). "Great differences in antioxidant properties exist between 56
apple cultivars and vegetation seasons." Journal of Agricultural and Food Chemistry 53:
8970-8978.
Lata, B. and K. Tomala (2007). "Apple peel as a contributor to whole fruit quantity of potentially
healthful bioactive compounds. Cultivar and year implication." Journal of Agricultural and
Food Chemistry 55: 10795-10802.
Le Marchand, L., S. P. Murphy, et al. (2000). "Intake of flavonoids and lung cancer." Journal of the
National Cancer Institute 92(2): 154-160.
Lee, K. W., Y. J. Kim, et al. (2003). "Major phenolics in apple and their contribution to the total
antioxidant capacity." Journal of Agricultural and Food Chemistry 51: 6516-6520.
Lee, K. W., J. K. Kundu, et al. (2006). "Cocoa polyphenols inhibit phorbol ester-induced superoxide
anion formation in cultured HL-60 cells and expression of cyclooxygenase-2 and activation of
NF-kappaB and MAPKs in mouse skin in vivo." Journal of Nutrition 136(5): 1150-1155.
Linseisen, J., S. Rohrmann, et al. (2007). "Fruit and vegetable consumption and lung cancer risk:
updated information from the European Prospective Investigation into Cancer and Nutrition
(EPIC)." International Journal of Cancer 121(5): 1103-1114.
Lipinski, C. A., F. Lombardo, et al. (2001). "Experimental and computational approaches to estimate
solubility and permeability in drug discovery and development settings." Advanced Drug
Delivery Reviews 46(1-3): 3-26.
Loke, W. M., A. M. Jenner, et al. (2009). "A metabolite profiling approach to identify biomarkers of
flavonoid intake in humans." Journal of Nutrition 139(12): 2309-2314.
Lotito, S. B. and B. Frei (2004). "Relevance of apple polyphenols as antioxidants in human plasma:
Contrasting in vitro and in vivo effects." Free Radical Biology and Medicine 36(2): 201-211.
Mainla, L., U. Moor, et al. (2011). "The effect of genotype and rootstock on polyphenol composition
of selected apple cultivars in Estonia." Zemdirbyste-Agriculture 98(1): 63-70.
Manach, C., A. Scalbert, et al. (2004). "Polyphenols: food sources and bioavailability." American
Journal of Clinical Nutrition 79: 727-747.
Manach, C., O. Texier, et al. (1999). "Comparison of the bioavailability of quercetin and catechin in
rats." Free Radical Biology and Medicine 27(11-12): 1259-1266.
Manach, C., G. Williamson, et al. (2005). "Bioavailability and bioefficacy of polyphenols in humans.
I. Review of 97 bioavailability studies." American Journal of Clinical Nutrition 81(suppl):
230S-242S.
98
LITERATURE REVIEW
REFERENCES
Mangas, J. J., R. Rodriguez, et al. (1999). "Study of the phenolic profile of cider apple cultivars at
maturity by multivariate techniques." Journal of Agricultural and Food Chemistry 47: 40464052.
Mari, A., I. Tedesco, et al. (2010). "Phenolic compound characterisation and antiproliferative activity
of "Annurca" apple, a southern Italian cultivar." Food Chemistry 123: 157-164.
Marks, S. C., W. Mullen, et al. (2009). "Absorption, metabolism, and excretion of cider
dihydrochalcones in healthy humans and subjects with an ileostomy." Journal of Agricultural
and Food Chemistry 57(5): 2009-2015.
Maskarinec, G. (2009). "Cancer protective properties of cocoa: a review of the epidemiologic
evidence." Nutrition and Cancer 61(5): 573-579.
Masumoto, S., Y. Akimoto, et al. (2009). "Dietary phloridzin reduces blood glucose levels and
reverses Sglt1 expression in the small intestine in streptozotocin-induced diabetic mice."
Journal of Agricultural and Food Chemistry 57(11): 4651-4656.
Mateos-Martin, M. L., J. Perez-Jimenez, et al. (2012). "Profile of urinary and fecal proanthocyanidin
metabolites from common cinnamon (Cinnamomum zeylanicum L.) in rats." Molecular
Nutrition and Food Research 56: 671.
Mather, A. and C. Pollock (2010). "Renal glucose transporters: novel targets for hyperglycemia
management." Nature Reviews Nephrology 6(5): 307-311.
Matsui, N., R. Ito, et al. (2005). "Ingested cocoa can prevent high-fat diet-induced obesity by
regulating the expression of genes for fatty acid metabolism." Nutrition 21(5): 594-601.
McGhie, T. K., M. Hunt, et al. (2005). "Cultivar and growing region determine the antioxidant
polyphenolic concentration and composition of apples grown in New Zealand." Journal of
Agricultural and Food Chemistry 53: 3065-3070.
Mellor, D. D., T. Sathyapalan, et al. (2010). "High-cocoa polyphenol-rich chocolate improves HDL
cholesterol in Type 2 diabetes patients." Diabetic Medicine 27(11): 1318-1321.
Mennen, L., D. Sapinho, et al. (2007). "Urinary excretion of 13 dietary flavonoids and phenolic acids
in free-living healthy subjects – variability and possible use as biomarkers of polyphenol
intake." European Journal of Clinical Nutrition: 1-7.
Michels, K. B., E. Giovannucci, et al. (2006). "Fruit and vegetable consumption and colorectal
adenomas in the Nurses' Health Study." Cancer Research 66(7): 3942-3953.
Monagas, M., M. Urpi-Sarda, et al. (2010). "Insights into the metabolism and microbial
biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites." Food and
Function 1(3): 233-253.
Montagnana, M., E. J. Favaloro, et al. (2012). "Coffee intake and cardiovascular disease: virtue does
not take center stage." Seminars in Thrombosis and Hemostasis 38(2): 164-177.
Monteiro, M., A. Farah, et al. (2007). "Chlorogenic acid compounds from coffee are differentially
absorbed and metabolized in humans." Journal of Nutrition 137(10): 2196-2201.
Morand, C., C. Manach, et al. (2000a). "Quercetin 3-O-beta-glucoside is better absorbed than other
quercetin forms and is not present in rat plasma." Free Radical Research 33(5): 667-676.
Morand, C., C. Manach, et al. (2000b). "Respective bioavailability of quercetin aglycone and its
glycosides in a rat model." Biofactors 12(1-4): 169-174.
Mullen, W., C. A. Edwards, et al. (2006). "Absorption, excretion and metabolite profiling of methyl-,
glucuronyl-, glucosyl- and sulpho-conjugates of quercetin in human plasma and urine after
ingestion of onions." British Journal of Nutrition 96(1): 107-116.
Muniyappa, R., G. Hall, et al. (2008). "Cocoa consumption for 2 wk enhances insulin-mediated
vasodilatation without improving blood pressure or insulin resistance in essential
hypertension." American Journal of Clinical Nutrition 88(6): 1685-1696.
Murphy, K. J., A. K. Chronopoulos, et al. (2003). "Dietary flavanols and procyanidin oligomers from
cocoa (Theobroma cacao) inhibit platelet function." American Journal of Clinical Nutrition
77(6): 1466-1473.
Mursu, J., S. Voutilainen, et al. (2004). "Dark chocolate consumption increase HDL cholesterol
concentration and chocolat fatty acids may inhibit lipid peroxidation in healthy humans." Free
Radical Biology and Medicine 37(9): 1351-1359.
Napolitano, A., A. Cascone, et al. (2004). "Influence of variety and storage on the polyphenol
composition of apple flesh." Journal of Agricultural and Food Chemistry 52: 6526-6531.
99
LITERATURE REVIEW
REFERENCES
Nardini, M., E. Cirillo, et al. (2002). "Absorption of phenolic acids in humans after coffee
consumption." Journal of Agricultural and Food Chemistry 50: 5735-5741.
Neilson, A. P. and M. G. Ferruzzi (2011). "Influence of formulation and processing on absorption and
metabolism of flavan-3-ols from tea and cocoa." Annual Review of Food Science and
Technology 2: 125-151.
Neukam, K., W. Stahl, et al. (2007). "Consumption of flavanol-rich cocoa acutely increases
microcirculation in human skin." European Journal of Nutrition 46(1): 53-56.
Neveu, V., J. Perez-Jiménez, et al. (2010). Phenol-Explorer: an online comprehensive database on
polyphenol contents in foods, online Database. Version 1.5.2, available at http://www.phenolexplorer.eu.
Njike, V. Y., Z. Faridi, et al. (2011). "Effects of sugar-sweetened and sugar-free cocoa on endothelial
function in overweight adults." International Journal of Cardiology 149(1): 83-88.
Nöthlings, U., S. P. Murphy, et al. (2007). "Flavonols and pancreatic cancer risk The Multiethnic
Cohort Study." American Journal of Epidemiology 166(8): 924-931.
O’Reilly, J. D., A. I. Mallet, et al. (2001). "Consumption of flavonoids in onions and black tea: lack of
effect on F2-isoprostanes and autoantibodies to oxidized LDL in healthy humans." American
Journal of Clinical Nutrition 73: 1040-1044.
Oghbaei, M. and J. Prakash (2012). "Bioaccessible nutrients and bioactive components from fortified
products prepared using finger millet (Eleusine coracana)." Journal of the Science of Food and
Agriculture 92(11): 2281-2290.
Okoko, B. J., P. G. Burney, et al. (2007). "Childhood asthma and fruit consumption in South London."
European Respiratory Journal 29: 1161-1168.
Olthof, M. R., P. C. H. Hollman, et al. (2003). "Chlorogenic acid, quercetin-3-rutinoside and black tea
phenols are extensively metabolized in humans." Journal of Nutrition 133(6): 1806-1814.
Olthof, M. R., P. C. H. Hollman, et al. (2001). "Chlorogenic acid and caffeic acid are absorbed in
humans." Journal of Nutrition 131: 66–71.
Ong, K. W., A. Hsu, et al. (2012). "Chlorogenic acid stimulates glucose transport in skeletal muscle
via AMPK activation: A contributor to the beneficial effects of coffee on diabetes." PLOS One
7(3): e32718.
Ortega, N., J. Reguant, et al. (2009). "Effect of fat content on the digestibility and bioaccessibility of
cocoa polyphenol by an in vitro digestion model." Journal of Agricultural and Food
Chemistry 57(13): 5743-5749.
Ostertag, L. M., N. O'Kennedy, et al. (2010). "Impact of dietary polyphenols on human platelet
function--a critical review of controlled dietary intervention studies." Molecular Nutrition and
Food Research 54(1): 60-81.
Oszmianski, J., M. Wolniak, et al. (2007). "Comparative study of polyphenolic content and antiradical
activity of cloudy and clear apple juices." Journal of the Science of Food and Agriculture 87:
573-579.
Ottaviani, J. I., C. Kwik-Uribe, et al. (2012). "Intake of dietary procyanidins does not contribute to the
pool of circulating flavanols in humans." American Journal of Clinical Nutrition 95(4): 851858.
Ottaviani, J. I., T. Y. Momma, et al. (2011). "The stereochemical configuration of flavanols influences
the level and metabolism of flavanols in humans and their biological activity in vivo." Free
Radical Biology and Medicine 50(2): 237-244.
Palafox-Carlos, H., J. F. Ayala-Zavala, et al. (2011). "The role of dietary fiber in the bioaccessibility
and bioavailability of fruit and vegetable antioxidants." Journal of Food Science 76(1): R6R15.
Panchal, S. K., H. Poudyal, et al. (2012). "Quercetin ameliorates cardiovascular, hepatic, and
metabolic changes in diet-induced metabolic syndrome in rats." Journal of Nutrition 142(6):
1026-1032.
Pearson, D. A., R. R. Holt, et al. (2005). "Flavanols and platelet reactivity." Clinical and
Developmental Immunology 12(1): 1-9.
Pearson, D. A., T. G. Paglieroni, et al. (2002). "The effects of flavanol-rich cocoa and aspirin on ex
vivo platelet function." Thrombosis Research 106(4-5): 191-197.
100
LITERATURE REVIEW
REFERENCES
Pierini, R., P. A. Kroon, et al. (2008). "Procyanidin effects on oesophageal adenocarcinoma cells
strongly depend on flavan-3-ol degree of polymerization." Molecular Nutrition and Food
Research 52(12): 1399-1407.
Pinent, M., C. Blade, et al. (2006). "Procyanidin effects on adipocyte-related pathologies." Critical
Reviews in Food Science and Nutrition 46(7): 543-550.
Podsedek, A., J. Wilska-Jeszka, et al. (2000). "Compositional characterisation of some apple
varieties." European Food Research and Technology 210: 268-272.
Poquet, L., M. N. Clifford, et al. (2008a). "Effect of dihydrocaffeic acid on UV irradiation of human
keratinocyte HaCaT cells." Archives of Biochemistry and Biophysics 476(2): 196-204.
Poquet, L., M. N. Clifford, et al. (2008b). "Investigation of the metabolic fate of dihydrocaffeic acid."
Biochemical Pharmacology 75(5): 1218-1229.
Prasain, J. K. and S. Barnes (2007). "Metabolism and bioavailability of flavonoids in
chemoprevention: current analytical strategies and future prospectus." Molecular
Pharmaceutics 4(6): 846-864.
Praticò, D. (2005). "Antioxidants and endothelium protection." Atherosclerosis 181: 215-224.
Prior, R. L., X. Wu, et al. (2005). "Standardized methods for the determination of antioxidant capacity
and phenolics in foods and dietary supplements." Journal of Agricultural and Food Chemistry
53(10): 4290-4302.
Puel, C., A. Quintin, et al. (2005). "Prevention of bone loss by phloridzin, an apple polyphenol, in
ovariectomized rats under inflammation conditions." Calcified Tissue International 77(5):
311-318.
Ramirez-Moreno, E., D. Hervert-Hernandez, et al. (2011). "Intestinal bioaccessibility of polyphenols
and antioxidant capacity of pulp and seeds of cactus pear." International Journal of Food
Science and Nutrition 62(8): 839-843.
Ramos, S. (2007). "Effects of dietary flavonoids on apoptotic pathways related to cancer
chemoprevention." Journal of Nutritional Biochemistry 18(7): 427-442.
Rawel, H. M. and S. E. Kulling (2007). "Nutritional contribution of coffee, cacao and tea phenolics to
human health." Journal Für Verbraucherschutz Und Lebensmittelsicherheit-Journal of
Consumer Protection and Food Safety 2(4): 399-406.
Re, R., N. Pellegrini, et al. (1999). "Antioxidant activity applying an improved ABTS radical cation
decoloration assay." Free Radical Biology and Medicine 26(9/10): 1231–1237.
Rein, D., T. G. Paglieroni, et al. (2000). "Cocoa inhibits platelet activation and function." American
Journal of Clinical Nutrition 72(1): 30-35.
Renard, C. M. G. C., N. Dupont, et al. (2007). "Concentrations and characteristics of procyanidins and
other phenolics in apples during fruit growth." Phytochemistry 68: 1128-1138.
Renouf, M., P. A. Guy, et al. (2010). "Measurement of caffeic and ferulic acid equivalents in plasma
after coffee consumption: Small intestine and colon are key sites for coffee metabolism."
Molecular Nutrition and Food Research 54(6): 760-766.
Rios, L. Y., R. N. Bennett, et al. (2002). "Cocoa procyanidins are stable during gastric transit in
humans." American Journal of Clinical Nutrition 76(5): 1106-1110.
Robbins, R. J. (2003). "Phenolic acids in foods:An overview of analytical methodology." Journal of
Agricultural and Food Chemistry 51: 2866-2887.
Rodriguez-Mateos, A., M. Jose Oruna-Concha, et al. (2012). "Influence of sugar type on the
bioavailability of cocoa flavanols." British Journal of Nutrition: 1-8.
Romieu, I., R. Varraso, et al. (2006). "Fruit and vegetable intakes and asthma in the E3N study."
Thorax 61(3): 209-215.
Rossetti, L., G. I. Shulman, et al. (1987a). "Effect of chronic hyperglycemia on in vivo insulin
secretion in partially pancreatectomized rats." Journal of Clinical Investigation 80(4): 10371044.
Rossetti, L., D. Smith, et al. (1987b). "Correction of hyperglycemia with phlorizin normalizes tissue
sensitivity to insulin in diabetic rats." Journal of Clinical Investigation 79(5): 1510-1515.
Roura, E., M. P. Almajano, et al. (2007). "Human urine: Epicatechin metabolites and antioxidant
activity after cocoa beverage intake." Free Radical Research 41(8): 943 - 949.
101
LITERATURE REVIEW
REFERENCES
Saha, S., W. Hollands, et al. (2012). "Human O-sulfated metabolites of (-)-epicatechin and methyl-(-)epicatechin are poor substrates for commercial aryl-sulfatases: Implications for studies
concerned with quantifying epicatechin bioavailability." Pharmacological Research.
Sano, A., J. Yamakoshi, et al. (2003). "Procyanidin B1 is detected in human serum after intake of
procyanidin-rich grape seed extrac." Bioscience, Biotechnology, and Biochemistry 67(5):
1140-1143.
Sanoner, P., S. Guyot, et al. (1999). "Polyphenol profiles of French cider apple varieties (Malus
domestica sp.)." Journal of Agricultural and Food Chemistry 47: 4847-4853.
Saura-Calixto, F., J. Serrano, et al. (2007). "Intake and bioaccessibility of total polyphenols in a whole
diet." Food Chemistry 101(2): 492-501.
Scalbert, A., C. Manach, et al. (2005). "Dietary polyphenols and the prevention of diseases." Critical
Reviews in Food Science and Nutrition 45(4): 287-306.
Scalbert, A. and G. Williamson (2000). "Dietary intake and bioavailability of polyphenols." Journal of
Nutrition 130: 2073S-2085S.
Schewe, T., Y. Steffen, et al. (2008). "How do dietary flavanols improve vascular function? A position
paper." Archives of Biochemistry and Biophysics 476: 102-106.
Scholz, S. and G. Williamson (2007). "Interactions affecting the bioavailability of dietary polyphenols
in vivo." International Journal for Vitamin and Nutrition Research 77(3): 224-235.
Scow, J. S., C. W. Iqbal, et al. (2011). "Absence of evidence of translocation of GLUT2 to the apical
membrane of enterocytes in everted intestinal sleeves." Journal of Surgical Research 167(1):
56-61.
Serra, A., A. Macià, et al. (2010). "Bioavailability of procyanidin dimers and trimers and matrix food
effects in in vitro and in vivo models." British Journal of Nutrition 103(07): 944-952.
Sesso, H. D., J. M. Gaziano, et al. (2003). "Flavonoid intake and the risk of cardiovascular disease in
women." American Journal of Clinical Nutrition 77(6): 1400-1408.
Shaheen, S. O., J. A. C. Sterne, et al. (2001). "Dietary antioxidants and asthma in adults . Populationbased case-control study." American Journal of Respiratory and Critical Care Medicine
164(10): 1823-1828.
Sharma, S. D., S. M. Meeran, et al. (2010). "Proanthocyanidins inhibit in vitro and in vivo growth of
human non-small cell lung cancer cells by inhibiting the prostaglandin E(2) and prostaglandin
E(2) receptors." Molecular Cancer Therapeutics 9(3): 569-580.
Shiina, Y., N. Funabashi, et al. (2009). "Acute effect of oral flavonoid-rich dark chocolate intake on
coronary circulation, as compared with non-flavonoid white chocolate, by transthoracic
Doppler echocardiography in healthy adults." International Journal of Cardiology 131(3):
424-429.
Sies, H. (2010). "Polyphenols and health: Update and perspectives." Archives of Biochemistry and
Biophysics 501(1): 2-5.
Silveira, A. C., C. K. Sautter, et al. (2007). "Determination of some quality parameters of the fuji
cultivar and their mutants at harvest." Ciência e Tecnologia de Alimentos 27(1): 149-153.
Song, Y., J. E. Manson, et al. (2005). "Associations of dietary flavonoids with risk of type 2 diabetes,
and markers of insulin resistance and systemic inflammation in women: a prospective study
and cross-sectional analysis." Journal of the American College of Nutrition 24(5): 376-384.
Spadafranca, A., C. Martinez Conesa, et al. (2010). "Effect of dark chocolate on plasma epicatechin
levels, DNA resistance to oxidative stress and total antioxidant activity in healthy subjects."
British Journal of Nutrition 103(7): 1008-1014.
Spencer, J. P. (2008). "Flavonoids: modulators of brain function?" British Journal of Nutrition 99 E
Suppl 1: ES60-77.
Spencer, J. P., H. Schroeter, et al. (2001). "Epicatechin is the primary bioavailable form of the
procyanidin dimers B2 and B5 after transfer across the small intestine." Biochemical and
Biophysical Research Communications 285(3): 588-593.
Spencer, J. P. E., F. Chaudry, et al. (2000). "Decomposition of cocoa procyanidins in the gastric
milieu." Biochemical and Biophysical Research Communications 272(1): 236-241.
Stalmach, A., W. Mullen, et al. (2009). "Metabolite profiling of hydroxycinnamate derivatives in
plasma and urine after the ingestion of coffee by humans: identification of biomarkers of
coffee consumption." Drug Metabolism and Disposition 37(8): 1749-1758.
102
LITERATURE REVIEW
REFERENCES
Stalmach, A., W. Mullen, et al. (2010a). "Absorption, metabolism, and excretion of green tea flavan-3ols in humans with an ileostomy." Molecular Nutrition and Food Research 54(3): 323-334.
Stalmach, A., H. Steiling, et al. (2010b). "Bioavailability of chlorogenic acids following acute
ingestion of coffee by humans with an ileostomy." Archives of Biochemistry and Biophysics
501(1): 98-105.
Stoclet, J. C., T. Chataigneau, et al. (2004). "Vascular protection by dietary polyphenols." European
Journal of Pharmacology 500(1-3): 299-313.
Stoner, L. and M. J. Sabatier (2012). "Use of ultrasound for non-invasive assessment of flow-mediated
dilation." Journal of Atherosclerosis and Thrombosis 19(5): 407-421.
Stopar, M., U. Bolcina, et al. (2002). "Lower crop load for Cv. Jonagold apples (Malus x domestica
Borkh.) increases polyphenol content and fruit quality." Journal of Agricultural and Food
Chemistry 50(6): 1643-1646.
Stoupi, S., G. Williamson, et al. (2010). "In vivo bioavailability, absorption, excretion, and
pharmacokinetics of [14C]procyanidin B2 in male rats." Drug Metabolism and Disposition
38(2): 287-291.
Stracke, B. A., C. E. Rufer, et al. (2009). "Three-year comparison of the polyphenol contents and
antioxidant capacities in organically and conventionally produced apples (Malus domestica
Bork. cultivar 'Golden Delicious')." Journal of Agricultural and Food Chemistry 57(11):
4598-4605.
SwissFIR.
(2012,
27.10.2012).
"Apfel,
roh."
Retrieved
27.10.2012,
from
http://nwdb.ethz.ch:8080/nwdb/request?query=ProductDetails&xml=MessageData&xml=Met
aData&xsl=ListDetails&productId=1000841&searchString=apfel&choice=all&lan=de&page
Key=ListDetails.
Szliszka, E., Z. P. Czuba, et al. (2010). "Chalcones and dihydrochalcones augment TRAIL-mediated
apoptosis in prostate cancer cells." Molecules 15(8): 5336-5353.
Tabak, C., I. C. Arts, et al. (2001). "Chronic obstructive pulmonary disease and intake of catechins,
flavonols, and flavones: the MORGEN Study." American Journal of Respiratory and Critical
Care Medicine 164(1): 61-64.
Tarko, T., A. Duda-Chodak, et al. (2009). "Transformations of phenolic compounds in an in vitro
model simulating the human alimentary tract." Food Technology and Biotechnology 47(4):
456-463.
Taubert, D., R. Roesen, et al. (2007a). "Effects of low habitual cocoa intake on blood pressure and
bioactive nitric oxide - A randomized controlled trial." Journal of the American Medical
Association 298(1): 49-60.
Taubert, D., R. Roesen, et al. (2007b). "Effect of cocoa and tea intake on blood pressure - A metaanalysis." Archives of Internal Medicine 167(7): 626-634.
Theodoratou, E., J. Kyle, et al. (2007). "Dietary flavonoids and the risk of colorectal cancer." Cancer
Epidemiology, Biomarkers and Prevention 16(4): 684-693.
Tokede, O. A., J. M. Gaziano, et al. (2011). "Effects of cocoa products/dark chocolate on serum lipids:
a meta-analysis." European Journal of Clinical Nutrition 65(8): 879-886.
Tomás-Barberán, F. A. and M. N. Clifford (2000). "Flavanones, chalcones and dihydrochalcones –
nature, occurrence and dietary burden." Journal of the Science of Food and Agriculture 80:
1073-1080.
Treutter, D. (2001). "Biosynthesis of phenolic compounds and its regulation in apple." Plant Growth
Regulation 34(1): 71-89.
Treutter, D. (2005). "Significance of flavonoids in plant resistance and enhancement of their
biosynthesis." Plant Biology 7(6): 581-591.
Treutter, D. (2010). "Managing phenol contents in crop plants by phytochemical farming and
breeding-visions and constraints." International Journal of Molecular Sciences 11(3): 807857.
Tsao, R., R. Yang, et al. (2005). "Which polyphenolic compounds contribute to the total antioxidant
activities of apple?" Journal of Agricultural and Food Chemistry 53(12): 4989-4995.
Tsao, R., R. Yang, et al. (2003). "Polyphenolic profiles in eight apple cultivars using highperformance liquid chromatography (HPLC)." Journal of Agricultural and Food Chemistry
51: 6347-6353.
103
LITERATURE REVIEW
REFERENCES
Tsuda, S., T. Egawa, et al. (2012). "Coffee polyphenol caffeic acid but not chlorogenic acid increases
5'AMP-activated protein kinase and insulin-independent glucose transport in rat skeletal
muscle." Journal of Nutritional Biochemistry.
Urpi-Sarda, M., M. Monagas, et al. (2009). "Epicatechin, procyanidins, and phenolic microbial
metabolites after cocoa intake in humans and rats." Analytical and Bioanalytical Chemistry
394(6): 1545-1556.
Urpi-Sarda, M., E. Ramiro-Puig, et al. (2010). "Distribution of epicatechin metabolites in lymphoid
tissues and testes of young rats with a cocoa-enriched diet." British Journal of Nutrition
103(10): 1393-1397.
Valavanidis, A., T. Vlachogianni, et al. (2009). "Polyphenolic profile and antioxidant activity of five
apple cultivars grown under organic and conventional agricultural practices." International
Journal of Food Science and Technology 44: 1167-1175.
van der Sluis, A. A., M. Dekker, et al. (2001). "Activity and concentration of polyphenolic
antioxidants in apple: Effect of cultivar, harvest year, and storage conditions." Journal of
Agricultural and Food Chemistry 49: 3606-3613.
van Der Sluis, A. A., M. Dekker, et al. (2004). "Activity and concentration of polyphenolic
antioxidants in apple juice. 2. Effect of novel production methods." Journal of Agricultural
and Food Chemistry 52: 2840-2848.
van der Sluis, A. A., M. Dekker, et al. (2002). "Activity and concentration of polyphenolic
antioxidants in apple juice. 1. Effect of existing production methods." Journal of Agricultural
and Food Chemistry 50: 7211-7219.
van der Sluis, A. A., M. Dekker, et al. (2005). "Activity and concentration of polyphenolic
antioxidants in apple juice. 3. Stability during storage." Journal of Agricultural and Food
Chemistry 53: 1073-1080.
van Dijk, A. E., M. R. Olthof, et al. (2009). "Acute effects of decaffeinated coffee and the major
coffee components chlorogenic acid and trigonelline on glucose tolerance." Diabetes Care
32(6): 1023-1025.
Veberic, R., M. Trobec, et al. (2005). "Phenolic compounds in some apple (Malus domestica Borkh)
cultivars of organic and integrated production." Journal of the Science of Food and
Agriculture 85: 1687-1694.
Vidal, S., L. Francis, et al. (2003). "The mouth-feel properties of grape and apple proanthocyanidins in
a wine-like medium." Journal of the Science of Food and Agriculture 83(6): 564-573.
Vinson, J. A., B. R. Burnham, et al. (2012). "Randomized, double-blind, placebo-controlled, linear
dose, crossover study to evaluate the efficacy and safety of a green coffee bean extract in
overweight subjects." Diabetes, Metabolic Syndrome and Obesity 5: 21-27.
Vrhovsek, U., A. Rigo, et al. (2004). "Quantitation of polyphenols in different apple varieties."
Journal of Agricultural and Food Chemistry 52(21): 6532-6538.
Walle, T., U. K. Walle, et al. (2001). "Carbon dioxide is the major metabolite of quercetin in humans."
Journal of Nutrition 131: 2648-2652.
Wan, Y., J. A. Vinson, et al. (2001). "Effects of cocoa powder and dark chocolate on LDL oxidative
susceptibility and prostaglandin concentrations in humans." American Journal of Clinical
Nutrition 74(5): 596-602.
Wang-Polagruto, J. F., A. C. Villablanca, et al. (2006). "Chronic consumption of flavanol-rich cocoa
improves endothelial function and decreases vascular cell adhesion molecule in
hypercholesterolemic postmenopausal women." Journal of Cardiovascular Pharmacology 47
Suppl 2: S177-186; discussion S206-179.
Wang, J., M. H. Chung, et al. (2010). "Estrogenic and antiestrogenic activities of phloridzin."
Biological and Pharmaceutical Bulletin 33(4): 592-597.
Watanabe, T., Y. Arai, et al. (2006). "The blood pressure-lowering effect and safety of chlorogenic
acid from green coffee bean extract in essential hypertension." Clinical and Experimental
Hypertension 28(5): 439-449.
Weng, C. J. and G. C. Yen (2012). "Flavonoids, a ubiquitous dietary phenolic subclass, exert extensive
in vitro anti-invasive and in vivo anti-metastatic activities." Cancer and Metastasis Reviews.
Wikipedia.
(2012a,
02.10.2012).
"Apple."
Retrieved
02.10.2012,
from
http://en.wikipedia.org/wiki/Apple.
104
LITERATURE REVIEW
REFERENCES
Wikipedia. (2012b, 02.10.2012). "List of apple cultivars." Retrieved 02.10.2012, from
http://en.wikipedia.org/wiki/List_of_apple_cultivars.
Willers, S. M., G. Devereux, et al. (2007). "Maternal food consumption during pregnancy and asthma,
respiratory and atopic symptoms in 5-year-old children." Thorax 62(9): 773-779.
Williams, S., S. Tamburic, et al. (2009). "Eating chocolate can significantly protect the skin from UV
light." Journal of Cosmetic Dermatology 8(3): 169-173.
Williamson, G. and A. Carughi (2010). "Polyphenol content and health benefits of raisins." Nutrition
Research 30(8): 511-519.
Williamson, G., F. Dionisi, et al. (2011). "Flavanols from green tea and phenolic acids from coffee:
critical quantitative evaluation of the pharmacokinetic data in humans after consumption of
single doses of beverages." Molecular Nutrition and Food Research 55(6): 864-873.
Wiswedel, I., D. Hirsch, et al. (2004). "Flavanol-rich cocoa drink lowers plasma F(2)-isoprostane
concentrations in humans." Free Radical Biology and Medicine 37(3): 411-421.
Wittemer, S. M., M. Ploch, et al. (2005). "Bioavailability and pharmacokinetics of caffeoylquinic
acids and flavonoids after oral administration of Artichoke leaf extracts in humans."
Phytomedicine 12: 28-38.
Wojdylo, A., J. Oszmianski, et al. (2008). "Polyphenolic compounds and antioxidant activity of new
and old apple varieties." Journal of Agricultural and Food Chemistry 56: 6520-6530.
Wu, J., H. Gao, et al. (2007). "Chemical compositional characterization of some apple cultivars." Food
Chemistry 103(1): 88-93.
Yang, C. S., S. Sang, et al. (2008). "Bioavailability issues in studying the health effects of plant
polyphenolic compounds." Molecular Nutrition and Food Research 52(S): S139 - S151.
Yang, K. C., C. Y. Tsai, et al. (2009). "Apple polyphenol phloretin potentiates the anticancer actions
of paclitaxel through induction of apoptosis in human hep G2 cells." Molecular
Carcinogenesis 48(5): 420-431.
Yuri, J. A., A. Neira, et al. (2009). "Antioxidant activity and total phenolics concentration in apple
peel and flesh is determined by cultivar and agroclimatic growing regions in Chile." Journal
of Food, Agriculture and Environment 7(3-4): 513-517.
Zhang, L., Z. Zuo, et al. (2007). "Intestinal and hepatic glucuronidation of flavonoids." Molecular
Pharmaceutics 4(6): 833-845.
Zhu, Q. Y., R. R. Holt, et al. (2002). "Stability of the flavan-3-ols epicatechin and catechin and related
dimeric procyanidins derived from cocoa." Journal of Agricultural and Food Chemistry 50(6):
1700-1705.
105
LITERATURE REVIEW
REFERENCES
106
PAPERS
107
108
PAPER 1
ANALYTICAL METHOD
PAPER 1
(published in: Analytical Methods, 2011, 3, 1774)
Rapid high performance screening method using UHPLC-MS to quantify 12 polyphenol
compounds in fresh apples
Maria Ceymann*,a, Eva Arrigonia, Hans Schärera, Daniel Baumgartnera, Anna Bozzi Nisinga,
Richard F. Hurrellb
a
Research Station Agroscope Changins-Wädenswil ACW, Schloss, 8820 Wädenswil,
Switzerland
b
ETH Zürich, Institute of Food, Nutrition and Health, Schmelzbergstrasse 7, 8092 Zürich,
Switzerland
*
Corresponding author: Fax: +41 44 783 6224; Tel: +41 44 783 6437
E-mail: [email protected]
109
PAPER 1
ANALYTICAL METHOD
Abstract
A rapid screening method for quantifying different polyphenol compounds in fresh apples
using UHPLC-MS was developed. Apples were frozen and ground to a fine powder, which
was extracted with methanol containing 1% formic acid. The resulting supernatant was used
directly for total polyphenol determination with the Folin–Ciocalteu reagent and after dilution
and filtration for UHPLC-MS analysis. Quantification of individual polyphenols was achieved
with an external standard calibration. Four different apple cultivars were analysed with both
methods. Braeburn contained the lowest amount of polyphenols (calculated as sum of
polyphenols by UHPLC-MS) followed by Gala Galaxy and Golden Reinders with average
amounts while Fuji contained the highest concentrations ranging from 24.2 to 50.0 mg per
100 g of edible fresh matter. Total polyphenol content by the Folin-Ciocalteu method showed
the same trend, but was 3–4 times higher. Using the rapid high performance screening method
the 12 most important polyphenols of apples (catechin, epicatechin, procyanidin B1 and B2,
chlorogenic acid, coumaroylquinic acid, phloridzin, phloretin-xyloglucoside, quercetingalactoside/-glucoside, rutin, quercetin rhamnoside) were quantified.
110
PAPER 1
ANALYTICAL METHOD
Introduction
In Western diets apples are one of the most consumed fruits. Apples provide consumers not
only with a good taste, but also with a variety of health relevant food components. In addition
to vitamins and dietary fibres, apples contain phenolic compounds (MANACH et al. 2004),
which have been reported to have beneficial health effects (BOYER et al. 2004). The four main
groups of polyphenols present in apples are flavan-3-ols, phenolic acids, dihydrochalcones
and flavonols. Each group varies in bioaccessibility, bioavailability and supposed beneficial
health effects. Chlorogenic acid is reported to have inhibitory effects on fat accumulation,
body weight and modulation of glucose metabolism (THOM 2007), whereas flavan-3-ols have
been shown to decrease blood pressure and platelet aggregation (WILLIAMSON et al. 2005).
Additionally, the dihydrochalcone phloridzin may affect the plasma glucose concentration and
gastrointestinal hormone secretion (JOHNSTON et al. 2002). For flavonols, various beneficial
health effects have been described depending on the foodstuffs (WILLIAMSON et al. 2005) and
the amounts present.
As a prerequisite to studying the potential of polyphenols for health in apples, it is necessary
to quantify total polyphenol content by methods such as Folin-Ciocalteu, but also to quantify
the individual polyphenols. According to TSAO et al. (2003) and VALAVANIDIS et al. (2009),
flavan-3-ols are present in apples both as monomers and oligomers. Epicatechin and catechin
are the only monomeric flavan-3-ols and various condensation products of these two
monomers result in the oligomeric flavan-3-ols, procyanidins or condensed tannins. Phenolic
acids detected in apples are mainly chlorogenic and coumaroylquinic acid, the
dihydrochalcones are phloridzin and phloretin-xyloglucoside, and the flavonols are quercetingalactoside, -glucoside and -rhamnoside and rutin. Quantifying this multiplicity of substances
is an analytical challenge and various analytical methods have been proposed and reviewed
(ALONSO-SALCES et al. 2004b; LATA 2007; MERKEN et al. 2000; OSZMIANSKI et al. 2008;
STALIKAS 2007; TURA et al. 2002). In general, for polyphenol analysis, apples are first peeled,
cut, frozen and freeze-dried in preparation for extraction. Analysis is often done by HPLC. All
these steps are time consuming and reduce the number of samples to be analysed in a day.
The aim of this paper was to quantify the 12 major polyphenols in apples, which are
absorbable after ingestion (MANACH et al. 2004; MANACH et al. 2005) and no effort was made
to integrate polyphenols with higher molecular weight within this method. Both, sample
preparation and extraction time were reduced to a minimum of treatment steps. Additionally,
the use of an UHPLC-MS (Ultra High Pressure Liquid Chromatography-Mass Spectrometry)
111
PAPER 1
ANALYTICAL METHOD
lowers the time of analysis substantially compared to other analytical procedures as described
for example by NOVAKOVA et al. (2010), who used an UHPLC with PDA detection.
112
PAPER 1
ANALYTICAL METHOD
Materials and methods
Reagents
Liquid nitrogen for sample preparation was purchased from Messer Schweiz AG (Lenzburg,
Switzerland). Methanol and formic acid for the extraction (analytical grade) were obtained
from Acros Organics (Chemie Brunschwig, Basel, Switzerland) and from Carbagas
(Gümligen, Switzerland), respectively.
Methanol and formic acid (99 %) of ULC/MS grade for preparing the mobile phase were
acquired from Biosolve (Valkenswaard, The Netherlands). Water was distilled and filtered
through a 0.2 µm nylon filter (OPTI – Flow®, WICOM, Heppenheim, Germany). FolinCiocalteu reagent and anhydrous sodium carbonate from Merck (Darmstadt, Germany) were
used for total polyphenol determination.
Standards
Catechin, epicatechin, chlorogenic acid, quercetin rhamnoside, quercetin glucoside, quercetin
galactoside and rutin were purchased from Fluka (Buchs, Switzerland), phloridzin from
Sigma (St. Louis, USA) and procyanidin B1 and B2 from Extrasynthèse (Genay, France). For
UHPLC-MS analysis 1 mg of each standard was dissolved in 1 ml of methanol for stock
solutions, which were afterwards combined into one mixed stock solution and diluted to
different working concentrations (0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, 5, 7.5 and 10 mg/L
48 % aqueous methanol (v/v) containing 1 % ascorbic acid from Fluka (Buchs, Switzerland)
and 1% formic acid from Merck (Darmstadt, Germany)). Aliquots were stored in UHPLC
vials at -20 °C until analysis. For total polyphenol content analysis by Folin-Ciocalteu
standard concentrations of 50, 100, 150 and 200 mg catechin/L methanol were freshly
prepared.
Samples
The four apple cultivars Braeburn, Fuji, Gala Galaxy (mutant of Gala), Golden Reinders
(mutant of Golden Delicious) were chosen to evaluate the analytical procedure. Each sample
is composed of 20 fruits, which were sliced into 10 pieces and the core area with an apple
divider (DIVISOREX, Famos-Westmark GmbH, Lennestadt Elspe, Germany). Two opposite
cuts of every fruit were immediately frozen in liquid nitrogen and all 40 slices were pooled as
representative sample. The frozen apple pieces were abrasively crushed in a dry ice mill
(Meidinger AG, Kaiseraugst, Switzerland) and ground to fine powder with a cutter (La
113
PAPER 1
ANALYTICAL METHOD
Moulinette DPA 1, Moulinex, Germany). Afterwards, the samples were stored at -20 °C until
extraction.
Extraction
Aliquots (2.50 g) of the frozen powder were mixed with 45 ml of methanol containing 1 %
formic acid (v/v), flushed with nitrogen for 30 sec and homogenized with a Polytron (Polytron
PT 3100, MERCK, Zug, Switzerland) at maximum speed for another 30 sec. The extracts
were made up to 50 ml with additional methanol containing formic acid (1 % v/v) and
allowed to stand for one hour. An aliquot of the supernatant was used directly for total
polyphenol content determination by Folin-Ciocalteu. For UHPLC-MS analysis the
supernatant was filtered through 0.7 µm glass fibre filters (OPTI – Flow®, WICOM,
Heppenheim, Germany), diluted 1:1 (v/v) with distilled water and filtered a second time
through 0.2 µm nylon filters (OPTI – Flow®, WICOM, Heppenheim, Germany) directly in
UHPLC vials. Extractions were done in duplicate.
Total polyphenol content analysis
The total polyphenol content method described by SINGLETON et al. (1999) was adapted for
the Konelab ARENA 20XT analyser (Thermo Fisher Scientific OY, Vantaa, Finland). Ten µL
of Folin-Ciocalteu reagent and 100 µL of distilled water were automatically pipetted to 10 µL
of methanolic extract in a 200 µL cuvette. After 1 min, 40 µL sodium carbonate solution
(200 g/L) and 40 µL distilled water were added, thoroughly mixed and incubated for 30 min
at 37 °C. Absorption was measured automatically at 700 nm. Total polyphenol content was
calculated by means of an external calibration with methanolic catechin standards and
expressed as mg catechin equivalents/100 g.
UHPLC-MS analysis
UHPLC-MS analysis was carried out using an ACQUITY Ultra Performance LC™ system
(UPLCTM) with binary solvent manager and single quadrupole micromass ZQ Mass Detector
(Waters Corporation, Milford, USA) equipped with an electrospray ionization (ESI) source
operating in negative mode. For instrument control, data acquisition and processing
MassLynx™ software (Version 4.1) was used.
A reversed phase column (BEH C18, 1.7 µm, 2.1 x 50 mm with a BEH C18, 1.7 µm
VanGuardTM Pre-Column, 2.1 x 5 mm, Waters Corporation, Milford, USA) at 40 °C was used
for separation of individual polyphenols. Samples (2.5 µL) were injected and elution
completed in 10 min with a sequence of linear gradients and isocratic flow rates of
114
PAPER 1
ANALYTICAL METHOD
0.3 ml/min. Solvent A was composed of water containing 0.1 % formic acid and solvent B of
methanol with 0.1 % formic acid. Separation was achieved using the following gradient: 02.5 min 5-25 % B, 2.5-5.0 min 25-30 % B, 5.0-6.5 min 30-95 % B, 6.5-8.0 min 95 % B, 8.08.01 min 95-5 % B and 8.01-10.00 min 5 % B.
The effluent was led directly to an electrospray source with a source block temperature of
130 °C, desolvation temperature of 350 °C, capillary voltage of 2.5 kV and cone voltage of
20 V. Nitrogen was used as desolvation gas (300 L/h). For quantitative analysis, single ion
recording (SIR) with necessary parameters for detection the single polyphenols listed in
Table 1 were used. Quantification was calculated by using an external standard calibration
and the software QuantLynx™ (Waters Corporation, Milford, USA, Version 4.1). All
UHPLC-MS analysis was done in duplicate.
Table 1
Chromatographic and calibration parameters for standard polyphenols in apples;
tR=retention time; R2=correlation coefficient (n=7)
RT (min)
[M-H]- (m/z)
Catechin
Epicatechin
Procyanidin B1
Procyanidin B2
Chlorogenic acid
p-Coumaroylquinic acid
Phloridzin
Phloretin-xyloglucoside
2.63
3.47
2.31
2.91
2.96
3.63
6.49
6.16
289.16
289.16
577.37
577.37
353.28
337.42
435.44
567.30
Quercetin-Galactoside/Glucoside
Rutin
Quercetin-Rhamnoside
5.98
6.02
6.54
463.15
609.52
447.42
Compound
Linear range (mg/L)
R2,
0.05-5
0.9947
0.75-10
0.9903
0.05-5
0.9962
0.5-10
0.9925
0.75-10
0.9905
calibrated as chlorogenic acid
0.05-5
0.9970
calibrated as phloridzin
0.05-5
0.05-5
0.05-5
0.9922
0.9958
0.9976
115
PAPER 1
ANALYTICAL METHOD
Results and discussion
Sample preparation
Sample preparation was adapted as closely as possible to consumer behaviour. The edible part
of the apple with peel but without the core area was analysed. Literature data however
commonly refer to apples analysed after separating peel from the flesh (KHANIZADEH et al.
2008; MCGHIE et al. 2005; TSAO et al. 2003; VEBERIC et al. 2005). In addition to closely
simulating how apples are recommended to be eaten, analysis with the peel provided a better
prevention of oxidation. By using an apple divider, sample treatment was more easily
standardised and removing the core area included a traditional domestic treatment in the
preparation. To our knowledge this consumer adapted apple preparation procedure is
described here for the first time. In addition freezing in liquid nitrogen, grinding to a fine
powder and storage at -20 °C prior to analysis was a time saving and more simple approach
compared to freeze-drying.
Extraction
Different extraction methods described in literature were considered (AWAD et al. 2000; LATA
2007; NAPOLITANO et al. 2004; OSZMIANSKI et al. 2008; TSAO et al. 2003;
VAN DER SLUIS
et
al. 2001), tested and finally combined to the optimised approach described. Methanol was
chosen as extraction solvent, because of its frequent use in previous studies (TURA et al. 2002;
VEBERIC et al. 2005) and its suitability as UHPLC eluent. The addition of formic acid was an
adaption to the UHPLC eluent and 1 % showed the best results (MCGHIE et al. 2005;
OSZMIANSKI et al. 2008). A series of experiments were carried out to optimise the extraction
conditions. In the first studies, the methanol concentration was kept constant at 99 %, and
extracts made with apple powder to solvent ratios of 1:1 (TSAO et al. 2003), 1:5 (NAPOLITANO
et al. 2004) 1:10 and 1:20 (VAN
DER
SLUIS et al. 2001). The ratio 1:20 showed the best
extraction and handling qualities. Additionally, with this ratio one extraction step was found
to be timesaving and sufficient, because no relevant amounts of polyphenols were extracted in
following extraction steps. Therefore, the 1:20 ratio of apple powder to solvent was used for
optimising the methanol concentration. Extracts were made with 50 % aqueous methanol up
to pure methanol and 90 % methanol led to the highest sensitivity in UHPLC-MS signals. For
simplification 99 % methanol with 1 % formic acid as extraction solvent was tested and
replaced the 90 % methanol solution for any further extractions.
The resulting extracts were used to quantify total polyphenol content by Folin-Ciocalteu
without any further treatment. Prior to UHPLC-MS, however, a dilution with water was
116
PAPER 1
ANALYTICAL METHOD
necessary, because of the high water content on the equilibrated column. Extract to water
ratios of 2:1, 1:1 and 1:3 were tested. The highest sensitivity in UHPLC-MS signals were
achieved with the ratio 1:1.
The optimised extraction procedure used to quantify the apple polyphenols included a 1:20
powder to solvent ratio, 99 % methanol with 1 % formic acid as the extraction solvent, and an
additional 1:1 dilution with water for UHPLC-MS analysis. The described procedure is rapid
and well adapted to quick-frozen apple samples.
Calibration
The calibration was adapted to the expected polyphenol concentrations in the apple extracts
(Table 1). Each standard solution was measured seven times over a period of 24 h from two
different vials and data were combined into one calibration curve. Repeatability for n=7 was
found to be within the normal limits and no time-dependent deviation could be detected. The
lowest standard concentration was 0.05 mg/L and values below this concentration were not
quantified. Table 1 show, that all calibrations were linear over a wide range and had very high
correlation coefficients. This calibration range is similar to that reported by CHINNICI et al.
(2004b) and NOVAKOVA et al. (2010), but wider than reported by MARI et al. (2010). The
described UHPLC-MS screening method allows to quantify concentrations 10-fold lower than
the HPLC-photodiode array detection system of KAHLE et al. (2005a).
Chromatography
Separation of the most abundant apple polyphenols was achieved within 8 min (Figure 1).
Two additional min were necessary for equilibration. The same analysis time was reported by
COOPER et al. (2007), who analysed the major polyphenols in chocolate including epicatechin,
catechin and procyanidins, but not dihydrochalcones or phenolic acids. In addition to the short
analysis time, another advantage of this screening method is the use of a water-methanol
gradient instead of water-acetonitril as used by COOPER et al. (2007), because of both lower
cost and toxicity of methanol. SPACIL et al. (2008) analysed chlorogenic acid, the two
monomeric flavan-3-ols and two flavonols by UHPLC-UV in three different runs. To our
knowledge, the simultaneous chromatographic analysis of these polyphenol groups by
UHPLC-MS is reported here for the first time.
While the separation of catechin and epicatechin as well as Procyanidin B1 and B2 was
satisfactorily achieved (Figure 1), no effort was made to separate quercetin-galactoside and glucoside. These compounds are mainly present in apple peels and in much lower amounts
117
PAPER 1
ANALYTICAL METHOD
than flavan-3-ols. Since apples contain approximately three times more quercetin-galactoside
than quercetin-glucoside (AWAD et al. 2000; KHANIZADEH et al. 2008; TSAO et al. 2003;
WOJDYLO et al. 2008), concentrations were calculated as galactoside.
091201_Braeburn_Conthey_1_a
2
100
3.61
%
1
1.14
7.54
2.77
0
1.00
2.00
091201_Braeburn_Conthey_1_a
3.00
4.00
5.00
6.00
7.00
4.00
5.00
6.00
7.00
4.00
5.00
6.00
7.00
5.00
6.00
7.00
min
4
100
3.05
%
3
2.45
0.91
0
1.00
2.00
091201_Braeburn_Conthey_1_a
3.00
min
5
100
3.10
%
3.75
0
1.00
2.00
091201_Braeburn_Conthey_1_a
3.00
100
min
6
3.82
%
2.98
1.02
0
1.00
2.00
091201_Braeburn_Conthey_1_a
3.00
4.00
min
7
100
%
1.07
0
1.00
2.00
091201_Braeburn_Conthey_1_a
4.48
3.17
3.00
4.00
5.00
6.00
7.00
min
8
100
%
5.60
0
1.00
2.00
091201_Braeburn_Conthey_1_a
3.00
4.00
5.00
6.00
7.00
min
9
100
%
0
1.00
2.00
091201_Braeburn_Conthey_1_a
3.00
4.00
5.00
100
6.00
7.00
10
6.30
%
min
7.19
6.82
0.42
1.03
3.32
1.91
0
1.00
2.00
091201_Braeburn_Conthey_1_a
3.92
3.00
4.00
4.81
5.00
6.00
7.00
min
11
100
%
0.93
0
Figure 1:
3.46
1.00
2.00
3.00
4.00
5.00
6.00
7.00
min
UHPLC-MS chromatogram of a Braeburn apple extract. Extracted ion chromatograms
of 1 catechin, 2 epicatechin, 3 procyanidin B1, 4 procyanidin B2, 5 chlorogenic acid, 6
coumaroylquinic acid, 7 phloridzin, 8 phloretin-xyloglucoside, 9 quercetingalactoside/glucoside, 10 rutin and 11 quercetin-rhamnoside
Polyphenol content of selected apple cultivars
Table 2 illustrates the polyphenol contents and patterns of four common apple cultivars. The
data represent mean values of two extracts, which were analysed in duplicate and a mean
standard deviation of approximately 6 % was found for all compounds.
The cultivar Braeburn showed the lowest sum of polyphenols in UHPLC-MS analysis and
Fuji the highest. Intermediate amounts were analysed in Gala Galaxy and Golden Reinders.
The flavan-3-ols catechin and procyanidin B1 were present in low amounts (0.4-2 mg/100 g
fresh matter) in all apples, whereas epicatechin with 3-7 mg/100 g and procyanidin B2 with 58 mg/100 g contribute most to the total flavan-3-ol content. The phenolic acids were
dominated by chlorogenic acid with amounts of 6-21 mg/100 g, being equivalent to the total
118
PAPER 1
ANALYTICAL METHOD
flavan-3-ols content. Both dihydrochalcones (phloridzin and phloretin-xyloglucoside) were
found in all four cultivars in similar and relatively low amounts (1-2 mg/100 g and 24 mg/100 g, respectively). Flavonols were present in the range of 2 to 6 mg/100 g. Quercetingalactoside and -glucoside accounted for approximately half of the flavonols (2-3 mg/100 g)
whereas rutin was present in small amounts only in Braeburn, Fuji and Gala Galaxy.
Comparing the polyphenol patterns of the four cultivars, it can been seen that the total levels
of flavan-3-ols and phenolic acids are similar but that Fuji and Gala Galaxy have slightly
higher amounts of phenolic acids than flavan-3-ols, whereas the other cultivars have slightly
higher amounts of flavan-3-ols than phenolic acids. Dihydrochalcones and flavonols
contribute almost equal with low proportions of approximately 11 % to the polyphenol pattern
of all cultivars analysed.
Summing up these polyphenol groups as analysed by UHPLC-MS, the amounts were between
24 and 50 mg/100 g FM which is clearly lower than the total polyphenol content by the FolinCiocalteu method with 102 to 155 mg catechin equivalents/100 g (Table 2). This comparison
showed that the latter was 3-4 times higher than the sum of polyphenols calculated from
UHPLC-MS data. This trend was found across all cultivars. The difference can be explained
by losses due to solubility changes on dilution with water and by filtration before UHPLC-MS
analysis. For the mono- and dimeric polyphenol standards used in our methodology, these
losses amounted to approximately 10 %, however losses up to 57 % were found with apple
extracts (data not shown). Whether oligomeric procyanidins were removed during filtration
remains to be elucidated, since they would not be detected in the SIR mode anyway. Large
amounts of oligomeric procyanidins have been reported in apples (GUYOT et al. 2002) and,
according to VRHOVSEK et al. (2004), oligomeric procyanidins represent 64% of the total
polyphenol value as measured by Folin-Ciocalteu. This value is consistent with the 57 %
losses in total polyphenols by Folin-Ciocalteu we found after precipitation and filtration.
VRHOVSEK et al. (2004) used 3 different chromatographic runs and were able to explain the
complete composition of the total polyphenol content as measured by Folin-Ciocalteu by
quantifying additionally the oligomeric procyanidins in Golden Delicious, Royal Gala (mutant
of Gala), Braeburn and Fuji and other cultivars.
Another contribution to the large difference we found between the sum of polyphenols as
measured by UHPLC-MS and the Folin-Ciocalteu measurement can be attributed to the poor
specificity of the Folin-Ciocalteu assay, which is known to measure additional components
present in the extract (SINGLETON et al. 1999). VRHOVSEK et al. (2004) excluded these
119
PAPER 1
ANALYTICAL METHOD
substances by an additional cleanup step on a C-18 cartridge. The consequence of this
additional clean up step is that the total polyphenol values reported by VRHOVSEK et al.
(2004) are lower than reported by other studies. For example, SANONER et al. (1999) reported
128 mg epicatechin equivalents/100 g in the cortex area of Golden Delicious using
epicatechin as a reference, compared to 86 mg catechin equivalents/100 g in the same cultivar
reported by VRHOVSEK et al. (2004). In Table 2, the Folin-Ciocalteu total polyphenol content
in Golden Reinders (mutant of Golden Delicious) (127 mg catechin equivalents/100 g)
conforms to the value reported by SANONER et al. (1999).
Although our procedure is considerably faster than other published procedures, the measured
values for individual polyphenols in Braeburn, Fuji, Gala Galaxy and Golden Reinders apple
varieties (Table 2) are similar to those reported by VRHOVESK et al. (2004). On the other
hand, GUYOT et al. (2002) reported higher values for catechin, epicatechin, procyanidin B2,
chlorogenic acid, coumaroylquinic acid, phloridzin and phloretin-xyloglucoside in Braeburn
apple flesh without peel. These higher values could be explained by the methodology used or
by differences in growing region, harvest year and other pre-harvest factors. Our values for
catechin, epicatechin, procyanidin B1 and B2, chlorogenic acid, phloridzin as well as
quercetin-galactoside and quercetin-glucoside (Table 2) for the Fuji, Golden Reinders and
Jonagold cultivars are more or less within the ranges reported by other authors (NEVEU et al.
2010; USDA 2007; VALAVANIDIS et al. 2009). Comparisons between the values generated
using our new method and values reported in the literature are difficult due to the effects of
different pre-harvest factors and methodological differences. Most previous studies have
peeled and freeze-dried apples prior to polyphenol analysis and polyphenol data of whole
apples are scarce. Additionally, polyphenol composition data is published in different formats.
For example, the USDA database (USDA 2007) provide information on flavonoids in specific
cultivars whereas the new Polyphenol Explorer database (NEVEU et al. 2010) provided data on
all relevant polyphenols in apples but does not include cultivar-specific data. These results
thus support the aim of our study which was to develop a faster and simpler method than
previously published (GUYOT et al. 2002; VALAVANIDIS et al. 2009; VRHOVSEK et al. 2004)
which accurately quantifies within one simple extraction step and one chromatographic run,
all the major absorbable polyphenols in apples.
120
PAPER 1
Table 2
ANALYTICAL METHOD
Polyphenol content of four different apple cultivars analysed by UHPLC-MS and total
polyphenol content. Results given in mg/100 g edible fresh matter (mean values of two
extracts analysed in duplicate)
compound
Catechin
Epicatechin
Procyanidin B1
Procyanidin B2
Chlorogenic acid
Coumaroylquinic acid
Phloridzin
Phloretin-xyloglucoside
Quercetin-Galactoside/-Glucoside
Rutin
Quercetin-Rhamnoside
Sum of polyphenols by UHPLC-MS
Braeburn
0.4
4.1
0.8
4.9
5.7
0.9
1.1
2.2
2.3
0.6
1.2
Fuji
0.9
7.4
1.4
8.2
20.5
0.9
2.0
2.7
2.7
1.0
2.3
Gala Galaxy
1.2
6.7
2.1
6.8
17.2
1.7
1.5
3.4
2.2
0.5
1.5
Golden Reinders
0.5
5.9
1.2
8.0
13.4
1.1
1.9
2.7
1.2
0.0
2.3
24.2
50.0
44.8
38.3
a
Total polyphenol content
102
154
155
127
Determined by Folin-Ciocalteu method, expressed as mg catechin equivalents/100 g.
a
Conclusion
This paper describes a screening method to analyse 12 mono- and dimeric polyphenols in
fresh apples without any concentration procedure. The different steps, including sample
preparation, extraction and quantification are high performance and rapid. The procedure
described here represents a good screening method for a consumer adapted analysis of those
polyphenol compounds present in apples, that are reported to have beneficial health effects.
Such a method could be extended to other foodstuffs and also be used in breeding programs to
select varieties high in specific health promoting polyphenol compounds.
Acknowledgement
The authors thank Dr. Astrid Bachmann for their support during the development of this
method.
121
PAPER 1
ANALYTICAL METHOD
References
Alonso-Salces, R. M., C. Herrero, et al. (2004b). "Technological classification of Basque cider apple
cultivars according to their polyphenolic profiles by pattern recognition analysis." Journal of
Agricultural and Food Chemistry 52(26): 8006-8016.
Awad, M. A., A. Jager de, et al. (2000). "Flavanoid and chlorogenic acid levels in apple fruit:
characterisation of variation." Scientia Horticulturae 83: 249-263.
Boyer, J. and H. L. Rui (2004). "Apple phytochemicals and their health benefits." Nutrition
Journal(3): 5.
Chinnici, F., A. Gaiani, et al. (2004b). "Improved HPLC determination of phenolic compounds in cv.
golden delicious apples using a monolithic column." Journal of Agricultural and Food
Chemistry 52: 3-7.
Cooper, K. A., E. Campos-Gimenez, et al. (2007). "Rapid reversed phase ultra-performance liquid
chromatography analysis of the major cocoa polyphenols and inter-relationships of their
concentrations in chocolate." Journal of Agricultural and Food Chemistry 55: 2841-2847.
Guyot, S., C. L. Bourvellec, et al. (2002). "Procyanidins are the most abundant polyphenols in dessert
apples at maturity." LWT- Food Science and Technology 35: 289-291.
Johnston, K. L., M. N. Clifford, et al. (2002). "Possible role for apple juice phenolic compounds in the
acute modification of glucose tolerance and gastrointestinal hormone secretion in humans."
Journal of the Science of Food and Agriculture 82: 1800-1805.
Kahle, K., M. Kraus, et al. (2005a). "Polyphenol profiles of apple juices." Molecular Nutrition and
Food Research 49: 797 – 806.
Khanizadeh, S., R. Tsao, et al. (2008). "Polyphenol composition and total antioxidant capacity of
selected apple genotypes for processing." Journal of Food Composition and Analysis 21: 396401.
Lata, B. (2007). "Relationship between apple peel and the whole fruit antioxidant content: year and
cultivar variation." Journal of Agricultural and Food Chemistry 55: 663-671.
Manach, C., A. Scalbert, et al. (2004). "Polyphenols: food sources and bioavailability." American
Journal of Clinical Nutrition 79: 727-747.
Manach, C., G. Williamson, et al. (2005). "Bioavailability and bioefficacy of polyphenols in humans.
I. Review of 97 bioavailability studies." American Journal of Clinical Nutrition 81(suppl):
230S-242S.
Mari, A., I. Tedesco, et al. (2010). "Phenolic compound characterisation and antiproliferative activity
of "Annurca" apple, a southern Italian cultivar." Food Chemistry 123: 157-164.
McGhie, T. K., M. Hunt, et al. (2005). "Cultivar and growing region determine the antioxidant
polyphenolic concentration and composition of apples grown in New Zealand." Journal of
Agricultural and Food Chemistry 53: 3065-3070.
Merken, H. M. and G. R. Beecher (2000). "Measurement of food flavonoids by High-Performance
Liquid Chromatography: A Review." Journal of Agricultural and Food Chemistry 48: 577599.
Napolitano, A., A. Cascone, et al. (2004). "Influence of variety and storage on the polyphenol
composition of apple flesh." Journal of Agricultural and Food Chemistry 52: 6526-6531.
Neveu, V., J. Perez-Jiménez, et al. (2010). Phenol-Explorer: an online comprehensive database on
polyphenol contents in foods, online Database. Version 1.5.2, available at http://www.phenolexplorer.eu.
Novakova, L., Z. Spacil, et al. (2010). "Rapid qualitative and quantitative ultra high performance
liquid chromatography method for simultaneous analysis of twenty nine common phenolic
compounds of various structures." Talanta 80: 1970-1979.
Oszmianski, J., M. Wolniak, et al. (2008). "Influence of apple purée preparation and storage on
polyphenol contents and antioxidant activity." Food Chemistry 107: 1473-1484.
Sanoner, P., S. Guyot, et al. (1999). "Polyphenol profiles of French cider apple varieties (Malus
domestica sp.)." Journal of Agricultural and Food Chemistry 47: 4847-4853.
Singleton, V. L., R. Orthofer, et al. (1999). "Analysis of total phenols and other oxidation substrates
and antioxidants by means of Folin-Ciocalteu reagent." Methods in Enzymology 299: 152-178.
122
PAPER 1
ANALYTICAL METHOD
Spacil, Z., L. Novakova, et al. (2008). "Analysis of phenolic compounds by high performance liquid
chromatography and ultra performance liquid chromatography." Talanta 76: 189-199.
Stalikas, C. D. (2007). "Extraction, separation, and detection methods for phenolic acids and
flavonoids." Journal of separation science 30: 3268-3295.
Thom, E. (2007). "The effect of chlorogenic acid enriched coffee on glucose absorption in healthy
volunteers and its effect on body mass when used long-term in overweight and obese people."
Journal of International Medical Research 35: 900-908.
Tsao, R., R. Yang, et al. (2003). "Polyphenolic profiles in eight apple cultivars using highperformance liquid chromatography (HPLC)." Journal of Agricultural and Food Chemistry
51: 6347-6353.
Tura, D. and K. Robards (2002). "Sample handling strategies for the determination of biophenols in
food and plants." Journal of Chromatography A 975: 71-93.
USDA (2007). USDA database for the flavonoid content of selected foods, U.S. Department of
Agriculture. Release 2.1, available at http://www.ars.usda.gov/nutrientdata.
Valavanidis, A., T. Vlachogianni, et al. (2009). "Polyphenolic profile and antioxidant activity of five
apple cultivars grown under organic and conventional agricultural practices." International
Journal of Food Science and Technology 44: 1167-1175.
van der Sluis, A. A., M. Dekker, et al. (2001). "Activity and concentration of polyphenolic
antioxidants in apple: Effect of cultivar, harvest year, and storage conditions." Journal of
Agricultural and Food Chemistry 49: 3606-3613.
Veberic, R., M. Trobec, et al. (2005). "Phenolic compounds in some apple (Malus domestica Borkh)
cultivars of organic and integrated production." Journal of the Science of Food and
Agriculture 85: 1687-1694.
Vrhovsek, U., A. Rigo, et al. (2004). "Quantitation of polyphenols in different apple varieties."
Journal of Agricultural and Food Chemistry 52(21): 6532-6538.
Williamson, G. and C. Manach (2005). "Bioabailability and bioefficacy of polyphenols in humans. II.
Review of 93 intervention studies." American Journal of Clinical Nutrition 81: 243S-255S.
Wojdylo, A., J. Oszmianski, et al. (2008). "Polyphenolic compounds and antioxidant activity of new
and old apple varieties." Journal of Agricultural and Food Chemistry 56: 6520-6530.
123
PAPER 1
ANALYTICAL METHOD
124
PAPER 2
SCREENING OF APPLES
PAPER 2
(published in: Journal of Food Composition and Analysis, 2012, 26, 128)
Identification of apples rich in health-promoting flavan-3-ols and phenolic acids by
measuring the polyphenol profile
Maria Ceymanna*, Eva Arrigonia, Hans Schärera, Anna Bozzi Nisinga, Richard F. Hurrellb
a
Research Station Agroscope Changins-Wädenswil ACW, Schloss, 8820 Wädenswil,
Switzerland
b
ETH Zürich, Institute of Food, Nutrition and Health, Schmelzbergstrasse 7, 8092 Zürich,
Switzerland
*
Corresponding author: Tel.: +41 44 783 6437; Fax: +41 44 783 6224.
E-mail address: [email protected]
125
PAPER 2
SCREENING OF APPLES
Abstract
It has been reported that polyphenolic compounds from various plant foods produce
physiological effects beneficial to health. Nevertheless, a comprehensive evaluation of the
polyphenol content and profile of different apple cultivars are scarce.
This study examined 104 European apple cultivars for polyphenols by UHPLC-MS, total
polyphenol content (TPC) by Folin-Ciocalteu and antioxidative potential by Trolox®
Equivalent Antioxidative Capacity (TEAC) and Ferric Reducing Antioxidant Power (FRAP).
The greatest concentrations of individual polyphenols were found for epicatechin, procyanidin
B2 and chlorogenic acid. Individual apple polyphenols ranged from below the limit of
detection to 70 mg/100 g FM and varied strongly between the different cultivars. The TPC
varied from 49 mg to 377 mg catechin equivalents (CE) per 100 g and was much higher than
the sum of the 12 individual polyphenols. TPC as well as the antioxidative potential correlated
well with the sum of individual polyphenols as quantified by UHPLC-MS.
Based on this polyphenol profiling, apples can be divided into flavan-3-ol dominated or
phenolic acid dominated cultivars. Both classes of polyphenols have been reported to have
physiological effects beneficial to health.
126
PAPER 2
SCREENING OF APPLES
Introduction
Apples are the most popular fruits in Europe and are grown all over the world. From 2003 to
2005, the average per capita apple consumption in Europe was 61 g per day, which is twice as
high as the per capita consumption worldwide and represents one quarter of the total
European fruit consumption. The price, convenience and positive health image are all reasons
for this popularity (HARKER et al. 2003). The beneficial health effects of apples have been
attributed to the polyphenolic compounds, a group of secondary plant metabolites, of which
several thousand different compounds have been identified (SIES 2010). The four polyphenol
classes which predominate in apples are the flavan-3-ols, phenolic acids, dihydrochalcones
and flavonols. These compounds are present in the monomeric forms, but occur mostly as
high molecular weight polymers. Only low molecular weight, mainly monomeric polyphenols
are reported to be absorbed from polyphenol-containing foods (MANACH et al. 2004;
MANACH et al. 2005), whereas breakdown products from high molecular weight polyphenols
may be absorbed after degradation by the microflora in the colon (DEPREZ et al. 2000;
MONAGAS et al. 2010). The potential health benefits of polyphenols have been reviewed by
SCALBERT et al. (2005). The major apple polyphenols, flavan-3-ols and chlorogenic acid have
been investigated for their health effects in other foodstuffs. The flavan-3-ols from cocoa have
been reported to have a vasodilatory effect which improves blood flow (FARIDI et al. 2008).
Chlorogenic acid, which is present in high amounts in coffee, is reported to have beneficial
effect on cardiovascular disease (BONITA et al. 2007) and to decrease the risk of type II
diabetes (BIDEL et al. 2008; JOHNSTON et al. 2003).
A detailed knowledge of the polyphenol profile and content in different apple cultivars is
necessary in order to evaluate their potential beneficial health effects. At present, there are
literature reports on the contents of selected polyphenols in a small number of cultivars (IMEH
et al. 2002; LEE et al. 2003; NEVEU et al. 2010; PODSEDEK et al. 2000; VRHOVSEK et al. 2004;
WOJDYLO et al. 2008) and studies reporting the influence of pre- and post-harvest factors
(D'ABROSCA et al. 2007; MARI et al. 2010; MCGHIE et al. 2005) or farming methods
(CHINNICI et al. 2004a; HECKE et al. 2006; LAMPERI et al. 2008; VALAVANIDIS et al. 2009) on
their levels, but there is no comparison of individual low molecular weight polyphenols in a
comprehensive range of different apple cultivars. Polyphenols in apples have usually been
measured in the flesh after separation of peel (LATA et al. 2005; TSAO et al. 2003;
VAN DER
SLUIS et al. 2001), after processing into a puree (DRAGOVIC-UZELAC et al. 2005; OSZMIANSKI
et al. 2008), or more frequently as a juice from cider apples (ALONSO-SALCES et al. 2004a;
KAHLE et al. 2005a; MANGAS et al. 1999; OSZMIANSKI et al. 2007; WU et al. 2007). The aim
127
PAPER 2
SCREENING OF APPLES
of this study was to quantify potentially absorbable low molecular weight polyphenols, with
potential health benefits, in 104 different apple cultivars grown in Switzerland. The
polyphenols were measured by UHPLC-MS in the combined peel and flesh so as to be more
representative of normal eating habits. For comparison, total polyphenol content (TPC) and
antioxidative potential were measured by simpler colorimetric methods.
128
PAPER 2
SCREENING OF APPLES
Materials and methods
Materials
Reagents
Liquid nitrogen for sample preparation was purchased from Messer Schweiz AG (Lenzburg,
Switzerland). Methanol and formic acid (analytical grade) for extraction of polyphenols were
obtained from Acros Organics (Chemie Brunschwig, Basel, Switzerland) and from Merck
(Darmstadt, Germany), respectively.
Methanol and formic acid (99 %) for preparing the mobile phase were acquired from Biosolve
(Valkenswaard, The Netherlands). Water was distilled and filtered through a 0.2 µm nylon
filter (WICOM, Heppenheim, Germany). Folin-Ciocalteu reagent and anhydrous sodium
carbonate from Merck (Darmstadt, Germany) were used for total polyphenol determination.
For
measuring
the
antioxidative
potential
diammonium
2,2’-azinobis[3-
ethylenbenzothiazoline]-6-sulfonic acid (ABTS, >99 %), potassium persulfate (>99 %) and
2,4,6-tripyridyl-s-triazine (TPTZ) were purchased from Fluka (Buchs, Switzerland). Sodium
acetate and acetic acid (100 %, water free) from Merck (Darmstadt, Germany) and ferric
chloride hexahydrate (Riedel de Haën, Seelze, Germany) were used for the Ferric Reducing
Antioxidant Power (FRAP) assay. Potassium phosphate (Sigma-Aldrich, Buchs, Switzerland)
and 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox®, Sigma, Steinheim,
Germany) were used for the Trolox® Equivalent Antioxidative Capacity (TEAC) assay.
Catechin (C), epicatechin (E), chlorogenic acid (CA), quercetin rhamnoside (QR), quercetin
glucoside, quercetin galactoside and rutin (R) were purchased from Fluka (Buchs,
Switzerland), phloridzin (P) from Sigma (St. Louis, USA) and procyanidin B1 (PCB1) and B2
(PCB2) from Extrasynthèse (Genay, France). Coumaroylquinic acid (CQA) was quantified as
chlorogenic acid and phloretin-xyloglucoside (PXG) as phloridzin.
Apple cultivars
The 104 apple cultivars analyzed (Table 1) were harvested at optimal maturity in 2008, 2009
and 2010 from different locations in Switzerland except for cultivar PRI 159 and Red
Delicious, which were a gift from Plant Research International (Wageningen, The
Netherlands) and the Research Centre for Agriculture and Forestry Laimburg (Italy),
respectively. The tested cultivars included 15 cider cultivars and 89 dessert apples, of which
46 were new cultivars.
129
PAPER 2
SCREENING OF APPLES
Methods
Sample preparation
Sampling and sample treatment was done as described by CEYMANN et al. (2011). Briefly, at
harvest, 20 fruits out of the whole production were randomly chosen and pooled together to
one sample. The 20 fruits were sliced with an apple divider (DIVISOREX, Famos-Westmark
GmbH, Lennestadt-Elspe, Germany) into 10 pieces and the core area. The core area was
discarded and two opposite cuts of each fruit were randomly chosen and immediately frozen
in liquid nitrogen and all 40 slices were pooled as representative sample. The frozen apple
pieces were ground to fine powder with a dry ice mill (Meidinger AG, Kaiseraugst,
Switzerland) and a cutter (La Moulinette DPA 1, Moulinex, Germany). Afterwards, the
samples were stored at -20 °C until extraction.
Extraction
Extraction was carried out as described earlier (CEYMANN et al. 2011). Briefly, within three
months after harvest aliquots (2.50 g) of the frozen powder were mixed with 50 mL of
methanol containing 1 % formic acid (v/v) and homogenized. For TPC, TEAC and FRAP the
supernatants were used directly. For UHPLC-MS analysis the supernatants were filtered
through 0.7 µm glass fiber filters (OPTI – Flow®, WICOM, Heppenheim, Germany), diluted
1:1 (v/v) with distilled water and filtered a second time through 0.2 µm nylon filters (OPTI –
Flow®, WICOM, Heppenheim, Germany) directly in UHPLC vials. All extractions were
done in duplicate and analyzed twice.
Polyphenol analysis by UHPLC-MS
Immediately after extraction, UHPLC-MS analysis was carried out as described earlier
(CEYMANN et al. 2011) by using an ACQUITY Ultra Performance LC™ system (UPLCTM)
with binary solvent manager and single quadrupole micromass ZQ Mass Detector (Waters
Corporation, Milford, USA) equipped with an electrospray ionization (ESI) source operating
in negative mode. For instrument control, data acquisition and processing MassLynx™
software (Version 4.1) was used.
A reversed phase column (BEH C18 1.7 µm, 2.1 mm x 50 mm with a BEH C18 1.7 µm
VanGuardTM Pre-Column, 2.1 mm x 5 mm, Waters Corporation, Milford, USA) at 40 °C was
used for separation of individual polyphenols. Elution was completed in 10 min with a
sequence of linear water-methanol gradient (CEYMANN et al. 2011) and a flow rate of
0.3 mL/min. The software QuantLynx™ (Waters Corporation, Milford, USA, Version 4.1)
was used for integration and calculations based on external standards. Samples were analyzed
130
PAPER 2
SCREENING OF APPLES
twice and mean values were used for calculation.
TPC by Folin-Ciocalteu
The analysis of TPC was carried out as described earlier (CEYMANN et al. 2011). Ten
microliters of Folin-Ciocalteu reagent and 100 µL of distilled water were automatically
pipetted to 10 µL of methanolic extract in a 300 µL cuvette. After 1 min, 40 µL sodium
carbonate solution (200 g/L) and 40 µL distilled water were added, thoroughly mixed and
incubated for 30 min at 37 °C. Absorption was measured automatically at 700 nm. Total
polyphenol content was calculated by means of an external standard calibration with
methanolic catechin standards and expressed as mg catechin equivalents/100 g FM.
Antioxidative potential by TEAC
TEAC was measured by using a Konelab ARENA 20XT analyzer (Thermo Fisher Scientific
OY, Vantaa, Finland) with a method adapted from RE et al. (1999). To measure TEAC,
200 µL of a mixture of ABTS (7 mM) and potassium persulfate (2.45 mM) in a saline
phosphate buffer (prepared the day before, pH 6.7), 4 µL of extract and 4 µL of distilled water
were pipetted into a 300 µL cuvette, mixed and incubated for 15 min at 37 °C. The
absorbance at 700 nm was recorded automatically. Quantification was achieved with an
external Trolox® standard calibration and results were expressed as mg Trolox® equivalents
(TE)/100 g FM.
Antioxidative potential by FRAP
FRAP was measured by using a Konelab ARENA 20XT analyzer (Thermo Fisher Scientific
OY, Vantaa, Finland) with methods adapted from TSAO et al. (2005). The FRAP reagent was
prepared by mixing 50 mL acetate buffer (300 mM, pH 3.6), 5 mL of a solution of 10 mM
2,4,6-tripyridyl-s-triazine (TPTZ) in 40 mM HCl, and 5 mL of 20 mM ferric (III) chloride
solution. In a 300 µL cuvette, 150 µL of the FRAP reagent, 30 µL of distilled water and
10 µL of extract were mixed and incubated for 4 min at 37 °C until measurement of
absorbance at 600 nm. An external catechin standard calibration was used for quantification
and results expressed as mg CE/100 g FM.
131
PAPER 2
SCREENING OF APPLES
Results and discussion
Cultivar screening
The content of the 12 individual polyphenols in 104 dessert and cider apple cultivars are given
in Table 1. The polyphenol levels varied from below the limit of detection to
70 mg/100 g FM. With respect to the flavan-3-ols (i.e. mono- and dimeric flavan-3-ols), the
ranges for catechin and procyanidin B1 in the different cultivars varied from below the limit
of detection to 11 mg/100 g FM, whereas epicatechin and procyanidin B2 were present in
concentrations from 0.5 to 34 mg/100 g FM. These ranges were large with the highest
catechin concentration being 49 times greater than the lowest. The corresponding values for
the magnitude of the differences in epicatechin, procyanidin B1 and B2 concentrations were
66, 55 and 51, respectively. High amounts of epicatechin correlated well with high amounts of
procyanidin B2 which can be explained by procyanidin B2 being composed of two molecules
epicatechin in contrast to procyanidin B1, which contains one molecule of both catechin and
epicatechin.
With respect to phenolic acids, the levels of chlorogenic acid and coumaroylquinic acid
ranged from 1.4 to 70 mg/100 g FM and up to 18 mg/100 g FM, respectively. As for the
flavan-3-ols, the range of values was large with the highest chlorogenic acid concentration
being 49-fold the lowest, whereas the highest coumaroylquinic acid value was 92 times higher
than the limit of detection. Slightly lower ranges were found for dihydrochalcones. Phloridzin
varied from 0.4 to 13 mg/100 g FM (34-fold) and phloretin-xyloglucoside from 0.7 to
20 mg/100 g FM (27-fold). The polyphenol group showing the smallest variation in
concentrations were the flavonols (quercetin-galactoside, -glucoside, -rhamnoside and rutin).
The quercetin-glycosides concentrations varied between 0.4 and 4.3 mg/100 g FM (11-fold),
the quercetin-rhamnoside from 0.4 to 5 mg/100 g FM (12-fold) and rutin concentration varied
7-fold from the limit of detection to 1.4 mg/100 g FM. In absolute terms, the amount of both
flavan-3-ols and phenolic acids in apples was always higher than that of dihydrochalcones and
flavonols.
A comparison of the data given in Table 1 with that given by the phenol explorer (NEVEU et
al. 2010) showed good agreement for the mean content and range of the different individual
polyphenols in apples, except for the procyanidin B2 value (14.56 mg/100 g FM) (NEVEU et
al. 2010). However, the range of 0.90 - 38.46 mg/100 g FM (NEVEU et al. 2010) is very
similar to our data. This difference is presumably due to the mean values of the phenol
explorer being based on only 23 different cultivars compared to 104 cultivars in Table 1.
132
PAPER 2
SCREENING OF APPLES
Table 1:
Content of individual polyphenols [data given in mg/100 g edible FM] of different
apple cultivars analyzed by UHPLC -MS; C catechin, E epicatechin, PCB1 procyanidin
B1, PCB2 procyanidin B2, CA chlorogenic acid, CQA coumaroylquinic acid, P
phloridzin, PXG phloretin-xyloglucoside, QG quercetin-galactoside and -glucoside, R
rutin, QR quercetin-rhamnoside, total polyphenol content (TPC) in mg catechin
equivalents/100 g edible FM
Cultivar
Year
C
E
R
QR
TPC
ACW 11303a
ACW 11907a
ACW 13652a
ACW 14995a
ACW 6104a
ACW 6375a
Alter Engländerb
Ariane
Arlet
Beffertapfelb
Berlepschb
Bohnapfelb
Boskoop
Boskoop, Roter
Braeburn
Braeburn
Redfield
Braeburn
Rosabel®
Burgunderb
Caudle
civG198
Civni
Cox Cherry
Cripps Pink
Delblush
Discovery
Elstar
Elstar Reinhard
Empire
Engishoferb
Fiesta
Fuji
Fuji KIKU
Fubrax®
FxD 1005c
FxD 1020c
FxD 1033c
FxD 1068c
FxD 1118c
FxD 1119c
FxD 1120c
2010
2010
2008
2010
2009
2008
2008
2010
2009
2008
2009
2009
2010
2008
2010
1.50
1.10
0.65
0.80
0.56
1.16
8.00
0.58
0.80
2.98
0.27
1.31
0.64
1.28
0.30
14.15
7.12
17.26
7.14
4.56
5.63
20.19
5.65
5.23
9.56
1.35
16.75
6.74
8.40
4.10
1.55
2.04
0.93
1.97
1.50
1.85
9.61
2.38
2.27
8.41
1.06
3.86
2.31
3.48
0.99
2.64 2.43 0.81
1.59 1.44 1.01
1.05 0.88 1.82
0.96 1.80 0.53
1.61 1.59 4.10
1.06 1.03 2.77
2.41 7.98 2.79
2.01 1.39 0.37
2.84 0.99 1.54
5.72 14.57 2.11
4.18 8.31 2.32
2.80 5.95 3.04
6.53 3.90 0.42
7.12 5.91 0.86
1.22 1.88 1.34
<lod
<lod
0.37
<lod
1.36
0.47
0.37
<lod
0.48
0.62
0.79
0.79
0.27
<lod
<lod
3.85
1.92
1.72
1.52
2.72
2.09
1.61
2.25
0.97
1.53
1.40
2.85
0.72
0.41
0.73
214
166
154
99
141
131
223
119
120
319
92
258
210
202
106
2010
0.86
6.15
0.84
0.87
1.58
0.79
<lod
0.49
115
2010
0.54
5.27
6.84
0.78
1.42
1.94
1.61
0.25
0.80
104
2008
2010
2010
2010
2010
2009
2009
2009
2009
2010
2008
2008
2009
2010
8.26 19.77 11.03 20.47 20.46
0.48 6.73 1.36 7.29 16.49
2.59 6.98 2.79 4.81 7.22
1.25 7.36 2.47 5.92 11.31
0.91 8.52 1.76 8.79 5.93
0.79 5.69 1.64 7.61 15.76
0.80 5.51 2.15 6.93 11.17
3.39 9.22 3.39 8.66 44.00
0.44 4.60 1.27 5.05 4.05
1.10 8.72 2.87 9.41 6.96
0.50 1.45 0.67 1.83 11.49
1.26 14.36 4.66 24.84 11.71
0.41 4.43 1.47 4.75 8.43
1.33 9.66 1.89 7.20 18.74
9.88
1.39
<lod
0.62
0.60
0.57
0.34
3.68
0.81
1.30
0.72
10.26
0.43
1.16
7.45 11.92 2.52
1.75 1.88 1.28
1.12 1.09 1.58
1.09 1.43 1.31
0.78 1.98 1.54
1.45 2.50 2.26
1.91 3.92 2.39
1.47 2.13 0.56
1.65 1.37 1.06
1.78 1.81 1.30
1.63 1.01 1.91
5.19 4.22 2.11
0.88 1.36 0.73
2.42 1.92 1.63
0.53
0.43
0.41
0.33
<lod
0.55
0.45
<lod
0.47
0.47
0.44
0.66
<lod
1.05
1.26
2.76
2.19
0.94
1.13
1.98
1.75
0.54
0.61
1.13
1.21
1.07
0.53
1.90
379
126
134
102
146
128
111
171
90
115
75
335
62
167
2010
1.33
2.62
2008
2008
2008
2008
2008
2008
2008
3.80 15.08 5.92 14.93 37.13 0.85
1.06 3.77 1.81 4.02 19.85 0.27
5.03 12.66 8.06 13.42 18.24 0.75
<lod 0.83 0.37 1.14 19.89 <lod
<lod 0.51 <lod 0.64 17.02 <lod
0.42 2.05 0.73 2.29 22.34 0.58
2.55 12.25 5.11 15.41 32.17 1.29
8.18
PCB1 PCB2
CA
CQA
10.62
6.86
17.06
6.24
6.27
7.24
21.94
10.04
7.09
13.52
2.39
18.61
13.18
13.47
3.38
23.19
16.46
7.21
3.35
12.62
19.74
15.39
16.36
13.77
46.93
25.05
26.04
25.63
27.05
4.54
0.65
1.14
0.59
<lod
1.74
1.85
12.02
2.25
2.36
3.48
0.59
1.19
1.54
2.17
0.92
1.38
5.53
7.76
1.37
4.95
2.41
7.13 17.31 0.77
P
PXG
QG
1.87
1.23
0.37
1.07
148
2.20 5.20
1.94 2.52
3.74 3.85
1.62 4.00
0.92 1.76
3.27 5.28
5.71 12.15
0.65
1.11
1.22
1.71
0.77
1.03
1.33
<lod
<lod
<lod
<lod
<lod
<lod
<lod
0.53
0.74
0.62
0.73
0.48
0.58
0.72
164
113
169
84
53
61
150
133
PAPER 2
Table 1:
SCREENING OF APPLES
(continued)
Cultivar
Year
C
E
FxD 1121c
FxD 1143c
FxD 1171c
FxD 1180c
FxD 1184c
FxD 1188c
FxD 1206c
FxD 1252c
FxD 1253c
FxD 1299c
FxD 1360c
FxD 1371c
FxD 1378c
Gala
Gala Galaxy
Gala Schniga
Galiwa
Galmac
Golden
Delicious
Golden
Delicious B
Golden Orange
Goldparmäne
Goldrush
Grauer
Hordapfelb
Gravensteiner
Grüner
Fürstenapfelb
Hessenreuther
Blauacherb
Idared
Iduna
Jonagold
Jonagold
Jonagored
Jonagold
Jonagored 9613
Jonagold T 1270
Jonagold T 1272
Jonagold van de
Poel
Jonamac
Jonathan
Jonared
Kanada Reinette
Katzengrindlerb
2008
2008
2008
2008
2008
2008
2008
2008
2008
2008
2008
2008
2008
2010
2010
2010
2010
2010
<lod
0.21
3.40
<lod
<lod
<lod
<lod
2.47
1.06
1.96
2.32
5.32
3.84
0.68
0.32
0.94
1.14
1.27
0.84
1.49
14.47
0.94
0.88
0.77
0.65
11.36
10.02
7.42
11.49
12.47
13.08
5.87
3.65
7.70
11.85
6.46
2010
0.31
2009
PCB1 PCB2
CA
P
PXG
QG
R
QR
TPC
0.21 0.99 22.72 0.31
0.88 2.62 19.91 0.41
6.48 13.81 18.05 0.40
0.46 1.22 23.64 0.22
0.44 1.14 21.36 0.42
0.29 1.13 33.35 <lod
0.28 1.14 42.86 0.73
2.93 7.53 37.17 1.10
2.07 9.65 26.07 0.20
3.85 8.49 17.97 <lod
2.51 9.52 26.36 0.61
7.29 18.10 52.37 2.53
6.19 18.30 35.35 1.60
1.68 6.48 13.40 1.39
1.03 2.57 6.04 0.47
2.13 6.56 14.80 1.15
1.81 6.37 5.78 0.70
2.16 6.83 25.67 4.78
2.35
2.94
1.19
3.08
1.17
1.29
2.39
2.48
3.06
1.41
2.51
2.35
2.29
1.30
0.90
0.81
1.00
1.62
2.18
4.84
2.68
4.55
3.24
3.88
2.87
4.91
6.44
3.41
2.84
4.35
4.02
3.74
1.96
2.51
2.09
2.51
0.53
1.98
1.29
0.65
0.40
1.34
0.76
0.69
1.81
1.53
1.32
1.36
0.40
1.74
0.69
2.08
1.18
2.08
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
<lod
0.31
0.27
1.00
0.51
1.05
0.64
0.53
0.46
0.63
0.46
0.67
0.74
0.90
0.59
1.10
0.43
1.22
0.75
1.38
0.81
1.32
61
78
158
62
57
69
68
162
150
91
183
241
193
137
102
172
166
252
3.77
1.65
8.29 10.97 1.16
2.10
1.36
0.42
0.25
1.43
102
0.31
6.10
1.22
7.92 10.78 1.89
1.91
1.90
1.36
<lod
1.48
129
2009
2009
2009
<lod
1.65
0.59
0.89
4.70
6.70
0.34
3.68
2.07
1.42 9.79 <lod
5.93 14.27 1.21
7.85 10.95 0.30
2.30
6.82
1.80
6.27
8.67
3.56
2.39
2.59
2.05
0.48
<lod
<lod
2.83
0.56
2.00
82
159
141
2009
0.45
5.57
1.42
8.30 42.59 7.59
6.13
3.57
1.45
<lod
1.68
237
2009
2.37
5.18
3.82
6.17 16.23 1.41
2.56
3.42
1.37
0.54
2.40
143
2008
8.25 21.98 9.14 15.69 70.02 18.36 12.77 12.34 0.90
0.21
0.50
307
2008
1.39
6.79
2.81
8.56 21.54 3.07
1.51
3.16
0.94
0.27
0.78
175
2008
2010
2008
2.38
0.35
0.70
6.39
5.06
3.83
3.56
0.87
0.83
6.29 16.21 <lod
5.94 9.22 0.42
4.85 9.42 0.87
2.14
1.79
1.02
0.90
2.63
1.56
4.00
0.66
2.08
<lod
<lod
0.36
1.14
0.74
2.27
136
94
135
2010
0.30
5.78
0.87
8.01 14.08 0.90
1.55
2.71
0.74
<lod
2.96
151
2010
0.39
6.06
0.90
7.59 14.60 0.83
1.30
2.36
1.03
<lod
2.99
179
2010
2010
0.44
0.29
6.69
5.32
1.01
0.71
9.06 19.22 1.09
7.47 14.38 0.77
2.44
1.65
3.28
2.41
1.67
1.37
0.20
<lod
4.08
3.21
216
168
2010
0.23
4.65
0.68
6.69 11.20 0.68
1.39
2.56
0.77
<lod
2.43
161
2009
2009
2010
2008
2008
0.91 4.85 2.10 5.60
1.20 8.74 2.55 10.92
1.71 10.58 1.92 9.13
2.02 12.68 4.80 16.32
2.99 14.56 6.33 20.22
1.53
3.11
1.55
4.70
4.70
1.67
6.26
2.73
8.02
9.80
1.08
0.83
1.11
1.22
1.56
0.47
<lod
<lod
0.50
0.52
0.59
1.44
1.88
0.78
1.69
114
143
148
228
218
3.39
6.27
7.35
27.11
9.98
CQA
1.50
0.90
0.70
2.20
3.52
134
PAPER 2
Table 1:
Cultivar
SCREENING OF APPLES
(continued)
Year
C
E
PCB1 PCB2
CA
CQA
P
PXG
QG
R
QR
TPC
Kidds Orange
2008 2.50 6.73 5.22 6.95 6.75 3.03 1.38 2.59
La Flamboyante 2010 0.57 4.81 1.38 5.41 15.75 1.28 0.95 3.88
Maigold
2008 0.86 4.42 1.32 6.07 27.30 3.41 1.70 5.12
McIntosh
2009 2.32 5.60 3.39 6.40 15.53 4.05 1.82 1.52
Milwa
2010 0.65 8.44 1.34 6.49 16.23 1.57 1.49 2.51
Nevson
2009 0.69 5.10 1.80 5.12 14.47 0.93 1.16 2.49
Nicogreen
2010 0.90 8.66 1.29 6.01 1.44 <lod 0.41 1.90
Nicoter
2010 0.47 3.82 1.19 4.86 4.56 <lod 0.38 1.50
Opal
2010 1.66 9.97 2.49 8.80 4.18 0.98 1.89 0.74
Otava
2010 0.99 8.78 2.96 12.95 9.38 1.34 2.20 1.15
Pinova
2009 0.27 5.95 1.43 9.43 12.41 0.20 2.09 1.98
PRI 159d
2010 0.23 4.14 0.72 4.07 8.07 0.21 0.70 1.27
Red Deliciouse 2008 1.46 7.01 2.92 7.59 7.51 1.29 3.50 1.51
Redfieldb
2008 9.76 33.92 9.01 32.43 20.32 8.97 2.38 11.73
b
Rewena
2010 1.11 9.51 1.68 9.49 15.28 0.31 0.96 1.43
Rubinola
2010 1.01 8.37 2.45 9.79 22.76 0.49 2.10 2.30
Santana
2010 <lod 0.80 <lod 0.82 11.38 1.38 2.09 1.86
Schneiderapfel
2009 4.85 11.55 6.12 14.43 11.03 12.41 2.63 7.77
Scifresh
2009 0.37 3.92 0.86 5.56 22.91 1.01 0.96 2.14
Shalimar
2010 1.63 8.73 2.80 7.01 3.47 <lod 1.64 1.90
Spartan
2009 1.77 8.04 3.38 8.61 13.55 1.26 3.15 1.16
b
Surschibech
2008 2.35 14.53 3.47 17.99 24.56 7.78 5.17 19.81
Tobiässlerb
2008 3.34 10.13 6.56 13.82 14.43 6.66 1.90 2.21
Topaz
2010 1.07 9.89 2.55 10.66 10.49 <lod 0.74 1.07
b
Wilerrot
2009 1.28 7.12 2.72 9.42 10.87 4.98 4.60 4.32
Meanf
1.66 7.72 2.71 8.58 17.44 2.18 2.38 3.63
Minimum
<lod 0.51 <lod 0.64 1.44 <lod 0.38 0.74
Maximum
9.76 33.92 11.03 32.43 70.02 18.36 12.77 19.81
<lod: below limit of detection (<0.2 mg/100 g)
a
New cultivar, crossed at ACW Wädenswil.
b
Cider apples.
c
Progeny of the cross breeding of Fiesta and Discovery.
All cultivars were grown in Switzerland except d in The Netherlands, e in Italy.
f
Mean values of n=104 cultivars.
1.77
1.04
1.81
1.26
1.08
1.73
0.80
1.14
0.98
1.04
4.28
0.47
2.16
2.33
0.79
1.07
0.75
1.19
2.81
1.15
1.33
2.40
1.52
1.51
0.90
1.45
0.37
4.28
<lod
<lod
0.27
0.47
0.26
0.62
<lod
<lod
<lod
0.68
0.65
<lod
0.31
0.42
0.31
0.34
0.36
<lod
0.68
0.20
0.51
0.36
0.63
0.35
<lod
0.48
<lod
1.36
0.66
0.63
1.19
1.81
0.48
0.78
1.17
0.87
0.93
4.46
1.83
0.67
1.01
1.04
3.28
5.04
1.35
1.18
1.25
4.81
0.92
2.37
1.01
4.54
0.74
1.45
0.41
5.04
110
132
143
130
124
114
137
93
108
159
147
79
133
337
156
166
52
208
123
161
149
232
276
141
160
150
52
379
Table 2 gives a summary of literature data for polyphenols determined in selected apple
cultivars. These values are in reasonable agreement with our values for the corresponding
cultivars as shown in Table 1, except for quercetin-galactoside/-glucoside values of Golden
Delicious, which are much lower. Additionally, PODSEDEK et al. (2000) reported less flavan3-ols and VALAVANIDIS et al. (2009) more flavan-3-ols than found in our cultivars (Table 1).
Differences in extraction procedures or quantification methods could explain the different
results for the same cultivar as could influences of pre- and post-harvest factors (AWAD et al.
2000; LAMPERI et al. 2008; MCGHIE et al. 2005; VALAVANIDIS et al. 2009). To our
135
PAPER 2
SCREENING OF APPLES
knowledge, these are the first polyphenol profile measurements for more than half of the
cultivars included in our study.
Table 2:
Cultivar
Content of individual polyphenols [data given in mg/100 g edible FM] of selected apple
cultivars analyzed by different authors; C catechin, E epicatechin, PCB1 procyanidin
B1, PCB2 procyanidin B2, CA chlorogenic acid, CQA coumaroylquinic acid, P
phloridzin, PXG phloretin-xyloglucoside, QG quercetin-galactoside/-glucoside, R rutin,
QR quercetin-rhamnoside
Reference
C
E
Boskoop (PODSEDEK et al. 2000)
1.30
8.90
1.60 25.00
Braeburn (VRHOVSEK et al. 2004)
0.45
5.40
5.77
4.48
Elstar
(ARTS et al. 2000a)
1.24
8.17
(PODSEDEK et al. 2000)
0.40
4.10
1.00
3.10
2.28
Empire
(LEE et al. 2003)
Fuji
(VRHOVSEK et al. 2004)
Golden (LEE et al. 2003)
Delicious (VRHOVSEK et al. 2004)
Idared
CQA
P
PXG
QG
R
QR
1.67
1.01
1.04
4.86
0.22
0.78
3.44 11.52
2.80
1.72
6.60
6.11
6.50 12.34 0.76
1.31
0.68
2.26
7.12
6.28
8.48
1.80
1.92
6.60
6.76
9.18
1.49
1.26
3.55
6.40
8.30
0.47
5.18
(ARTS et al. 2000a)
0.53
7.42
(VALAVANIDIS et al.
2009)
(PODSEDEK et al. 2000)
0.80
9.20
0.60
3.90
0.40
6.71
(PODSEDEK et al. 2000)
0.80
6.20
(VALAVANIDIS et al.
2009)
(VRHOVSEK et al. 2004)
2.10
7.80
(VALAVANIDIS et al.
2009)
Jonagold (ARTS et al. 2000a)
Red
Delicious
0.82
PCB1 PCB2 CA
1.50
1.35
4.30
2.80
0.60
1.20
3.84
0.25
0.54
3.84
0.17
1.39
0.19
0.59
1.10 12.60
2.60 12.00
0.40
6.00
3.80
2.37 10.55
7.91
7.09
3.10 14.80 5.60
9.70 11.20
2.46
3.23
9.60
0.85
2.64
3.20
Correlation of different methods
In addition to the individual polyphenol data presented in Table 1, the 104 cultivars were
screened for TPC (Folin-Ciocalteu), and antioxidant potential (TEAC, FRAP) so as to
evaluate the usefulness of these simple colorimetric methods as predictors of possible health
benefits. Table 1 contains also the TPC values measured for all cultivars screened. The
correlation between the sum of individual polyphenols as determined by UHPLC-MS and
TPC was reasonably good (0.61), but the TPC values were higher than the sum of individual
polyphenols. This difference of 60 % is due to precipitation and filtration losses in UHPLCMS sample preparation of mainly oligomeric procyanidins in contrast to crude extracts for the
colorimetric assays as shown by CEYMANN et al. (2011). The TPC values in Table 1 range
between 52 mg and 379 mg CE/100 g FM which is consistent with the ranges given in the
136
PAPER 2
SCREENING OF APPLES
phenol explorer (NEVEU et al. 2010). They are also in accordance with other literature values
(IMEH et al. 2002; LAMPERI et al. 2008; VRHOVSEK et al. 2004) even though comparison with
other investigations is difficult, because of the use of different standards (for example
quercetin (MARI et al. 2010) or gallic acid (VALAVANIDIS et al. 2009)). While the TPC
method can adequately differentiate between apple varieties that are high and low in
polyphenols, it is less useful as a predictor of potentially positive health effects. This is
because the TPC measurement includes the non-absorbable polymeric polyphenols as well as
the smaller, potentially absorbable polyphenolic compounds which are thought to be mainly
responsible for the observed physiological effects. Although some degradation products of the
polymeric polyphenols are absorbed in the colon, it is still not clear whether they would have
physiological effects beneficial to health.
Figure 1:
Correlations (p<0.0001) between sum of individual polyphenols determined by
UHPLC-MS and antioxidant potential of 104 apple cultivar, (A) determined by TEACassay, (B) determined by FRAP-assay
TEAC and FRAP are prescreening assays widely used in literature to determine antioxidative
capacity. The usefulness of these assays in predicting health benefits in humans is limited
(HERMANS et al. 2007; HERRERA et al. 2009; HOLST et al. 2008; KROON et al. 2004; SIES
2007), because the antioxidant properties measured in vitro are greatly changed in the human
body after absorption (FERNANDEZ-PANCHON et al. 2008). Bioavailability, metabolic
137
PAPER 2
SCREENING OF APPLES
transformations and interactions change the antioxidant capacity in vivo (FERNANDEZPANCHON et al. 2008; SCALBERT et al. 2005), and extrapolation from in vitro to in vivo is not
possible.
Although the antioxidant capacity as measured by TEAC and FRAP is not useful to predict
health benefits, it may still be useful as a screening method to predict total polyphenol
content. The correlation coefficient between TEAC and FRAP with UHPLC-MS data was
approximately 0.6 (Figure 1), which is acceptable for a prescreening method. The TEAC and
FRAP range from 24 mg to 919 mg TE/100 g FM and 42 mg to 429 mg CE/100 g FM,
respectively. The discrepancy between the absolute values of TEAC and FRAP depends on
the standards used and the reaction mechanism. This has been discussed elsewhere (WOJDYLO
et al. 2008). A direct comparison of the TEAC and FRAP values presented in Figure 1 with
those from the literature data is thus difficult. Although the individual polyphenol standards
showed somewhat different responses in the photometric tests, all individual polyphenols and
polyphenol groups resulted in comparable correlations with TPC, TEAC and FRAP (data not
shown). Nevertheless, the many different apple cultivars tested represent a wide range of
polyphenol contents, whose magnitude was detected by the more simple methods such as
Folin-Ciocalteu, TEAC and FRAP. Whether the magnitude of these values has any relevance
to the health properties of apples must then be tested by measuring the individual small
molecular weight polyphenols.
Classification of apples based on their polyphenol profile
The analysis of individual polyphenols using UHPLC-MS provides information on potential
health benefits. There are large differences between apple cultivars with regard to polyphenol
content and profile. Two major classes of polyphenols, the flavan-3-ols and the phenolic
acids, dominate the apple polyphenol profile and by calculating the flavan-3-ol to phenolic
acid ratio, apple cultivars can be classified into flavan-3-ol rich or phenolic acid rich. Phenolic
acid dominated apples (Figure 2, left) are rich in chlorogenic acid and coumaroylquinic acid
with a flavan-3-ol to phenolic acid ratio of 0.1 - 0.9. Santana with a ratio of 0.1 and Jonagold
with a ratio of 0.8 belong to this group. In contrast, flavan-3-ol dominated apples are rich in
catechin, epicatechin and both procyanidin B1 and B2 with ratios of 1.6 - 11.7 (Figure 2,
right). Two cultivars from this group are Topaz and Nicogreen with flavan-3-ol to phenolic
acid ratios of 2.3 and 11.7, respectively. Cultivars listed in Table 1 with similar contents of
flavan-3-ol and phenolic acids are not shown in Figure 2. The often consumed Gala and
Golden Delicious belong to this group. This classification based on the polyphenol pattern
138
PAPER 2
SCREENING OF APPLES
could be a tool to link potential beneficial health effects to specific apple cultivars. Some
cultivars contain similar or even higher amounts of chlorogenic acid (see Table 1) as brewed
coffee (50 mg/100 mL) (NARDINI et al. 2002). Assuming an apple serving size of 150 g and,
taking 150 mL for a cup of coffee (NEVEU et al. 2010), the chlorogenic acid intake from one
serving of Grüner Fürstenapfel, FxD 1371 and Beffertapfel would be at least as high and
maybe higher than a cup of brewed coffee (NARDINI et al. 2002) (70-105 mg/serving). Eight
of the cultivars (Table 1) contained 48-66 mg chlorogenic acid in one serving which is about
two thirds of the chlorogenic acid content of a cup of brewed coffee, whereas half of the apple
cultivars of Table 1 contain at least one third as much chlorogenic acid as a cup of coffee.
Chlorogenic acid in coffee has been reported to have beneficial health effects in relation to
cardiovascular disease (BONITA et al. 2007) and to decrease the risk of type II diabetes (ITO et
al. 2005). The mechanism is unclear but both caffeinated and decaffeinated coffee delay
glucose absorption in comparison to the placebo glucose in water possibly via chlorogenic
acid-mediated Na+-electrochemical gradient dissipation (JOHNSTON et al. 2003). Other
possible mechanisms, including the inhibition or retardation of the action of α-glucosidase,
have been reviewed (BIDEL et al. 2008). In the same way, the amounts of epicatechin in
Tobiässler, Schneiderapfel, Engishofer and Burgunder cultivars (10-20 mg/100 g FM,
Table 1) are as high as those reported for milk chocolate (12.5 mg/100 g) (ARTS et al. 2000b)
and the epicatechin content of the red-fleshed cultivar Redfield (34 mg/100 g FM) was as high
as in dark chocolate (32.7 mg/100 g) (ARTS et al. 2000b). In fact the amount of epicatechin
ingested with one 150 g serving of the red-fleshed cultivar Redfield would be almost twice the
amount contained in a 20 g portion of dark chocolate (NEVEU et al. 2010). More than one
third of the cultivars listed in Table 1 would provide as much as or more epicatechin per 150 g
apple than in a 20 g bar of dark chocolate. Chocolate is associated with reduced inflammation
and reduced heart disease risk (COOPER et al. 2008) and the acute ingestion of dark chocolate
(21.5 mg epicatechin per serving) significantly improved endothelial function and lowered
blood pressure in overweight adults (FARIDI et al. 2008) by elevating the plasma epicatechin
level (ENGLER et al. 2004b). In addition, the daily consumption of dark chocolate containing
30 mg cocoa flavanols over 18 weeks in pre-hypertensive elderly adults increased the
formation of vasodilatory nitric oxide, which resulted in reduced blood pressure (TAUBERT et
al. 2007a). Whether similar effects can be observed after apple consumption, remains to be
elucidated. The polyphenol profile of apples with flavan-3-ols, phenolic acids,
dihydrochalcones and flavonols however is more diverse than that of coffee which contains
different phenolic acids (NEVEU et al. 2010) or chocolate which contains mainly flavan-3-ols
139
PAPER 2
SCREENING OF APPLES
of different chain length, quercetin and ferulic acid (NEVEU et al. 2010). A phenolic acid
dominated apple of 150 g has equivalent concentrations of polyphenols as a combination of a
cup of coffee and 20 g of chocolate.
The matrix of an apple is also different from that of roasted and ground coffee or the
fermented, toasted and ground chocolate. While the processing might improve the
bioavailability of the coffee and cocoa polyphenols, the poorer release of polyphenols from
the cellular structure of raw apples during digestion may lead to lower absorption. However,
the many different apple cultivars with a wide range of polyphenol contents and profiles
provide a large assortment for human studies. A recently published study (AUCLAIR et al.
2010) however with two apple cultivars with different amounts of polyphenols failed to show
an influence on endothelial function in humans. The polyphenol profiles from different apple
cultivars evaluated in the present study could help in selecting cultivars with different
polyphenol patterns for future studies designed to measure the physiological effects of flavan3-ols or phenolic acid rich apples.
Figure 2:
Classification of apple cultivars based on their polyphenol profile (flavan-3-ols: sum of
catechin, epicatechin, procyanidin B1 and B2; phenolic acids: sum of chlorogenic acid
and coumaroylquinic acid; dihydrochalcones: sum of phloridzin and phloretinxyloglucoside; flavonols: quercetin- galactoside, -glucoside, -rhamnoside and rutin)
140
PAPER 2
SCREENING OF APPLES
Conclusions
Apple cultivars grown in Switzerland vary widely in the content of monomeric and dimeric
polyphenols which are potentially absorbable and may have physiological activity which
promotes health. Nevertheless, much still remains to be discovered in relation to the
absorption, metabolism and physiological action of these individual polyphenols. Two main
classes of apples were identified based on to their polyphenol profile: those rich in flavan-3ols and those rich in phenolic acids. Epicatechin and chlorogenic acid have been reported to
be beneficial to cardiovascular health, when consumed in cocoa and coffee, respectively, and
levels of these polyphenolic compounds in some apple cultivars are predictive of similar
health benefits. The next step is to confirm the absorption of physiologically active
polyphenolic compounds from apples and demonstrate a beneficial health effect in humans.
Acknowledgment
The authors thank Melanie Erzinger, Jennifer Kläui, Martin Heiri, Dominik Friedmann, Viola
Cassina, Marion Scheidegger and Dominic Ritler for their support during sample preparation
and analysis and Reto Leumann, Otto Läubli, Dr. Ton den Nijs and Dr. Irene Höller for apple
supply. Thanks are given to Dr. Markus Kellerhals and Simon Egger for scientific discussion.
141
PAPER 2
SCREENING OF APPLES
References
Alonso-Salces, R. M., A. Barranco, et al. (2004a). "Polyphenolic profiles of Basque cider apple
cultivars and their technological properties." Journal of Agricultural and Food Chemistry 52:
2938-2952.
Arts, I. C. W., B. van de Putte, et al. (2000a). "Catechin contents of foods commonly consumed in The
Netherlands. 1. Fruits, vegetables, staple foods, and processed foods." Journal of Agricultural
and Food Chemistry 48(5): 1746-1751.
Arts, I. C. W., B. van de Putte, et al. (2000b). "Catechin contents of foods commonly consumed in The
Netherlands. 2. Tea, wine, fruit juices, and chocolate milk." Journal of Agricultural and Food
Chemistry 48: 1752-1757.
Auclair, S., G. Chironi, et al. (2010). "The regular consumption of a polyphenol-rich apple does not
influence endothelial function: a randomised double-blind trial in hypercholesterolemic
adults." European Journal of Clinical Nutrition 64(10): 1158-1165.
Awad, M. A., A. Jager de, et al. (2000). "Flavanoid and chlorogenic acid levels in apple fruit:
characterisation of variation." Scientia Horticulturae 83: 249-263.
Bidel, S., G. Hu, et al. (2008). "Coffee consumption and type 2 diabetes - An extensive review."
Central European Journal of Medicine 3(1): 9-19.
Bonita, J. S., M. Mandarano, et al. (2007). "Coffee and cardiovascular disease: In vitro, cellular,
animal, and human studies." Pharmacological Research 55: 187-198.
Ceymann, M., E. Arrigoni, et al. (2011). "Rapid high performance screening method using UHPLCMS to quantify 12 polyphenol compounds in fresh apples." Analytical Methods 3: 1774.
Chinnici, F., A. Bendini, et al. (2004a). "Radical scavenging activities of peels and pulps from cv.
golden delicious apples as related to their phenolic composition." Journal of Agricultural and
Food Chemistry 52(15): 4684-4689.
Cooper, K. A., J. L. Donovan, et al. (2008). "Cocoa and health: a decade of research." British Journal
of Nutrition 99(01): 1-11.
D'Abrosca, B., S. Pacifico, et al. (2007). "`Limoncella' apple, an Italian apple cultivar: Phenolic and
flavonoid contents and antioxidant activity." Food Chemistry 104: 1333-1337.
Deprez, S., C. Brezillon, et al. (2000). "Polymeric proanthocyanidins are catabolized by human
colonic microflora into low-molecular-weight phenolic acids." Journal of Nutrition 130(11):
2733-2738.
Dragovic-Uzelac, V., J. Pospisil, et al. (2005). "The study of phenolic profiles of raw apricots and
apples and their purees by HPLC for the evaluation of apricot nectars and jams authenticity."
Food Chemistry 91: 373-383.
Engler, M. B., M. M. Engler, et al. (2004b). "Flavonoid-rich dark chocolate improves endothelial
function and increases plasma epicatechin concentrations in healthy adults." Journal of the
American College of Nutrition 23(3): 197-204.
Faridi, Z., V. Y. Njike, et al. (2008). "Acute dark chocolate and cocoa ingestion and endothelial
function: a randomized controlled crossover trial." American Journal of Clinical Nutrition
88(1): 58-63.
Fernandez-Panchon, M. S., D. Villano, et al. (2008). "Antioxidant activity of phenolic compounds:
From in vitro results to in vivo evidence." Critical Reviews in Food Science and Nutrition
48(7): 649 - 671.
Harker, F. R., F. A. Gunson, et al. (2003). "The case for fruit quality: an interpretive review of
consumer attitudes, and preferences for apples." Postharvest Biology and Technology 28(3):
333-347.
Hecke, K., K. Herbinger, et al. (2006). "Sugar-, acid- and phenol contents in apple cultivars from
organic and integrated fruit cultivation." European Journal of Clinical Nutrition 60: 11361140.
Hermans, N., P. Cos, et al. (2007). "Challenges and pitfalls in antioxidant research." Current
Medicinal Chemistry 14(4): 417-430.
Herrera, E., R. Jimenez, et al. (2009). "Aspects of antioxidant foods and supplements in health and
disease." Nutrition Reviews 67(5): S140-S144.
Holst, B. and G. Williamson (2008). "Nutrients and phytochemicals: from bioavailability to
bioefficacy beyond antioxidants." Current Opinion in Biotechnology 19(2): 73-82.
142
PAPER 2
SCREENING OF APPLES
Imeh, U. and S. Khokhar (2002). "Distribution of conjugated and free phenols in fruits: Antioxidant
activity and cultivar variations." Journal of Agricultural and Food Chemistry 50(22): 63016306.
Ito, H., M. Gonthier, et al. (2005). "Polyphenol levels in human urine after intake of six different
polyphenol-rich beverages." British Journal of Nutrition 94(4): 500-509.
Johnston, K. L., M. N. Clifford, et al. (2003). "Coffee acutely modifies gastrointestinal hormone
secretion and glucose tolerance in humans: glycemic effects of chlorogenic acid and caffeine."
American Journal of Clinical Nutrition 78(4): 728-733.
Kahle, K., M. Kraus, et al. (2005a). "Polyphenol profiles of apple juices." Molecular Nutrition and
Food Research 49: 797 – 806.
Kroon, P., M. Clifford, et al. (2004). "How should we assess the effects of exposure to dietary
polyphenols in vitro?" American Journal of Clinical Nutrition 80: 15-21.
Lamperi, L., U. Chiuminatto, et al. (2008). "Polyphenol levels and free radical scavenging activities of
four apple cultivars from integrated and organic farming in different Italian areas." Journal of
Agricultural and Food Chemistry 56(15): 6536-6546.
Lata, B., M. Przeradzka, et al. (2005). "Great differences in antioxidant properties exist between 56
apple cultivars and vegetation seasons." Journal of Agricultural and Food Chemistry 53:
8970-8978.
Lee, K. W., Y. J. Kim, et al. (2003). "Major phenolics in apple and their contribution to the total
antioxidant capacity." Journal of Agricultural and Food Chemistry 51: 6516-6520.
Manach, C., A. Scalbert, et al. (2004). "Polyphenols: food sources and bioavailability." American
Journal of Clinical Nutrition 79: 727-747.
Manach, C., G. Williamson, et al. (2005). "Bioavailability and bioefficacy of polyphenols in humans.
I. Review of 97 bioavailability studies." American Journal of Clinical Nutrition 81(suppl):
230S-242S.
Mangas, J. J., R. Rodriguez, et al. (1999). "Study of the phenolic profile of cider apple cultivars at
maturity by multivariate techniques." Journal of Agricultural and Food Chemistry 47: 40464052.
Mari, A., I. Tedesco, et al. (2010). "Phenolic compound characterisation and antiproliferative activity
of "Annurca" apple, a southern Italian cultivar." Food Chemistry 123: 157-164.
McGhie, T. K., M. Hunt, et al. (2005). "Cultivar and growing region determine the antioxidant
polyphenolic concentration and composition of apples grown in New Zealand." Journal of
Agricultural and Food Chemistry 53: 3065-3070.
Monagas, M., M. Urpi-Sarda, et al. (2010). "Insights into the metabolism and microbial
biotransformation of dietary flavan-3-ols and the bioactivity of their metabolites." Food and
Function 1(3): 233-253.
Nardini, M., E. Cirillo, et al. (2002). "Absorption of phenolic acids in humans after coffee
consumption." Journal of Agricultural and Food Chemistry 50: 5735-5741.
Neveu, V., J. Perez-Jiménez, et al. (2010). Phenol-Explorer: an online comprehensive database on
polyphenol contents in foods, online Database. Version 1.5.2, available at http://www.phenolexplorer.eu.
Oszmianski, J., M. Wolniak, et al. (2007). "Comparative study of polyphenolic content and antiradical
activity of cloudy and clear apple juices." Journal of the Science of Food and Agriculture 87:
573-579.
Oszmianski, J., M. Wolniak, et al. (2008). "Influence of apple purée preparation and storage on
polyphenol contents and antioxidant activity." Food Chemistry 107: 1473-1484.
Podsedek, A., J. Wilska-Jeszka, et al. (2000). "Compositional characterisation of some apple
varieties." European Food Research and Technology 210: 268-272.
Re, R., N. Pellegrini, et al. (1999). "Antioxidant activity applying an improved ABTS radical cation
decoloration assay." Free Radical Biology and Medicine 26(9/10): 1231–1237.
Scalbert, A., C. Manach, et al. (2005). "Dietary polyphenols and the prevention of diseases." Critical
Reviews in Food Science and Nutrition 45(4): 287-306.
Sies, H. (2007). "Total antioxidant capacity: Appraisal of a concept." Journal of Nutrition 137: 14931495.
143
PAPER 2
SCREENING OF APPLES
Sies, H. (2010). "Polyphenols and health: Update and perspectives." Archives of Biochemistry and
Biophysics 501(1): 2-5.
Taubert, D., R. Roesen, et al. (2007a). "Effects of low habitual cocoa intake on blood pressure and
bioactive nitric oxide - A randomized controlled trial." Journal of the American Medical
Association 298(1): 49-60.
Tsao, R., R. Yang, et al. (2005). "Which polyphenolic compounds contribute to the total antioxidant
activities of apple?" Journal of Agricultural and Food Chemistry 53(12): 4989-4995.
Tsao, R., R. Yang, et al. (2003). "Polyphenolic profiles in eight apple cultivars using highperformance liquid chromatography (HPLC)." Journal of Agricultural and Food Chemistry
51: 6347-6353.
Valavanidis, A., T. Vlachogianni, et al. (2009). "Polyphenolic profile and antioxidant activity of five
apple cultivars grown under organic and conventional agricultural practices." International
Journal of Food Science and Technology 44: 1167-1175.
van der Sluis, A. A., M. Dekker, et al. (2001). "Activity and concentration of polyphenolic
antioxidants in apple: Effect of cultivar, harvest year, and storage conditions." Journal of
Agricultural and Food Chemistry 49: 3606-3613.
Vrhovsek, U., A. Rigo, et al. (2004). "Quantitation of polyphenols in different apple varieties."
Journal of Agricultural and Food Chemistry 52(21): 6532-6538.
Wojdylo, A., J. Oszmianski, et al. (2008). "Polyphenolic compounds and antioxidant activity of new
and old apple varieties." Journal of Agricultural and Food Chemistry 56: 6520-6530.
Wu, J., H. Gao, et al. (2007). "Chemical compositional characterization of some apple cultivars." Food
Chemistry 103(1): 88-93.
144
PAPER 3
PRE-HARVEST FACTORS
PAPER 3
(in preparation)
Effects of pre-harvest factors on polyphenol content and profile in fresh apples
Maria Ceymanna, Eva Arrigonia*, Hans Schärera, Daniel Baumgartnera, Laura Nyströmb,
Richard F. Hurrellb
a
Research Station Agroscope Changins-Wädenswil ACW, Schloss, 8820 Wädenswil,
Switzerland
b
ETH Zürich, Institute of Food, Nutrition and Health, Schmelzbergstrasse 7, 8092 Zürich,
Switzerland
*
Corresponding author: Tel.: +41 44 783 6661; Fax: +41 44 783 6224.
E-mail address: [email protected]
145
PAPER 3
PRE-HARVEST FACTORS
Abstract
Apples contain polyphenol compounds with potential health benefits. Their level and profile
is dependent on the cultivar and to some extent on pre-harvest factors. This study investigated
the influence of the growing year, production method, mutant variety, growing environment,
rootstock and crop load on the polyphenol content and profile of different apple cultivars.
Compared to the major differences in polyphenol level and profile in different cultivars, preharvest factors had a relatively small influence on polyphenol content and no influence on
profile. Depending on the cultivar, the mutant variety and the growing year influenced
polyphenol levels to some extent but other pre-harvest factors evaluated had little or no effect.
We conclude that health promoting polyphenols can be best increased in the diet by carefully
selecting the apple cultivar and modestly by modifying other pre-harvest factors.
146
PAPER 3
PRE-HARVEST FACTORS
Introduction
In 2009, the global apple production was 71 Mio tons, which represented about 12 % of the
global fruit production; only banana production, at 97 Mio tons, was higher (FAOSTAT
2012b). Apple quality has been mainly associated with sensory characteristics such as texture,
taste and flavour (HARKER et al. 2003) and to a lesser extent with nutrition and health. In
relation to nutrition quality and composition, apples are low in energy, but nutrient-dense.
They contain mainly carbohydrates, with low amounts of fat and protein, and relatively high
amounts of minerals and vitamins. They also contain non-nutrients, especially polyphenol
compounds, and these secondary plant metabolites are reported to have a positive impact on
health.
The four polyphenol classes predominating in apples are flavan-3-ols, phenolic acids,
dihydrochalcones and flavonols. Flavan-3-ols and phenolic acids are physiologically active
after absorption and have been reported to have beneficial health effects. The flavan-3-ols
catechin and epicatechin, which occur at equally high levels in cocoa as in some apple
varieties, have been reported to improve the vascular endothelium, insulin resistance, satiety,
cognitive function and mood (KATZ et al. 2011). Chlorogenic acid, which is also a major
phenolic acid in coffee, as well as in some apple varieties, is reported to inhibit fat
accumulation and decrease body weight through its modulation of glucose metabolism (THOM
2007). Although the beneficial health effects of catechin, epicatechin and chlorogenic acid in
apples still remain to be confirmed in human intervention studies, there would appear to be a
great potential for apples to add to the health value of the daily diet. In order to exploit the
health value of apples, a detailed knowledge of their polyphenol content and profile is needed.
We have recently reported that the apple cultivar is a major determinant of both polyphenol
level and profile (CEYMANN et al. 2012), and have suggested that apple cultivars can be
classified into three groups according to the flavan-3-ols to phenolic acid ratio. Apples can be
rich in flavan-3-ols, rich in phenolic acids, or contain similar levels of both. Pre-harvest
factors and storage conditions have been reported to influence the polyphenol content to some
extent. The effect of organic compared to conventional production has been investigated by
STRACKE et al. (2009) and VALAVANIDIS et al. (2009), whereas CHINNICI et al. (2004a),
HECKE et al. (2006), LAMPERI et al. (2008) and VEBERIC et al. (2005) studied the influence of
organic versus integrated production. Whether mutant variety affects polyphenols in apples,
was examined by AWAD et al. (2000) and SILVEIRA et al. (2007) and the effect of crop load
was tested by AWAD et al. (2001a) and STOPAR et al. (2002). MCGHIE et al. (2005) and YURI
147
PAPER 3
PRE-HARVEST FACTORS
et al. (2009) examined growing region and climate in various apple cultivars, while LAMPERI
et al. (2008) combined growing region with production method in their study. MAINLA et al.
(2011) checked the influence of rootstock together with that of the growing year. The latter
has been explored in many studies (GUYOT et al. 2003; LATA et al. 2007; MAINLA et al. 2011;
STRACKE et al. 2009;
VAN DER
SLUIS et al. 2001) along with other pre-harvest factors, most
often in combination with the effect of the cultivar. To our knowledge, however, a thorough
investigation taking into account all these factors has not been published yet. Therefore, in the
present study we have systematically looked at the influence of production method, mutant
variety, growing environment, rootstock, crop load and growing year on the polyphenol level
and profile in apple cultivars containing different polyphenol profiles. Quantification of
polyphenols was achieved by applying a recently published rapid UHPLC-MS method, which
allows to measure potentially absorbable low molecular weight polyphenols with potential
health benefits (CEYMANN et al. 2011). Based on the results, we estimated the effective preharvest effects by cross-comparing all factors.
148
PAPER 3
PRE-HARVEST FACTORS
Materials and Methods
Apples
Apple cultivars (cv.) Golden Delicious, Ariane, Otava, Topaz, Gala Schniga, Gala Galaxy,
Golden Reinders, Braeburn Royal, Braeburn Redfield, Braeburn Rosewell, Braeburn Hinwell,
Berlepsch, Jonathan, Caudle, Fuji KIKU 8, La Flamboyante, Nicogreen, Nicoter, Civni,
Scifresh and Milwa were produced and collected at optimal harvest at the Research Station
Agroscope Changins-Wädenswil in Wädenswil, Switzerland in the growing years 2008, 2009
and 2010 unless stated otherwise. The fruits were grown on trees with normal crop load and,
unless otherwise mentioned, with an integrated plant protection strategy. The following
experiments were set-up to investigate the different pre-harvest factors:
Production method (Experiment 1)
Fruits of the scab sensitive cv. Golden Delicious and the scab resistant cvs. Ariane, Otava and
Topaz were all produced in one orchard. Each cultivar was cultivated with four different plant
protection strategies: no treatment, organic treatment (excluding synthetic pesticides and
fertilizers), integrated production (IP; economical production of high quality with priority to
ecologically safe methods) and low input treatment (combination of organic and IP methods).
Within a single field, buffer zones were placed between the different production methods.
Fruits were harvested in 2008, 2009 and 2010.
Mutant variety (Experiments 2 and 3)
Fruits of three different cultivars were used to investigate the mutant effect. The Gala
mutants, Gala Schniga and Gala Galaxy, were harvested in 2008, 2009 and 2010 for
experiment 2. The mutants of the cv. Golden, Golden Delicious and Golden Reinders and of
the cv. Braeburn, Braeburn Royal, Braeburn Redfield, Braeburn Rosewell and Braeburn
Hillwell, were harvested in 2010 for experiment 3.
Growing environment (Experiment 4)
Fruits of the cvs. Berlepsch and Jonathan were harvested in 2009 in Arn, Switzerland, 5 km
from Wädenswil. Cvs. Caudle, Fuji KIKU 8, Golden, La Flamboyante, Nicogreen, Nicoter,
Civni, Scifresh and Topaz fruits were grown in 2009 in Conthey, Valais, Switzerland, 152 km
from Wädenswil. Fruits from cv. Gala Schniga in 2010 and cv. Milwa in 2008 were harvested
in Güttingen, 63 km from Wädenswil. Additionally, all cultivars were compared with apples
grown in Wädenswil in the corresponding growing year.
149
PAPER 3
PRE-HARVEST FACTORS
Rootstock (Experiment 5)
Samples of cv. Gala Galaxy of the same orchard were taken in 2008, 2009 and 2010 grown on
the rootstocks B9, CG 4013, J-TE-E, M9 T337 and P59 respectively.
Crop Load (Experiment 6)
Milwa fruits were collected one month before and at optimal harvest in 2009 at Strickhof,
Switzerland and Wädenswil with low, normal and high crop load. At Strickhof (46 km from
Wädenswil), the tree alternated already at blossom, i.e. the bloom densities were low, normal
and high. At Wädenswil, the blossom load for all crop loads was the same. The crop load was
adjusted to low, normal and high levels in June after cell proliferation.
Methods
Sample preparation
Immediately after harvest, apples were pretreated as described earlier (CEYMANN et al. 2011).
Briefly, each sample was composed of 20 fruits, which were sliced into 10 pieces and the core
area with an apple divider (DIVISOREX, Famos-Westmark GmbH, Lennestadt-Elspe,
Germany). Two opposite cuts of each fruit were immediately frozen in liquid nitrogen and all
40 slices were pooled as representative sample. The frozen apple pieces were ground to fine
powder with a dry ice mill (Meidinger AG, Kaiseraugst, Switzerland) and a cutter (La
Moulinette DPA 1, Moulinex, Germany). Afterwards, the samples were stored at -20 °C until
extraction.
Extraction
Extraction and UHPLC-MS analysis were carried out as described earlier (CEYMANN et al.
2011). Briefly, for extraction, aliquots (2.50 g) of the frozen powder were mixed and
homogenized with 50 ml of methanol (analytical grade, Acros Organics, Chemie Brunschwig,
Basel, Switzerland) containing 1 % formic acid (v/v) (analytical grade, Merck, Darmstadt,
Germany). An aliquot of the supernatant was filtered through 0.7 µm glass fiber filters
(OPTI – Flow®, WICOM, Heppenheim, Germany), diluted 1:1 (v/v) with distilled water and
filtered a second time through 0.2 µm nylon filters (OPTI – Flow®, WICOM, Heppenheim,
Germany) directly in UHPLC vials. All extractions were made in duplicate and analyzed
twice.
Polyphenol analysis by UHPLC-MS
UHPLC-MS analysis was carried out using an ACQUITY Ultra Performance LC™ system
(UPLCTM) with a binary solvent manager and a single quadrupole micromass ZQ Mass
150
PAPER 3
PRE-HARVEST FACTORS
Detector (Waters Corporation, Milford, USA) equipped with an electrospray ionization source
operating in negative mode. For instrument control, data acquisition and processing
MassLynx™ software (Version 4.1) was used.
A reversed phase column (BEH C18 1.7 µm, 2.1 x 50 mm with a BEH C18 1.7 µm
VanGuardTM Pre-Column, 2.1 x 5 mm, Waters Corporation, Milford, USA) at 40 °C was used
for separation of individual polyphenols. Samples were injected and analyzed twice and mean
values were used for calculations. Elution was completed in 10 min with a sequence of linear
water-methanol gradients and flow rates of 0.3 ml/min. Solvents and gradient were described
elsewhere (CEYMANN et al. 2011). The software QuantLynx™ (Waters Corporation, Milford,
USA, Version 4.1) was used for integration and calculations based on external standards.
Results are given as polyphenol groups: flavan-3-ols contain catechin, epicatechin,
procyanidin B1 and B2; phenolic acids are composed of chlorogenic acid and
coumaroylquinic acid; dihydrochalcones are represented by phloridzin and phloretinxyloglucoside; and the flavonols correspond to the sum of quercetin-galactoside/glucoside, rhamnoside and rutin. Moreover, the ratio of flavan-3-ols to phenolic acids was used to
classify apples into flavan-3-ol dominated or phenolic acid dominated cultivars. Cultivars
with a ratio close to 1:1 belong to the group with an equilibrated flavan-3-ol to phenolic acid
ratio (CEYMANN et al. 2012).
Statistical analysis
All statistical analyses were conducted with XLSTAT Pro Version 2011.2.04 (Addinsoft,
Andernach, Germany) using analysis of variance (ANOVA, post-hoc test Tukey) to evaluate
significant differences (p<0.05). If more than two factor levels within one factor were
available, only the significantly highest level is indicated.
151
PAPER 3
PRE-HARVEST FACTORS
Results and Discussion
Influence of pre-harvest factors on the polyphenol content of apples
Influence of the growing year (Experiment 1)
Figure 1 shows the polyphenol content of the cvs. Golden Delicious, Ariane, Otava and
Topaz, grown with four different production methods over three years. Regarding flavan-3-ol
content, the highest amount with an average of 24 mg/100 g FM over the three years was
obtained by the flavan-3-ol dominated cv. Otava. In contrast, the highest average amount of
phenolic acids with 21 mg/100 g FM was analyzed for cv. Ariane, a cultivar with an
equilibrated ratio of flavan-3-ols to phenolic acids. The highest average amounts for
dihydrochalcones were 5 mg/100 g FM, detected in cv. Golden Delicious, also a cultivar with
an equilibrated ratio. For flavonols, the highest average values with 7 mg/100 g FM were
obtained with the flavan-3-ol dominated cv. Topaz.
The highest polyphenol contents were reached in 2008 for the flavan-3-ols, dihydrochalcones
and flavonols for all cultivars (see Figure 1, indicated by #). For phenolic acids, however, the
differences between the growing years were small and the highest amounts were detected in
2010 for Otava and Topaz. Of all the polyphenol groups, flavonols were the most strongly
influenced by the growing year. The extent of sun exposure could be a reason, because
flavonols are located in the skin of the apple and their production is induced by sunlight
(AWAD et al. 2001b). However, the available climate data (not shown) were not sufficiently
strong to confirm the link between sun exposure and flavonol content. Dihydrochalcones were
influenced more strongly by the cultivar than by the growing year.
In cvs. Golden Delicious and Ariane, the two cultivars with a ratio of flavan-3-ols:phenolic
acids close to 1:1 (see Figure 1), the influence of the growing year on flavan-3-ols and
phenolic acids was not as pronounced as for the flavan-3-ol dominated cvs. Otava and Topaz,
which showed the highest flavan-3-ol and phenolic acid contents in 2008. However,
polyphenol profiles remained fairly constant over the three years for all four cultivars. This is
in agreement with GUYOT et al. (2003), who concluded an overall stability of polyphenol
composition from one year to another, but also found significant year-to-year variations in the
levels of individual polyphenol compounds, comparable to our own results (Figure 1).
Consistently, KEVERS et al. (2011) and
VAN DER
SLUIS et al. (2001) reported similar
variabilities in concentrations for regional cultivars investigated over two or three years.
152
PRE-HARVEST FACTORS
Golden Delicious
25
20
15
10
5
0
#
*
#
*
#
#
*
* *
20
*
#
#
*
#
#
30
#
#
#
#
#
*
2010
2009
2008
Otava
#
*
#
#
*
#
*
*#
Topaz
Flavanols
Phenolic Acids
*#
Dihydrochalcones
#
#
# #
2010
2009
2008
*#
low input
*#
integrated production
low input
organic
no treatment
integrated production
low input
organic
no treatment
#
#
#
*
#
integrated production
#
low input
#
organic
#
*
#
no treatment
#
integrated production
#
*#
#
2010
2009
2008
#
organic
10
0
mg/100 g FM
#
#
*
#
20
Figure 1:
#
#
#
*
*
#
#
*
#
40
*
#
Ariane
*
#
10
25
20
15
10
5
0
#
#
2010
2009
2008
*
0
mg/100 g FM
*
#
*
30
mg/100 g FM
#
no treatment
mg/100 g FM
PAPER 3
Flavonols
Effect of growing year (2008 black, 2009 grey, 2010 white) and production method (for
details see section Materials and Methods) on polyphenol content of different apple
cultivars; values represent means of extraction duplicates; # significantly highest
among years, * significantly highest among production method
LATA et al. (2007) measured phenolics spectrophotometrically in 19 different cultivars over
two growing years. They detected either no variation in individual polyphenol classes for cvs.
153
PAPER 3
PRE-HARVEST FACTORS
Gala and Red Rome or up to three-fold differences for cv. Granny Smith. This compares to
differences of 30-70 % in the four cultivars over the three years of our studies. Cv. Golden
Delicious showed a variability of 40 % between the two years in their trial compared to only
10 % in our study. The larger variations observed by LATA et al. (2007) could be due to the
analytical
methods
used
as
they
measured
the
phenolics
with
an
unspecific
spectrophotometric method, whereas we quantified them from UHPLC-MS analysis.
STRACKE et al. (2009) reported similar polyphenol values to our own for conventionally
grown cv. Golden Delicious apples in three growing years. The levels of flavan-3-ols were
13 mg/100 g FM, phenolic acids 10 mg/100 g FM, dihydrochalcones 2 mg/100 g FM and
flavonols 5 mg/100 g FM. We found 16 mg, 13 mg, 4 mg and 3 mg/100 g FM of these
specific polyphenol groups, respectively. In the study by STRACKE et al. (2009) differences
over the three years in cv. Golden Delicious were 9 mg/100 g FM for flavan-3-ols,
4 mg/100 g FM for phenolic acids, 1 mg/100 g FM for dihydrochalcones and 2 mg/100 g FM
for flavonols. This is in reasonable agreement with the differences we found over the growing
seasons which were 6 mg, 1 mg, 2 mg and 3 mg/100 g FM, respectively. As with our own
study, few of the differences due to growing season reported by STRACKE et al. (2009) were
statistically significant.
The growing season has been reported to lead to much higher differences in polyphenol
compounds in the peel. An increase of up to 9-fold over two growing years was reported for
the same polyphenol groups in six different apples cultivars from Estonia (MAINLA et al.
2011), but the influence of the growing year was dependent on cultivar.
The growing year can thus have a quantitative as opposed to a qualitative influence on
polyphenols in apples, but the extent of the effect strongly depends on the cultivar and has
different impacts on the different polyphenol groups. The maximum change in polyphenols
due to growing season is less than the maximum difference between cultivars. While the
growing season does have such a small influence on polyphenol content, this is not an option
to use this parameter to influence polyphenol levels.
Influence of the production method (Experiment 1)
Figure 1 also shows the polyphenol content of apples grown under different production
methods. We noted few significant differences (indicated by *) between the production
methods. Moreover, no clear tendency towards one treatment was detected. For example, cv.
Ariane grown in 2008 contained the highest amount of phenolic acids when produced under
154
PAPER 3
PRE-HARVEST FACTORS
IP conditions. However in 2009, the highest phenolic acid content was in organic fruits and in
2010 in apples grown with no treatment.
The influence of production method on the polyphenol levels in the flavan-3-ol dominated
cvs. Otava and Topaz, was similar. The highest levels of flavan-3-ol and phenolic were found
with IP in 2010. On the other hand, cvs. Golden Delicious and Ariane, both cultivars with a
balanced polyphenol profile, showed highest values for flavan-3-ols following organic
treatment in 2009. In our studies, the mean values for the different polyphenol classes in
apples grown under the different production methods over the three growing years varied in
general by less than 20%. Somewhat higher variations were found in cv. Golden Delicious for
dihydrochalcones (low input: 26 %; IP: 27 %) and for flavonols (organic treatment: 45 %).
Overall, flavan-3-ols and phenolic acids were less influenced by production methods than
dihydrochalcones and flavonols. This could be explained by lower absolute amounts for the
last two groups. Quantification closer to the detection limit caused higher relative deviations.
Combining the results for all cultivars over the three growing years, no significant differences
between the production methods were observed.
In recent years, several studies have compared organically and conventionally produced
apples. Significantly higher amounts in polyphenol contents have been reported in organically
produced cv. Roter Boskoop compared to conventionally produced cv. Golden Delicious
(HECKE et al. 2006; VEBERIC et al. 2005). These differences are most likely due to the
different cultivars, because our earlier results showed that cv. Roter Boskoop contained twice
the amount of polyphenols as cv. Golden Delicious (CEYMANN et al. 2012) both with IP
treatment.
Other studies have reported no significant effect of organic versus conventional production on
the polyphenol content of cvs. Annurca and Golden Delicious (LAMPERI et al. 2008) or cvs.
Red Delicious Starking, Golden Delicious, Royal Gala, Granny Smith and Jonagold
(VALAVANIDIS et al. 2009). STRACKE et al. (2009) examined also the polyphenol content in
cv. Golden Delicious fruits over three years and reported for two years no effect of growing
method, but for one year significantly higher concentrations in organically produced fruits.
These findings are in accordance to our results.
One reason why polyphenols increase slightly under some production conditions could be an
increase in metabolic stress (TREUTTER 2005). For cv. Golden Delicious, organic production
seems to have caused more stress in some years, because it is a cultivar that is well adapted to
conventional production methods. It is possible that the organically produced cv. Golden
155
PAPER 3
PRE-HARVEST FACTORS
Delicious, which is less protected by plant protection products such as insecticides, defends
itself by increasing its polyphenol level. For other apple cultivars, other production methods
may similarly elicit more stress and therefore higher polyphenol content. For example for cv.
Otava in Figure 1, the highest significant values were analyzed for IP treatment for flavan-3ols, phenolic acids and flavonols in 2010. Cv. Otava is thought to be more stressed under IP
treatment, due to the fact that cv. Otava is adapted to the organic production methods. But
general recommendations for all cultivars are not possible. CHINNICI et al. (2004a) reported
higher amounts of polyphenols for cv. Golden Delicious produced with conventional
treatment than in organically produced fruits, which contrasts to the finding of STRACKE et al.
(2009) but not the other publications mentioned above.
Theoretically, it might be possible to increase the polyphenol concentration in apples by
avoiding plant protection and thus forcing the plant to increase its own protection against
insects or microbiological attack by producing more polyphenols. However, this seems to
depend on the cultivar, and natural protection is less effective than conventional protection.
For the producer, productivity and marketability are more important than nutritional aspects
such as a higher polyphenol level. For consumers, the appearance of the fruits is a buying
criterion and fruits with black spots as described by VALAVANIDIS et al. (2009), and as found
in our no treatment group of cv. Golden Delicious fruits, would be rejected by the consumer.
Consequently, consuming organically or conventionally produced apple fruits seems to be an
environmental choice rather than a way of increasing the polyphenol intake.
Influence of the mutant variety (Experiment 2 and 3)
In apple breeding, mutants may offer a possibility to improve fruit quality. Mutants, which are
more blushed (red colored) than the original variety, are often cultivated. For consumers,
these variations within the cultivar are unimportant, because apples are typically offered with
the cultivar labeling only. Nevertheless, since genotypes vary slightly between mutants, the
polyphenol content might differ between mutants.
Comparing the mutants in Figure 2, cv. Gala Schniga showed significantly higher amounts in
flavan-3-ols and phenolic acids (indicated by *) for two of the three growing years. However,
this was not consistent and in 2009 the phenolic acid content of cv. Gala Galaxy was
significantly higher than that of cv. Gala Schniga. For dihydrochalcones and flavonols, no
significant differences were found between the mutants over the three growing years. Figure 2
also shows that the influence of the growing year was as important as the influence of mutant
156
PAPER 3
PRE-HARVEST FACTORS
variety. A 2-fold variability was observed between mutants for the flavan-3-ol value in 2010
*
*
*
* #
#
*
Figure 2:
Phenolic acids
2010
Galaxy
Schniga
Galaxy
Schniga
Galaxy
Schniga
Galaxy
Flavan-3-ols
2009
#
#
2008
20
15
10
5
0
Schniga
mg/100 g FM
and for phenolic acids in mutant Galaxy between the growing years 2009 and 2010.
Dihydrochalcones Flavonols
Effect of growing year (2008 black, 2009 grey, 2010 white) and mutant variety on
polyphenol content of cv. Gala. #: significantly highest among year, *: significantly
higher among mutant variety; values represent means of extraction duplicates
In contrast, the mutants of cv. Golden, i.e. Golden Delicious and Golden Reinders, produced
only small but significant differences for flavonols (Figure 3 A). The polyphenol levels in the
different mutants of Braeburn however differed considerably (Figure 3 B). The cv. Braeburn
Redfield showed the highest levels of both flavan-3-ols and phenolic acids of all Braeburn
mutants. The greatest differences between mutants were obtained for flavan-3-ols with twice
as much in Redfield than in Hillwell (14 mg versus 7 mg/100 g FM). For dihydrochalcones
the highest levels were measured for Rosewell and for flavonols the highest levels were in
Rosewell and Hillwell.
A 20
Delicious
Reinders
mg/100 g FM
15
10
*
5
0
B 20
Royal
Redfield
Rosewell
Hillwell
*
mg/100 g FM
15
Figure 3:
10
5
*
*
**
0
Effect of mutant variety on polyphenol content of cvs. Golden (A) and Braeburn (B)
grown in 2010. *: significantly highest among mutant variety; values represent means
of extraction duplicates
157
PAPER 3
PRE-HARVEST FACTORS
Although we have shown some differences in polyphenol content between mutants in two of
the three varieties tested, the differences were not large compared to the differences between
cultivars. The observation that the mutation may or may not influence the polyphenol level
has been reported by other authors. SILVEIRA et al. (2007) detected no differences between
mutants for cv. Fuji, whereas AWAD et al. (2000) found differences in anthocyanin levels in
mutants of Jonagold and Elstar without influencing the levels of other flavonoids. It would
seem possible to increase polyphenol levels in apples by mutation, followed by selection and
cultivation. Consequently, comparison of polyphenol literature data without exact knowledge
of the mutant, may lead to uncertain conclusions. However, the differences between mutants
within a single cultivar are small compared to the differences between cultivars. In addition
the consumer is offered the cultivar not the mutant.
Influence of the growing environment (Experiment 4)
The growing environment is determined by the type of soil, altitude, temperature, sunshine
duration, water availability and pruning, i.e. conditions which can only partly be influenced
by producers. Accordingly, the apple growers choose apple trees that are adapted to the
growing environment so as to achieve the optimum yield. To investigate the growing
environment, 13 different cultivars with total polyphenol concentration varying from 21 mg to
66 mg/100 g FM were grown in Wädenswil and three other locations.
Figure 4 compares the polyphenol content of these 13 cultivars grown in different locations.
In general, location caused differences less than 15%. Significant differences in polyphenol
content were detected in only 4 of the 13 comparisons. Cvs. Jonathan, La Flamboyante and
Milwa grown in Wädenswil had higher total polyphenol concentrations than the same apples
grown in Arn, Conthey and Güttingen, respectively, whereas in cv. Scifresh polyphenols were
higher in Conthey apples than in Wädenswil apples.
Differences in polyphenol content are presumably related to some extent to climate and the
growing environment conditions of Wädenswil, which may have been sub-optimal and thus
may have induced metabolic stress (TREUTTER 2005). As mentioned above, this is known to
increase polyphenol biosynthesis. It may be especially true for cv. La Flamboyante, which
was bred in Conthey, where both sunshine duration and temperature are higher than in
Wädenswil.
As expected, there were greater differences in polyphenol contents between cultivars than
between growing environments, and the polyphenol profile changed little with respect to
growing environment.
158
Milwa
Gala
Schniga
Topaz
Scifresh
Civni
Nicoter
Nicogreen
La Flamboyante
Golden
Phenolic acids
Dihydrochalcones
Wädenswil
Güttingen
Wädenswil
Güttingen
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Conthey
Wädenswil
Arn
*
*
Flavan-3-ols
Figure 4:
Fuji
KIKU ®
Caudle
Jonathan
*
*
Wädenswil
Arn
70
60
50
40
30
20
10
0
Berlepsch
PRE-HARVEST FACTORS
mg/100 g FM
PAPER 3
Flavonols
Effect of growing environment on polyphenol content of different cultivars grown in
2009 (except Gala Schniga: grown in 2012 and Milwa: grown in 2008). *: significantly
higher among growing environment; values represent means of extraction duplicates
Comparable experiments have been carried out in New Zealand (MCGHIE et al. 2005), Italy
(LAMPERI et al. 2008) and Chile (YURI et al. 2009) for one or more cultivars and in these
studies growing environment more consistently influenced the polyphenol content of the
apples. MCGHIE et al. (2005) found significant differences between three growing
environments in New Zealand for 10 different apple cultivars. They concluded that the
growing environment influenced the polyphenol content, but the level of change was highly
cultivar-dependent. Similarly, four apple cultivars were grown in two different regions in
Italy. However, in this study significant differences were detected only in the apple peel and
not in the apple flesh. The authors explained the differences in polyphenol content as being
due to the unequal maturity status of the fruits, which depends on climatic conditions
(LAMPERI et al. 2008). In Chile, significant differences were reported in five cultivars grown
at different sites. The authors related the differences to different climatic conditions,
especially UV radiation (YURI et al. 2009).
In conclusion, environment can influence polyphenol content but not profile. The extent of the
changes depends on the cultivar and is generally small compared to differences between
cultivars. Further detailed studies could help find what factors in the environment are
important.
Influence of the rootstock (Experiment 5)
Grafting on specific rootstocks is a common treatment for apple trees to optimize their
growth, requirement for space, start of fruit production, stability and adaption to soil and
climate. The influence of five different rootstocks on the polyphenol content of cv. Gala
Galaxy was tested in 2010, 2008 and 2009 (Figure 5). The effect of rootstock on polyphenol
159
PAPER 3
PRE-HARVEST FACTORS
levels was modest and variable, and much less than the impact of the growing year.
Rootstocks B9, P59 and J-TE-E all produced apples with significantly higher amounts of
polyphenols than the other rootstocks (indicated by *) in some years but not others. The
differences were small and remained below 40 % except for flavonols (50 %). In contrast to
the polyphenol level, the profile remained unaffected by the different rootstocks.
# #
#
#
#
*
#
#
*
#
*
*
*
#
B9
CG 4013
J-TE-E
M9 T337
P59
#
Flavan-3-ols
Phenolic acids
#
*
Dihydrochalcones
#
#
*
#
*
*
B9
CG 4013
J-TE-E
M9 T337
P59
5
2010
2008
2009
10
0
Figure 5:
* #
#
#
B9
CG 4013
J-TE-E
M9 T337
P59
15
#
B9
CG 4013
J-TE-E
M9 T337
P59
mg/100 g FM
20
Flavonols
Effect of growing year (2008 black, 2009 grey, 2010 white) and rootstock on
polyphenol content of cv. Gala Galaxy. #: significantly highest among year, *:
significantly highest among rootstock; values represent means of extraction duplicates
Much greater differences in polyphenol content were observed between the growing years
(indicated by #), with the highest amounts in 2009. The greatest difference was obtained
between 2009 and 2010 for phenolic acids with rootstock M9 T337 (2.5 fold). A reason for
the higher variability observed with flavonols could be analytical and due to their lower
values, which were more influenced by detection fluctuations as mentioned earlier.
These results are similar to other reports in the literature. MAINLA et al. (2011) detected
twofold variations in the polyphenol content in the peel of cv. Talvenauding apples grown on
three different rootstocks over two growing years. The influence of different rootstocks on
polyphenol content seems to be also explained by metabolic stress, as different rootstocks
provide the fruit with higher or lower amounts of nutrients, which could influence the
biosynthesis of polyphenols. A rootstock with a less than optimal fit to the cultivar could lead
to more metabolic stress (TREUTTER 2005). A similar explanation is given by JAKOBEK et al.
(2009), who investigated the influence of rootstock on polyphenol content in sweet cherry
fruits.
According to our apple growers, the rootstock M9 T337 provided the best production
characteristics. On this rootstock, no increase of polyphenol contents was observed compared
160
PAPER 3
PRE-HARVEST FACTORS
to the other investigated rootstocks (Figure 5), which may indicate a lower level of metabolic
stress.
In conclusion the combination of rootstock and cultivar can have a small influence on the
polyphenol content depending on the extent of metabolic stress. The polyphenol fluctuations
with different rootstocks observed in this experiment would by themselves be too small to
induce the reported health benefits.
Influence of the crop load (Experiment 6)
Apple trees, where neither bloom nor fruit have been thinned, can fall into alternation, which
leads to a harvest with high numbers of small fruits in one growing year and a small number
of big fruits in the following growing year. An experiment was conducted with cv. Milwa in
the two different orchard locations Wädenswil and Strickhof, so as to investigate the influence
of the crop loads on the polyphenol content. The crop loads were adjusted to low, normal and
high levels. In Wädenswil, small but significant differences in flavan-3-ols only were detected
between the highest and the lowest crop load (Figure 6 A). These differences were not yet
apparent one month before harvest (data not shown). In the orchard at Strickhof, with a
comparable altitude and climate, low crop load significantly increased both flavan-3-ols and
phenolic acids compared to normal and high crop loads (Figure 6 B). The maximum increases
were 7.7 mg for flavan-3-ols and 6.4 mg/100 g FM for phenolic acids. Dihydrochalcones
remained constant between the three crop loads, whereas for flavonols, higher levels were
detected in the high crop load only at Strickhof. These differences were already observed one
month before harvest (data not shown). A reason for the inverse correlation of flavonols
compared to flavan-3-ols and phenolic acids might be the surface to volume ratio. Fruits of
high crop load were smaller with an average weight of 144 g compared to 171 g of low crop
load and therefore the surface to volume ratio was higher in high crop load. Since flavonols
are mainly located in the skin of apples, the higher surface of the high crop load apples would
contain more flavonols.
A reason for the slightly different outcomes of the two experiments could be the different
bloom density for the three crop loads at Strickhof. These trees showed an alternation already
at blossom and therefore the differences in crop loads occurred naturally. Consequently, less
fruits were developed on trees with a low crop load than on trees with high crop load. In
Wädenswil however, all trees were flowering to the same intensity and comparable numbers
of fruits were produced. When cell proliferation had finished, the trees were thinned to reach
the three different crop loads. After termination of the cell proliferation only cell expansion
161
PAPER 3
PRE-HARVEST FACTORS
occurs, which leads to a dilution of polyphenol concentration during the growing of apples as
reported by RENARD et al. (2007). From these results, it can be concluded that adjusting the
crop load after cell proliferation has little influence on the polyphenol content at harvest, but
large influence on the bloom density and consequently on next season’s harvest, as it was
demonstrated at Strickhof. Therefore, crop load seems to influence the polyphenol content of
the fruits of the following harvest. However, as with all other interventions, the polyphenol
profile remains stable during all crop loads.
mg/100 g FM
A 30
20
low
a
ab
b
medium
normal
aaa
high
10
aaa
aaa
0
B 30
a
mg/100 g FM
a
Figure 6:
20
b
c
b
b
10
aaa
bb
a
0
Effect of crop load on polyphenol content of cv. Milwa grown in 2009. A location
Wädenswil, same bloom density for all crop loads; B location Strickhof, different
bloom densities for different crop loads; values represent means of extraction
duplicates
Data from similar studies in the literature is variable. The data presented in Figure 6 A are
comparable to AWAD et al. (2001a), who detected no significant differences in the skin
polyphenols of cvs. Jonagold and Red Elstar between crop loads, whereas STOPAR et al.
(2002) similarly showed increased polyphenol contents at lower crop loads in cv. Jonagold,
despite the same bloom density. A reason for this difference could be the cultivar-dependent
behavior on crop load.
It would seem that a low crop load can increase the level of polyphenols to a small extent.
From an economical point of view, however, a high crop load delivers a higher yield and a
greater profit, but also reduces fruit flesh firmness (STOPAR et al. 2002). Nevertheless, it
should be noted that the consumption of bigger fruits containing the same polyphenol content
per 100 g FM as smaller fruits, results in a higher absolute intake of polyphenols per apple.
162
PAPER 3
PRE-HARVEST FACTORS
A comparison of the different factors influencing polyphenols in apples
A direct comparison of all aforementioned factors influencing the polyphenol content in
apples is difficult because of their many interactions. To better quantify the effective impact
of all pre-harvest factors, additional experiments would be necessary with a cross-comparison
of all factors. However, Figure 7 illustrates the influence of the pre-harvest factors on the
polyphenol content experiment-wise. For that purpose, mean values of all data within one
experiment (represented by lines in Figure 7) as well as mean values of the different variables
(represented by symbols) were calculated. Data obtained from Experiment 1 indicate, that the
influence of the cultivar was greater than the influence of the growing year, which in turn was
higher than the impact of the production method. The higher impact of the cultivar on the
polyphenol content was also seen in Experiment 4, where differences in polyphenol levels
between cultivars were greater than those induced by the growing environment. The
differences in polyphenol levels induced by different crop loads were also more than those
induced by the growing environment (Experiment 6). The growing year however did cause
small changes in polyphenol content, comparable to those resulting from using different
mutants (Experiment 2) and greater than those resulting from changing the rootstock
(Experiment 5). The influence of mutant variety (Experiment 3) was difficult to estimate as
Figure 7 visualizes. Cv. Golden mutants had a small effect similar to other pre-harvest factors,
whereas cv. Braeburn mutants showed a variability comparable to the cultivar effect observed
in Experiment 1. The latter indicates that it might be possible to increase polyphenol levels in
apples by mutation, followed by selection and cultivation.
Concerning the polyphenol profile, the pre-harvest factors investigated in our study had little
or no effect. Therefore, the classification of cultivars into flavan-3-ol dominated, phenolic
acids dominated cultivars, and cultivars with an equilibrated polyphenol profile respectively is
not influenced by pre-harvest factors.
163
PAPER 3
PRE-HARVEST FACTORS
60
mg/100 g FM
50
40
30
20
Experiment 1
Figure 7:
Experiment 2
Experiment 3
Experiment 4
Experiment 5
Crop load
Growing
environment
Rootstock
Year
Growing
environment
Cultivar
Braeburn
mutant
Golden
mutant
Galaxy
mutant
Year
Production
method
Year
0
Cultivar
10
Experiment 6
Sum of polyphenols as analyzed by UHPLC-MS; horizontal lines are the means of all
data of the corresponding experiment; symbols represent means of the different
variables
164
PAPER 3
PRE-HARVEST FACTORS
Conclusion
Overall, the cultivar itself has the highest influence on the polyphenol content of apples, very
likely followed by the mutant variety. All other factors individually had only a small impact
on the polyphenol content. The growing year resulted in greater changes than the pre-harvest
factors rootstock, production method, growing environment and crop load. The influence of
production methods on the polyphenol content was small compared to most other pre-harvest
factors. Therefore, the omnipresent discussion concerning organically produced fruit
containing more healthy substances than when conventionally grown appears less important,
as long as comparisons are based on identical cultivars.
Apple producers grow fruits, adapted to meet consumer’s expectance of sensory and visual
quality. In the future, consumers may look for apples with an added health benefit and apple
growers could produce fruits with higher concentrations in polyphenols. Choosing optimal
cultivars and mutant varieties would be the first step to both increase the polyphenol content
and select the profile, but then it should be possible to modify pre-harvest factors to further
modestly increase the selected polyphenols. Individually, the rootstock, growing environment,
crop load and production methods cause small changes in polyphenol content of apples but, if
each were optimized, useful additional levels of health promoting polyphenols could be
produced.
Acknowledgment
The authors thank Reto Leumann, Claudia Good, Andreas Naef, Danilo Christen, Otto Läubli,
Michael Gölles, Martin Kockerols, Isabel Mühlenz for apple supply and Melanie Erzinger,
Jennifer Kläui, Martin Heiri and Dominic Ritler for their support during sample preparation
and analysis. Thanks are given to Dr. Markus Kellerhals and Simon Egger for scientific
discussion.
165
PAPER 3
PRE-HARVEST FACTORS
References
Awad, M. A., A. De Jager, et al. (2001a). "Formation of flavonoids and chlorogenic acid in apples as
affected by crop load." Scientia Horticulturae 91(3-4): 227-237.
Awad, M. A., A. Jager de, et al. (2000). "Flavanoid and chlorogenic acid levels in apple fruit:
characterisation of variation." Scientia Horticulturae 83: 249-263.
Awad, M. A., P. S. Wagenmakers, et al. (2001b). "Effects of light on flavonoid and chlorogenic acid
levels in the skin of 'Jonagold' apples." Scientia Horticulturae 88(4): 289-298.
Ceymann, M., E. Arrigoni, et al. (2011). "Rapid high performance screening method using UHPLCMS to quantify 12 polyphenol compounds in fresh apples." Analytical Methods 3: 1774.
Ceymann, M., E. Arrigoni, et al. (2012). "Identification of apples rich in health-promoting flavan-3-ols
and phenolic acids by measuring the polyphenol profile." Journal of Food Composition and
Analysis 26(1-2): 128–135.
Chinnici, F., A. Bendini, et al. (2004a). "Radical scavenging activities of peels and pulps from cv.
golden delicious apples as related to their phenolic composition." Journal of Agricultural and
Food Chemistry 52(15): 4684-4689.
FAOSTAT. (2012b, 01.10.2012). "Food production data." Retrieved 01.10.2012, from
http://faostat.fao.org/site/567/DesktopDefault.aspx?PageID=567#ancor.
Guyot, S., N. Marnet, et al. (2003). "Variability of the polyphenolic composition of cider apple (Malus
domestica) fruits and juices. (vol 51, pg 6240, 2003)." Journal of Agricultural and Food
Chemistry 51: 7522-7522.
Harker, F. R., F. A. Gunson, et al. (2003). "The case for fruit quality: an interpretive review of
consumer attitudes, and preferences for apples." Postharvest Biology and Technology 28(3):
333-347.
Hecke, K., K. Herbinger, et al. (2006). "Sugar-, acid- and phenol contents in apple cultivars from
organic and integrated fruit cultivation." European Journal of Clinical Nutrition 60: 11361140.
Jakobek, L., M. Seruga, et al. (2009). "Flavonol and phenolic acid composition of sweet cherries (cv.
Lapins) produced on six different vegetative rootstocks." Scientia Horticulturae 123(1): 2328.
Katz, D. L., K. Doughty, et al. (2011). "Cocoa and chocolate in human health and disease."
Antioxidants and Redox Signaling.
Kevers, C., J. Pincemail, et al. (2011). "Influence of cultivar, harvest time, storage conditions, and
peeling on the antioxidant capacity and phenolic and ascorbic acid contents of apples and
pears." Journal of Agricultural and Food Chemistry 59(11): 6165-6171.
Lamperi, L., U. Chiuminatto, et al. (2008). "Polyphenol levels and free radical scavenging activities of
four apple cultivars from integrated and organic farming in different Italian areas." Journal of
Agricultural and Food Chemistry 56(15): 6536-6546.
Lata, B. (2007). "Relationship between apple peel and the whole fruit antioxidant content: year and
cultivar variation." Journal of Agricultural and Food Chemistry 55: 663-671.
Lata, B. and K. Tomala (2007). "Apple peel as a contributor to whole fruit quantity of potentially
healthful bioactive compounds. Cultivar and year implication." Journal of Agricultural and
Food Chemistry 55: 10795-10802.
Mainla, L., U. Moor, et al. (2011). "The effect of genotype and rootstock on polyphenol composition
of selected apple cultivars in Estonia." Zemdirbyste-Agriculture 98(1): 63-70.
McGhie, T. K., M. Hunt, et al. (2005). "Cultivar and growing region determine the antioxidant
polyphenolic concentration and composition of apples grown in New Zealand." Journal of
Agricultural and Food Chemistry 53: 3065-3070.
Renard, C. M. G. C., N. Dupont, et al. (2007). "Concentrations and characteristics of procyanidins and
other phenolics in apples during fruit growth." Phytochemistry 68: 1128-1138.
Silveira, A. C., C. K. Sautter, et al. (2007). "Determination of some quality parameters of the fuji
cultivar and their mutants at harvest." Ciência e Tecnologia de Alimentos 27(1): 149-153.
Stopar, M., U. Bolcina, et al. (2002). "Lower crop load for Cv. Jonagold apples (Malus x domestica
Borkh.) increases polyphenol content and fruit quality." Journal of Agricultural and Food
Chemistry 50(6): 1643-1646.
166
PAPER 3
PRE-HARVEST FACTORS
Stracke, B. A., C. E. Rufer, et al. (2009). "Three-year comparison of the polyphenol contents and
antioxidant capacities in organically and conventionally produced apples (Malus domestica
Bork. cultivar 'Golden Delicious')." Journal of Agricultural and Food Chemistry 57(11):
4598-4605.
Thom, E. (2007). "The effect of chlorogenic acid enriched coffee on glucose absorption in healthy
volunteers and its effect on body mass when used long-term in overweight and obese people."
Journal of International Medical Research 35: 900-908.
Treutter, D. (2005). "Significance of flavonoids in plant resistance and enhancement of their
biosynthesis." Plant Biology 7(6): 581-591.
Valavanidis, A., T. Vlachogianni, et al. (2009). "Polyphenolic profile and antioxidant activity of five
apple cultivars grown under organic and conventional agricultural practices." International
Journal of Food Science and Technology 44: 1167-1175.
van der Sluis, A. A., M. Dekker, et al. (2001). "Activity and concentration of polyphenolic
antioxidants in apple: Effect of cultivar, harvest year, and storage conditions." Journal of
Agricultural and Food Chemistry 49: 3606-3613.
Veberic, R., M. Trobec, et al. (2005). "Phenolic compounds in some apple (Malus domestica Borkh)
cultivars of organic and integrated production." Journal of the Science of Food and
Agriculture 85: 1687-1694.
Yuri, J. A., A. Neira, et al. (2009). "Antioxidant activity and total phenolics concentration in apple
peel and flesh is determined by cultivar and agroclimatic growing regions in Chile." Journal
of Food, Agriculture and Environment 7(3-4): 513-517.
167
PAPER 3
PRE-HARVEST FACTORS
168
PAPER 4
IN VITRO DIGESTION OF APPLES
PAPER 4
(in preparation)
Influence of matrix on in vitro accessibility of apple polyphenols
Maria Ceymanna, Pina Ernia, Eva Arrigonia* and Richard F. Hurrellb
a
Research Station Agroscope Changins-Wädenswil ACW, Schloss, 8820 Wädenswil,
Switzerland
b
ETH Zürich, Institute of Food, Nutrition and Health, Schmelzbergstrasse 7, 8092 Zürich,
Switzerland
*
Corresponding author: Tel.: +41 44 783 6661; Fax: +41 44 783 6224.
E-mail address: [email protected]
169
PAPER 4
IN VITRO DIGESTION OF APPLES
Abstract
Apples are popular fruits in Western countries and, due to their polyphenol content, are
reported to be beneficial for human health. Apple polyphenols however, must be released
from the food matrix before they can be absorbed and exert a physiological effect. We have
used an in vitro procedure simulating digestion in the stomach and small intestine to predict
the bioaccessibility of polyphenol compounds in fresh apples and apple juices. Flavan-3-ols
and chlorogenic acid recovered from the food matrix were quantified by UHPLC-UV. The
mean recovery of monomeric flavan-3-ols from fresh apples was 57 % after simulated
stomach and 77 % after simulated stomach plus small intestine digestion, but lower for the
dimers procyanidin B1 and B2 with means of 37 % and 45 % respectively. As for apple
juices, 65 % of monomers and 55 % of dimers were recovered after gastric digestion falling to
53 % and 27 % respectively after combined gastric and intestinal digestion. Additionally,
mean chlorogenic acid recovery from apple juices declined from 44 % after simulated
stomach to 30 % after combined digestion, whereas a slight increase from 46 % to 49 % was
found for fresh apples. The recovery of the monomeric flavan-3-ols is a composite value
including their release from the apple matrix, their degradation during the in vitro digestion
procedure, and their formation from the breakdown of dimers and oligomers. Pure catechin
and epicatechin were 60-70 % degraded during in vitro digestion, the dimers procyanidin B1
and B2 were broken down to monomers leading to a very low recovery, and epimerisation
occurred for all flavan-3-ols. The results indicate that the bioaccessibility of polyphenols from
apples is relatively high with nearly 50 % recovered on simulated gastric digestion. The
recovery of polyphenols from apple juice after simulated gastric digestion was similar
indicating 50% degradation during the in vitro procedure, and suggesting that release of
polyphenols from apples was almost complete. Bioaccessibility of apple polyphenols
therefore does not appear to be an issue when evaluating potential health effects of apples
170
PAPER 4
IN VITRO DIGESTION OF APPLES
Introduction
Apples constitute the most popular fruit in many Western countries. From 2005 to 2007, the
average per capita consumption of apples in EU countries was 57-60 g per day representing
nearly one fourth of the European fruit consumption (FAOSTAT 2012a). In Switzerland,
apples are reported to be the most often consumed fruit with an average of 43 g per capita per
day (SWISSFRUITASSOCIATION 2012). Reasons for this high consumption are cultivar
diversity, all-year availability, reasonable price and high convenience (HARKER et al. 2003;
SWISSFRUITASSOCIATION 2012). From a nutritional point of view, apples are characterised by
a low energy density, but high nutrient density. Moreover, they are rich in polyphenols which
are considered to have beneficial effects on health (SIES 2010). Predominant polyphenol
classes in apples are flavan-3-ols (i.e. catechin, epicatechin and procyanidins B1 and B2),
phenolic acids (mainly chlorogenic acid and coumaroylquinic acid), dihydrochalcones
(phloridzin and phloretin-xyloglucoside) and flavonols (various quercetin glycosides) which
vary in bioaccessibility, bioavailability and potential health effects. The mechanisms by which
they cause these beneficial effects are still under investigation. Studies made with flavan-3-ols
from cocoa have shown a preventive effect on cardiovascular disease by improving
endothelial function (HOOPER et al. 2012), whereas chlorogenic acid mainly present in coffee
has been reported to decrease the risk of type II diabetes (BIDEL et al. 2008). Additionally, the
polyphenol concentrations in different apple cultivars (ALONSO-SALCES et al. 2004a; AWAD et
al. 2000; MANGAS et al. 1999; TSAO et al. 2003; VRHOVSEK et al. 2004; WOJDYLO et al. 2008)
and apple juices (KAHLE et al. 2005a; OSZMIANSKI et al. 2007; RYAN et al. 2010) have been
shown to be highly variable (NEVEU et al. 2010), whereas polyphenol profiles are mainly
influenced by the genotype (CEYMANN et al. 2012).
One concern however is the bioaccessibility of apple polyphenols, that is the extent to which
they are released from the cellular matrix into the intestinal lumen during digestion and thus,
if not degraded during digestion, available for absorption. Absorption studies in humans with
cocoa products (BABA et al. 2000; FOGLIANO et al. 2011; HOLT et al. 2002; NEILSON et al.
2011; RIOS et al. 2002), cider (DUPONT et al. 2002) and apple juice (KAHLE et al. 2005a)
indicate a relatively low absorption of flavan-3-ols (ca. 30%) based on low amounts detected
in body fluids. One explanation of such a result could be due to a poor bioaccessibility. There
are no published studies estimating the bioaccessibility of apple polyphenols either in vivo or
in vitro. The in vitro measurement of bioaccessibility is rather simple but has some major
drawbacks because it includes not only the release of flavan-3-ols from the food matrix, but
also their degradation during the digestion procedure and the formation of monomers and
171
PAPER 4
IN VITRO DIGESTION OF APPLES
dimers from dimers and oligomers respectively. The free monomeric and dimeric
procyanidins are reported to be stable during passage of the human stomach (RIOS et al.
2002). In contrast, in vitro digestions showed degradation of procyanidins due to the low pH
and the oxidative environment (SPENCER et al. 2000; ZHU et al. 2002) and depolymerisation
of procyanidins is reported to occur in acid milieu or by heating (GUYOT et al. 1998).
In apples, polyphenols are stored in vacuoles of intact cells (TREUTTER 2010). Fresh apples
are mainly eaten unprocessed with mechanical impact only from mastication. Consequently,
the destruction of the matrix could be a major influencing factor for polyphenol release. When
producing apple juice, however, the matrix is broken down leading to a higher release of
polyphenols, which could be degraded or removed during processing and filtration.
The aim of this study was to estimate the bioaccessibility of apple polyphenols by measuring
the recovery of flavan-3-ols and chlorogenic acid after in vitro digestion, and by quantifying
the degradation of pure apple flavan-3-ols during the digestion procedure and the formation of
monomers from procyanidin dimers. We conducted in vitro digestion experiments to evaluate
the bioaccessibility of flavan-3-ols and chlorogenic acid from various apple cultivars differing
in their polyphenol profile (CEYMANN et al. 2012) and, in order to assess a possible matrix
effect, fresh apples and freshly pressed apple juices were compared.
172
PAPER 4
IN VITRO DIGESTION OF APPLES
Materials and Methods
Sample preparation
Apple cultivars (cvs.) grown in Switzerland and covering a broad range of polyphenol content
and profile were used in the digestion studies. These included the dessert apple cvs. Golden
and Topaz, the cider apple cv. Berlepsch, as well as FxD 1253 and FxD 1184 (both crossbreds
of cvs. Fiesta and Discovery), and the red-fleshed cider and baking apple cv. Redfield which
was picked in 2008.
Samples were prepared directly after harvest as described by CEYMANN et al. (2011). Briefly,
20 fruits of each cultivar were sliced into 10 pieces and the core area with an apple divider
(Divisorex, Famos-Westmark GmbH, Lennestadt-Elspe, Germany). Two opposite slices of
each fruit were immediately frozen in liquid nitrogen. The frozen pieces were crushed in a dry
ice mill (Meidinger AG, Kaiseraugst, Switzerland) and ground into a fine powder with a
cutter (La moulinette DPA 1, Moulinex, Germany). Homogenous aliquots of frozen powder
(subsequently indicated as “fresh apple”) were stored in amber plastic bottles at -20 °C until
further use.
The identical frozen apple powder was used to prepare apple juice. After defrosting, the
material was squeezed in a hand squeezer equipped with a filter paper. The resulting juice was
centrifuged and the supernatant (subsequently indicated as “apple juice”) was stored in amber
plastic bottles at -20 °C until further use.
Extraction of polyphenols
Extraction of fresh apple was done as described previously (CEYMANN et al. 2011). Forty-five
mL of methanol containing 1 % formic acid (v/v) (both analytical grade) were added to 2.5 g
of frozen apple powder. The slurry was flushed with nitrogen and homogenized using a
Polytron (Polytron PT 3100, MERCK, Zug, Switzerland). The extracts were made up to
50 mL with extraction solution and allowed to stand for 1 h. The resulting supernatant was
used for quantification of individual polyphenols by UHPLC after being filtered through a 0.7
µm glass-fibre filter (OPTI-Flow®, WICOM, Heppenheim, Germany). Afterwards, samples
were diluted 1:1 with water containing 1 % formic acid (v/v) and filtered through a 0.2 µm
nylon filter (OPTI-Flow®, WICOM, Heppenheim, Germany) into a UHPLC vial. Extractions
were done in duplicate. Likewise, apple juice samples were filtered, diluted with water and
filtered again as described above.
173
PAPER 4
IN VITRO DIGESTION OF APPLES
In vitro gastric and intestinal digestion
To mimic the release of polyphenols from apple matrices, an in vitro digestion protocol was
elaborated based on the method as described by OOMEN et al. (2003). It could be shown in
pre-experiments that salivary incubation representing digestion in mouth did not improve the
release of polyphenols (results not shown). Therefore, this incubation step was omitted to
simplify the protocol.
Briefly, either 10 g of fresh apple or 15 mL of apple juice were added to 9 mL of artificial
gastric juice described by OOMEN et al. (2003) containing 13.6 mg bovine serum albumin
(BSA; Roth, Arlesheim, Switzerland), 13.6 mg of porcine pepsin ( ≥ 2500 units/mg protein,
Sigma-Aldrich, Buchs, Switzerland) and 10.6 mg mucin (Roth, Arlesheim, Switzerland). The
sample was incubated for 1 h under exclusion of light in a shaking water bath (Salvis,
Reussbuehl, Switzerland) at 37 °C and 90 strokes/min. To determine the recovery of
polyphenols during gastric incubation, an aliquot was removed for analysis. Subsequently,
6 mL of demineralised water and 18 mL duodenal juice containing 27 mg BSA as well as
81.1 mg porcine pancreatin (activity equivalent to 8 x UPS specifications) and 13.6 mg
porcine lipase (type II), both from Sigma-Aldrich (Buchs, Switzerland) were added. pH was
adjusted to pH 6.5 ± 0.5 and samples were incubated for 2 h as described above. Afterwards,
gastric incubation aliquots and small intestine digesta were centrifuged at 4000 g (Heraeus
Multifuge 3 SR+, Thermo Fisher Scientific, Osterode, Germany) for 5 min and the
supernatants were freeze dried overnight. Freeze dried residues were re-dissolved in methanol
containing 1 % formic acid (v/v) and further prepared for UHPLC analysis as described
below. For evaluating the effect of the digestion juices on the analytical methods, a sample
using water instead of apple juice was treated as described above. For determining flavan-3-ol
stability, 0.325 mg of catechin, epicatechin, procyanidin B1 and B2 respectively dissolved in
methanol and made up with water to 15 ml were subjected to the in vitro digestion as apple
juice. All digestions were done in duplicate.
Analytical methods
UHPLC analysis was carried out on an ACQUITY Ultra Performance LC™ system
(UPLC™) from Waters Corporation (Milford, USA) with binary solvent manager and
tuneable UV (TUV) detector. For instrument control, data acquisition and processing
Empower Pro 2 Software from Waters was used.
For separation a reversed phase column (BEH C18 1.7 μm, 2.1 x 50 mm with a BEH C18
1.7 μm VanGuardTM Pre-Column, 2.1 x 5 mm, Waters Corporation, Milford, USA) at 40 °C
174
PAPER 4
IN VITRO DIGESTION OF APPLES
was used. Samples (2.5 µL) were injected and the elution completed within 15 min. The
mobile phase consisted of (A) water with formic acid 0.1 % (v/v) and (B) methanol. Methanol
and formic acid (99 %) of UPLC/MS grade for preparing the mobile phase were acquired
from Biosolve (Valkenswaard, The Netherlands). Separations were carried out with a constant
flow rate of 0.35 mL/min using the following gradient: 0-8.5 min 5-13 % B; 8.5-9 min 1315 % B; 9-11.5 min 15-60 % B; 11.5-13 min, 60-95 % B; 13-15 min 5 % B. Flavan-3-ols
were monitored at 280 nm and chlorogenic acid at 320 nm.
Catechin, epicatechin (both Fluka, Buchs, Switzerland), procyanidin B1 and B2 (both
Extrasynthèse, Genay, France) as well as chlorogenic acid (Fluka, Buchs, Switzerland) were
used as standards. Quantification was achieved by an external calibration (linear range of 0.520 mg/L and correlation coefficients of 0.999). Detection limit was set at 0.05 mg/L and limit
of quantification at 0.5 mg/L for all five polyphenols. All samples were analysed twice and
data are reported in mg per 100 g fresh apple and 100 mL apple juice respectively.
Calculation of in vitro accessibility
Data are expressed as means of duplicates. In vitro accessibility was calculated as the amount
of polyphenols recovered in the supernatants after stomach as well as after stomach and small
intestine digestion respectively relative to the total amount measured in the starting material
(Table 1). Likewise, the results of in vitro accessibility determinations of pure flavan-3-ols are
given as recoveries (Table 2).
175
PAPER 4
IN VITRO DIGESTION OF APPLES
Results and discussion
Flavan-3-ol and chlorogenic acid content in apples and apple juices
The 6 apples used in the described experiments are characterised in Table 1 and showed a
high variability in polyphenol profile and content. The values of individual polyphenols
ranged from 0.5 mg/100 g FM for catechin in cv. Berlepsch to 33.2 mg/100 g FM for
procyanidin B2 in cv. Redfield. With the exception of cv. Redfield, catechin values of 0.50.8 mg/100 g FM, epicatechin values of 0.7-5.9 mg/100 g FM, procyanidin B1 values of 1.42.2 mg/100 g FM, procyanidin B2 values of 1.7-8.4 mg/100 g FM and chlorogenic acid
values of 6.7-21.9 mg/100 g FM were analysed by UHPLC-UV. Cv. Redfield, a cider and
baking apple with red flesh, contained exceptionally high levels of flavan-3-ols, whereas the
chlorogenic acid level of the cider cv. Berlepsch exceeded the value of cv. Redfield. These
values determined by UHPLC-UV are comparable to the values reported earlier by CEYMANN
et al. (2012) for the same cultivars analysed with a similar UHPLC-MS method CEYMANN et
al. (2011). The polyphenol levels of cv. Golden reported in Table 1 were also close (< 20 %
deviation) to the values reported by VRHOVSEK et al. (2004) with exception of the catechin
value, which was close to the quantification limit. The largest differences in the values
reported here compared to those reported in the earlier paper (CEYMANN et al. 2012) were for
cv. Berlepsch. One reason for this could be the different production years because pre-harvest
factors may influence the polyphenol content to some extent (TREUTTER 2010). On the other
hand, the flavan-3-ol to chlorogenic acid ratio in the different apple varieties was similar in
the present study to that reported by CEYMANN et al. (2012). Cvs. Topaz and Redfield are
flavan-3-ol dominated cultivars and contained a ratio higher than 2:1, cvs. Golden and
FxD 1253 belong to the group of cultivars with an equilibrated flavan-3-ol to chlorogenic acid
ratio, and cvs. FxD 1184 and Berlepsch are part of the cultivar group with a chlorogenic acid
dominated profile characterised by a ratio below 0.4:1 (CEYMANN et al. 2012). These
classifications did not change during pressing into apple juice (Table 1).
The contents of individual polyphenols in apple juices compared to fresh apples were variable
(Table 1). In commercial apple juices, they are clearly lower due to oxidation and absorbance
onto the pomace (ALONSO-SALCES et al. 2004a), and due to losses during clarification
(KAHLE et al. 2005a). During juice preparation in the present investigation (see sample
preparation) oxidation reactions might have occurred as well, but clarification losses were
mainly avoided. This could explain why cvs. Redfield and FxD 1184 showed comparable
amounts of polyphenols in both fresh apple and juices and why higher amounts were
determined in the juice of cv. Topaz with epicatechin, procyanidin B2 and chlorogenic acid
176
PAPER 4
IN VITRO DIGESTION OF APPLES
being nearly doubled. Chlorogenic acid is known to be easily water soluble. In the cv. Golden
juice only about half of the polyphenols of fresh apples were recovered. However, data
presented in Table 1 are similar to the amounts reported by KAHLE et al. (2005a).
Table 1:
Apples
Juices
Content of flavan-3-ols and chlorogenic acid in apples and apple juices. Data given in
mg/100 g apple and mg/100 ml apple juice, respectively (n=2, mean values, variability
of the duplicates < 32 %)
Topaz
Redfield*
Golden
FxD 1253
FxD 1184
Berlepsch
Topaz*
Redfield*
Golden*
FxD 1184*
Catechin
0.80
10.58
0.71
0.82
0.58
0.50
0.81
6.72
0.29
0.18
Epicatechin
5.83
32.36
4.52
5.89
1.63
0.65
8.69
29.00
2.40
1.84
Procyanidin B1 Procyanidin B2 Chlorogenic acid
2.14
8.44
6.74
10.20
33.24
19.59
1.41
8.01
9.99
1.42
5.11
13.82
1.42
2.20
15.83
2.18
1.68
21.91
2.71
13.28
10.64
9.59
27.45
17.76
0.61
2.73
6.85
1.51
1.96
17.20
*: n=1
Stability test with pure flavan-3-ols
Although the oligomeric procyanidins appear to be stable during digestion in vivo (RIOS et al.
2002) they are extensively degraded in vitro (SPENCER et al. 2001). In order to compare the
influence of pH and digestion enzymes on flavan-3-ols, we digested in vitro the main
monomeric and dimeric apple flavan-3-ols without matrix. Table 2 shows their recovery after
stepwise in vitro digestion, i.e. gastric digestion, and gastric digestion followed by intestinal
digestion. The monomers catechin and epicatechin were approximately 30 % recovered after
gastric digestion and 40 % recovered after gastric plus intestinal digestion. As reported
previously by SPENCER at al. (2000) and ZHU et al. (2002), the procyanidin dimers were
extensively degraded during digestion. In our study some 10-15 % of catechin or epicatechin
were generated on the breakdown of these compounds possibly due to the low gastric pH or
as a result of oxidation reactions. Relatively high amounts of epicatechin were detected
following the digestion of catechin, which indicates an epimerisation between the different
flavan-3-ols. Surprisingly, we could also detected traces of dimers after the incubation of both
monomeric flavan-3-ols. Epimerisation has also been reported in simulated intestinal juice by
ZHU et al. (2002), but the possible formation of procyanidin B2 from monomers is described
here for the first time. Hence, the in vitro digestion procedure appeared to influence the
procyanidin dimers differently. While both were extensively degraded, procyanidin B1 was
additionally epimerised to procyanidin B2. ZHU et al. (2002) described a comparable
observation with procyanidin B2 being produced after incubation of procyanidin B5 but not
177
PAPER 4
IN VITRO DIGESTION OF APPLES
vice versa. Additionally, they reported that in vitro incubation of oligomeric procyanidins
(dimer to hexamer) resulted in the recovery of mainly dimers, which were not further
examined.
Table 2:
Recovered as:
Recovery of individual flavan-3-ols in simulated stomach and in simulated small
intestine after in vitro accessibility test. Data given % (n=2, mean values, variability of
the duplicates < 11 %)
After simulation of:
Stomach
Small intestine
Stomach
Epicatechin
Small intestine
Stomach
Procyanidin B1
Small intestine
Stomach
Procyanidin B2
Small intestine
nd: not detected; tr: traces (< 5 %)
Catechin
Catechin
34
41
nd
16
nd
nd
tr
tr
Incubation of:
Epicatechin
Procyanidin B1
nd
11
tr
7
nd
30
14
42
nd
nd
nd
tr
tr
tr
tr
tr
Procyanidin B2
nd
4
15
14
nd
nd
tr
6
Recovery of flavan-3-ols and chlorogenic acid from apples compared to apple juices
In order to evaluate matrix effects, all apple cultivars and apple juices were subjected to the in
vitro digestion. Table 3 represents the amount of individual polyphenols recovered after
simulated stomach and simulated stomach plus small intestine incubation respectively. The
recoveries of the flavan-3-ol monomers represent a combination of the monomers released
from the matrix plus monomers produced by degradation of di- and oligomeric procyanidins
minus the monomers degraded during the simulated digestions plus potential epimerisation
products. Based on the experiments with pure catechin and epicatechin, approximately 60 %
of the monomers were degraded after the digestion process, but whether epimerisation
products are included remains open. Accordingly, monomer recoveries from fresh apples and
apple juices might comprise different fractions. For the dimers, a comparable assumption can
be made, i.e. the recoveries might be composed of dimers released from the apple matrix and
stable during digestion, and dimers produced by degradation of oligomers during digestion.
When incubating fresh apples, the mean recoveries after simulated stomach and simulated
stomach plus small intestine respectively were 70 % and 81 % for catechin, 44 % and 73 %
for epicatechin, 33 % and 43 % for procyanidin B1 and 40 % and 47 % for procyanidin B2,
i.e. a non-significant increase in mono- and dimeric flavan-3-ols during small intestine
incubation was observed. An increase if it occurred could be explained by the further
digestion of the cellular structures by the small intestine milieu. Similar observations were
reported by BOUAYED et al. (2011), who also found an increase of small intestine total
178
PAPER 4
IN VITRO DIGESTION OF APPLES
phenolic values compared to simulated stomach when investigating four apple cultivars with a
comparable in vitro method. They concluded that the main release of apple polyphenols takes
place in the stomach with a slight further release in small intestine.
Table 3:
In vitro accessibility of polyphenols from fresh apples and juices after in vitro
simulated stomach and small intestine digestion respectively. Data given as % of
amount determined in starting material (n=2, mean values, variability: 0-106 %)
Topaz
Redfield
Golden
FxD 1253
FxD 1184
Berlepsch
fresh apples
160
58
53
38
39
catechin a
101
36
80
69
93
b
45
53
41
43
40
epicatechin a
69
41
53
48
65
b
38
60
27
38
33
procyanidin B1 a
54
44
29
38
63
b
38
43
35
51
30
procyanidin B2 a
54
30
32
45
41
b
53
51
39
40
41
chlorogenic acid a
58
30
51
51
60
b
juice
87
73
91
94
catechin a
58
16
106
145
b
51
48
40
34
epicatechin a
32
15
35
34
b
74
72
43
55
procyanidin B1 a
36
18
19
43
b
52
48
41
36
procyanidin B2 a
27
12
26
31
b
48
44
47
37
chlorogenic acid a
29
18
40
32
b
a after simulated gastric digestion, b after simulated gastric and duodenal digestion, nd: not detected
74
105
46
165
nd
32
46
79
55
44
It should be noted, however, that the concentrations of catechin and procyanidin B1 (except
cv. Redfield) were close to the quantification limit, (< 2 mg/100 g apple FW and < 3 mg/100
ml juice respectively, Table 1) and may explain the wide variability and recoveries >100 %
(Table 3).
In contrast, after the in vitro digestion of apple juice, mean recoveries were higher after
simulated stomach than after simulated stomach plus small intestine with 86 % and 81 % for
catechin, 43 % and 29 % for epicatechin, 61 % and 29 % for procyanidin B1 and 44 % and
24 % for procyanidin B2. The bioaccessibility of polyphenols in beverages has been reported
to be 100% (PALAFOX-CARLOS et al. 2011) therefore apple juice polyphenols are expected to
be completely released from the cellular structures during pressing. The recoveries of our
digested juices were lower, which can be largely explained by progressively more degradation
of the polyphenols during the in vitro digestion procedure. As reported for pure flavan-3-ols,
179
PAPER 4
IN VITRO DIGESTION OF APPLES
the recoveries tend to be higher for monomers than for dimers, which could be due to
degradation of dimers to monomers. Nevertheless, procyanidin B1 and B2 embedded in a
fresh apple or juice matrix showed higher recoveries (Table 3) than the pure flavan-3-ol
dimers (Table 2). This could be due to the protective effect of the matrices, but also due to the
degradation of oligomeric procyanidins. Monomeric flavan-3-ols showed also higher
recoveries with matrix (Table 3) than without matrix (Table 2) presumably due to the
generation of monomers from oligomers. This degradation seems to occur in vitro (SPENCER
et al. 2000; TARKO et al. 2009; ZHU et al. 2002) but in vivo cocoa monomeric flavan-3-ols and
procyanidins have been reported to be stable during passage of the human stomach sampled
with a nasogastric tube (RIOS et al. 2002). OTTAVIANI et al. (2012) confirmed this findings by
feeding flavan-3-ols with different polymerisation degrees to humans. Monomeric flavan-3ols resulted in high plasma levels, whereas dimers to decamers were absent in plasma.
Another study (SERRA et al. 2010) investigating grape seed procyanidins in vitro and in vivo,
reported stability in stomach, but a decomposition of the highly polymerised procyanidins in
the small intestine. This is in accordance with the reduced polymerisation degree of
procyanidins analysed in the ileostomy effluent after ingestion of 1 L of a cloudy apple juice
compared to the juice itself (KAHLE et al. 2007). The authors suggested that the oligomeric
procyanidins were cleaved during digestion in stomach and small intestine. Therefore, it
seems possible that monomeric and dimeric procyanidins remain stable during passage of the
stomach and the small intestine whereas higher oligomeric procyanidins are depolymerised.
This leads to increasing amounts of monomers and dimers in the recoveries after simulated
small intestine in vitro.
The mean recoveries of chlorogenic acid from fresh apples after simulated stomach and after
stomach followed by small intestine were similar at 46 % and 49 %, respectively, whereas the
corresponding mean recoveries of apple juices were 44 % and 30 %. If we assume that
practically all the chlorogenic acid in apple juice is accessible then the some 60-70% is
degraded by the digestion procedure. As the recoveries of chlorogenic acid after simulated
gastric digestion of fresh apples and apple juice are similar, we can assume that most of the
chlorogenic acid in fresh apples is also accessible. However, there would appear to be a slight
matrix effect as chlorogenic acid recovery from fresh apples increased after the additional
intestinal digestion step. In the in vitro digestion procedure, polyphenols embedded in the
fresh apple matrix are better protected than in apple juice, but are susceptible to degradation
when released, first during simulated gastric digestion and then during simulated intestinal
digestion.
180
PAPER 4
IN VITRO DIGESTION OF APPLES
Because of the extensive polyphenol degradation occurring during the in vitro procedure but
not in vivo it is difficult to extrapolate the results from polyphenol recovery in our in vitro
studies to bioaccessibility in humans. Additionally, it is not sure whether transformation of
flavan-3-ol oligomers to monomers and dimers, or flavan-3-ol epimerization, which occur in
vitro, also occurs in vivo. SERRA et al. (2010) reported degradations of highly polymerised
procyanidin in vivo but oligomeric ones remained stable during digestion. Nevertheless, if we
assume that the polyphenols in apple juice are more or less 100% accessible, as would be
expected since they are released during pressing from the apple cellular structures, we can
make some conclusions concerning the in vitro procedure and bioaccessibility in vivo. Unlike
during digestion in vivo, the present in vitro procedure leads to extensive degradation of the
monomeric and dimeric flavan-3-ols and chlorogenic acid. Modifying the in vitro procedure
to exclude oxygen may overcome this problem. However, as the recoveries of polyphenols
from apples and apple juice were similar after the simulated gastric digestion, as was the
recovery of the control monomeric flavan-3-ols, we can also predict that the release of
polyphenols from apple flesh was substantial although perhaps not complete. There was a
further release of polyphenols from apple flesh after the simulated gastric digestion. One
reason for the high recovery of the polyphenols from apple after gastric digestion, and the
relatively small matrix effect, could be the extensive homogenization which the apples
underwent before the in vitro digestion. An additional experiment with fresh apples crushed
into small pieces rather than homogenized resulted in lower recoveries after gastric digestion
as compared to the finely ground fresh apple but similar recoveries after additional intestinal
digestion (Erni unpublished data).
In conclusion, our results indicate that predicting accessibility using the current in vitro
method is difficult because the recovery of polyphenols after in vitro digestion is a
combination of their release from the food structures, degradation during the in vitro
procedure, formation by the breakdown of oligomers, and epimerization. However, as
recovery of flavan-3-ols and chlorogenic acid from apples was similar to that from apple
juice, after simulate gastric digestion we can assume that accessibility of the polyphenols is
high and that they are mostly released in the stomach. Bioaccessibility of apple polyphenols
therefore does not appear to be an issue when evaluating potential health effects of apples.
181
PAPER 4
IN VITRO DIGESTION OF APPLES
References
Alonso-Salces, R. M., A. Barranco, et al. (2004a). "Polyphenolic profiles of Basque cider apple
cultivars and their technological properties." Journal of Agricultural and Food Chemistry 52:
2938-2952.
Awad, M. A., A. Jager de, et al. (2000). "Flavanoid and chlorogenic acid levels in apple fruit:
characterisation of variation." Scientia Horticulturae 83: 249-263.
Baba, S., N. Osakabe, et al. (2000). "Bioavailability of (-)-epicatechin upon intake of chocolate and
cocoa in human volunteers." Free Radical Research 33(5): 635-641.
Bidel, S., G. Hu, et al. (2008). "Coffee consumption and type 2 diabetes - An extensive review."
Central European Journal of Medicine 3(1): 9-19.
Bouayed, J., L. Hoffmann, et al. (2011). "Total phenolics, flavonoids, anthocyanins and antioxidant
activity following simulated gastro-intestinal digestion and dialysis of apple varieties:
Bioaccessibility and potential uptake." Food Chemistry 128(1): 14-21.
Ceymann, M., E. Arrigoni, et al. (2011). "Rapid high performance screening method using UHPLCMS to quantify 12 polyphenol compounds in fresh apples." Analytical Methods 3: 1774.
Ceymann, M., E. Arrigoni, et al. (2012). "Identification of apples rich in health-promoting flavan-3-ols
and phenolic acids by measuring the polyphenol profile." Journal of Food Composition and
Analysis 26(1-2): 128–135.
DuPont, M. S., R. N. Bennett, et al. (2002). "Polyphenols from alcoholic apple cider are absorbed,
metabolized and excreted by humans." Journal of Nutrition 132(2): 172-175.
FAOSTAT. (2012a). "Food consumption data." Retrieved 21 March 2012, from
http://faostat.fao.org/site/609/DesktopDefault.aspx?PageID=609#ancor.
Fogliano, V., M. L. Corollaro, et al. (2011). "In vitro bioaccessibility and gut biotransformation of
polyphenols present in the water-insoluble cocoa fraction." Molecular Nutrition and Food
Research 55 Suppl 1: S44-55.
Guyot, S., N. Marnet, et al. (1998). "Reversed-phase HPLC following thiolysis for quantitative
estimation and characterisation of the four main classes of phenolic compounds in different
tissue zones of a French cider apple variety (Malus domestica Var. Kermerrien)." Journal of
Agricultural and Food Chemistry 46: 1698-1705.
Harker, F. R., F. A. Gunson, et al. (2003). "The case for fruit quality: an interpretive review of
consumer attitudes, and preferences for apples." Postharvest Biology and Technology 28(3):
333-347.
Holt, R. R., S. A. Lazarus, et al. (2002). "Procyanidin dimer B2 [epicatechin-(4beta-8)-epicatechin] in
human plasma after the consumption of a flavanol-rich cocoa." American Journal of Clinical
Nutrition 76(4): 798-804.
Hooper, L., C. Kay, et al. (2012). "Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular
health: a systematic review and meta-analysis of randomized trials." American Journal of
Clinical Nutrition 95(3): 740-751.
Kahle, K., W. Hümmer, et al. (2007). "Polyphenols are intensively metabolized in the human
gastrointestinal tract after apple juice consumption." Journal of Agricultural and Food
Chemistry 55: 10605-10614.
Kahle, K., M. Kraus, et al. (2005a). "Polyphenol profiles of apple juices." Molecular Nutrition and
Food Research 49: 797 – 806.
Mangas, J. J., R. Rodriguez, et al. (1999). "Study of the phenolic profile of cider apple cultivars at
maturity by multivariate techniques." Journal of Agricultural and Food Chemistry 47: 40464052.
Neilson, A. P. and M. G. Ferruzzi (2011). "Influence of formulation and processing on absorption and
metabolism of flavan-3-ols from tea and cocoa." Annual Review of Food Science and
Technology 2: 125-151.
Neveu, V., J. Perez-Jiménez, et al. (2010). Phenol-Explorer: an online comprehensive database on
polyphenol contents in foods, online Database. Version 1.5.2, available at http://www.phenolexplorer.eu.
Oomen, A. G., C. J. M. Rompelberg, et al. (2003). "Development of an in vitro digestion model for
estimating the bioaccessibility of soil contaminants." Archives of Environmental
Contamination and Toxicology 44(3): 281-287.
182
PAPER 4
IN VITRO DIGESTION OF APPLES
Oszmianski, J., M. Wolniak, et al. (2007). "Comparative study of polyphenolic content and antiradical
activity of cloudy and clear apple juices." Journal of the Science of Food and Agriculture 87:
573-579.
Ottaviani, J. I., C. Kwik-Uribe, et al. (2012). "Intake of dietary procyanidins does not contribute to the
pool of circulating flavanols in humans." American Journal of Clinical Nutrition 95(4): 851858.
Palafox-Carlos, H., J. F. Ayala-Zavala, et al. (2011). "The role of dietary fiber in the bioaccessibility
and bioavailability of fruit and vegetable antioxidants." Journal of Food Science 76(1): R6R15.
Rios, L. Y., R. N. Bennett, et al. (2002). "Cocoa procyanidins are stable during gastric transit in
humans." American Journal of Clinical Nutrition 76(5): 1106-1110.
Ryan, L. and S. L. Prescott (2010). "Stability of the antioxidant capacity of twenty-five commercially
available fruit juices subjected to an in vitro digestion." International Journal of Food Science
and Technology 45(6): 1191-1197.
Serra, A., A. Macià, et al. (2010). "Bioavailability of procyanidin dimers and trimers and matrix food
effects in in vitro and in vivo models." British Journal of Nutrition 103(07): 944-952.
Sies, H. (2010). "Polyphenols and health: Update and perspectives." Archives of Biochemistry and
Biophysics 501(1): 2-5.
Spencer, J. P., H. Schroeter, et al. (2001). "Epicatechin is the primary bioavailable form of the
procyanidin dimers B2 and B5 after transfer across the small intestine." Biochemical and
Biophysical Research Communications 285(3): 588-593.
Spencer, J. P. E., F. Chaudry, et al. (2000). "Decomposition of cocoa procyanidins in the gastric
milieu." Biochemical and Biophysical Research Communications 272(1): 236-241.
SwissFruitAssociation. (2012). "Wissens-Wertes Früchte." Retrieved 21 March 2012.
Tarko, T., A. Duda-Chodak, et al. (2009). "Transformations of phenolic compounds in an in vitro
model simulating the human alimentary tract." Food Technology and Biotechnology 47(4):
456-463.
Treutter, D. (2010). "Managing phenol contents in crop plants by phytochemical farming and
breeding-visions and constraints." International Journal of Molecular Sciences 11(3): 807857.
Tsao, R., R. Yang, et al. (2003). "Polyphenolic profiles in eight apple cultivars using highperformance liquid chromatography (HPLC)." Journal of Agricultural and Food Chemistry
51: 6347-6353.
Vrhovsek, U., A. Rigo, et al. (2004). "Quantitation of polyphenols in different apple varieties."
Journal of Agricultural and Food Chemistry 52(21): 6532-6538.
Wojdylo, A., J. Oszmianski, et al. (2008). "Polyphenolic compounds and antioxidant activity of new
and old apple varieties." Journal of Agricultural and Food Chemistry 56: 6520-6530.
Zhu, Q. Y., R. R. Holt, et al. (2002). "Stability of the flavan-3-ols epicatechin and catechin and related
dimeric procyanidins derived from cocoa." Journal of Agricultural and Food Chemistry 50(6):
1700-1705.
183
184
CONCLUSIONS
Epidemiological studies have indicated that an increased intake of polyphenols is related to
improve health. Apples contain four main groups of polyphenols, which are thought to be
associated with beneficial health effects. These are flavan-3-ols, phenolic acids,
dihydrochalcones and flavonols. The main postulated health effects of flavan-3-ols are an
improvement of CVD, and phenolic acids are considered to support weight reduction by
improving the glucose metabolism. Dihydrochalcones have been reported to inhibit glucose
transporters and thus may reduce the development of type 2 diabetes, whereas flavonols are
thought to prevent various cancers. Therefore the saying “An apple a day keeps the doctor
away” seems to be plausible but, due to the extensive diversity in the composition of apples,
general statements about beneficial effects are not possible. This thesis was designed to
evaluate the factors influencing both polyphenol contents and profiles of different apple
cultivars so that potential health effects could be defined based on apple composition.
To determine the polyphenol composition of apples, a rapid screening method using UHPLCMS was developed. A simple extraction method without any concentration step during sample
preparation allows the identification and quantification of the 12 most important individual
polyphenols, i.e. the flavan-3-ols catechin, epicatechin, procyanidin B1 and B2, the phenolic
acids chlorogenic acid and coumaroylquinic acid, the dihydrochalcones phloridzin and
phloretin-xyloglucoside and the flavonols quercetin-galactoside, -glucoside, -rhamnoside and
rutin. In the same extracts, the total polyphenol content by Folin-Ciocalteu and the
antioxidative potential by TEAC and FRAP can be measured and used as pre-screening
method. The sample preparation to investigate fresh apples was adapted to consumer
behaviour by removing the core area, but not the skin. The analysis of more than 100 different
apples cultivars mainly grown in Switzerland demonstrated a significant variability between
the polyphenol contents and profiles in apples. The amount of individual polyphenols ranged
from below the limit of detection to 70 mg/100 g FM, whereby epicatechin, procyanidin B2
and chlorogenic acid are the predominate polyphenols in apples. The highest flavan-3-ol
(catechin, epicatechin, procyanidin B1 and B2) concentration was in average 55 times higher
than the lowest one and for phenolic acids, the mean variability was 71. The amount of
dihydrochalcones varied 31-fold, whereas the variability of flavonols was lower with only
ten-fold.
By choosing the correspondent cultivars from this wide range, it appears possible to reach
levels of the different polyphenol classes which have been reported to have positive health
185
effects. For flavan-3-ols, a daily ingested dose of 22 mg epicatechin has been reported to be
sufficient to improve FMD. This level could be achieved by eating daily one apple (150 g) of
the cultivars Grüner Fürstenapfel or Burgunder, both cider apples. Moreover, also the
common dessert apple cultivars Milwa, Elstar Reinhard, Fuji, Gala Schniga, Otava,
Schneiderapfel and Topaz were found to exceed the 22 mg epicatechin, when two fruits per
day were ingested. To obtain weight reduction by the ingestion of 700 mg chlorogenic acid
per day, seven fruits of the cultivar Grüner Fürstenapfel have to be eaten per day. The
minimum level reported for achieving this effect is rather high, but so far only one study
investigated the weight reduction in humans after chlorogenic acid intake. Conducting an
intervention study with two apples per day of the cultivar Maigold or Boskoop, leading to an
intake of 74-80 mg/d of chlorogenic acid, would be interesting to study, if a weight reduction
in overweight or obese humans would be obtained. Moreover, cultivars Boskoop and
Schneiderapfel could be considered to reach sufficient dihydrochalcone levels (12 mg) to
reduce the risk of type 2 diabetes, when a fruit per day is eaten and with two apples per day,
this level could be obtained with half of the other investigated cultivars. For reducing the risk
of pancreatic cancer, smokers could be advised to eat two fruits of cultivars Idared or Pinova
per day, which contained quercetin-glycoside levels (10 mg/d) reported to obtain this effect.
In general, to reduce the risk of cancer, red-skin apples should be chosen, because the
biosynthesis of quercetin-glycosides correlates with the red colour of the apple skin.
Depending on their polyphenol profile, apples can be divided into flavan-3-ol dominated and
phenolic acid dominated cultivars or in cultivars with equilibrated amounts of flavan-3-ols
and phenolic acids. Phenolic acid dominated cultivars have a flavan-3-ol to phenolic acid ratio
of 0.1-0.9. Examples for these cultivars are the cvs. Santana and Jonagold. In contrast, the
cvs. Topaz and Nicogreen are flavan-3-ol dominated cultivars with ratios of 2.3 and 11.7
respectively. Examples for cultivars with an equilibrated ratio are cvs. Gala and Golden
Delicious. A flavan-3-ol dominated apple cultivar can contain equal amounts of epicatechin in
one serving as cocoa and chocolate, whereas a phenolic acid dominated cultivar can reach
levels of chlorogenic acid comparable to one serving size of coffee. It therefore seems
possible, to predict the main health effect of an apple cultivar with the knowledge of flava-3ol to phenolic acid ratio.
Pre-harvest factors reported to influence the polyphenol content of apples are production
method, growing environment and year, rootstock, crop load and mutant variety.
Investigations of these pre-harvest factors showed a minor impact on apple polyphenol
186
contents. The main influencing factor was the cultivar, but mutant variety and the growing
year had some influence in the investigated apple cultivars as well. The other pre-harvest
factors appear negligible, including the often discussed comparison of organically and
conventionally produced fruits. Higher polyphenol values in organically produced fruits
reported in literature are often cultivar dependent rather than caused by the organic treatment.
Moreover, the polyphenol profile remains unaffected by all pre-harvest factors except for the
cultivar. Therefore, breeding new apple cultivars appears to be the method of choice to
increase polyphenol content and to vary the polyphenol profile. Apple growers should be
advised to choose their apple cultivar with regard to the polyphenol content and profile due to
the low influence of pre-harvest factors. The year-to-year variation and the influence of other
pre-harvest factors could be analysed by simple photometric methods such as Folin-Ciocalteu,
TEAC and FRAP, due to the fact that these methods correlate well with the sum of individual
polyphenols determined by UHPLC-MS. These methods are simple to conduct and allow to
screen plenty of new cultivars and thus the determination of individual polyphenols may only
be necessary in interesting cultivars.
To evoke beneficial health effects, apple polyphenols have to be released from the apple
matrix. By means of an in vitro digestion method, it has been shown that 57 % of the
monomeric and 37 % of the dimeric flavan-3-ols were recovered from fresh apple matrix after
the simulated stomach and 77 % and 45 % after simulated stomach plus small intestine. For
apple juice, recoveries after simulated stomach and simulated stomach plus small intestine
were found to be inverted with 65 % and 53 % for monomers and 55 % and 27 % for dimers,
respectively. Overall, the recovery of dimeric procyanidins was reduced compared to
monomeric catechin and epicatechin, because degradation and epimerisation occurred. For
chlorogenic acid, comparable results were obtained. The prediction of in vivo bioaccessibility
from the in vitro data is difficult due to the in vitro recovery being a combined value of
released polyphenols from the food, their degradation during simulated digestion, breakdown
products from oligomers and epimerization. In addition, during in vivo digestion, flavan-3-ols
appear to be more stable, which indicates, that the exposure to oxygen and light may be
responsible for the degradations in simulated digestion experiments. To improve the
usefulness of in vitro digestion, it should be carried out under exclusion of light and oxygen.
The latter could be obtained by conducting the whole digestion in a bag flushed with nitrogen
without opening it until analysis. However, epicatechin has been shown to be the most stable
flavan-3-ol, which correlates well with its contribution to the postulated health effects of
flavan-3-ols.
187
Bioaccessibility of apples and apple juice was high (50 %) with the main release in the
simulated stomach indicating that the knowledge of bioaccessibility values is not a limiting
factor in evaluating beneficial health effects of apples. However, the literature data indicated
fractional absorption for both flavan-3-ols and phenolic acids being 30 %, whereas for
dihydrochalcones and flavonols the fractional absorption was higher with 60 % and 50 % due
to their glycoside moiety. These values should be considered for the administration of
polyphenols to investigate health effect so that the polyphenols could reach physiological
levels in the body.
In summary, polyphenol contents and profiles in apples can be modulated by selecting
cultivars and to a smaller extent by some pre-harvest factors. By choosing an apple with a
flavan-3-ol dominated profile, for example cvs. Topaz or Schneiderapfel, prevention of CVD
appears possible, if this apple cultivar is consumed twice a day. To reduce weight, cultivars
with high chlorogenic acid levels, for example cv. Grüner Fürstenapfel might be chosen,
whereby seven apples a day are needed. Reduction of type 2 diabetes appears possible to be
achieved with the ingestion of one apple of the cultivars Boskoop or Schneiderapfel due to
their high dihydrochalcone levels. Furthermore, apples with a red skin are supposed to reduce
the risk of various cancers.
The above mentioned saying should therefore be adapted to “Two apples a day keep the
doctor away”. Regarding apple consumption data, the Swiss population should be advised to
increase their apple consumption four- to five-fold to reach this recommendation and to
possibly obtain any beneficial health effect. Nevertheless, since the postulated health effects
are mainly investigated with other foods such as chocolate and coffee, studies investigating
bioavailability and health effects of apple polyphenols are still needed to confirm the
postulated health benefits of cultivars rich in polyphenols.
188
CURRICULUM VITAE
Maria Ceymann
born 26.05.1982
in Krumbach (Schwaben), Germany
Education
2007-2013
PhD Thesis in the field Human Nutrition
Research Station Agroscope Changins-Wädenswil in
Wädenswil, Switzerland
in collaboration with
Laboratory of Human Nutrition
Institute of Food Science and Nutrition, ETH Zurich,
Switzerland
2006-2007
2. Staatsexamen in Food Chemistry
Bavarian Health and Food Safety Authority, Erlangen, Germany
2001-2006
1. Staatsexamen in Food Chemistry
Julius-Maximilians-Universität Würzburg, Germany
1992-2001
High School (Gymnasium)
Simpert-Kraemer-Gymnasium Krumbach (Schwaben), Germany
1988-1992
Primary School
Grundschule Deisenhausen, Germany
189