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C H A P T E R
84
c0084
The Use of Bergamot-derived Polyphenol
Fraction in Cardiometabolic Risk Prevention
and its Possible Mechanisms of Action
Ross Walker*, Elzbieta Janda† and Vincenzo Mollace‡
*Consultant Cardiologist, Sydney Adventist Hospital, Sydney, Australia †Department of Health Sciences, University
“Magna Graecia,” Campus Salvatore Venuta, Germaneto, Catanzaro, Italy ‡Department of Health Sciences, University
“Magna Graecia” of Catanzaro, Complesso Nini’ Barbieri, Roccelletta di Borgia, Catanzaro, Italy
s0010
p0010
p0015
p0020
1. INTRODUCTION
Hypercholesterolemia (increased cholesterol levels),
hyperlipidemia (increased triglycerides (TG) and cholesterol) and hyperglycemia (blood glucose over
100 mg/dL) are important risk factors for the development of atherosclerosis and coronary artery disease.1,2
Increased concentrations of total blood cholesterol
(tChol, $ 200 mg/dL) as well as high ratio of cholesterol bound to low-density lipoprotein (cLDL) with
respect to the cholesterol bound to high-density lipoprotein (cHDL) are considered to be the main pathogenic blood parameters. However, conditions of
insulin resistance such as impaired glucose tolerance
or prediabetes are also characterized by a high risk of
atherosclerotic vascular diseases (AVD).3 The presence
of all cardiometabolic risk factors that occur in the metabolic syndrome particularly accelerate atherosclerotic
changes, and are treated with polypharmacy, as discussed in Section 3.1.
Experimental and epidemiological evidence suggests that dietary polyphenols, such as flavonoids,
may prevent atherosclerosis by counteracting its risk
factors.46 Accordingly, better clinical results by
increasing the dosage and quality of consumed flavonoids should be expected. This idea has been exploited
in the case of bergamot juice flavonoids and proved to
work in clinical practice.
Bergamot (Citrus bergamia Risso et Poiteau) belongs
to the family Rutaceae, genus Citrus, and is an endemic
Polyphenols in Human Health and Disease.
DOI: http://dx.doi.org/10.1016/B978-0-12-398456-2.00084-0
plant from the Calabrian region in Italy. The plant is
very sensitive to the pedoclimatic conditions of the soil
and it grows almost exclusively in the narrow coastal
area from Reggio Calabria to Locri, in the most southern part of the Italian Peninsula (Figure 84.1).
Bergamot juice was traditionally recognized by the
local population of Calabria, as a remedy for “fatty
arteries” and heart problems. The medicinal use of bergamot derivatives, forgotten for decades, is now being
rediscovered. This started from a demonstration in animal models of hypolipemic properties of bergamot
juice.7 Next, Mollace and co-workers8 addressed the
efficacy of a concentrated mixture of bergamot flavonoids, so-called Bergamot Polyphenol Fraction (BPF),
in experimental and clinical settings. These authors
showed, using animal models and through human
studies, that BPF is effective in combating several
symptoms of metabolic syndrome such as increased
tChol, cLDL and TG, increased blood glucose levels
and endothelial dysfunction in a group of metabolic
syndrome patients. The effects on mean cholesterol
parameters were comparable with a moderate dose of
simavastatin (20 mg daily), including a marked
increase in cHDL levels. In addition, 1000 mg BPF
taken for 30 days reduced moderate hyperglycemia by
more than 20%.8
The brilliant performance of BPF on MS and dyslipi- p0025
demia is astonishing and needs a mechanistic explanation. Unfortunately, so far there is very limited
experimental evidence proving the modulation of one
1085
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84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
FIGURE 84.1 Mature bergamot fruits of Femminello cultivar (left) and the geographical distribution of bergamot cultivars in Calabria.
Bergamot plantations are restricted to a narrow costal area in the most south part of Italian peninsula, between Reggio Calabria and Locri.
This is due to high sensitivity to pedoclimatic conditions of bergamot trees.
p0030
or another metabolic pathway by BPF itself. However,
as discussed in Section 5, the vast scientific literature
suggests that certain individual flavonoids present in
BPF are implicated in the regulation of several metabolic enzymes, expressed in the liver, blood and endothelial cells. In addition, natural polyphenols show less
specific, antioxidant properties, that depend on the
free radical scavenging ability of hydroxyl groups
linked to carbon aromatic rings.911 Together with
antioxidant properties, dietary flavonoids and their
metabolites may modulate basic signal transduction
pathways of every cell leading to anti-proliferative,
anti-aging and immune responses as well as other beneficial effects for human health, as discussed in other
chapters of this volume and in the vast literature to be
found on the subject.9,10 Finally, besides intracellular,
molecular effects, flavonoids, in cooperation with pectins, can work at the level of intestine and liver to stimulate fat excretion and reduce fat absorption, which
augments the direct activity on enzymes, involved in the
regulation of carbohydrate and lipid metabolism.7,12,13
In this chapter, we will discuss chemical and pharmacological properties of flavonoids, present in bergamot
fruits (Section 2). Section 3 will examine the experimental evidence, along with clinical and observational stud-
ies demonstrating efficacy of BPF in combating
cardiometabolic risk factors. The use of BPF, its advantages and limitations will be discussed in the context of
conventional statin therapy (Section 4). Section 5 will
focus on possible molecular mechanisms of action of
bergamot polyphenols that could account for the therapeutic effects of BPF.
2. CHEMICAL AND FUNCTIONAL
CHARACTERIZATIONS OF BERGAMOT
FLAVONOIDS
s0015
Bergamot juice is particularly rich in flavanone- p0035
O-glycosides (see Table 84.1) and is characterized by a
unique profile of flavonoids, showing partial similarity
to Citrus x myrtifolia Raf. (chinotto)14 and Citrus aurantium L.15 It contains relatively rare neoeriocitrin and
neohesperidin and, as recently discovered, rare brutieridin
and
melitidin
bearing
3-hydroxy3-methylglutaric acid (HMG) moiety16 (Figure 84.2, see
also Plate 13). High-throughput analysis of flavonoid
content in 45 citrus species and cultivars, reported by
Nogata et al.,17 shows that the amount of the flavonoids per volume unit of juice, or per mass unit of
8. CARDIAC HEALTH AND POLYPHENOLS
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2. CHEMICAL AND FUNCTIONAL CHARACTERIZATIONS OF BERGAMOT FLAVONOIDS
t0010
TABLE 84.1
1087
Flavonoid Content in Bergamot Fruits (Juice and Albedo).
Content
Juice
mg/L
Albedo
mg/kg
Position on the
Ranking List
for Flavonoid
Content*
Naringin
248b, 523a
670a
3a
Neohesperidin
295b, 763a
686a
1a
Neoeriocitrin
296b, 293a
435a
1a
Bruteridin
230c
250300d
1e
Several times higher than in C. aurantium
and C. mirtifolia.
Melitidin
133c
150200d
1e
Several times higher than in C. aurantium
and C. mirtifolia.
Poncirin (7-O-neohesperidoside
of isosakuranetin)
64f, 1870 (?)a
777a
1a
Big difference, because of comparing
hand-squeezed juicea with industrial juiceb.
Eriocitrin
13.415.6b 13.4a
6.3a
Relatively low content compared
to other Citrus fruits
Narirutin
17.7a
13.9a
Relatively low content compared
to other Citrus fruits
Rhoifolin
53.268.1b 39.9a
21a
1a,f
Industrial juicef
Neodiosmin
19.027.1b 48.1a
13.8a
1f2a
Industrial juicef
Rutin
28.3a
0a
3a
Vicenin-2 (Apigenin 6,8-di-C-glucoside)
58.363.2b,f
1f
Industrial juicef
Diosmetin 6,8-di-C-glucoside
(Lucenin-2 40 -methyl ether)
37.949.7b
2f
Industrial juice, second conc. after Citrus limonf
Chrysoeriol 7-O-neohesperoside
40 glucoside
11.613.2b
1f
Industrial juicef
Stellarin-2 (Chrysoeriol 6,
8-di-C-glucoside)
5.87.3b
Lucenin-2 (Luteolin 6,8-di-C-glucoside)
6.47.5b
4.55.3
b
Scoparin (Chrysoeriol 8-C-glucoside)
7.27.9
b
Diosmetin 8-C-glucoside (Orientin
40 -methyl ether)
7.68.8b
Compound
Remarks
MAJOR FLAVANONES O-GLYCOSIDES
MAJOR FLAVONES
Isovitexin (Apigenin 6-C-glucoside)
*Position in the classification of Citrus juices according the content of a specific flavonoid, based on indicated references.
a
Nogata et al.17
b
Gattuso et al.18
c
Unpublished results, obtained in samples of industrial juice, courtesy of D. Malara, Reggio Calabria, Italy.
d
Di Donna et al.16
e
Barreca et al.14
f
Gattuso et al.20
solid parts of the fruit, is absolutely the highest in bergamot compared to other Citrus species. These observations were then confirmed for industrial bergamot
juice obtained by fruit crashing.18 A lower content of
flavonoids is present in manually squeezed bergamot
juice.19,20 This discrepancy can be explained by
differences in processing and different distribution of
flavonoids between the vacuole juice, albedo (the inner
white part of the peel), flavedo (external peel rich in
aromatic oils), and inner fibers of the fruits. Indeed,
the majority of flavonoids are several times more
abundant in albedo than in the juice,17 and industrial
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84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
2500
400
350
2000
Absorbance (mAU)
300
250
200
150
1500
1000
100
500
50
0
0
0
f0015
2
4
6
8
Time (min)
10
12
14
0
2
4
6
8
10
12
Time (min)
14
16
18
FIGURE 84.2 Typical High Performance Liquid Chromatography (HPLC) chromatograms of flavonoid components in bergamot juice
(left panel) and in BPF (Bergamot Polyphenol Fraction (right panel) used in clinical practice as food supplement to treat metabolic syndrome,
hypercholesterolemia and hyperlipidemia. Below, chemical structures of the most abundant flavanones. Bold, sugar portion of the flavanones
glucosides; Grey, 3-hydroxy-3- methylglutaryl portion of brutieridin and melitidin. Note that the profile of polyphenols in BPF is comparable
to the HPLC profile of the bergamot juice (see also Plate 13).
8. CARDIAC HEALTH AND POLYPHENOLS
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3. PHARMACOLOGICAL EFFECTS OF BERGAMOT POLYPHENOL FRACTION ON CARDIOVASCULAR RISK FACTORS
p0040
p0045
pressing causes a release of albedo compounds. This
processing enriches the total flavonoid content of the
juice to more than 1600 mg/L in the industrial juices
from the initial 373 up to 512 mg/L, present in the
“hand-squeezed” juices of two different Bergamot cultivars Fantastico and Feminello, respectively.19 Thus, the
industrial bergamot juice likely shows the highest concentration of polyphenols among non-concentrated
natural juices.
Bergamot fruits (juice, albedo, flavedo, and fibers)
show the highest concentrations of flavanones: neoeriocitrin, neohesperidin, naringin, poncirin, melitidin
and brutieridin, and the highest content of certain flavones: rhoifolin and neodiosmin among 45 different
Citrus species17 (see Table 84.1). A high content was
confirmed for rhoifolin and neodiosmin by another
independent study that also identified other highly
abundant flavones in bergamot juice: vicenin-2, diosmetin di-C-glucoside and chryseriol 7-O-neohesperoside and lower amounts of other flavonoids: lucenin-2,
stellarin-2, isovitexin, scoparin, diosmetin 8-C-glucoside, chrysoeriol 7-O-neohesperoside-40 -O-glycoside
and others.18 Another peculiar feature of bergamot
juice is a high content of unsaturated fatty acids,
accounting for 80% of total acids, such as oleic acid,
linoleic acid and palmitic acid.21
As a medicinal food, bergamot juice is not always
available and is subjected to fast deterioration. In the
search of a more stable and possibly more powerful
pharmaceutical surrogate of bergamot juice, a method
to concentrate and exsiccate bergamot juice was developed. According to this method, bergamot juice is concentrated
by
a
preparative
size
exclusion
chromatography based on polystyrene gel filtration.8
Polyphenols and other macromolecules of the molecular size over 300 Da are retained on the column, eluted
with acids and exsiccated with other clarified juice
components to give rise to a flavonoid-enriched bergamot powder or BPF.8 BPF contains over 40% flavonoids and the remaining 60% are carbohydrates, fatty
acids, pectins and other compounds as well as maltodextrins which are added to allow exsiccation
(D. Malara, personal communication). In contrast to
BPF, bergamot juice derivatives obtained by spry-drier
method rich maximum 1% polyphenols concentration
(D. Malara, personal communication). The main polyphenol components of BPF are flavonoids and their
composition basically mirrors the bergamot juice polyphenol profile (see Figure 84.2, and Plate 13), with the
only difference that flavonoids are over 200 times
more concentrated in BPF. Flavanones glycosides
account for 95% of total flavonoids present in BPF
(and in bergamot juice), while flavones can be found
in the remaining 5% (Table 84.1 and D. Malara and
C. Malara, unpublished observations).
1089
Currently, there are no published bioavailability and p0050
pharmacokinetic studies for BPF. However, absorption,
metabolism and excretion parameters have been
described for several individual flavonoids present in
bergamot juice. It is well known that flavonoid glycosides are hydrolyzed to aglycones by baterial flora of
the gut. Gut microflora hydrolysis is thought to favor
flavonoid glycoside bioavailability.22 When sugar unit
is removed, the resulting aglycone can be absorbed
more readily.23 Indeed, flavonoid aglycones of diosmin,
hesperidin and naringin, diffuse usually easily through
the plasma membrane of Caco-2 cells, in contrast to the
respective glycosides. Moreover, naringin, hesperidin,
rutin and poncirin are hydrolyzed to their respective
aglycones by human intestinal microflora, and the
resulting aglycones are absorbed better.24 However, the
bioavilability of flavonoids is low and it is estimated
that only 10% of total consumed polyphenols are
absorbed, although these numbers vary between species and individuals.23 When inside the enterocytes part
of aglycones are subjected to intestinal metabolism,
such as glucuronidation and sulfation. Phase II metabolism is the main route of metabolism for polyphenols.
Conjugation of free phenolic groups via glucuronidation and/or sulfation will increase their polarity and
water solubility, enabling their elimination from the
body.23 Generally, citrus and other flavonoids have a
short life-time and metabolites are thought to lose their
biological activity. Nevertheless, recent data suggest
that sulfonated or methylated and much less glucuronidated metabolites of resveratrol maintain full or partial
activity of the parent compound. In addition, sulfonation has been shown to increase the activity for some
molecular targets of resveratrol.25
Therefore although not properly investigated, it is p0055
likely aglycones of bergamot flavonoids and their
metabolites contribute to the modulation of the intracellular targets and the subsequent biological response.
3. PHARMACOLOGICAL EFFECTS OF
BERGAMOT POLYPHENOL FRACTION
ON CARDIOVASCULAR RISK FACTORS
3.1 Metabolic Syndrome and Cardiovascular
Risk Factors
s0020
s0025
AVD is the leading cause of death in developed p0060
countries.26 Coronary artery disease is the commonest
form of cardiovascular disease accounting for around a
third of all-cause mortality in people over the age of 35.
AVD is caused by atherosclerosis, which is defined as
the deposition of lipids, inflammatory cells and mediators in the subendothelial layer of blood vessels,
eventually leading to thickening and hardening of
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1090
p0065
p0070
p0075
o0010
o0015
o0020
o0025
o0030
o0035
o0040
o0045
o0050
p0125
84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
medium- to large-sized arteries. Atherosclerosis has a
very long pre-symptomatic phase and can begin even
in-utero in predisposed individuals. The presymptomatic phase of atherosclerosis averages between
4060 years, although clinical presentation seldom
occurs before this time frame and commonly thereafter.
The presentation, thus depends on the rates of atherosclerotic deposition in the blood vessel wall, often lasting decades, while common clinical manifestation of
AVD, such as acute myocardial infarction,
unstable angina, stroke or sudden cardiac death, usually occur following the rupture of a large plaque in the
blood vessel wall.
It is well-established that the abundance of fat and
sugar in diets found in industrialized western society is
the most important risk factor for developing AVD.
Indeed, as many of the developing nations adopt more
affluent life styles, an increasing rates of cardiovascular
disease is seen.27 This is because the diet influences blood
parameters that may become AVD risk factors if out of
established limits. For example, the average cholesterol
levels in malnourished regions (for example, rural China)
are
between
7797 mg/dL
(22.5 mmol/L).
Atherosclerosis is a rarity in these regions.28 Once food
becomes available and in plentiful supply, the cholesterol
level rises above 3 mmol/L and it is above this level that
atherosclerosis occurs. In fact, in the majority of cases,
people with lifelong cholesterol levels below 4 mmol/L
are rarely troubled by significant complications of
atherosclerosis.28
Excessive fat and sugar consumption, linked to
genetic predisposition and poor lifestyle behaviors
may lead to the development of a pathological state,
called metabolic syndrome (MS). MS is very common
and 1030% of individuals in industrialized countries
suffer from this condition. MS is defined as a cluster of
cardiovascular risk factors, including atherogenic dyslipidemia, insulin resistance or glucose intolerance, visceral
obesity,
hypertension
and
endothelial
dysfunction.
There are four basic components of MS:
1. Hyperglycemia (fasting plasma glucose $ 110 mg/dl
(6.1 mmol/L))
2. Hypertension (systolic blood pressure .130 or
diastolic blood pressure .85 mmHg)
3. Dyslipidemia
a) Increased cholesterol .200 mg/dL (5 mmol/L)
b) Increased Triglyceride .150 mg/dL (1.5 mmol/L)
c) Reduced cHDL ,55 mg/dL, (1.3 mmol/L)
4. Abdominal obesity
a) Males .95 cm
b) Females .80 cm
The presence of all components of MS is associated
with a particularly increased risk of accelerated
atherosclerosis and cardiovascular events.29 However,
the probability of developing AVD (cardiovascular
risk) is also increased in individuals with isolated metabolic dysfunctions, such as increased cholesterol
levels (hypercholesterolemia) or increased level of triglycerides (hypertriglyceridemia) and in patients with
type-1 and -2 diabetes as well as in patients with glucose intolerance and hypertension.
Pharmacological treatment of any cardiometabolic p0130
risk factors is clearly a preventive medicinal approach,
since it slows down the process of atherosclerosis and
reduces the probability of cardiovascular events. For
this reason, it would be preferable that such a treatment is based on diet and natural drugs, and starts as
early as possible in adult age; and not on conventional
and aggressive polypharmacy started when MS or
even ADV is well-advanced.
The experimental and clinical evidence will be pre- p0135
sented here, showing that BPF—a concentrate of natural bergamot polyphenols—is a suitable treatment for
MS and other metabolic pathologies associated with an
increased cardiovascular risk of atherosclerosis and
life-threatening cardiovascular events.
3.2 Hypolipidemic Effects
s0030
The first evidence of the cholesterol-lowering prop- p0140
erties of bergamot polyphenols comes from experimental studies on rats fed a high-cholesterol diet.7 In this
laboratory model of diet-induced hypocholesterolemia,
rats on a 2% cholesterol and 20% oil diet developed
extremely high levels of total blood cholesterol (tChol,
700 mg/dL). The animals treated daily with 1 ml bergamot juice for 28 days showed a marked reduction in
total blood cholesterol (tChol) by (29.3%) , triglicerides
(46.2%) and cLDL (51.7%). These results were confirmed in an independent work by Mollace et al.,8
addressing the efficacy of the bergamot polyphenol
mixture, BPF. Another important pharmacological
finding observed by Miceli et al.7 was evident reduction of lipid steatosis in the liver, defined as a decrease
in the number and size of hepatic lipid droplets and a
reduction in inflammatory changes due to fat accumulation in the liver. In addition, the anatomical analysis
of arteries indicated some protective effect of bergamot
juice against mild endothelial thickening. Next
Mollace’s group confirmed these observations and
demonstrated a prevention of accelerated stenosis with
the use of Bergamot extract. In this model of stenosis/
atherosclerosis, the balloon injury is induced to the
carotid artery in laboratory rats.30 Bergamot extract (in
this case a non-volatile part of bergamot oil) uniformly
prevented rapid thickening and inflammatory changes
in the arteries subjected to balloon injury.
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1091
3. PHARMACOLOGICAL EFFECTS OF BERGAMOT POLYPHENOL FRACTION ON CARDIOVASCULAR RISK FACTORS
p0145
AU:1
t0015
The final evidence of hypolipemic effects of bergamot polyphenols comes from an extensive placebocontrolled human clinical trial.8 This study recruited
237 patients who had been divided into three groups:
hypercholesterolemic (HC, increased tChol, cLDL, low
cHDL), mixed dyslipidemic (HC and increased TG),
and metabolic syndrome patients (HC, HT and
increased blood glucose HG). The patients received 500
or 1000 mg BPF daily. After 30 days, 90% of the patients
in all three groups responded with a significant reduction in lipid blood parameters ranging from 5 up to
45% decrease (Table 84.2). In particular, more than a
45% decrease for cLDL was achieved in 10% patients.
The most striking reduction (mean 41.0 6 2.6%) was
observed for plasma triglyceride levels. Excellent mean
reduction for other blood parameters was achieved:
228.1% for totChol and 233.2% for cLDL and 130.3%
for cHDL in a group of MS patients (Table 84.2 and
Figure 84.3). Patients with isolated HC and HC 1 HT
responded in a similar way to treatment with BPF
(Table 84.3, groups A and B vs C). Placebo-treated
patients showed a maximum 5% reduction in
cholesterol parameters and triglycerides. Less striking,
but significant results were obtained in a small group of
statin-intolerant patients (group D), who started BPF
therapy after a considerable “wash-out period” (4
weeks) to reduce the rebound effect that takes place
after statin withdrawal (Table 84.2, group D).
In order to verify pharmacological properties of p0150
BPF, an independent observational study has been performed in Australia on over 600 patients, with MS or
hyperlipidemia. All patients were subjected to 1300 mg
BPF therapy. Although, with all treatments, there was
a group of non-responders, when all patients were
considered, there was (on average) a 29% reduction in
cholesterol, a 36% reduction in cLDL, up to a 40%
reduction in triglycerides and up to a 40% increase in
HDL levels (R. Walker, unpublished observations).
3.3 Hypoglycemic Effects and Improvement of
Endothelial Function
The clinical study by Mollace et al.8 showed other p0155
important pharmacological properties of BPF. Indeed,
TABLE 84.2 A summary of the Results from Clinical Trial Testing of the Efficacy of BPF Treatment (30 days) Against tChol, cLDL,
cHDL in Patients with Hypercholesterolemia (HC, Group A), Mixed Hyperlipidemia (HC/HT, Group B), MS (HC/HT/HG, Group C) and
Patients Intolerant to Statins (Group D).
Patients
Group
SubGroupa
Nr
Pb
BPF
Dose
Daily
(mg)
A
A1
35
500
2 20.7 6 1.9
6
234.6
A2
37
1000
2 30.9 6 1.5
2
240.0
APL
32
0
2 0.4 6 0.4
32
2
B1
14
500
2 21.9 6 1.8
1
B2
14
1000
2 27.7 6 3.4
BPL
14
0
2 0.5 6 0.5
B
tChol
cLDL
% Δ6
SEMc
No
Resp.d
Best
10%e
% Δ6
SEM
cHDL
No
Resp.
Best
10%
2 23.0 6 1.9
4
237.2
2 38.6 6 1.5
0
249.1
2 1.7 6 0.5
27
2
228.3
2 25.3 6 2.0
0
2
241.5
2 33.4 6 3.9
14
2
2 0.5 6 0.7
% Δ6
SEM
No
Resp.
Best
10%
25.9 6 2.3
0
50.0
39.0 6 2.8*
0
68.6
0.5 6 1.1
22
2
234.6
17.3 6 1.4
0
26.7
2
243.6
35.8 6 4.2*
0
66.7
13
2
2 1.3 6 1.8
11
2
C1
20
500
2 24.7 6 2.6
2
241.7
2 26.8 6 3.6
1
253.6
16.5 6 1.6
0
42.9
C2
19
1000
2 28.1 6 2.6
1
241.1
2 33.2 6 3.0
1
247.0
29.6 6 1.8*
0
64.6
CPL
20
0
20
2
2 0.9 6 1.4
18
2
2.9 6 2.0
15
2
D
2
32
1500
2 25.0 6 1.6
2
239.8
2 27.6 6 0.5
0
232.4
23.8 6 1.7
0
41.1
A1B1C
2
69
500
2 21.8 6 1.4
9
237.8
2 24.1 6 1.5
5
245.0
22.3 6 1.3
0
248.6
2
70
1000
2 29.4 6 1.3
5
240.6
2 36.0 6 1.4*
3
247.9
40.1 6 1.9*
0
266.4
2
66
0
2 0.1 6 0.3
66
58
2
48
2
C
0.5 6 0.5
s0035
2 1.1 6 0.5
a
1.2 6 0.9
For division of all recruited patients in groups and subgroups, see Section 2.3.1.
Nr P, number of patients recruited for each treatment group (AD) and subgroup: 1 (low dose BPF), 2 (high dose BPF) or PL (placebo).
Mean changes in blood parameters for each group or subgroup of patients were calculated by adding the changes recorded for individual patients and dividing them by the
number of patients (Nr P).
d
No response Number of patients that show a smaller than 5% reduction in totChol, cLDL or a lower than 5% increase in cHDL after 30 days treatment with BPF. Note that
in some cases bigger than 5% changes were recorded in the placebo treated patients.
e
Best 10%, mean value in the subgroup of the best 10% responders.
*Statistically significant difference compared to 500 mg BPF dose.
The table was prepared according to the data published in Mollace et al.8 Copyright Elsevier Inc.
b
c
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1092
84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
f0020
FIGURE 84.3 Reduction of total cholesterol (tCho), LDL cholesterol (cLDL), triglycerides (TG) and glucose levels and increase of HDLcholesterol (cHDL) in the blood of patients with metabolic syndrome. A group of 59 metabolic syndrome patients was divided in two
groups. Twenty-nine patients were asked to take 500 mg BPF once or twice a day while 30 patients took equivalent amounts of placebo for 30
days. Note the significant reduction in all blood parameters accompanied by the increase in “good cholesterol” cHDL. The graphs show the
mean values for indicated blood parameters from the relative treatment group. Group C1, n 5 20, 500 mg/day, group C2, n 5 19, 1000 mg/
day, group CPL, n 5 20, placebo. The indicated patients’ blood parameters, all expressed in mg/dLl, were analyzed on day 0 (before) and day
30 (after) of treatment. Error bars show the standard deviation (SD). *Indicates a statistically significant change compared to the placebo group
at p ,0.0001; #Indicates a statistically significant change between C1 and C2 subgroups at p ,0.05. From Mollace et al.8
t0020
TABLE 84.3 The Percent Variations in TG and Blood Glucose in Patients Suffering from HC/HT and HC/HT/HG Subjected to the 30
Day BPF Treatment.
Triglycerides
Glucose
Patients
Group
Subgroup
Nr
Pa
BPF Dose Daily
(mg)
%Δ 6
SEMb
No
Responsec
Best
10%d
%Δ 6 SEM
No
Response
Best
10%
B
B1
14
500
2 28.2 6 3.9
2
246.8
234.6
B2
14
1000
2 37.9 6 3.3
1
247.9
243.6
BPL
14
0
0.1 to 0.5
14
C1
20
500
2 32.7 6 2.5
0
241.6
2 18.9 6 1.2
0
227.1
C2
19
1000
2 41.0 6 2.6
1
249.6
2 22.4 6 1.0
0
232.2
CPL
20
0
19
2 0.5 6 0.7
20
C
0.0 6 0.6
a
Nogata et al.17
Gattuso et al.18
c
Unpublished results, obtained in samples of industrial juice, courtesy of D. Malara, Reggio Calabria, Italy.
d
Di Donna et al.16
Prepared according to the data published in Mollace et al.8 Copyright Elsevier.
Treatment groups or subgroups of patients are indicated B-C and . . .1,. . .2, . . .PL according to codes described in the title of Table 84.1.
b
p0160
the hypolipemic effect in patients with MS was accompanied by a significant reduction in blood glucose
levels. In particular, the mean reduction in glucose was
218.9% and 222.4% in patients taking 500 and
1000 mg BPF daily, respectively8 (Table 84.2 and
Figure 84.3). This observation was unexpected and
clearly suggests that the pharmacological effects of
BPF go beyond statin-like activity. This interesting
metformin-like property of BPF was partially confirmed in the Australian observational study.
The patients with elevated cardiometabolic risk factors usually suffer from endothelial stiffness, i.e., the
reduction of flow-mediated vasodilatation. This problem is particularly pronounced in MS patients with
elevated blood glucose. Indeed, the study by Mollace
et al.8 showed that patients with mixed
hyperlipidemia and hyperglycemia (HC/HT/HG)
with lowest flow-mediated vasodilatation before the
treatment, showed a particular B50% improvement
of endothelial function after 30 days of 1000 mg BPF
therapy. Significant improvement was also observed
in other groups patients (see Figure 84.4).
4. STATIN THERAPY AND BPF
4.1 Advantages and Disadvantages of
Conventional Therapy Based on Statins
s0040
s0045
The most commonly prescribed drug across the p0165
world against hypercholesterolemia is Atorvastatin,
which belongs to statin family of cholesterol-lowering
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1093
4. STATIN THERAPY AND BPF
f0025
p0170
u0010
u0015
u0020
p0190
FIGURE 84.4 The effect of BPF (500 and 1000 mg/die) on reactive vasodilation in patients suffering from hypercholesterolemia
(HC), mixed hyperlipidemia (HC/HT) or metabolic syndrome (HC/
HT/HG). After 30 days of treatment with placebo or BPF (500 or
1000 mg/diet), endothelial function was assessed in three groups of
patients (14 to 30 patients per group). Flow-mediated vasodilation
was measured from brachial artery diameter during reactive hyperemia. *Indicates a statistically significant change compared to the
respective placebo group of patients at p ,0.05; #Indicates statistically significant changes compared to the 500 mg BPF group at p
,0.01; 1 Indicates statistically significant changes compared to HC
group at p ,0.01. From Mollace et al.8
drugs. Statins have been one of the greatest advances
in cardiology over the past 50 years.
The advantages of statin therapy are:
• Proven clinical efficiency. Depending on dose, statins
reduce LDL anywhere between 3060%, based on
the particular statin and dose. Typically the
reduction in cardiovascular events is somewhere
2030%.31 The recent Jupiter trial suggested a 50%
reduction in cardiac events with a moderate dose of
Rosuvastatin (20 mg) but this was in an
intermediate risk group with an overall low number
of events, albeit statistically significant according to
the Jupiter study.32
• Tolerability and compliance. Most patients tolerate low
to moderate doses, which only needs to be taken as
a daily dose. However, a number of studies have
clearly shown that compliance is poor with only
50% of patients still taking their medications twelve
months after the initial prescription.33
• Pleiotropic effects. Not only do statins have powerful
total cholesterol and LDL lowering effects, there is
also good evidence to support non-lipid benefits
such as anti-inflammatory, antioxidant and antiplatelet effects. Statins have also been shown to
reduce plaque size and burden.34
There are, however, disadvantages to the long-term
use of statins. Although the medical profession has
had a “long-term love affair” with statins, there has
been much disquiet amongst the complementary medical world and the general public. Several of the
patients attribute their many side effects, both subtle
and not so subtle, to their statin therapy. Statins are
well-described as contributing to myalgia and other
muscle related issues, including an elevated CPK.
Although the clinical trials suggests the incidence of
muscle problems is only 12%, in real world clinical
practice this occurs in around 1020% of patients.35
Twenty percent of patients taking (especially fat soluble) statins experience neuro-cognitive symptoms.
Liver abnormalities have been described and abnormal
liver function is commonly seen.35
For a number of years, cancer concerns have been p0195
raised in regard to chronic statin therapy but there has
been no consistent data to prove or deny this relationship.36 There is no doubt that statins are effective
agents with a central role in the management of AVD,
but it is, in fact, this powerful, metabolic regulatory
effect that also raises some concerns.
Some people who take statins, often commencing in p0200
their fourth or fifth decade of life, may potentially be
maintained on these agents for anywhere between 20
to 50 years, based on their longevity.
Cholesterol is a vital component of cell membrane p0205
structure and function, along with a key component of
steroid, bile salts and vitamin D metabolism. To date,
there have been no data regarding the potential deleterious effects of prolonged statin therapy, the majority
of clinical trials running for less than 10 years. In fact,
four studies over the past 2 years have suggested a
50% increased risk for diabetes in patients ingesting
statins long-term.37 Finally, recent studies have raised
doubts as to the benefits of statin use in primary prevention. A recent meta-analysis of 65,229 patients
reviewing 11 studies demonstrated no statistically significant reduction in all-cause mortality.38
A recent Cochrane review of 14 trials, involving p0210
34,272 patients showed no evidence of improved quality of life or cost effectiveness in primary prevention
patients.39
Thus, in the authors’ opinion, there needs to be a p0215
different approach to the management of hypercholesterolemia and associated lipid disorders. The evidence
strongly suggests the routine use of statins as part of
the management of all patients with established vascular disease or with strong evidence of a significant atherosclerotic load on appropriate non-evasive screening
tools (e.g., coronary calcium scoring, carotid intimal—
medical thickness measurements, etc.). To date, however, there are no scientific data supporting the use of
statins for isolated lipid abnormalities, in the absence
of other significant cardiac risk factors, especially
when, for example, a coronary calcium score places
the individual either at 0 or in a low percentile risk
range (e.g., ,50th percentile for score and age). In
these individuals, the current data suggests no benefit
and possibly harm from long-term exposure, such as
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1094
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84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
the risk for diabetes, myopathy and neuro-cognitive
problems.39
As argued in this chapter, the much maligned cholesterol is a vital component of many metabolic and
structural processes, but the evidence clearly shows
that in the long-term, hypercholesterolemia in many
cases is associated with an increase in vascular risk.
4.2 Therapeutic Indications for the Use of BPF
p0225
The specific indications for the use of Bergamot extract
in a dose of between 10001500 mg daily include:
o0055
1. Statin intolerant patients
• A significant minority of patients cannot tolerate
statins, even at low doses
• A more substantial group suffer mild side effects,
Bergamot extract allows the statin dose to be
reduced but maintaining the global lipid
lowering, HDL raising benefits.
2. Increasing statin potency
• Even in those patients experiencing no side
effects from statin therapy, Bergamot extract
allows lower doses to be used with an increase in
benefit in regard to the potency of the overall
lipid effects
• As already stressed, there is increasing evidence
for concerns with long-term use (10 years or
greater) with (especially) higher doses of statins
and therefore any strategy that allows long-term
use of lower statin doses should be encouraged.40
3. Triple therapy for lipid abnormalities in patients
with proven vascular disease.
4. The evidence is overwhelming that all patients with
proven vascular disease (secondary prevention)
should be prescribed aggressive lipid lowering
therapy.This not only includes patients with a
clinical history of cardiovascular disease (i.e., post
myocardial infarction, angina pectoris, coronary
stent or coronary artery bypass graft (CABG) and
also cerebrovascular disease, peripheral vascular
disease or disease in other vascular beds), but also
asymptomatic patients with evidence of significant
sub-clinical atherosclerosis, such as patients with a
high coronary artery calcium score (CACS).
In the authors’ opinion, derived from current
available clinical evidence that all patients in this
category should be treated with statins or drug
combinations to target lipid values:
Chol , 4 mmol/L
LDL ,2 mmol/L
Triglycerides ,1 mmol/L
HDL .1.5 mmol/L
These goals may be achieved with an appropriate
dose of statin therapy, Bergamot extract
u0025
u0030
o0060
u0035
u0040
o0065
o0070
u0045
u0050
u0055
u0060
(10001500 mg daily), and the judicious use of
nicotinic acid with gradually introduced doses
aiming for somewhere between 10002000 mg
daily.
Although the recent AIM-HIGH data41,42
suggests that nicotinic acid sustained release adds
little to high-dose statin therapy, this contradicts the
majority of data that demonstrate a significant
benefit over statin therapy alone.43 A full discussion
of these issues is outside of the scope of this
chapter.
It is important to note that the long-term safety
and clinical results of adding Bergamot extract to
statin and nicotinic acid has not yet been
determined.
5. Metabolic Syndrome
Bergamot extract (10001500 mg daily) is the
only current therapy available that has
demonstrated efficiency in all parameters of
metabolic syndrome8 Thus, it is logical that
Bergamot extract should be standard therapy as
prevention and treatment for patients with features
of metabolic syndrome.
6. Low Risk Patients
With the recent data suggesting no major benefit
for long-term statin therapy, as well as the potential
for significant long-term side effects such as
diabetes in low risk patients, it is important that the
medical profession becomes more selective in those
patients who are offered treatment. However,
numerous data over the last 2030 years have
confirmed a reduction in cardiovascular events in
those with lower cholesterol levels, both in
epidemiological and intervention studies. Thus, a
natural therapy such as bergamot extract with no
significant, reported side effects, should be seen as a
useful adjunct in those patients with an abnormal
lipid profile but otherwise deemed at low risk
through commonly accepted risk scores (e.g.,
Framingham Risk Score).44,45 One must always be
mindful of the commonly stated aphorism, “First,
do no harm.”
4.3 Limitations of the Use of BPF
o0075
o0080
s0055
Firstly, it is important to stress that Bergamot extract p0330
is not a replacement for statins in those clinical situations where statin therapy is clearly indicated.
It is also important to point out that statin therapy p0335
does have 5 to 10 years of data taken from over
100,000 patients, demonstrating clear efficacy in the
reduction of cardiovascular events.39
As the evaluation of Bergamot extract is only rela- p0340
tively recent, no large mortality/morbidity studies
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5. POSSIBLE MOLECULAR MECHANISMS OF ACTION OF BERGAMOT POLYPHENOLS
have yet been performed, despite the very robust double blind controlled human trials demonstrating:
u0065
u0070
u0075
p0360
p0365
o0085
o0090
o0095
o0100
• a significant hypolipidemic/hypoglycemic effect
• animal data suggesting a substantial reduction in
atherosclerosis
• human data suggesting an improvement in
endothelial function. Endothelial dysfunction being
one of the initial indicators of early atherosclerosis.
In an observational study performed on a group of
patients in Australia (R. Walker, unpublished observations), benefits were noted in all patients treated in at
least one of the vascular parameters, i.e., lipids, blood
glucose, hypertension, weight and arterial stiffness.
The Australian patients were not recruited in a
blinded, clinical trial, but they did represent “real
world” medical therapy, and the overall results
obtained in Australia supported the Italian data. Some
concerns, however, may be:
1. Local effects
Bergamot trees only produce this vascular benefit
when grown in the specific southern Ionic Coast of
Calabria. It is possible that those people with a
southern Italian heritage benefit most from this
treatment due to local genetic and environmental
factors. One is very mindful, for example, of the
Apo A1 Milano effect.46
This aspect of Bergamot extract requires further
study.
2. Random clinical trials (RCTs) versus observational
data
Patients in RCTs tend to have much more
rigorous supervision and are more compliant with
therapy. A number of patients have demonstrated
poor compliance, variable dosing, ill-timing of
dosing, etc., which may have contributed to lesser
benefits.
3. Variable batch strength
The initial extract sent to Australia had only
25% purity of active ingredient. When Bergamet
Mega 650 mg, manufactured from a 40% purity
extract, was introduced (twice daily dosing) there
was a much greater benefit in all vascular
parameters.
4. Pleiotropic effects
Statins have numerous pleiotropic (non-lipid)
benefits and this has been well-described for a
number of years.
There is substantial evidence that this is also true
for BPF. In the Australian observational study, a
minority of patients with mild to no significant lipid
lowering demonstrated a substantial reduction in
arterial stiffness, suggesting that the BPF-mediated
improvement of the endothelial function is
1095
dominant over the lipid lowering effect (R. Walker,
unpublished observations).
There needs to be substantial research into the
effects of Bergamot extract on lipid subfractions,
lipoprotein (a), anti-inflammatory, antioxidant,
anti-platelet and its direct effect on all parameters
of endothelial function and arterial stiffness.
5. Statin Rebound
A poorly described and poorly studied
phenomenon is that of statin rebound. In a
significant number of patients who ceased statins
for a variety of reasons (typically significant side
effects), their cholesterol rebounds well above their
initial baseline values. To date, it has not been
described as to how long (if ever), the cholesterol
values return to their baseline levels. This
phenomenon may, however, affect the perceived
efficacy of bergamot extract as it is dominant over
the moderate effect of bergamot polyphenols on
lipid metabolism. Again, “rebound effect” needs to
be studied further to develop safe pharmacological
approaches aimed at avoiding a potent cholesterol
increase during statin withdrawal.
5. POSSIBLE MOLECULAR MECHANISMS
OF ACTION OF BERGAMOT
POLYPHENOLS
5.1 Interference with Cholesterol Synthesis by
HMG-CoA Reductase Inhibition
o0105
s0060
s0065
Bergamot juice and albedo and derivatives as BPF p0435
contains two rare flavonoids, brutieridin and melitidin
that have been suggested to act as direct HMG-CoA
reductase inhibitors.16,47 Lower amounts of melitidin
and brutieridin are also found in sour orange (Citrus
aurantium)15 and chinotto (Citrus myrtifolia).14
Computational studies have demonstrated that both
molecules bind efficiently to the catalytic site of
HMG-CoA reductase.47 This is because these two flavonoids contain HMG -moiety covalently O-linked to
sugar residue of naringenin (melitidin) or hesperitin
(brutieridin), which is identical to S-linked 3-hydroxy3-methyl-glutarlyl residue of HMG-coenzyme A
(HMG-CoA), a substrate of HMG-CoA reductase (see
Figure 84.2, 84.5, and Plates 13 and 14). This enzyme
reduces HMG-CoA to mevalonate, which is the only a
precursor of the later steps in cholesterol synthesis
(Figure 84.5 and Plate 14). Thus, this reaction is ratelimiting the entire biochemical pathway of cholesterol
synthesis. HMG-CoA reductase cannot hydrolase Olinked HMG which competes with the S-linked substrate for catalytic pocket of the enzyme47 as depicted
in Figure 84.5 and Plate 14. However, beside convinc-
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1096
f0030
84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
FIGURE 84.5 Cholesterol synthesis pathway and a proposed model of its inhibition by brutieridin and melitidin. Acetyl-coenzyme A
(Acetyl-CoA) is a product of the metabolism of any source of energy—be it protein, fat, or carbohydrate. Acetyl-CoA is then first converted
into 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA), which is a substrate of the rate-limiting reaction in the cholesterol synthesis pathway, catalyzed by HMG-CoA reductase. This enzyme is a target of statins, which inhibit HMG-CoA and melitidin and bruteridin, two prominent bergamot flavanone O-glycosides, that contain HMG moiety covalently linked to glucose via esterification of carbon 60 , hydroxyl group (see left
scheme, and Plate 8). According to molecular modeling studies HMG-flavanone O-glycosides or relative metabolites containing HMG moiety
can fit into catalytic pocket of HMG-CoA reductase and mimic the substrate, thereby blocking the reduction of the carbonyl group, since Olinked HMG cannot be reduced. If HMG is a modification of the thiol group of CoA the reduction reaction can catalyze. The product of HMGCoA reductase, mevalonic acid is subsequently converted into various other products by numerous enzymes, until it is converted into several
important end products. Beside cholesterol, this enzymatic pathway generates coenzyme Q10 (ubiquinone), farnesyl groups of farnesylated
proteins, steroid hormones and other products not indicated in this scheme (see Plate 14).
ing theoretical consideration regarding the mechanisms of inhibition of HMG-CoA reductase by these
two HMG-modified polyphenols, proposed by
Sindona and co-workers,16,47 the direct demonstration
of statin-like activity in vitro and in assays for HMGCoA activity and their experimental comparison to statins is still missing. It is also not clear if brutieridin
and melitidin exert any activity if administered to animals as purified compounds. In fact, the potent HMGCoA inhibition shown in blood samples of patients
receiving BPF in the clinical trial reported by Mollace
et al.8 can result from the activation of AMPK, as discussed in the following sections. In addition, the
potent hypolipidemic effects of a citrus peel extract
from tangerines, lemons or oranges, that do not contain (as far as we know), any brutieridin and melitidin,
but mainly polymetoxylated flavonoids plus hesperidin, naringin and pectins, causes a significant inhibition of HMG-CoA reductase and another enzyme of
cholesterol biosynthesis pathway, which is ACAT
(Acyl-CoA:cholesterol acyltransferase) in animal livers.48
This suggests that an inhibition of HMG-CoA reductase
can be achieved in vivo by citrus polyphenol mixtures,
devoid of brutieridin and melitidin. Naringenin has
been proposed to act as the active component in these
mixtures, responsible of HMG-CoA reductase inhibition,
but it has not been formally shown in vitro,49,50 although
experimental demonstration for HMG-CoA reductase
inhibition by flavonoids in vitro is available for soy isoflavones such as genistein, daidzein and glycitein.51
However, pure preparations of naringin and hesperidin
did not reduced cholesterol, cLDL and tryglicerides
blood levels in moderately hyperlipemic patients in
more recent human trials.52 In fact, it is possible that
HMG-CoA reductase inhibition in vivo is a complex
effect of cooperation of AMPK inhibitory properties of
naringin with other properties of flavonoids, including
phosphodiesterases (PDE) inhibition, ROS scavenging
and other molecular and physiological effects, as discussed below. Thus, although brutieridin and melitidin
appear as plausible HMG-CoA reductase inhibitors and
can explain the potent cholesterol-lowering effects
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1097
5. POSSIBLE MOLECULAR MECHANISMS OF ACTION OF BERGAMOT POLYPHENOLS
bergamot derivatives,7,8 it is likely that other flavonoids
and biochemical pathways play a vital role in achieved
pharmacological effects, as discussed below.
s0070
p0440
p0445
for cAMP and play critical roles in regulating energy
metabolism and lipolysis.6 The rate of TG hydrolysis
(lipolysis) is positively regulated by increase in intracellular cAMP. In fact, cAMP activates, a cAMP dependent
protein kinase, PKA and indirectly, as recently demonstrated, AMP (adenosine monophosphate)-activated
protein kinase (AMPK), a central regulator of lipolysis
and glucose metabolism.54 These recent findings identifying PDEs as direct molecular targets of resveratrol,54
put an end on a long dispute regarding mechanisms of
action of resveratrol and related compounds, i.e., citrus/bergamot flavonoids, as discussed in the next paragraph. The cascade of molecular events leading to the
activation of AMPK from the inhibition of a cAMPspecific PDE, is depicted schematically in the
Figure 84.6 (see also Plate 15).
The structureactivity relationships of flavonoids p0450
with regard to their inhibitory effects on PDE isozymes
5.2 Phosphodiesterases as Other Potential
Molecular Targets of Bergamot Flavonoids
Other potential targets of bergamot flavonoids could
be cyclic nucleotide PDEs.
PDEs catalyze hydrolysis of cAMP (cyclic adenosine
monophosphate) or cGMP, regulate their intracellular
concentrations and, consequently, the physiological
effects of these second messengers. PDEs belong to a
complex and diverse superfamily of at least 11 structurally related, highly regulated, and functionally distinct
gene families (PDE1PDE11).53 PDE3B, and certain
PDE4 isoforms are characterized by their high affinity
Resveratrol
Apigenin ?
Neoericitrin ?
Catecholamines
Glucose
Adiponectin
α−Adrenergic
GLUT4
Receptor
β
GLUT4
Vesicle
Insulin Receptor
cAMP
CaMKK
GLUT4
Translocation
Insulin
PDE
αGq
PLCβ
Naringin?
Rutin ?
Ca2+
PI3K
AKT
Epac
TSC1/2
AMPK γ
α
mTOR
PI3 Kinase
Classe III
4EBP1
P70
S6K
Synthesis Protein
eEF2K
Protein
ACC
Apoptosis
HNF4
SREBP1
Malonyl
CoA
GeF
EF2
HMG-CoA
Reductase
p53
Metabolism
MEF
TORC2
PFK2
CPT1
PGC1a
Fatty
Acid
Syntase
Hepatic
Acid VLDL
Synthesis
Glut4
Gene Expression
Glycolysis
GS
Glyconeogenesis Expression of
Mytochondrial Genes in Muscle
Fatty Acid
Oxidation
Glycogen Syntesis
Sterol/Isoprenoil
Synthesis
Lipid Metabolism
Carbohydrate Metabolism
f0035
FIGURE 84.6 Metabolic pathways and molecular players regulated by AMPK. Cascade of events following PDE inhibition by resveratrol
and other flavonoids and unknown alternative mechanisms lead to AMPK activation which is a central regulator of lipid and glucose metabolism, as well as negative regulator of oncogenic pathways. This is thanks to AMPK ability to inhibit or activate several enzymes involved in
these processes as discussed in the text (see Plate 15).
8. CARDIAC HEALTH AND POLYPHENOLS
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1098
84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
Regulation of lipolysis by phosphodiesterase (PDE) inhibition in the liver
PKB
IR
AC
PI3K
ATP
P
5’AMP
C/EBPα
P
PDE
cAMP
PKA
PKB
PDE
Flavonoid
Lipolysis
Products
TGH
CH
P
PNPLA4
LIPID
DROPLET
TAG CE
Fatty Acids
BILE
VLDL Secretion
β–Oxidation
cLDL
f0035
p0455
FIGURE 84.6 Continued
are subject of an intensive research. Computational
chemistry studies indicate that certain flavonoids can
sterically fit in the cyclic nucleotide binding pocket of
PDE and mediate competitive inhibition of the enzyme
activity.6 The positions of hydroxyl groups on polyphenol ring C of flavones and flavanones are important for
differential PDE inhibition. For example, the
C-40 hydroxyl group of flavones is very important for
PDE3 inhibition; hydroxylation at the C-5 position is
important for inhibition of PDE1, PDE2, PDE4, and
PDE5; and C-7 hydroxylation is important for inhibition
of PDE1, PDE3, and PDE4.55,56 The most potent phosphodiesterase (PDE) inhibitors are aglycones that have
a C2.3 double bond, a keto group at C4 and hydroxyls
at C30 and/or C40 . However, when the C-ring is opened
the requirement for the C2.3 double bond is eliminated.
Aglycone flavonoids such as apigenin, genistein, daidzein, and quercetin fit very well in the catalytic site of
x-ray crystallographic models of human PDE3B,
PDE4B, and PDE4D.1.6 Apigenin glycosides, such as
Vicenin-2 reach a relatively very high concentration in
bergamot fruits (see Table 84.1). In addition, computational chemistry considerations would suggest that
aglycone of neoeriocitrin, rich in bergamot juice and
albedo, can be also a potent inhibitor of PDE3 due to
the presence of hydroxyls at C30 position.
There are two different PDE3 isoforms A and B. They
exhibit cell-specific differences in expression. PDE3A is
highly expressed primarily in the cardiovascular system,
for example in platelets, smooth muscle cells and cardiac
myocytes.57 PDE3B is expressed in cells of importance
for energy metabolism including hepatocytes, brown
and white adipocytes, pancreatic β cells58,59 and it is also
found in the cardiovascular system.57
Interestingly, lack of functional of PDE3B in the p0460
liver leads to a severe dysregulation of glucose, triglyceride and cholesterol metabolism, suggesting that the
modulatory role of PDE3B is crucial for lipid and glucose homeostasis.60,61 Hepatocytes from PDE3B knockout mice display increased glucose, triglyceride and
cholesterol levels, which was associated with increased
expression of gluconeogenic and lipogenic genes/
enzymes including, HMG-CoA reductase, phosphoenolpyruvate carboxykinase, peroxisome proliferatoractivated receptor γ (PPAR g) and sterol regulatory
element-binding protein 1c (SREBP 1c). Dysregulation
of PDE3B can have a role in the development of fatty
liver, which is very common in metabolic syndrome
and type-2 diabetes patients.60
Thus it is very likely that bergamot flavonoids such p0465
as apigenin derivatives and neoeriocitrin mediate their
beneficial effects on lipid and glucose homeostasis by
PDE3B modulation, but this hypothesis needs to be
verified by experimental results.
5.3 AMP Kinase Activation and Glucose and
Lipid Metabolism
s0075
As discussed above, the consequence PDE inhibition p0470
and cAMP increase is AMPK activation (Figure 84.6,
8. CARDIAC HEALTH AND POLYPHENOLS
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5. POSSIBLE MOLECULAR MECHANISMS OF ACTION OF BERGAMOT POLYPHENOLS
p0475
p0480
Plate 15). AMPK plays a central role in regulating glucose and lipid metabolism and energy production in
several different organs working as a sensor of AMP/
ATP levels.62 The intracellular AMP increases, when
energy status is low and binds to AMPK to allow its
activation. At the molecular level, AMPK is a serine/
threonine protein kinase that can regulate activity of
many downstream targets. To ensure the energy production, AMPK, not only facilitates glucose uptake,
but it triggers several catabolic and blocks anabolic
pathways. First off all, AMPK prevents the accumulation of fat by modulating downstream-signaling
components like acetyl-CoA carboxylase (ACC) and
HMG-CoA reductase (Figure 84.6, Plate 15). AMPK
phosphorylates and inhibits ACC1 activity, which
blocks the rate-limiting step in fatty acid synthesis.63,64
On the other hand, phosphorylation of ACC2 by
AMPK augments fatty acid oxidation, which is a catabolic pathway for fat deposits (Figure 84.6, Plate 15).
More importantly, AMPK is an established target in
the treatment of type-2 diabetes. Metformin, the first
line anti-diabetic drug, currently prescribed to more
than 120 million people with type-2 diabetes worldwide, activates AMPK and this activation is required
for the blood glucose lowering effects of the drug.
As shown in Figure 84.6 (Plate 15), critical for the
therapeutic effects of AMPK activation is augmentation
of intracellular glucose uptake. This occurs via different
effectors, and leads to the activation of the glucose
transporter GLUT1 in all cells or upregulation and
translocation GLUT4 to the cell membrane in muscle
cells.65 Interestingly, in the absence of elevated levels of
blood glucose, AMPK prevents glycogen synthesis to
increase glucose availability for energy production and
promotes glycolysis. This occurs via direct inhibition of
glycogen synthase and activation of glycolytic enzymes
as 6-phosphofrukto-2-kinase (Figure 84.7).65
AMPK also plays a role in reducing cholesterol syn- p0485
thesis. Indeed, It has been shown that AMPK phosphorylates HMG-CoA reductase at Ser-871, thereby
lowering its catalytic activity.66,67
There is overwhelming evidence that different flavo- p0490
noids can activate AMPK, both in vitro and in vivo.68,69
The majority of experimental studies were performed
on resveratrol, which rapidly and potently activates
AMPK in model systems. Indeed, resveratrol in a mice
model of diabetes and dyslipidemia (LDL receptordeficient mice)70 is 200 times more potent than
metformin in the activation of AMPK kinase and its
downstream effects.
Beside resveratrol, AMPK activation is closely associ- p0495
ated with the action of several other polyphenols implicated in the prevention of various metabolic disorders,
including diabetes, obesity, cardiac hypertrophy, and
alcoholic and non-alcoholic fatty liver disease.65 For
example, flavonoids such as green tea epigallocatechin
(ECCG) and soy genistein,71 as well as polyphenols
belonging to different chemical groups (ginsenoside,
berberine, curcumin. and theaflavin)65 are also good
activators of AMPK. All of these compounds show antidiabetic and hypolipidemic effects in animal models
and, in some cases, in human studies.4,72
Citrus/Bergamot flavonoids such as naringenin (narin- p0500
gin aglycon) and rutin can also activate AMPK.73,74 In
+ pectins
Bruteridin
Melitidin
Sequestration
of bile acids
All flavonoids
ACAT
HMG-Co
Reductase
Rutin?
Naringin?
All flavonoids
AMPK
HMG-CoA
Red
DNA methylation
Epigenetic
modifications
PDE
1099
QR2
Glucokinase
8. CARDIAC HEALTH AND POLYPHENOLS
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00084
FIGURE 84.7 Summary of possible f0040
molecular mechanisms which are likely
triggered by bergamot polyphenols.
Based on the vast literature several
mechanisms of hypolipemic and hypoglycemic effects of bergamot juice and
BPF could be attributed to aglycone
forms of bergamot flavanones and flavones as discussed in Section 5.
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1100
p0505
p0510
p0515
84. THE USE OF BERGAMOT-DERIVED POLYPHENOL FRACTION IN CARDIOMETABOLIC RISK PREVENTION
particular, naringenin has been shown to induce AMPK
phosphorylation and to stimulate glucose uptake in L6
myotubes in a dose- and time-dependent manner.74
Maximum stimulation was seen with 75 μM naringenin
for 2 hours, a response comparable to maximum insulin
response. The translocation of GLUT4 glucose transporters
has been indicated as a mechanism underlying the
increased glucose uptake. Furthermore, silencing of
AMPK, using a siRNA approach, abolished the
naringenin-stimulated glucose uptake. The SIRT1 inhibitors nicotinamide and EX527 did not have an effect on
naringenin-stimulated AMPK phosphorylation and glucose uptake, suggesting that naringin aglycon may show
anti-diabetic effects via AMPK and in contrast to resveratrol, this is not mediated by SIRT1.74
Hypoglycemic effects of naringin and its aglycon
form have been confirmed in animal models of metabolic syndrome induced by high-fat diet,7577 but not
in rat models of type-1 diabetes induced by streptozotocin.78 Interestingly, in all models, the animals treated
with naringin showed a significant reduction in
plasma tChol and TG levels, which in one study was
directly associated with activation of AMPK.77 The
addition of naringenin to the high-fat diet in animals
with genetic ablation of LDL receptor, not only suppressed significantly hyperlipidemia, but also reduced
atherosclerotic lesions by more than 70%.76
The second example of bergamot flavonoids that
can activate AMPK is rutin. Rutin is a flavon which is
particularly abundant in bergamot juice and albedo.17
Rutin has been shown to induce AMPK in hepatocytes
and pancreatic β cells.73 In hepatocytes, beside AMPK
stimulation, rutin suppressed oleic acid-induced lipid
accumulation. The expression of a critical molecule
involved in lipid synthesis, SREBP-1 was also attenuated in rutin-treated cells. Moreover, long-term incubation of rutin inhibited the transcriptions of HMG-CoA
reductase, ACC, fatty acid synthase (FAS), and other
enzymes involved in lipogenesis. All these molecular
endpoints can be explained by activation of AMPK.73
These findings in vitro, correlate nicely with results of
an independent study in vivo with a rat model of metabolic syndrome. Rutin, supplemented to western diet,
reversed or prevented metabolic changes such as
abdominal fat accumulation and glucose tolerance,
normalized expression of liver markers and prevented
hepatic steatosis, reversed oxidative stress and inflammation in the liver and heart.79 AMPK was suggested
as the main target of rutin.79
Taken together, these results suggest that AMPK
activation, via PDE/cAMP and calcium pathway, is a
potential target of many natural polyphenols, including naringin, rutin, apigenin and neoericitrin, rich in
bergamot.
5.4 Quinone Oxidoreductase 2 as an Example of s0080
Known Targets of Polyphenols with Unknown
Functions
Another possible mediator of antioxidant and meta- p0520
bolic effects of flavonoids is quinone oxidoreductase 2
(QR2). QR2 is one of the highest affinity targets of flavonoids that has been identified to date. The physical interaction between resveratrol and QR2 has been
characterized by crystallography.80 In addition, resveratrol, quercetin and other flavonoids, including those
found in bergamot juice, such as diosmin and rhoifolin,
are potent inhibitors of QR2 with IC50 values between
50 nM and 2 μM.81 QR2, also known as NRH:quinone
oxidoreductase 2 (NQO2), is a highly flexible flavoenzyme in terms of substrate and co-substrate and may
catalyze many oxido-reduction reactions, involving
xenobiotic and biogenic substrates.82 The typical reaction
catalyzed by this enzyme is a two-electron reduction of
quinones and oxidation of N-alkyl and N-ribosyl nicotinamide (NADH) derivatives.83 Biological functions of
QR2 are largely unknown, but it has been suggested that
QR2 is involved in the detoxification processes of endogenous quinones.8486 However, this enzyme plays totally
opposite functions with respect to several exogenous
substrates (mainly quinone or nitrogen compounds) and
many reaction products of QR2 appear to be toxic. In
fact, QR2 mediates toxicity of menadione, a toxic variant
of vitamin K. It bioactivates the anticancer antibiotic
mitomycin C and a synthetic anticancer agent CB1954
that is currently in clinical trials.87 Interestingly, QR2
mediates also human and animal toxicity of certain herbicides like paraquat. However, in this case paraquat is
not a direct substrate of QR2-mediated oxidoreduction,
but QR2 is crucially involved in the generation of oxidative stress in the presence of the herbicide.85 Thus, QR2
is capable of metabolically activating various quinones
and other compounds, which generate oxidative stress
and can ultimately lead to cytotoxicity and cell
death.82,85 In addition, QR2 is likely involved in the regulation of ROS levels and metabolic oxidative stress in
the absence of toxic substrates.85 Therefore, inhibition of
QR2 by citrus flavonoids might protect cells from harmful metabolically activated compounds and resulting
oxidative stress, which is a common denominator of several metabolic dysfunctions, including metabolic syndrome, diabetes and atherosclerosis. This interesting
hypothesis should be verified in future studies.
6. CONCLUDING REMARKS
s0085
Citrus and bergamot polyphenols, in particular, p0525
may exert numerous effects on cellular and organism
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1101
REFERENCES
p0530
p0535
p0540
homeostasis, including the regulation of fat, cholesterol
and carbohydrate metabolism. Given the fact that
pharmacological effects of nutraceutical preparations
based on bergamot flavonoids appear to be comparable to low-dose statins and metformin, while sideeffects are negligible, they represent a valid alternative
for conventional drugs. In fact, bergamot BPF has a
potential to become a “one pill” natural therapy
against metabolic syndrome and dyslipidemias.
Although to date there is limited scientific evidence
on how bergamot polyphenols can act at the molecular
level, several potential mechanisms can be easily predicted. As summarized in Figure 84.7, bergamot polyphenols can exert their beneficial effects through
HMG-Co reductase, PDE inhibition and AMPK activation as well as other signaling or metabolic regulatory
proteins, including other QR2. Further studies are
needed to address molecular mechanism of action of
bergamot
polyphenols,
to
tested
empirically
predictable models presented in this chapter.
Finally, the pharmacological and molecular effects
of a “fitocomplex,” or a mixture of active compounds
as they occur in natural plants, may be quite different
from the sum of individual activities, due to compensatory or synergistic effects of the mixture. It is also
possible that other non-polyphenolic components, such
as poly-unsaturated fatty acids present in bergamot
juice and BPF play an important role in the overall
pharmacological activity together with flavonoids.
Thus, as well as the need for further testing of BPF
in clinical settings, a basic experimental approach
addressing possible mechanisms of action of BPF is
necessary in the future.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
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NON-PRINT ITEM
Abstract
Bergamot (Citrus bergamia Risso et Poiteau) fruits are characterized by a particularly high content and a unique
composition of flavonoids, such as neoeriocitrin, neohesperidin, naringin, melitidin and brutieridin. Bergamot
juice and its concentrate, highly enriched in polyphenols—here referred to as Bergamot Polyphenol Fraction
(BPF)—has been evaluated in experimental and clinical studies. Studies performed in Italy and Australia showed
that BPF treatment leads to an important reduction in lipid parameters in the blood of patients with hyperlipidemia ranging from 15 up to 40% for total cholesterol and cholesterol-LDL. A striking reduction (mean
41.0 6 2.6%) was also observed for plasma triglyceride levels, accompanied by a significant decrease in blood glucose (22.3 6 1.0%) in a subgroup of patients with metabolic syndrome. Although BPF cannot be proposed as a
substitute for statins in patients at high risk of cardiovascular events, it offers an excellent alternative for low-risk
and for statin-intolerant patients. The robust performance of BPF in clinical practice against cardiometabolic risk
factors can be explained in the light of scientific evidence showing that bergamot flavonoids influence lipid and
sugar metabolism acting as 3-hydroxy-3-methlglutaryl coenzyme A (HMG-CoA) reductase inhibitors and AMP
kinase (AMPK) activators.
Keywords: Bergamot; cardiovascular disease; cholesterol; flavonoids; natural drugs; molecular mechanisms
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