Food Chemistry 125 (2011) 660–664
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Phenolic composition and antioxidant activity of culms and sugarcane
(Saccharum officinarum L.) products
Joaquim Maurício Duarte-Almeida a,⇑, Antonio Salatino b, Maria Inés Genovese a, Franco M. Lajolo a
a
b
Departamento de Alimentos e Nutrição Experimental, FCF, Universidade de São Paulo, Av. Prof. Lineu Prestes 580, Bloco 14, CEP 05508-900 São Paulo, SP, Brazil
Departamento de Botânica, Instituto de Biociências, Universidade de São Paulo, C. Postal 11461, CEP 05422-970 São Paulo, SP, Brazil
a r t i c l e
i n f o
Article history:
Received 14 April 2010
Received in revised form 21 July 2010
Accepted 15 September 2010
Keywords:
Sugar cane products
Phenolic compounds
Antioxidant activity
Flavonoids
Tricin
Chlorogenic acid
a b s t r a c t
The present work reports amounts of flavonoids and phenylpropanoids of culms of three sugarcane varieties and of raw juice, syrup, molasse and VHP sugar. The antioxidant activity of those materials was evaluated by the DPPH and b-carotene/linoleic acid methods. The predominant phenolics in culms were
phenylpropanoids (caffeic, chlorogenic and coumaric acids), while flavones (apigenin, tricin and luteolin
derivatives) appeared in lower amounts. Differences were noted either among phenolic profiles of sugarcane culms or between culms and sugarcane products. The antioxidant activities of solutions from most
samples were similar or higher than a 80 lM Trolox solution.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
Sugarcane (Saccharum officinarum L.) is a source not only of sugar and raw material for alcohol production, but also for a variety
of other products. Raw juice is a liquid with 14–16° Brix obtained
upon squeezing of the culms. Concentration of the juice to 55–65°
Brix produces sugarcane syrup. Further evaporation up to about
80° Brix leads to molasse, a dark product almost as thick as bee
honey. ‘‘Rapadura” is a hard, dark green food consumed mainly
in Brazilian rural areas, obtained by solidification of molasse. It is
similar to kokuto, a sugarcane product consumed as candies in
Japan. VHP (Very High Polarisation) sugar, a dark brown product,
is sold in the sugar market for industrial production of refined,
white sugar. VHP processing may lead either to crystal, amorphous
or granulated refined sugar (Cosan, 2002). Conventional methods
for sugar production include thermal and chemical treatment of
the juice, syrup and molasse, aiming to increase the sucrose concentration and pigment removal.
Pigments present in sugarcane juice are mainly phenolic compounds. Paton and Duong (1992) reported the phenolic composition of sugarcane and its products. Such compounds are mainly
phenylpropanoids and flavonoids, major representatives of the
latter being derivatives of naringenin, tricin, apigenin and luteolin
(Smith & Paton, 1986; Williams, Harborne, & Clifford, 1974). Naka⇑ Corresponding author. Tel.: +55 11 4043 4412; fax: +55 11 5084 2793.
E-mail address: [email protected] (J.M. Duarte-Almeida).
0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2010.09.059
sone, Takara, Wada, Tanaka, and Yogi (1996) isolated five antioxidant compounds from a kokuto extract; Takara et al. (2002)
increased the number to thirteen, including several glycosylated
phenolic compounds. Some compounds showed antioxidant activity higher than a-tocoferol. Payet, Cheong, and Smadja (2005,
2006) reported antioxidant activity by different samples of brown
sugar and suggested that some phenolic acids and flavonoids may
account for at least part of the observed activity. Phenolic substances in sugarcane juice may exert biological activities
(Duarte-Almeida, Vidal Novoa, Fallarero Linares, Lajolo, & Genovese, 2006). An acylated tricin glycoside isolated from sugarcane
juice was shown to have antiproliferative activity (Duarte-Almeida, Negri, Salatino, Carvalho, & Lajolo, 2007).
The present work aims to analyse the phenolic composition of
culms of three varieties of sugarcane and products obtained during
sugar production, as well as the corresponding antioxidant
activities.
2. Materials and methods
2.1. Materials
Samples of sugarcane culms and sugarcane products from progressive stages of sugar production were obtained from Cosan
(Piracicaba, Brazil, a manufacturer enterprise of sugarcane products). Sugarcane culms analysed were of the varieties SP801842
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J.M. Duarte-Almeida et al. / Food Chemistry 125 (2011) 660–664
Care was taken to avoid the influence of differences in phenolic
concentrations among samples. After solid-phase extraction, all
nd
nd
nd
nd
nd
nd
1.41 ± 0.00a
nd
0.59 ± 0.01b
nd
1.21 ± 0.06c
nd
Caffeic
0.67 ± 0.00a
nd
0.51 ± 0.00b
nd
0.63 ± 0.00c
nd
0.56 ± 0.01a
nd
0.26 ± 0.00b
nd
0.60 ± 0.00c
nd
0.20 ± 0.00a
nd
0.10 ± 0.00b
nd
0.13 ± 0.00c
nd
Cinnamic acids
Tricin
Obs: V1, SP801842; V2, SP813250; V3, RB855486; nd, Not detected.
A
Data are expressed in mg/100 g as means ± SD from triplicate.
a,b,c,d,e
Values with different superscript letters within the same columns are significantly different (p < 0.05; one-way ANOVA and Newman Keuls test).
2.6. Antioxidant activity
Luteolin
Identification and quantification of phenolic substances in the
eluates were carried out in duplicates, using analytical reversedphase HPLC on an Agilent 1100 system with autosampler and quaternary pump coupled to a diode array detector. The column used
was a Prodigy 5 l ODS3 reversed phase silica (250 4.6 mm i.d.,
Phenomenex Ltd.) and elution solvents were: A, water:tetrahydrofuran:trifluoroacetic acid 98:2:0.1 and B, acetonitrile. Solvent gradient used was based on Duarte-Almeida et al. (2007). Identification
followed comparison of UV spectra and retention times with
authentic standards, and quantification based on external calibration. Standards used for phenylpropanoid analyses were caffeic,
coumaric, ferulic and chlorogenic acids, and for flavones, tricin,
luteolin and apigenin. Results were expressed as mg/100 g of
sample.
Apigenin
2.5. Analytical HPLC
Flavonoids
Purification of extracts, aiming to maximise phenolic contents,
was carried out by fractionation of 10 ml aliquots of the extracts
on polyamide columns (CC 6, Macherey–Nagel Gmbh and Co., Duren, Germany), soaked previously with 20 ml methanol and 60 ml
water. The columns were washed with water (20 ml) and eluted
with methanol (50 ml), followed by methanol/ammonia (99.5:0.5
v/v, 50 ml). Each eluate was evaporated to dryness under reduced
pressure at 40 °C, resuspended in 1 ml methanol and filtered
through a 0.22 lm PTFE filter (Millipore Ltd., Bedford, MA) prior
to HPLC analysis and evaluation of antioxidant activities.
Table 1
Phenolic compounds contentsA of sugarcane culm varieties determined by HPLC-DAD.
2.4. Solid-phase extraction
1.52 ± 0.00a
nd
1.60 ± 0.00a
nd
1.11 ± 0.00b
nd
Chlorogenic
Coumaric
Ferulic
Extraction was performed in triplicate according to Duarte-Almeida et al. (2007), with slight modifications. Solid samples were
thoroughly homogenised by powdering with liquid nitrogen.
Duplicate samples of the fresh powder (20 g) were extracted three
times with methanol/water 70:30, including the water conveyed
by the samples, at speed five on a Brinkmann homogenizer (Polytron-Kinematica GmbH, Kriens-Luzern, Sweden) for 1 min in an ice
bath. The homogenate was filtered under reduced pressure
through filter paper (Whatman No. 6). Other liquid samples were
extracted with methanol/water 70:30, including the volume of
water contained in the sample, at speed five for 1 min, as above.
The solvent of the extracts were evaporated almost to dryness at
40 °C under reduced pressure on a Rotavapor RE 120 (Büchi, Flawil,
Sweden). The concentrated extracts were diluted with water to the
final volume of 25 ml.
nd
0.34 ± 0.02a
nd
0.29 ± 0.03b
nd
0.35 ± 0.01a
2.3. Extraction of phenolic substances
1.24 ± 0.01a
0.34 ± 0.02b
0.77 ± 0.00c
0.29 ± 0.03d
1.23 ± 0.00e
0.35 ± 0.01b
Total flavonoids
All chemicals used were reagent or HPLC grade. DPPH (2,2-diphenyl-1-picrylhydrazyl free radical), Trolox, Tween 40, b-carotene, linoleic acid, apigenin, luteolin, and coumaric acids were
purchased from Sigma–Aldrich Chemical Co. (St. Louis, MO). Caffeic, ferulic and chlorogenic acids were purchased from Apin
Chemicals Ltd. (Abingdon, UK). Tricin was isolated as described
elsewhere (Duarte-Almeida et al., 2007).
V1m
V1a
V2m
V2a
V3m
V3a
2.2. Chemicals
3.12 ± 0.00a
nd
2.29 ± 0.01b
nd
2.46 ± 0.07c
nd
Total cinnamic acids
(V1), SP813250 (V2) and RB855486 (V3). Sugarcane products analysed were raw juice (RJ), syrup (SY), molasse (MO) and VHP sugar.
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J.M. Duarte-Almeida et al. / Food Chemistry 125 (2011) 660–664
Table 2
Phenolic compounds contentA of the sugarcane products obtained by HPLC-DAD.
Flavonoids
RJm
RJa
SYm
SYa
MOm
MOa
VHPm
VHPa
Cinnamic acids
Apigenin
Luteolin
Tricin
Caffeic
Chlorogenic
Coumaric
Ferulic
1.73 ± 0.16a
nd
2.31 ± 0.01a
nd
16.82 ± 0.96b
nd
0.71 ± 0.10c
nd
0.27 ± 0.05a
nd
1.05 ± 0.12b
nd
6.35 ± 0.51c
nd
0.42 ± 0.01a
nd
0.59 ± 0.06a
1.87 ± 0.48a
2.15 ± 0.14a
3.53 ± 0.22b
13.23 ± 1.59c
26.16 ± 2.38d
0.31 ± 0.11a
0.46 ± 0.12a
nd
nd
nd
nd
nd
nd
nd
nd
nd
0.51 ± 0.12a
2.93 ± 0.13b
0.77 ± 0.12a
24.29 ± 1.81c
3.29 ± 0.16b
0.57 ± 0.01a
nd
0.90 ± 0.06a
nd
5.55 ± 1.07b
nd
23.43 ± 0.4c
nd
0.38 ± 0.02a
nd
0.28 ± 0.13a
nd
1.41 ± 0.06b
nd
7.88 ± 1.34c
nd
0.13 ± 0.01a
nd
Total flavonoids
Total cinnamic acids
2.60 ± 0.18a
1.87 ± 0.48a,f
5.51 ± 0.02b
3.53 ± 0.22a,g
36.40 ± 0.50c
26.16 ± 2.38d
1.43 ± 0.09a,f
0.46 ± 0.12e
1.18 ± 0.19a
0.51 ± 0.12a,d
9.89 ± 1.18b
0.77 ± 0.12a,d
55.60 ± 3.03c
3.29 ± 0.16d
1.08 ± 0.04a,d
nd
Obs: RJ, raw juice; SY, syrup; MO, molasses; VHP, VHP sugar; nd, Not detected.
A
Data are expressed in mg/100 g as means ± SD from triplicate.
a,b,c,d,e,f
Values with different superscript letters within the same columns are significantly different (p < 0.05; one-way ANOVA and Newman Keuls test).
samples were diluted to the concentration of 100 lM of chlorogenic acid, based on estimates by HPLC analyses (Tables 1 and 2).
2.6.1. Radical scavenging activity (RSA)
The method determines the activity of substances at scavenging
free radicals (Brand-Williams, Cuvelier, & Berset, 1995). Analyses
were carried out using the DPPH (2,2-diphenyl-1-picrylhydrazyl)
method (Brand-Williams et al., 1995; Laskar, Sk, Roy, & Begum,
2010). Solutions of samples in methanol were individually added
to 0.1 mM DPPH in methanol. The mixture was incubated in the
dark at 25 °C for 30 min (Duarte-Almeida, Santos, Genovese, & Lajolo, 2006). Scavenging capacity was determined by monitoring the
absorbance at 517 nm. Lower absorbance indicates higher free radical scavenging activities. The RSA percent was calculated as
follows:
%RSA ¼
acontrol asample
100
acontrol
where acontrol is the absorbance of the control (methanol plus
DPPH), and asample is the absorbance of the sample plus DPPH. A
Trolox 80 lM methanol solution was used as reference for compar-
ison of relative RSA efficiency of the samples analysed. All assays
were run in five replicates.
2.6.2. b-Carotene/linoleic acid method (b-CLAM)
The method evaluates the antioxidant activity at inhibiting the
discolouration of b-carotene caused by free radicals yielded during
peroxidation of linoleic acid (Yanishilieva & Marinova, 1995). The
procedure used was based on Duarte-Almeida et al. (2006). Reactive solutions were prepared mixing 25 ll of 2 mg/ml chloroform
solution of b-carotene with 20 lg linoleic acid, 200 mg Tween 40
and 0.5 ml chloroform. Chloroform was then completely evaporated under nitrogen flow, and 25 ml distiled water saturated with
oxygen was added to the mixture. The absorbance was adjusted
with water to 0.6. For the oxidation reaction, 10 ll of the solutions
of concentrated extracts were mixed with 250 ll of the b-carotene/
linoleic acid solution in a microplate. The samples were submitted
to autoxidation at 45 °C for 120 min. The absorbance at 470 nm
was measured at time zero and at 15 min intervals using a microplate spectrophotometer (Benchmark Plus, Bio-Rad). Methanol was
used as control and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) as reference antioxidant. All assays were
run in five replicates. Antioxidant activity of the sample was calcu-
Fig. 1. HPLC chromatograms of sugarcane syrup from solid-phase extraction by elution with methanol (a) and methanol/ammonium hydroxide (b) monitored at 370 nm.
Peaks (1) chlorogenic acid; (2) apigenin derivative; (3) luteolin derivative; (4) ferulic acid derivative; (5) coumaric acid derivative; (6) tricin derivative.
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J.M. Duarte-Almeida et al. / Food Chemistry 125 (2011) 660–664
lated as percent inhibition of oxidation versus control, using the
equation
"
%Inhibition ¼ 100 1 #
ða0sample a120
sample Þ
ða0control a120
control Þ
where asample and acontrol are the absorbances of sample and control
at t = 0 min and t = 120 min, respectively.
2.7. Statistical analysis
Results are expressed as means ± standard deviation. The statistical analyses were carried out using Statistica version 5.0 (StatSoft
Inc., Tulsa, OK). Comparison of means of triplicate determinations,
using a significance level p < 0.05, was performed by one way analysis of variance (ANOVA). When significant differences were detected, Newman-Keuls post hoc test was used to determine the
degree of statistical significance of the differences.
Table 3
Antioxidant activity (% inhibition) of solutions of sugarcane products containing
chlorogenic acid at 100 lM concentration.
Products
RJm
RJa
SYm
SYa
MOm
MOa
VHPm
VHPa
TroloxC
RSAA
b-CLAMSB
a
28.6 ± 0.4
38.4 ± 0.4b
32.0 ± 0.6c
57.8 ± 1.2d
23.9 ± 0.6e
45.2 ± 2.6f
32.6 ± 1.3c
32.4 ± 1.9c
33.2 ± 2.8c
77.3 ± 1.4a
55.7 ± 2.4b
76.3 ± 1.4a,d
72.4 ± 6.0a
73.2 ± 3.8a
74,4 ± 0.5a
85.8 ± 0.6c
83.2 ± 1.6c,d
72.1 ± 3.2a
Obs.: RJ, raw juice; SY, syrup; MO, molasses; VHP, VHP sugar. Extracts m, obtained
with methanol and extracts a, obtained with methanol/ammonium hydroxide.
A
RSA, Radical Scavenging Activity.
B
b-CLAMS, b-carotene/linoleic acid method system.
C
Activity of solution of Trolox (at 80 lM for RSA and at 10 lM for b-CLAMS).
a,b,c,d,e,f
Values with different superscript letters within the same columns are
significantly different (p < 0.05; one-way ANOVA and Newman Keuls test). Data are
expressed as means ± SD from five replicates.
3. Results and discussion
3.1. Phenolic profiles of sugarcane culms and products
In the present paper, flavonoids, irrespective of being aglycones
or glycosides, are referred to by the respective aglycon. Thus, all
apigenin derivatives are designated ‘‘apigenin”. Although comprising several isomers, all chlorogenic acids are designated ‘‘chlorogenic acid”. Derivatives of caffeic and ferulic acids, conjugated or
not, are designated ‘‘caffeic” and ‘‘ferulic acid”, respectively. The
same applies to methoxycoumaric acid derivatives, irrespective
of being the o-, m- or p-isomer and being conjugated or not.
Most phenylpropanoids and flavones were obtained in the solid
phase by methanol elution. Tricin was eluted mostly with methanol/ammonium hydroxide. Fig. 1 contains chromatograms of
methanol and methanol/ammonium hydroxide eluates.
Phenolic compounds in culms are predominantly cinnamic
acids (caffeic, chlorogenic and coumaric acids). Flavonoid contents
are lower, represented exclusively by flavones (apigenin, luteolin
and tricin) (Table 1). To date, only flavones have been reported
for sugarcane (Colombo, Yariwake, Queiroz, Hdjoko, & Hostettmann, 2005; Paton, 1978; Takara et al., 2002). Phenolic profiles differ among the culm varieties analysed. For example, contents of
coumaric acid and apigenin are much lower in V2 than in V1 and
V3 (Table 1).
Phenolic profiles differ comparing sugarcane culms and derived
products (RJ, SY, MO and VHF). A remarkable difference is the mutual exclusiveness between caffeic and ferulic acids, the former
present only in culms and the latter in sugarcane products (Table
1 and 2). Seemingly, enzymatic processes take place after sugar
grinding, promoting methylation of caffeic acid from culms, giving
rise to ferulic acid in sugarcane products. Another difference is the
predominance of chlorogenic acid (a phenylpropanoid) in culms
and of tricin (a flavonoid) in sugarcane products. In addition, the
predominant flavonoid in culms is luteolin and in sugarcane products, tricin (Table 2).
Procedures for extraction and processing of sugarcane products
may account for some of the observed differences. RJ is produced
by grinding sugarcane culms. Thus, substances more water-soluble
tend to be washed away from culms into the juice flush. On the
other hand, extraction for chemical analysis of culms starts with
treatment of powdered material with 70% methanol, which favours
extraction of alcohol-soluble and fibre-associated phenylpropanoids (Pan, Bolton, & Leary, 1998). In addition, methanol probably
turns enzymes inactive (Paton, 1992) and high temperatures
aimed at accelerating concentration of solids may lead to thermal
degradation of more susceptible substances.
An increase of total solids implies higher contents of phenolic
substances: SY has more phenolic substances than RJ (p = 0.0017)
and MO has the highest values. At the latter phase, chlorogenic acid
becomes the predominant phenylpropanoid (Table 2). An inverse
relationship between browning and chlorogenic acid, some neutral
phenolic compounds and luteolin derivatives contents was previously observed (Paton & Duong, 1992). This finding suggests that
these compounds participate in the browning process, undergoing
enzymatic and oxidative reactions (Bucheli & Robinson, 1995).
Chlorogenic acid still predominates among phenylpropanoids in
VHP (Table 2). Results of Table 1 disagree with the data of Payet,
Cheong, and Smadja (2005), who reported neither chlorogenic
nor flavones in their analyses of seven samples of brown sugar.
Predominance of tricin is maintained throughout the sugar processing (Table 2). Tricin derivatives are relatively rare in nature
(Cai, Steward, & Gescher, 2005; Harborne & Hall, 1964; Hudson,
Dinh, Kokubun, Simmonds, & Gescher, 2000) and is probably related to resistance of plants to several illnesses (Harborne & Hall,
1964; McGhie, 1993).
3.2. Antioxidant activity
3.2.1. RSA
Some eluates from sugarcane products presented activity higher than Trolox 80 lM (Table 3). SYa had the highest activity (57.8%
of DPPH reduction), 74% over Trolox. Other products stood out,
such as MOa (45.2%) and RJa (38.4%) (Table 3), both with values
higher than Trolox (p < 0.01). Activities of eluates RJm, SYm, VHPa
and VHPm were not distinct from Trolox. Except for VHP, a eluates
had RSA higher than m eluates from the same product (Table 3)
(p < 0.05). Eluates a of RJ, MO and SY were 35%, 89% and 80% higher, respectively, than the m counterparts. No significant difference
was observed between the activities of VHPa and VHPm eluates.
3.2.2. b-Carotene/linoleic acid method (b-CLAM)
Except for RJa, antioxidant activity of eluates and Trolox varied
in the range 72–85% (Table 3). Contrary to results of RSA, differences between a and m eluates were not the rule. Activities of eluates RJa and RJm were significantly distinct; however, higher
activity corresponded to the m eluate (a eluates were more active
by RSA, Table 3). Activities of VHP eluates were low by RSA, but by
b-CLAM were the highest, being the only samples exceeding Trolox
activity (Table 3).
Antioxidant activity depends on many factors, such as contents
of phenolic compounds and structural details (Moure et al., 2001).
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J.M. Duarte-Almeida et al. / Food Chemistry 125 (2011) 660–664
Phenolic substances with o-dihydroxy systems tend to be highly
active (Cuyckens & Clayes, 2004; Hopia & Heinonem, 1999).
Often no correlation is observed between results by RSA and bCLAM (Tsao, Yang, Xie, Sockovie, & Khanizadeh, 2005). RAS is more
selective toward hydrophilic compounds, while b-CLAM reflects
contents of lipophilic substances (Sahreen, Khan, & Khan, 2010;
Xu, Tang, Liu, Li, & Dai, 2010). For these reasons, conclusions based
only on one method may lead to underestimation of antioxidant
activities. Hence it is prudent to evaluate antioxidant activities at
least by two different methods (Tsao et al., 2005; Verhagen et al.,
2003).
4. Conclusions
Sugarcane products are appreciated as food in social-economically disfavoured areas of Brazil and other countries. Results of
the present work indicate that consumption of these products
should be encouraged. Some of them, such as ‘‘rapadura”, are
cheap sources of antioxidant substances, vitamins (A, C and D)
and minerals (Fe, Ca, P, K, Mg) (Bernal, Guzman, & Jimenez,
2004). These facts lend support to programs, now in course, aiming
to stimulate or reintroduce the use of ‘‘rapadura” as complement
food for children in elementary school.
Acknowledgements
The authors thank CNPq (Conselho Nacional para o Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo
a Pesquisa de São Paulo) for financial support.
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