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Antioxidant Activity of Phenolics Compounds From Sugar Cane (Saccharum

officinarum L.) Juice

Article  in  Plant Foods for Human Nutrition · December 2006

DOI: 10.1007/s11130-006-0032-6 · Source: PubMed

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 Plant Foods for Human Nutrition


c 2006 Springer Science+Business Media, Inc.
DOI: 10.1007/s11130-006-0032-6
Antioxidant Activity of Phenolics Compounds From Sugar Cane (Saccharum officinarum L.)
Juice
JOAQUIM MAURÍCIO DUARTE-ALMEIDA,1 ALEXIS VIDAL NOVOA,2 ADYARY FALLARERO LINARES,2
FRANCO M. LAJOLO1 & MARIA INÉS GENOVESE1, ∗
1 Departamento de Alimentos e Nutrição Experimental, Universidade de São Paulo, Av. Prof. Lineu Prestes, 580, Bloco 14, Cep 05508-900, São Paulo,
SP, Brazil; 2 Departamento de Bioquı́mica, Universidad de La Habana, La Habana, Cuba (∗ author for correspondence; e-mail: genovese@usp.br)
Abstract. Phenolic compounds in sugar cane (Saccharum officinarum L.)
juice were identified and quantified by analytical high performance liq-
uid chromatography and photodiode array detection, showing the pre-
dominance of flavones (apigenin, luteolin and tricin derivatives), among
flavonoids, and of hydroxycinnamic, caffeic and sinapic acids, among
phenolic acids, representing a total content of around 160 mg/L. A tricin
derivative was present in the highest proportion ( >10% of the total). The
phenolic extract obtained from sugar cane juice showed a protective ef-
fect against in vivo MeHgCl intoxication and potent inhibition of ex vivo
lipoperoxidation of rat brain homogenates, indicating a potential use for
beneficial health effects and/or therapeutic applications.
Key words: Sugar cane, Polyphenolics profile, Antioxidant activity
Introduction
The sugar cane (Saccharum officinarum L.) is a widely dis-
tributed plant, and is one of the most important sources of
sugar. For Brazil, sugar cane has economic dimensions, as
the country is a leader of 80 main producers, and accounts
for the 25% of the worldwide production. Beside this, sugar
cane has other important uses as animal feed, as the raw ma-
terial for the production of alcohol, and other derivatives [1].
Recent reports have shed light into several biological
properties of sugar cane and its derived products. Some in-
vestigators [2, 3] have consistently proved on in vivo models
the antioxidant properties of high molecular weight alco-
hols contained in the wax. Sugar cane also contains pheno-
lic acids, flavonoids and other phenolic compounds [4, 5],
which could account for certain antioxidant activity. Indeed,
a direct relationship between antioxidant activity and phe-
nolics content in vegetable extracts has been convincingly
demonstrated [6].
In a 1996 report, Nakasone et al. [7] showed the presence
of five antioxidant compounds in a kokuto extract, followed
by reports of Takara et al. [8] on the isolation of several
glycosylated phenolic compounds from the same natural
extract. Kokuto is a food product similar to Brazilian “ra-
padura,” is highly appreciated as a candy and consisting
of a block of raw brown sugar, made from concentrated
sugar cane juice. In addition, Payet et al. [9] found antioxi-
dant activity in different samples of cane brown sugars and
proposed various phenolic acids and flavonoids compounds
to be at least partially responsible for the observed activ-
ity. However, the statistical correlation between phenolics
content and antioxidant activity was low. Hence, the au-
thors suggested on the possibility of other metabolites to
be also involved, possibly, those formed during the sugar
production.
Thus, although phenolic entities in sugar cane can act as
antioxidants, data on its actual polyphenolic composition
is scarce and very little is known about its antioxidant ac-
tivity and/or in vivo biological activity. In view of all the
above considerations, the aim of the current study was to
identify phenolic compounds present in sugar cane juice,
as consumed by humans, and possibly correlate it to the
antioxidant properties.
Materials and Methods
Materials
Fresh sugar cane juice was obtained from the Central Mar-
ket of São Paulo, Brazil (CEAGESP), transported under
refrigeration, immediately filtered through cheesecloth and
diluted 1:2 (v/v) with acidified (HCl) deionized water (pH
2.0).
Chemicals
All the chemicals were reagent or HPLC grade. DPPH•
(2,2-diphenyl-1-picrylhydrazyl radical), Trolox, Tween 40,
β-carotene, linoleic acid, apigenin, luteolin, catechin, caf-
feic, ferulic, coumaric and chlorogenic acids were pur-
chased from Sigma-Aldrich Chemical Co. (St. Louis, MO).
Preparation of Phenolic Extracts
The samples obtained as above were treated with Amberlite
XAD2 (Supelco, Bellefonte, PA, USA), according to
Andrade et al. [10]. Retained phenolic compounds were
recovered through elution with three portions (500 mL
each) of methanol:amonia (99.5:0.5 v/v). The eluates ob-
tained were kept together and concentrated until solvent
elimination on a Rotatory evaporator (Rotavapor RE 120,
Büchi, Flawil, Sweden) at ≤40◦ C. An aliquot of the con-
centrate was redissolved in methanol and filtered through
0.22 µm PTFE filters (Millipore Ltd., Bedford, USA) for
HPLC analysis. For in vivo rat administration, the extract
was lyophilized in a Edwards Super Modulyo freeze-drier
(Edwards High Vacuum, Crawley, Sussex, UK) at -52◦ C
for 12 h in vacuum (5 mbar).
Determination of Total Phenolic Concentrations
Total phenolic content was determined by means of the
Folin-Ciocalteu procedure [11] and expressed as chloro-
genic acid equivalents (CAE) per L of sample.
Analytical HPLC
Identification and quantification of phenolics was achieved
using analytical reversed-phase HPLC in a Hewlett-Packard
1100 system with autosampler and quaternary pump cou-
pled to a diode array detector. The column used was a
Prodigy 5 µ ODS3 reversed phase silica (250 mm × 4.6 mm
i.d., Phenomenex Ltd.) and elution solvents were: A, water
: tetrahydrofuran : trifluoroacetic acid 98:2:0.1 and B, ace-
tonitrile. Solvent gradient was similar to Arabbi et al. [12].
Identification was made based on the spectra and retention
time, and quantitation based on external calibration. Sam-
ples were injected in duplicate and flavones were quantified
using luteolin and apigenin chloride (Sigma Chemicals Co.,
St. Louis, U.S.A.) as external standards. For phenolic acids,
standard solutions of caffeic, sinapic and chlorogenic acids
(Apin Chemicals Ltd., Abingdon, UK) were used. Cali-
bration was performed injecting the standard three times
at five different concentrations. Results were expressed as
mg/100 mL of sugar cane juice.
Antioxidant Activity
Subchronic MeHgCl Intoxication Rat Model. The in vivo
antioxidant capacity was evaluated using a subchronic
MeHgCl intoxication rat model. Male Wistar rats (weight-
ing in the range of 190–215 g) were purchased from CEN-
PALAB (La Habana, Cuba) and used in this experiment.
Animals were kept at room relative humidity ( ∼ 79%)
and temperature ( ∼ 25◦ C) with free access to water and
standard diet (ALYco, La Habana, Cuba). For the exper-
iment, animals were randomly distributed into 3 groups.
Group II received a daily subcutaneous injection of ethano-
lic 10 mg/kg MeHgCl during 15 days. Group III received
sugar cane phenolic extract (10 mg of total phenolics/kg)
by gavage, 60 min before MeHgCl injection in alternate
days. Control group (I) received a daily sc. injection of
20% ethanol. Every day, the body weight and the food con-
sumption were assessed. Mortality was monitored up to 15
days after starting the treatment.
All the animal studies reported in this work have been
carried out in accordance with the Cuban regulations on
the protection of animals [13]. All experimental protocols
were revised by Animal Care and Use Committee of the
Faculty of Biology, University of Havana, and conducted
humanely.
Measurement of Thiobarbituric Reactive Substances
(TBARS) After Spontaneous Lipoperoxidation of Rat Brain
Homogenates. Measurement of TBARS after spontaneous
lipoperoxidation of rat brain homogenates was performed
according to Ohkawa et al. [14]. Male Wistar rats were
anesthetized with ethyl ether, decapitated, and the brain
was carefully removed from the skull, washed with ice-cold
0.9% NaCl solution, weighed and homogenized at 4◦ C in
50 mM phosphate buffer (pH 7.4), in a ratio of 1 g of wet
tissue to 9 mL of buffer, by using a Teflon Potter-Elvehjem
homogenizer. After centrifugation at 10,000 × g for 15 min,
the brain homogenate so obtained was kept at −70◦ C, for
up to a week, until use.
Measurement of thiobarbituric reactive substances
(TBARS) was performed as following: Aliquots of 25 µL
of brain homogenates were incubated with the same vol-
ume of different concentrations of sugar cane extracts, at
37◦ C during 40 min, in an oscillating bath. Incubations
were stopped by the addition of 350 µL of cold acetic acid
20% pH 3.5 and malondialdehyde (MDA) formation was
followed by the addition of 600 µL of TBA 0.5% in acetic
acid 20% pH 3.5. The mixtures were incubated at 90◦ C for
1 h, and allowed to cool. 50 µL of SDS were added, and
then tubes were centrifuged at 500 × g in a Kubota 1120
centrifuge for 15 min at room temperature ( ∼ 25◦ C). The
absorbance was measured at 532 nm. Antioxidant activity
was expressed as the percentage of inhibition of TBARS
formation related to the control undergoing maximum lipid
peroxidation on the assay conditions. All the values are
means of three determinations.
DPPH• Scavenging Activity. DPPH• (2,2-diphenyl-1-
picrylhydrazyl radical) scavenging activity of sugar cane
phenolics was assessed according to Brand-Williams et al.
[15], with some modifications [16]. Briefly, a 50 µL aliquot
of the extract previously diluted and 250 µL of a methanolic
solution of DPPH• (0.5 mM) were shaken and after 25 min
at 25◦ C the absorbance was measured at 517 nm using the
Microplate Spectrophotometer (Benchmark Plus, Biorad).
Results were expressed as percentage (%) of radical scav-
enging (MeOH as control).
β-Carotene/Linoleic Acid Bleaching Method (β-CLAMS).
The antioxidant capacity was also assessed by the
β-carotene bleaching method according to Marco [17], with
some modifications [16]. For the preparation of the reac- 100
tive solution, aliquots (25 µL) of β-carotene in chloroform
6
(2 mg/mL) were mixed with linoleic acid (20 µg), Tween

umA (328 nm)

40 (200 mg) and chloroform (0.5 mL). After this, chloro- 80

form was completely evaporated under nitrogen flow, and

25 mL of distilled water saturated with oxygen were added 60
to the mixture. The absorbance was adjusted with water to 1
0.6. For the oxidation reaction, an aliquot of the sample
(10 µL) was mixed with 250 µL of the β-carotene solution 40
in a microplate. The samples were submitted to autoxida-
3
tion at 45◦ C for 120 min, and the absorbance measured at 2

470 nm (methanol as control). Results were expressed as 20 4 5

3 5
percentage of inhibition of β-carotene bleaching compared
to the control (100% oxidation).
0
0 5 10 15 20 25
time (min)
Results and Discussion
Figure 1. HPLC chromatogram of the phenolic fraction from sugar cane
Phenolic Composition of Sugar Cane Juice juice: hydroxycinnamic acids (1); sinapic acid (2); apigenin derivatives
(3); caffeic acid (4); luteolin derivatives (5); tricin derivatives (6).
The total polyphenolic content of sugar cane juice, deter-
mined by the Folin-Ciocalteau procedure, was relatively
high, of 160 mg CAE/L. This means that the consumption hibit a potent activity against oxidative stress [21]. Ad-
of a glass of 250 mL would result in an intake of 40 mg of ditionally, recent studies in several systems have shown
phenolics, and in this way sugar cane juice would represent that apigenin induces tumor growth inhibition effects [22].
an important source of these antioxidant compounds in our Cinnamic acids such as caffeic acid are also very well
diet. Compared to commercial soy-based beverages, which known for their antioxidant properties in different model
present 18 to 83 mg of isoflavones/L, with a mean value systems [22]. It is important to consider that the bioactiv-
of 32 mg/L [18], sugar cane juice would be a cheaper and ity of tricin, together with apigenin and luteolin in sugar
better alternative to increase the ingestion of polyphenolics. cane juice, could be synergistic or additive. In this regard,
HPLC-DAD identification of the phenolic compounds from Lee et al. [24] screened approximately 700 plant extracts
sugar cane juice showed the presence of flavonoids (api- for antioxidant activity, from which 28 were found to be
genin, luteolin and tricin derivatives), and phenolic acids, active, and apigenin happened to be the most active among
mainly caffeic, sinapic, and isomers of chlorogenic acid the flavonoids constituents. Similarly, Cholbi et al. [25]
(Fig. 1, Table 1). Among the flavonoids detected, the highest investigated 35 phenolic compounds (most of them belong-
concentration corresponded to a tricin derivative accounting ing to the flavonoid group) for their ability to inhibit free
for approximately 10% of the total polyphenolic content. radical-induced microsomal lipid peroxidation, and they
These results are in accordance to that of Paton & Duong [4] found luteolin and apigenin to be the most potent. Hence,
who reported the presence of these three flavones in sugar in view of its polyphenolic composition and levels, sugar
cane extracts along with chlorogenic acid and chlorogenic cane juice may be regarded as a potential source of natural
acid isomers. antioxidants.
The identified flavonoids, tricin (5,7,4-trihydroxy-3,5-
dimethoxyflavone), apigenin (4 ,5,7-trihydroxy-flavone)
Table 1. Concentration of the main phenolic compounds
and luteolin (3 ,4 ,5,7-tetradihydroxy-flavone) are widely (mmol.L−1 ) in sugarcane juice
distributed in plants and fall into the classification of
flavones within polyphenolics. Commonly, flavonoids are Compound Concentration (mmol.L−1 )
consumed as natural component of vegetables, beans, fruits
Apigenin 27.8 ± 0.8
and/or as phytotherapeutics, and are generally regarded as Luteolin 9.2 ± 0.8
safe chemicals, thus displaying low toxicological activity Tricin 35.7 ± 1.1
[19]. Caffeic acid 4.1 ± 0.1
Polyphenolic compounds, and particularly flavonoids, Hydroxycinnamic acids 85.8 ± 1.3
Sinapic acid 25.4 ± 0.2
have been proposed as therapeutical alternatives for the
treatment of a variety of pathologies [20]. Flavonoids, par- Note. Values are expressed as average ± standard deviation
ticularly apigenin and luteolin, have been shown to ex- (n = 3).
 Apigenin, tricin and luteolin were present in sugar cane
as glycosylated derivatives, and glycosylation pattern is
widely known to influence the antioxidant capacity of
flavonoids [6]. However, the glycosides can undergo enzy-
matic hydrolysis in the gastrointestinal tract resulting in the
formation of the respective aglycones. The sugar moieties
may influence absorption and in vivo bioavailability of the
flavonoids [19]. Hence, in vivo systems for the evaluation
of food antioxidants is necessary and important.
Antioxidant Activity of Phenolics Compounds From Sugar
Cane Phenolic Extract
I. Subchronic MeHgCl Intoxication Rat Model. The in
vivo antioxidant activity was evaluated using a subchronic
MeHgCl intoxication rat model. The results demonstrated
that the administration of MeHgCl in Group II affected food
consumption, when compared to control animals (Group I).
Group III, which was also intoxicated but received sugar
cane extract by gavage, showed a lower decrease in the food
intake. At the end of the intoxication protocol, consump-
tion was statistically different among the groups as follows:
14.0 g/day/rat; 0.29 g/day/rat and 1.38 g/day/rat for Control
Group, II and III respectively.
Results concerning the effect of sugar cane phenolic ex-
tract treatment on the variations of body weights of rats
intoxicated with repeated doses of MeHgCl are presented
in Fig. 2. One week after starting the MeHgCl intoxication
protocol, a statistically significant loss of body weight was
observed in MeHgCl-treated animals (Group II) relative
to controls, and the difference was even higher at the end
of the experiment. The body weight loss was prevented
220
200
Body weight (g)
180
160
Group I
Group II
Group III
140
2 4 6 8 10 12 14 16
Treatment days
Figure 2. Effect of the extract from sugar cane juice on changes in body
weight of Wistar rats administered repeated doses of MeHgCl. Groups
II and III were daily injected ip with 10 mg/kg MeHgCl, Group III was
also treated with the sugar cane extract (10 mg of total phenolics/kg) by
gavage, Control group (Group I) was administered with the corresponding
solvents.
110
100
90
80
sorvival %
70
60
50
Group I
40 Group II
Group III
30
2 4 6 8 10 12 14 16
Treatment days
Figure 3. Effect of the extract from sugar cane juice on survival (%) of
Wistar rats administered repeated doses of MeHgCl. Groups II and III were
daily injected ip with 10 mg/kg MeHgCl, Group III was also treated with
the sugar cane extract (10 mg of total phenolics/kg) by gavage, Control
group (Group I) was administered with the corresponding solvents.
when the sugar cane phenolic extract was administered
to the MeHgCl-intoxicated rats (Group III) compared to
Group II, though the body weight gain in this group com-
pared to control animals was still lower. Body weight loss
is frequently observed in animals intoxicated by repeated
doses of MeHgCl and seems to be the result of MeHgCl-
induced anorexia, as reported by Magos [26]. As sugar cane
phenolic extract-treated animals were statistically different
when compared to control animals, it could be promising
to investigate the effect produced by higher dosages of the
phenolic extract.
The administration of the sugar cane phenolic extract
was able to significantly reduce the mortality of the animals
intoxicated with MeHgCl as seen in Fig. 3. These results
showed a protective effect for the phenolics present in sugar
cane extract, even at the low tested dose.
Different research evidences have illustrated the diversity
of perturbations induced by MeHg which make the identi-
fication of a specific event or pathway of primary relevance
difficult [27]. Several reports from in vitro and in vivo stud-
ies have indicated free radicals to play an important role in
MeHg-induced neurotoxicity, and so they can be targeted in
order to afford protection [28, 29]. Consequently, antioxi-
dants of natural origin can be a promising therapeutic alter-
natives for MeHg poisoning and other related-pathologies.
II. Measurement of Thiobarbituric Reactive Substances
(TBARS) After Spontaneous Lipid Peroxidation of Rat
Brain Homogenates. The assessment of thiobarbituric
reactive substances (TBARS) as an indirect index of lipid
peroxidation has been previously used to characterize
induced oxidative damage. Dose-response curve of
lipoperoxidation of rat brain homogenates for sugar cane
 120
Peroxidation inhibition (%)
100 y = 9.8886x + 1.8905
2
R = 0.9966
80
60
40
20
0 tract exhibited a low IC50 for inhibition of spontaneous
0 2 4 6 8 10 12
Phenolics (ug)
Figure 4. Ex vivo antioxidant activity of sugar cane phenolics determined
as inhibition of rat brain lipid peroxidation (%), measured through the
formation of TBA-reactive substances.
phenolic extract was then obtained (Fig. 4). As seen,
sugar cane phenolic extract displayed a dose-dependent
effect on the inhibition of spontaneous lipoperoxidation of
brain homogenates. The extract protected rat brain tissue
against free radicals- mediated damage and in this system,
sugar cane phenolic extract effectively inhibited TBARS
generation with estimated value of IC50 = 4.86 µg.
III. DPPH and β-CLAMS. Phenolic entities have already
been reported as neuroprotectants in models such as
MeHgCl intoxication, both in vitro and in vivo [26, 27].
Thus, phenolic compounds in sugar cane phenolic extract
may be proposed to have antioxidant roles and neuropro-
tective effects against MeHgCl-induced toxicity. In order
to evaluate their relative potency, in vitro antioxidant ac-
tivity of sugar cane phenolics was determined by two dif-
ferent methods, DPPH• radical scavenging activity and in-
hibition of β-carotene bleaching, and compared to that of
Trolox, a water soluble vitamin E analog. Table 2 shows
that sugar cane phenolics presented a similar efficiency in
both systems, while Trolox was more effective in avoiding
β-carotene bleaching. On the rough, 1 µmol of chloro-
genic acid equivalents, present in sugar cane juice, would
correspond to 0.5 µmol of Trolox in terms of antioxidant
activity.
Table 2. In vitro antioxidant activity of the sugar cane phenolic extract
determined through DPPH• scavenging capacity (%) and inhibition of
β-carotene bleaching (%)
DPPH β-CLAM
Sugar cane phenolics (150 µM EAC) 42.1 ± 1.8 49.4 ± 3.7
Trolox (80 µM) 33.7 ± 0.9 67.9 ± 1.3
Note. Values are expressed as average ± standard deviation (n = 3).
Conclusions
Sugar cane extract was shown to contain a range of pheno-
lic molecules such as flavonoids and cinnamic acids (api-
genin, luteolin, tricin derivatives, caffeic, sinapic acids and
isomers of chlorogenic acid). The extract decreased the ap-
pearance of clinical symptoms (food consumption weight
gain and mortality) in an in vivo model of MeHgCl neu-
rointoxication. Additionally, sugar cane polyphenolic ex-
peroxidation of rat brain homogenates. Altogether, current
results support the notion that natural phenolic antioxidants
present in sugar cane juice could be a useful alternative
therapy for relative oxidative stress.
Acknowledgement
The authors acknowledge CNPq (Conselho Nacional para
o Desenvolvimento Cientı́fico e Tecnológico) and CYTED
(Programa Iberoamericano de Ciencia y Tecnologia para
el Desarrollo—CYTED XI. 19. Aplicación de los nuevos
ingredientes funcionales em alimentación infantil y para
adultos), for financial support.
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