Food Chemistry 131 (2012) 441448

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Food Chemistry
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Effects of various solvents on the extraction of antioxidant phenolics

from the leaves, seeds, veins and skins of Tamarindus indica L.
Nurhanani Razali, Sarni Mat-Junit, Amirah Faizah Abdul-Muthalib, Senthilkumar Subramaniam,
Azlina Abdul-Aziz
Department of Molecular Medicine, Faculty of Medicine, University of Malaya, 50603 Kuala Lumpur, Malaysia

a r t i c l e i n f o a b s t r a c t

Article history: The effects of solvents, of varying polarities, on the extraction of antioxidant phenolics from the leaves,
Received 12 August 2010 seeds, veins and skins of Tamarindus indica (T. indica) were studied. The efciencies of the solvents for
Received in revised form 20 June 2011 extraction of the antioxidant phenolics were in the order: methanol > ethyl acetate > hexane. Phenolic
Accepted 1 September 2011
content ranged from 3.17 to 309 mg of gallic acid equivalents/g. Methanol leaf extract (MEL) had the
Available online 5 September 2011
highest phenolic content and was the most potent scavenger of DPPH and superoxide radicals. Methanol
vein extract had the highest ferric reducing activity whereas methanol seed extract was the most potent
Keywords:
ABTS radical-scavenger. A positive correlation existed between phenolic content and antioxidant activi-
Tamarindus indica
Tamarind
ties of the plant parts. HPLC analyses of MEL revealed the presence of catechin, epicatechin, quercetin and
Antioxidant activity isorhamnetin. Overall, methanol was the most effective solvent for extraction of antioxidant phenolics
Phenolics from T. indica. T. indica, particularly the leaf, can be a useful source of natural antioxidants.
Solvent extraction 2011 Elsevier Ltd. All rights reserved.
DPPH
ABTS

1. Introduction main phenolics studied, due to their documented potent antioxi-

Oxidative damage by free radicals is implicated in the aetiology
of many diseases, cancer and heart diseases being the more com-
mon ones (Azad, Rojanasakul, & Vallyathan, 2008; Heitzer, Schlin-
zig, Krohn, Meinertz, & Mnzel, 2001; Madamanchi, Vendrov, &
Runge, 2005).
Antioxidants are useful for providing protection against oxida-
tive damage. Antioxidants, such as glutathione, ubiquinone, uric
acid and the antioxidant enzymes glutathione peroxidase, superox-
ide dismutase and catalase, can be generated in the body; however,
the amounts maybe inadequate, particularly under conditions of
oxidative stress or inammation where production of free radicals
is increased. Hence, adequate amounts of antioxidants are impor-
tant to prevent build up of free radicals and oxidative damage in
the body.
Plants are rich alternative sources of natural antioxidants which
can complement the antioxidants produced by the human body.
Various studies have shown plants to be a rich source of antioxi-
dants. Compounds with antioxidant properties found in plants
include the vitamins A, E and C and phenolic compounds, including
avonoids, tannins and lignins (Boots, Haenen, & Bast, 2008; Valko,
Rhodes, Moncol, Izakovic, & Mazur, 2006). Flavonoids are one of the
Corresponding author. Tel.: +60 3 79674915; fax: +60 3 79674957.
E-mail address: azlina_aziz@um.edu.my (A. Abdul-Aziz).
dant activities (Rice-Evans, Miller, & Paganga, 1996), some are more
potent than the well known antioxidant vitamins. In addition, cor-
relation studies have demonstrated a link between antioxidant
activities in plants and their phenolic content, underlining the sig-
nicant contribution which phenolics can make to antioxidant
activities (Cai, Luo, Sun, & Corke, 2004; Kaur & Kapoor, 2002; Razab
& Abdul Aziz, 2010). In view of the potential of plants to provide a
natural source of antioxidants, studies are on-going in search of
plants with extracts of high phenolic content and antioxidant
activities.
Tamarindus indica (T. indica), commonly known as tamarind, is
ubiquitously found in tropical countries although it originated from
Africa. It is a tree from the family Fabaceae. The pulp of this plant is
used in cooking due to its sour taste and particularly to impart a-
vour to savoury dishes. T. indica is also used medicinally for gastric
and digestion . problems. An animal study, using hamsters, demon-
strated the hypolipidaemic effect of the seeds of T. Indica (Martinello
et al., 2006). The fruits and seeds of this plant showed anti-bacterial,
anti-inammatory and anti-diabetogenic effects (Maiti, Jana, Das, &
Ghosh, 2004; Paula et al., 2009). Most research on T. indica has con-
centrated on the fruits and seeds of this plant, mainly extracted
using polar solvents (Luengthanaphol et al., 2004; Razali, Abdul
Aziz, & Mat Junit, 2010; Siddhuraju, 2007; Soong & Barlow, 2004;
Sudjaroen et al., 2005). However, not much information is available
on the antioxidant potential of the other parts of T. indica or the

0308-8146/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.09.001
442 N. Razali et al. / Food Chemistry 131 (2012) 441448
effect of various types of solvents on extraction of antioxidants from
this plant. Hence, the aim of this study was to analyse the effective-
ness of methanol, ethyl acetate and hexane for the extraction of
antioxidant phenolics from the leaves, seeds, skins and veins of T.
indica. Such study can provide complete information on the
in vitro antioxidant potential of the other parts of this plant that
are less well researched but nonetheless important.
2. Materials and methods
2.1. Chemicals
All reagents used in the experiments were of analytical grade
and obtained mostly from Fluka and Sigma. Solvents used for
extraction of plant samples were purchased from Fisher Scientic.
The phenolic standards were obtained from Sigma. Water used was
of Millipore quality.
2.2. Plant material
Leaves, veins, skins and seeds of T. indica were collected from
Kedah, northern part of Malaysia. The material was identied by
a taxonomist of Rimba Ilmu Herbarium, University of Malaya,
Malaysia. A voucher specimen was deposited in Rimba Ilmu Her-
barium, having the voucher specimen number KLU 45976.
2.3. Preparation of plant extracts
The various parts of T. Indica were thoroughly cleaned and air-
dried and, once their weight was stable, the dried parts were
ground into powder forms, using a commercial blender. The pow-
ders were extracted, using methanol, ethyl acetate and hexane at
room temperature for 24 h (recording 0.05 g/ml). The solvents
were then removed using a rotary evaporator and the resulting res-
idues were re-dissolved in 10% DMSO. The extracts were stored at
20 C prior to further analyses.
2.4. Assay for phenolic content
Total phenolic contents of the extracts were measured using the
FolinCiocalteu assay developed by Singleton and Rossi (1965).
This is a colorimetric assay, involving production of a blue molyb-
denumtungsten complex in the presence of phenolics which can
be measured relative to gallic acid as the standard.
Aqueous FolinCiocalteu reagent (1:10) was added to the plant
extract or standard, incubated for 5 min before addition of
0.115 mg/ml of Na2CO3. After a 2 h incubation period, absorbance
was read at 765 nm. Gallic acid was used as a standard and a cal-
ibration curve was plotted in a concentration range of 50200 mg/
l. All analyses were performed in triplicate and results were ex-
pressed as mg of gallic acid equivalents/g dried plant.
2.5. Ferric reducing activity (FRAP)
The ferric reducing ability of plasma (FRAP) assay uses antioxi-
dants as reductants in a redox-linked colorimetric reaction, reduc-
ing a ferric-tripyridyltriazine (Fe (III)-TPTZ) complex to the ferrous,
Fe (II) form (Benzie & Strain, 1996), forming an intense blue colour
complex which can be measured colorimetrically.
Reagents for this assay consisted of 300 mM acetate buffer,
10 mM TPTZ in 40 mM of HCl and 20 mM FeCl3.6H2O. The respec-
tive solutions were mixed in a ratio of 10:1:1 as needed. The fresh
solution was warmed (37 C) for 5 min. After taking a blank read-
ing, plant extract or standard and water were added to the FRAP
reagent. Absorbances were taken at 0 and 4 min after the start of
the reaction. The differences between the two absorbance readings
were measured and compared to the standard graph. FeSO4 was
used as the standard and analysed as above.
2.6. DPPH radical-scavenging activity
The scavenging activity of the stable free radical; 2,2-diphenyl-
1-picrylhydrazyl (DPPH) was determined by the method described
by Cos et al. (2002). The principle of DPPH assay involves reaction
of the antioxidants with the stable DPPH radical, converting the
complex from a deep violet colour to a colourless complex. The
degree of discolouration indicates the scavenging potentials of
the samples.
DPPH solution (0.04 mg/ml) was added to varying concentra-
tions of the plant extracts and absorbance was read at 715 nm fol-
lowing a 20 min incubation period. Results were expressed as
mmol of Trolox equivalent antioxidant capacity (TEAC) per g of
dried plant material. Percentage inhibition of the DPPH radicals
was calculated using the formula below:
Percentage of inhibition% OD control  OD sample=
OD control  100%
where OD = optical density.
2.7. ABTS+ radical-scavenging activity
This assay is based on the inhibition, by antioxidants, of the
absorbance of the radical cation of 2,20 -azinobis(3-ethylbenzo-
thiazoline 6-sulphonate) (ABTS+) at a wavelength of 734 nm (Re
et al., 1998). This assay involves generation of the ABTS radical
chromophore by the oxidation of ABTS+ with potassium persul-
phate, which was conducted in the dark for 1216 h followed by
adjustment of the absorbance of the reactant to 0.700 0.02.
Briey, appropriate amounts of the radicals were mixed with
the plant extracts to give a 2080% inhibition of the absorbance.
Readings were taken at 1 and 15 min after the start of the reaction.
Trolox was used as the standard and a standard curve was plotted.
All analyses were done in triplicate and results were expressed as
TEAC values.
2.8. Superoxide anion radical-scavenging activity
The superoxide anion radicals were generated by the NADH
phenazine methosulphate (PMS) system (Siddhuraju & Becker,
2007). Scavenging activity was assessed by monitoring the ability
of antioxidants in the plant extracts to inhibit reduction of nitro-
blue tetrazolim (NBT) by the superoxide anion radicals.
The reaction mixture consisted of 0.1 M phosphate buffer (pH
7.4), 150 lM NBT, 60 lM PMS, 468 lM NADH and varying concen-
trations of the plant extracts. Absorbance was read at 560 nm after
a 10 min incubation period and percentage inhibition of the super-
oxide anion by the plant extracts was calculated. Quercetin and ru-
tin were used as positive controls and trolox was used as the
standard. All results were of triplicate analyses and expressed as
TEAC values.
2.9. Analyses of avonoid content using high performance liquid
chromatography (HPLC)
2.9.1. Acid hydrolysis of leaves
Acid hydrolysis of the dried leaf powder was performed accord-
ing to the method described by Aziz, Edwards, Lean, & Crozier, 1998.
Briey, 20 mg of dried leaves were hydrolysed at 90 C for 2 h in a
3 ml glass V-vial containing 1.2 M HCl in 50% aqueous methanol
and 20 mM sodium diethyldithiocarbamate as an antioxidant. The
 N. Razali et al. / Food Chemistry 131 (2012) 441448
vials were placed in a Reacti-Therm heating block (Pierce, Rockford,
USA) with a stirring capacity. Aliquots of the samples were taken
after the hydrolysis, centrifuged for 5 min at 5000g and diluted with
distilled water (pH 2.5) prior to analysis by HPLC. The hydrolysed
samples contained both free avonoids and aglycones released from
conjugated avonoids, following acid hydrolysis.
2.9.2. High performance liquid chromatography
Samples were analysed using a Shimadzu HPLC system com-
prising a system controller, a binary pump (LC 20AC), a manual
injector (Rheodyne 7725i manual injector with a 50 ll sample
loop), a column oven (CTO-10AS VP) and a dual channel (SPD-
20A UVVIS) UV detector. Separation of avonoids was achieved
using a NovaPak (end-capped) C18 reversed-phase column
(150  3.0 mm i.d, 4 lm) (Waters, USA) tted with a guard car-
tridge column, under controlled temperature (40 C) in a column
oven. The mobile phase consisted of water with triuoroacetic acid
(TFA) (pH 2.5) and acetonitrile, eluted at a ow rate of 0.5 ml/min.
Shimadzu Corporation LC solution software (version 1.23) was
used for the data acquisition through the dedicated personal
computer.
Stock standard solutions were prepared at a concentration of
1 mg/ml and stored at 20 C. Standard solutions, containing cate-
chin, epicatechin, rutin, genistin, myricetin, morin, quercetin, gen-
istein, kaempferol and isorhamnetin, were mixed together and
injected at a volume of 50 ll.
Separation of avonoids was achieved, based on the method de-
scribed by Aziz et al. (1998) with modications. Mobile phase A
consisted of water with TFA, adjusted to pH 2.5 and mobile phase
B was 100% acetonitrile. A linear gradient system, starting with 7%
B, increasing to 40% in 20 min, at a ow rate of 0.5 ml/min, was uti-
lised and absorbance was measured at a wavelength of 260 nm.
2.10. Statistical analyses
All analyses were done in triplicate and results were expressed
as means S.D. Students t-test (Microsoft Excel 972003 Work-
book) was performed to analyse for statistically signicant results.
Regression analyses (R) were performed to conrm correlation of
two data sets (Microsoft Excel 972003 Workbook).
3. Results and discussion
3.1. Extraction yield and phenolic content
Table 1 shows the extraction yield of the various plant parts
which range from 20 to 320 mg/g dried weight. The extraction
yields, in descending order, were: methanol > ethyl acetate > hex-
ane. This shows that methanol was the best solvent for extraction
of compounds from the various parts of the T. Indica plant.
Phenolic compounds are secondary plant metabolites with ben-
ecial biological effects, e.g. as anti-bacterial, anti-inammatory
and anti-allergic agents (Koshihara et al., 1983; Schramm & Ger-
man, 1998). Most important is their documented action as potent
antioxidants (Formica & Regelson, 1995; Rice-Evans et al., 1996).
Hence, it is common practice to measure both phenolic content
and antioxidant activities when investigating the antioxidant po-
tential of plants as various studies have shown that plants rich in
phenolics are also potent antioxidants (Maisuthisakul, Suttajit, &
Pongsawatmanit, 2007; Razali, Razab, Mat Junit, & Abdul Aziz,
2008).
There were varying phenolic contents in the tamarind extracts,
ranging from 3.17 to 308 mg GAE/g dried weight (Table 1) but,
generally, the methanol extracts had higher phenolic levels than
had the ethyl acetate and hexane extracts of the same plant part.
443
Overall, the methanol extract of the leaf (MEL) had the highest
phenolic content and the hexane extract of the skin (HESK) the
lowest. The methanol extracts of the seed (MES) and of the vein
(MEV) also contained considerable amounts of phenolics
(>200 mg GAE/g dried weight). The ethyl acetate extract of the leaf
(EAEL) and the methanol extract of the skin (MESK) had phenolic
contents in the range of 100200 mg GAE/g dried weight. The
remainder of the extracts had phenolic contents of less than
100 mg GAE/g dried weight.
The phenolic content of the T. Indica leaves, veins and skins has
not previously been reported. Most studies have concentrated on
the seeds and fruits (Luengthanaphol et al., 2004; Siddhuraju,
2007; Soong & Barlow, 2004). Soong and Barlow (2004) reported
the seeds, extracted with 50% ethanol to contain 94.5 mg GAE/g.
The phenolic content of the methanol seed extract in our study
was much higher.
3.2. Ferric reducing activity
FRAP assay provides a simple and effective method for measur-
ing the ability of antioxidants in plant samples to act as reducing
agents. We found MEV to contain the highest ferric reducing capac-
ity, followed by MEL and MES (Table 1). When compared with the
positive controls, quercetin was considerably more potent; how-
ever, the reducing activity was comparable to rutin.
In most instances, the methanol extracts of the different plant
parts contained substantial ferric reducing activities compared to
the ethyl acetate and hexane extracts. Among the plant parts, the
veins possessed the most ferric reducing activities, followed by
the leaves whereas the skins had the lowest activities. MEV and
MEL were found to have higher ferric reducing activities than sev-
eral Chinese medicinal plants, such as Scutellaria baicalensis and
Fraxinus rhynchophylla (Li, Wong, Cheng, & Chen, 2008). This sug-
gests the potency of the tamarind plant for medicinal use.
On the other hand, the ferric reducing activity of MES in this
study was lower than the T. Indica seed extract reported by Soong
and Barlow (2004) (1460 vs 2468 lmol/g, respectively). This could
be due to differences in the variety of plant, as well as location
where the plant is grown.
3.3. DPPH radical-scavenging activity
The three extracts with the highest DPPH radical-scavenging
capacity were in the order: MEL (3.17 0.00 mmol/g dried
weight) > MES (2.94 0.14 mmol/g dried weight) > EAEL (2.76
0.03 mmol/g dried weight) (Table 1). Radical-scavenging activities
of these extracts were comparable to quercetin and rutin, suggesting
their potency.
The DPPH radical-scavenging activities of the extracts depicted
a doseresponse relationship (Fig. 1). The inhibition reactions ap-
peared to be still occurring, even at the highest concentration
(100 lg/ml) tested. At this concentration, MEL achieved more than
80% inhibition of the DPPH radicals, almost matching that of rutin,
whereas quercetin was slightly higher, at 95%.
A weaker pattern of scavenging capacities was observed for the
ethyl acetate extracts of the various plant parts (compared to the
methanol extracts). Highest inhibition was demonstrated by the
EAEL (72%). The ethyl acetate extract of seed (EAES) was almost
non-reactive, with inhibition not exceeding 25%.
The hexane extracts of the various plant parts were generally
the least reactive with highest inhibition at 35% (HEL). The scav-
enging activities of the hexane extracts in most cases showed a
hyperbolic curve. This observation is fairly common in plants, as
not many non-polar compounds are able to act as potent antioxi-
dants (Kulkarni, Aradhya, & Divakar, 2004).
444 N. Razali et al. / Food Chemistry 131 (2012) 441448

Table 1
Phenolic content and antioxidant activities of methanol, ethyl acetate and hexane extracts of various parts of T. IndicaA.

Yield (mg/g Phenolic Ferric reducing DPPH radical-scavenging ABTS+ radical-scavenging Superoxide anion-scavenging
dried content (GAE activity (mmol/g dried activity (mmol Trolox/g dried activity (mmol Trolox/g dried activity (mmol trolox/g dried
weight) mg/g)B weight) weight)C weight)C weight)C
Leaves 320 309 3.78a 1.87 0.09a 3.17 0.00a 1.65 0.04a 4.64 0.003a
(M)
Leaves 40 101 12.2b 0.57 0.92b 2.76 0.03b 0.70 0.01b 4.54 0.14a
(E)
Leaves 40 31.8 3.70c 0.12 0.07c,i 1.35 0.04c,d 0.51 0.03c 3.99 0.01c
(H)
Seeds 280 272 18.2a 1.46 0.001d 2.94 0.14a,b 2.88 0.02d 3.96 0.35c,b
(M)
Seeds 80 26.6 13.6c,e 0.12 0.24c 0.98 0.00c,e,f,g,h,i 0.77 0.01e 0.76 0.02d
(E)
Seeds 40 95.3 14.9b,d 0.31 0.25e 1.13 0.01e,j,k 0.90 0.04f 3.14 0.10e
(H)
Veins 240 230 3.33f 2.05 0.22f 2.54 0.10l 1.41 0.003g 3.85 0.01b
(M)
Veins 40 65.6 10.0d 0.31 0.47e 1.41 0.03d,f 0.30 0.01h 2.53 0.01f
(E)
Veins 40 30.4 2.60c 0.01 0.49g 1.20 0.08g,j,m 0.16 0.01i 1.31 0.01g
(H)
Skins 240 116 1.08b 0.92 0.01h 2.34 0.0l1 1.32 0.004j 4.54 0.06a
(M)
Skins 40 17.2 2.89e 0.10 0.01i 1.19 0.04h,k,m 0.89 0.02f 2.93 0.02e
(E)
Skins 20 3.17 1.11l ND 0.73 0.01i 0.43 0.01k 1.50 0.03h
(H)
Rutin 3.36 0.003j 3.32 0.00n 1.72 0.01a,l 5.47 0.01i
Quercetin 13.3 0.002k 3.60 0.00o 4.18 0.03m 5.67 0.004j

Values not sharing the same letter within the same column were signicantly different at p < 0.01.
M Methanol, E Ethyl acetate, H Hexane, ND not detected.
A
Results were expressed as the averages of triplicates S.D.
B
Phenolic content was expressed as mg gallic acid equivalents (GAE) in 1 g of dry weight material S.D.
C
The antioxidant activities of the samples were compared with a trolox standard curve and results were expressed as trolox equivalent antioxidant capacity (TEAC).

Fig. 1. DPPH radical-scavenging capacity of various parts of T. indica extracted with methanol, ethyl acetate and hexane. All analyses were performed in triplicate and results
expressed as % inhibition of the absorbance of radicals S.D. MEL: methanol extract-leaves; EAL: ethyl acetate extract-leaves; HEL: hexane extract-leaves; MES: methanol
extract-seeds; EAES: ethyl acetate extract-seeds; HES: hexane extract-seeds; MEV: methanol extract-veins; EAEV: ethyl acetate extract-veins; HEV: hexane extract-veins;
MESK: methanol extract-skins; EAESK: ethyl acetate extract-skins; HESK: hexane extract-skins.
 N. Razali et al. / Food Chemistry 131 (2012) 441448 445

Fig. 2. ABTS+ radical-scavenging capacity of various parts of T. indica extracted with methanol, ethyl acetate and hexane. All analyses were performed in triplicate and results
expressed as % inhibition of the absorbance of radicals S.D. MEL: methanol extract-leaves; EAL: ethyl acetate extract-leaves; HEL: hexane extract-leaves; MES: methanol
extract-seeds; EAES: ethyl acetate extract-seeds; HES: hexane extract-seeds; MEV: methanol extract-veins; EAEV: ethyl acetate extract-veins; HEV: hexane extract-veins;
MESK: methanol extract-skins; EAESK: ethyl acetate extract-skins; HESK: hexane extract-skins.

Fig. 3. Superoxide anion radical-scavenging capacity of various parts of T. indica extracted with methanol, ethyl acetate and hexane. All analyses were performed in triplicate
and results expressed as % inhibition of the absorbance of radicals S.D. MEL: methanol extract-leaves; EAL: ethyl acetate extract-leaves; HEL: hexane extract-leaves; MES:
methanol extract-seeds; EAES: ethyl acetate extract-seeds; HES: hexane extract-seeds; MEV: methanol extract-veins; EAEV: ethyl acetate extract-veins; HEV: hexane extract-
veins; MESK: methanol extract-skins; EAESK: ethyl acetate extract-skins; HESK: hexane extract-skins.

Siddhuraju (2007) measured the DPPH radical-scavenging radical-scavenging capacity, implying that methanol was a
activities of T. indica seed coat, extracted with either methanol better solvent for the extraction of radical-scavenging antiox-
or 70% acetone and reported the former to contain the higher idants.
446 N. Razali et al. / Food Chemistry 131 (2012) 441448

3.4. ABTS+ radical-scavenging activity
The order of potency of the three most potent extracts was: MES
(2.88 0.02 mmol/g dried weight) > MEL (1.65 mmol/g dried
weight 0.04) > MEV (1.41 0.003 mmol/g dried weight) (Table 1).
The TEAC values were comparable to, if not higher, than those of
rutin but lower than those of quercetin. The ABTS+-scavenging
activity of MES is much higher than that of methanol extracts of
guava fruits (Thaipong, Boonprakob, Crosby, Cisneros-Zevallos, &
Byrne, 2006) and an array of Indian medicinal plants (Surveswaran,
Cai, Corke, & Sun, 2007). MES had a TEAC value several-fold higher
than those of several Chinese medicinal herbs, indicating their po-
tency and potential use as a source of antioxidants (Li et al., 2008).
When the ABTS+ radical-scavenging activity of MES was compared
with raw T. indica seed coats extracted with methanol (Siddhuraju,
2007), the former showed lower activity. This could be due to dif-
ferences in the variety of plant and the location where the plant is
grown.
When a doseresponse curve was plotted, MEL and MES could
inhibit 98% of the ABTS radicals at the highest concentration
(2000 lg/ml) used (Fig. 2), similar to rutin and quercetin. The
ethyl acetate and hexane extracts of all the plant parts had
doseresponse curves similar to the methanol extracts, albeit at
lower degrees of inhibition.
3.5. Superoxide anion-scavenging activity
Overall, the methanol extracts of the various parts of the plant
contained antioxidants with higher superoxide anion-scavenging
activities than did the ethyl acetate and hexane extracts (Table 1).
MEL had the highest superoxide anion radical-scavenging activ-
ity (4.64 0.003 mmol/g dried weight) which was comparable to
rutin (4.55 0.01 mmol/g dried weight) but lower than quercetin
(14.2 0.01 mmol/g dried weight). MESK (4.54 0.06 mmol/g
dried weight) and EAEL (4.54 0.14 mmol/g dried weight) had
similar superoxide anion-scavenging activities which were higher
than the other extracts but lower than MEL. Superoxide anion is
produced as a result of immune response although excess amounts
can lead to oxidative damage, causing conditions such as neuronal
death, ischemic reperfusion injury and cancer (Oku et al., 2008).
Tamarinds, particularly the leaves, have the potential to counteract
the oxidative damage effect of superoxide anion.
The methanol, ethyl acetate and hexane extracts of the plant
parts showed similar doseresponse relationships with an initial

uV (x100,000)
1.6

1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8
EC
0.7

0.6

0.5

0.4

0.3
C
0.2
(a)
0.1

0.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min

uV (x100,000)
1.6

1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4 EC Q
0.3

0.2
(b)
0.1

0.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min

uV (x10,000)
1.5

1.4

1.3

1.2

1.1

1.0

0.9

0.8

0.7

0.6

0.5

0.4

0.3
EC IR
0.2

0.1
(c)
0.0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 min

Fig. 4. (AC) Gradient reverse phase HPLC analysis of avonoids in leaf extracts of T. indica. HPLC analyses was performed on the acid-hydrolysed T. indica leaves extracted
with methanol (Fig. 4a), ethyl acetate (Fig. 4b) and hexane (Fig. 4c). Samples were separated on a NovaPak C18 reversed-phase column (150  3.0 mm i.d, 4 lm), using a linear
gradient system starting with 7% acetonitrile in water containing triuoroacetic acid (TFA) (pH 2.5), increasing to 40% in 20 min, at a ow rate of 0.5 ml/min. Absorbance was
measured at a wavelength of 260 nm. C: catechin; EC: epicatechin; Q: quercetin; IR: isorhamnetin.
 N. Razali et al. / Food Chemistry 131 (2012) 441448
rapid increase in superoxide anion-scavenging activities (up to a
concentration of 100 lg/ml) and, subsequently, a gradual decrease
in the rate of the inhibition of the superoxide anion (Fig. 3). Among
the three solvents, the methanol extracts showed higher inhibi-
tions of the superoxide anion.
3.6. Correlation analyses
Correlation analyses between phenolic content and antioxidant
activities of the various plant extracts were calculated using
regression analyses. Results showed positive correlations between
phenolic content and the ferric reducing activities (r = 0.8899);
phenolic content and DPPH radical-scavenging capacities
(r = 0.8849) and phenolic content and ABTS radical-scavenging
capacities (r = 0.8264). This shows the likelihood that the observed
antioxidant activities were mainly contributed by the phenolics in
the plant extracts. A lower, although positive, correlation was seen
between phenolic content and superoxide anion (r = 0.6444) of the
plant extracts. Various studies have reported positive correlations
between phenolic content and antioxidant activities of plants
(Cai et al., 2004; Kaur & Kapoor, 2002; Razab & Abdul Aziz,
2010), and results from this study further strengthen the impact
of phenolics as potent antioxidants.
3.7. HPLC analyses of the extracts of the leaves of T. indica
Samples were injected for HPLC, following acid hydrolysis. Acid
hydrolysis is a useful technique in avonoid analysis as it functions
to cleave sugars bound to avonoids (Aziz et al., 1998), thus allow-
ing easy identication of the released avonoid aglycones. For
comparison purposes, we show the HPLC chromatograms of the
methanol, ethyl acetate and hexane extracts of the leaves
(Fig. 4A-C). Reversed-phase HPLC of the extracts revealed the pres-
ence of several peaks corresponding to avonoids, detected at a
wavelength of 260 nm. The presence of avonoids was conrmed
by comparing the retention times of the samples with the stan-
dards and by adding a known concentration of the pure standards
to the extract and comparing the resulting peak heights.
Epicatechin could be detected in all three extracts, but more
noticeably in the methanol extracts (Fig. 4a). Catechin was also
present in MEL. Quercetin could be seen in EAEL (Fig. 4b) whereas
isorhamnetin was detected in HEL (Fig. 4c).
Several peaks, which did not correspond to the avonoid stan-
dards that we used, could be detected in the sample chromato-
grams, particularly at retention times between 15 and 23 min.
These peaks could potentially correspond to avonoids, as these
phenolics have maximal absorption at 260 nm (Careri, Elviri, Man-
gia, & Musci, 2000). The avonoids detected in the extracts could
potentially act synergistically in providing the antioxidant activi-
ties. Sudjaroen et al. (2005) analysed the phenolic content of the
seeds and pericarp of T. indica and reported that the pericarp
mainly contained proanthcyanidins, such as procyanidin tetramer,
()-epicatechin, procyanidin hexamer and catechin, while the
seeds are mainly dominated by oligomeric procyanidin tetramers
and lower levels of ()-epicatechin. In this study, we report the
presence of quercetin and isorhamnetin in the leaf extracts. The
presence of quercetin and isorhamnetin in the leaves of T. indica
has not previously been reported.
4. Conclusion
Analyses of the phenolic content and antioxidant activities of
the various parts of T. indica, extracted with solvents of varying
polarities, are useful in providing information on the potential of
this plant as a source of phenolic antioxidants. At the same time,
they also provide data on the characteristics of the antioxidants
447
present in the various parts of the plants. Overall, methanol was
the most effective solvent for extraction of antioxidant phenolics
from various parts of T. indica. From this study, it was found that
the methanol leaf extract contained the most antioxidant activities
and that the antioxidants were mainly polar compounds. The high
antioxidant potency of the leaves of T. indica implies the potential
of this plant for use as an alternative source of natural antioxidants.
In addition, this study also provides further scientic support for
the medicinal use of this plant. In vivo studies are required to fur-
ther conrm if the observed in vitro activities can be replicated
in vivo.
Acknowledgements
This research project was funded by research grants (RG127/
09HTM, FS353/2008C and H-20001-00-E000009) from University
of Malaya, Kuala Lumpur, Malaysia.
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