Hypolipemic and antioxidant activities from Tamarindus indica L.

pulp fruit extract in hypercholesterolemic hamsters

F. Martinello a, S.M. Soares a, J.J. Franco a, A.C. Santos a, A. Sugohara d,
S.B. Garcia c, C. Curti b, S.A. Uyemura a,*
a Faculdade de Ciencias Farmaceuticas de Ribeirao Preto, Departamento de Ana lises Clnicas,
Toxicolo gicas e Bromatolo gicas,
Universidade de Sao Paulo, Av. do Cafe , s/n, 14040-903 Ribeirao Preto, SP, Brazil
b Departamento de Fsica e Qumica, Universidade de Sao Paulo, Av. do Cafe , s/n, 14040-903
Ribeirao Preto, SP, Brazil
c Faculdade de Medicina de Ribeirao Preto, Departamento de Patologia, Universidade de Sao
Paulo, Av. Bandeirantes,
3900, 14049-900 Ribeirao Preto, SP, Brazil
d Faculdade de Ciencias Agra rias e Veterina ria, Departamento de Zootecnia, Universidade
Estadual Paulista Ju lio de Mesquita Filho,
Via de Acesso Prof. Paulo Donato Castellane, s/n, 14884-900 Jaboticabal, SP, Brazil
Received 3 May 2005; accepted 20 October 2005
Abstract
Dietary modifications may significantly reduce cardiovascular disease (CVD) risk factors, including
cholesterol and atherosclerosis.
The present study addressed the effects of the crude extract from the pulp fruit of Tamarindus indica
L. on lipid serum levels and early
atherosclerotic lesions in hypercholesterolemic hamsters in vivo, and the extract_s antioxidant
action, in vitro. Animals were fed on either
chow or atherogenic diet during 10 weeks and concomitantly received either water or T. indica L.
extract for drinking. Treatment of
hypercholesterolemic hamsters with the T. indica pulp fruit extract (5%) led to a decrease in the
levels of serum total cholesterol
(50%), non-HDL cholesterol (73%) and triglyceride (60%), and to an increase of high-density
lipoprotein (HDL) cholesterol levels
(61%). In vitro, the extract presented radical scavenging ability, as assessed by the 2,2-diphenyl-1-
picrylhydrazyl (DPPH) and superoxide
radicals assays, and led to decreased lipid peroxidation in serum, as assessed by the thiobarbituric
acid reactive substances (TBARS)
assay. In vivo, the extract improved the efficiency of the antioxidant defense system, as assessed by
the superoxide dismutase, catalase
and glutathione peroxidase activities. Together these results indicate the potential of tamarind
extracts in diminishing the risk of atherosclerosis
development in humans.
_ 2005 Elsevier Ltd. All rights reserved.

Keywords: Tamarind; Tamarindus indica L.; Hypercholesterolemic hamster; Antioxidant activity;

Free radical; Atherosclerosis

Abbreviations: LDL, low-density lipoprotein; oxLDL, oxidized lowdensity

lipoprotein; HDL, high-density lipoprotein; VLDL, very lowdensity
lipoprotein; IDL, intermediate-density lipoprotein; DMSO,
dimethyl sulfoxide; CAT, catalase; SOD, superoxide dismutase; GPx,
glutathione peroxidase; TBARS, thiobarbituric acid reactive substances;
DPPH, 2,2-diphenyl-1-picrylhydrazyl; EDTA, ethylenediaminetetraacetic
acid; NBT, nitro blue tetrazolium; AST, aspartate aminotransferase; ALT,
alanine amonotransferase; GSH, glutathione; GR, glutathione reductase;
t-BuOOH, tert-butyllhydroperoxide; MDA, malondialdehyde.
* Corresponding author. Tel.: +55 16 36024199; fax: +55 16 36331936.
E-mail address: suyemura@fcfrp.usp.br (S.A. Uyemura).

1. Introduction
Cardiovascular disease is a leading cause of global
mortality, accounting for almost 17 million deaths annually
(Smith et al., 2004); atherosclerosis, in particular, is
the main contributor for the pathogenesis of myocardial
and cerebral infarction. Elevated levels of plasma low-density
lipoprotein cholesterol (LDL) and triglycerides,
accompanied by reduced high-density lipoprotein (HDL)
levels, is often associated with an increased risk of coronary
heart disease (Smith et al., 2004).

Atherosclerosis represents a state of heightened oxidative

stress, characterized by lipid and protein oxidation
(Stocker and Keaney, 2004). Several studies indicate that
LDL oxidation is an early event of the process. Indeed,
oxLDL is cytotoxic to a variety of vascular cells (Morel
et al., 1983), induces the synthesis of monocyte chemotatic
protein-1 (Rajavashisth et al., 1990), recruits inflammatory
cells (Navab et al., 1991), and stimulates the production of
autoantibodies (Salonen et al., 1992).
Plant materials have long been used as traditional
medicines for the treatment of a wide variety of ailments
and diseases. Diets rich in vegetables and fruits can reduce
cardiovascular disease (Howard and Kritchevsky, 1997;
Hung et al., 2003; Mozaffarian et al., 2003; Katsube
et al., 2004), notably by inhibiting LDL oxidation (Gorinstein
et al., 1999; Marniemi et al., 2000; Landbo and
Meyer, 2001; Southon, 2001; Shafiee et al., 2003; Nicolle
et al., 2004).
The tamarind (Tamarindus indica L.) is a tree-type of
plant which belongs to the Leguminosae, caesalpiniaceae
family. It is indigenous to tropical Africa but has become
naturalized in North and South America from Florida to
Brazil, and is also cultivated in subtropical China, India,
Pakistan, Indochina, Philippines, Java and Spain. Initially,
the fruit shows a reddish-brown color that turns black or
black brown, becoming more aromatic and sour on ripening.
T. indica L. pulp fruit is used for seasoning, as a food
component and in juices. Its fruit is regarded as a digestive,
carminative, laxative, expectorant and blood tonic
(Komutarin et al., 2004). Other parts of the plant present
antioxidant (Tsuda et al., 1994), antihepatotoxic (Joyeux
et al., 1995), antiinflammatory (Rimbau et al., 1999), antimutagenic
(Ramos et al., 2003) and antidiabetic (Maiti
et al., 2004) activities. The antiatherosclerosis potential of
tamarind has not been previously investigated. In the present
study we characterize tamarind fruit extracts by assessing
the antiatherosclerosis potential in vivo, and the
antioxidant action in vitro.
2. Material and methods
2.1. Preparation of T. indica fruit extract
Approximately 100 g of the fruit pulp were placed in a conical flask
and soaked for three days in 400 ml of 70% alcohol, at 4 _C. The resulting
extract was filtered through a sieve and then roto-evaporated until complete
alcohol evaporation was obtained. In all experiments the extract was
diluted in water.
2.2. Characterization of T. indica fruit extract
2.2.1. HPLC analysis
HPLC analysis was performed in a Shimadzu LC-6AD apparatus with
a Diode Array Detector (SPD-M10 Avp, Shimadzu), coupled with an auto
injector (SIL-10AF, Shimadzu) and using the software CLASS-VP 6.14. A
Shim-pack ODS column (5 lm, 4.6/250 mm; Shimadzu) was employed
coupled to the respective guard-column, using the following elution
profile: 010 min: 2% B (isocratic), 1090 min: 220% B (linear gradient),
90120 min: 20100% B (linear gradient); solvents: A = aqueous acetic

acid 2% (v/v); B = MeCN with 2% acetic acid (v/v). Flow rate: 1 ml/min.
UV detection between 190 and 450 nm.
2.2.2. Total phenolic
Total soluble phenolics derivatives were determined according to the
FolinCiocalteau method (Singleton and Rossi, 1965) using a reaction
medium containing 50 ll of sample, blank or gallic acid standard,
respectively, 50 ll of 7% (v/v) acetic acid, 50 ll of FolinCiocalteaus
reagent, 50 ll of 35% (w/v) sodium carbonate and 800 ll of distilled water.
After mixing, the reaction was incubated during 90 min, at room temperature,
in the dark. Light absorbance was measured at 725 nm in a
Beckman DU-70 spectrophotometer, and the total phenolic content was
expressed as gallic acid equivalents per mg of extract.
2.2.3. Total sugars
Total sugars in crude extract were determined according to Dubois
et al. (1956), by the phenol-sulfuric method. The reaction mixture contained,
respectively, 500 ll of sample, blank or a glucose standard, 500 ll
of an aqueous phenol solution 5% (w/v) and 2.5 ml of fuming sulfuric acid.
After mixing and chilling, absorbance was measured at 490 nm to determine
the content of hexoses.
2.2.4. Total flavonoids
Total flavonoids present in the crude extract were determined in
medium containing 200 ll of sample, blank or a quercetin standard, 60 ll
of glacial acetic acid, 1 ml of pyridine:water: 12% aluminum chloride
solution (17:80:3), 1.24 ml of DMSO:H2O (1:1). The reaction product was
spectrophotometrically determined at 420 nm (Costa, 1972).
2.3. Determination of in vitro antioxidant activity of crude
extract
2.3.1. Inhibition of lipid peroxidation induced by the Fe2+/citrate system
Total lipid peroxidation in serum was determined by measuring thiobarbituric
acid reactive substances (TBARS), following the fluorescence
method described by Yagi (1998), using 515 nm and 553 nm as excitation
and emission wavelengths, respectively. Briefly, 250 ll of pooled serum was
added to a reaction mixture containing 125 mmol/l KCl and 50 mmol/l
Hepes-KOH (pH 7.4), and incubated at 37 C, for 6 h without addition of
extract (control), or in presence of 2.5 and 5.0% (w/v) of extract, respectively,
added 5 min prior to the beginning of the oxidation reaction. Peroxidation
was induced by adding 50 lmol/l Fe(NH4)2(SO4)2/2 mmol/l
sodium citrate. Forty microlitre were taken at zero time and every 1-h from
the incubation mixture. Lipids and proteins were precipitated by the
addition of 12 N H2SO4 and 10% (w/v) phosphotungstic, and centrifuged
at 1900g for 10 min. The pellet was resuspended in water and 1% (w/v)
thiobarbituric acid. The reaction mixture was kept at 95 C for 1 h, cooled,
and the reaction product extracted with butanol and measured in a Hitachi
F4500 fluorimeter, using tetramethoxypropane as a standard. The results
were obtained from three different experiments performed in triplicate.
2.3.2. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay
Different concentrations of the extract were measured for hydrogen
donating or radical scavenging ability, using the stable radical 2,
2-diphenyl-1-picrylhydrazyl (DPPH), according to Parejo et al. (2002).
The reaction mixture containing 1.5 ml of a DPPH methanolic solution
(0.2 mg/ml) plus 0.75 ml of the crude extract at different concentrations
(methanol for the control) was incubated at 37 C for 20 min,
and the absorbance was measured spectrophotometrically at 517 nm.
The percent of DPPH discoloration of the sample was then calculated.
Butylated hydroxytoluene (BHT, 1 mg/dl) was used as a positive
control.

2.3.3. Superoxide anion scavenging activity

Superoxide anion scavenging activity of the T. indica extract was
determined according to Parejo et al. (2002), by measuring the superoxide
radicals generated in vitro by the xanthine/xanthine oxidase system.
The reaction mixture, at a final volume of 1 ml, containing 50 mmol/l
sodium phosphate buffer (pH 7.5), EDTA (0.05 mmol/l), xanthine
(0.2 mmol/l), NBT (1 mmol/l), and 0.1 ml of crude extract at different
concentrations was incubated at 25 C, for 10 min. The reaction was
initiated by the addition of xanthine oxidase (1 U/ll) and carried out at
25 C, for 20 min. After this period, the reaction was stopped by
the addition of CuCl (50 lmol/l), and absorbance was measured at
560 nm. The results are expressed as the percentage inhibition of the
NBT reduction in relation to the reaction mixture without sample (buffer
only). Butylated hydroxytoluene (BHT, 1 mg/dl) was used as a positive
control.
2.4. Experimental animals and diets
Twenty-four Golden Syrian male hamsters weighing between 100
and 130 g were randomly divided into four groups, and acclimatized
for two weeks in colony cages (six hamsters per cage), at 25 2 C and
under a 12-h light:dark cycle. Group 1 was fed with a standard diet
(normal control), group 2 was fed with a standard diet and tamarind
extract, group 3 was fed with a standard diet plus 1% cholesterol
(hypercholesterolemic control), and group 4 was fed with a standard diet
plus 1% cholesterol and tamarind extract. Food and liquid intakes were
monitored daily in all groups. The water in groups 2 and 4 was replaced by
5% tamarind extract after complete resuspension in water, for a period of
10 weeks.
The dose of T. indica used in this study (5%) was chosen based on a
concentrationresponse study, which determined the antioxidant activity
of the extract regarding the lipid peroxidation process. Also, 5% concentration
is the concentration usually present in juice consumed by the
human population.
2.5. Measurement of body weight and biochemical parameters
Animals were anesthetized using a CO2 chamber and the blood
samples obtained by cardiac puncture; the body weight of each hamster
was determined prior and after treatment. AST, ALT, glucose, serum total
cholesterol, high-density lipoprotein (HDL) cholesterol, and triglyceride
levels from each animal were determined in triplicate using an Abbott VP
Super System Autoanalyser (Abbott, Irving, TX) in conjunction with
commercial enzymatic kits (Labtest, Montes Claros, MG, Brazil). Measurement
of HDL cholesterol in serum was carried out after precipitation
of apo-B containing lipoproteins, with dextran sulfate and magnesium
chloride (Warnick et al., 1982). Results were expressed as non-HDL
(VLDL + IDL + LDL) cholesterol instead of LDL cholesterol, because
the Friedewald equation (Friedewald et al., 1972) is not applicable to
hamsters. Thus, the concentration of lipoprotein (non-HDL) cholesterol
was calculated by subtracting HDL cholesterol concentrations from total
serum cholesterol.
2.6. Preparation of tissue samples
Livers were rapidly removed, washed in cold 0.15 mol/l NaCl solution,
blotted onto filter paper, weighed and homogenized in 9 ml ice-cold
potassium phosphate buffer. After homogenization in PotterElvehjen, the
homogenates were centrifuged at 13,000g for 4 min at 4 C and the
supernatant assayed.
2.7. Determination of TBARS levels in serum and liver
The levels of TBARS were determined as described above (Section
2.3.1) in hamster sera and liver homogenates.

2.8. Antioxidants enzymes assay in serum and liver

2.8.1. Glutathione peroxidase (GPx)
GPx activity was measured spectrophotometrically at 340 nm using a
technique based on the Flohe and Gu nzler (1984) procedure, in which
GSH formation is followed by measurement of oxidation of the reduced
form of nicotinamide adenine dinucleotide phosphate (NADPH) to the
oxidized form, nicotinamide adenine phosphate (NADP+). Final concentrations
in the reaction mixture, in 0.1 mol/l potassium phosphate
buffer (pH 7.0) were 0.2 mmol/l NADPH, 1 mmol/l diethylenetriaminepentaacetic
acid, 1.5 mmol/l reduced glutathione (GSH) and 7.3 106 U/
ml reductase glutathione (GR). The reaction was started with 0.12 mmol/l
tert-butyl hydroperoxide (t-BuOOH). GPx activity is expressed as
lmol min1 g1 or ml1.
2.8.2. Superoxide dismutase (SOD)
SOD activity was measured as described by Paoletti and Mocali
(1972). Samples (25 ll) were added to the reaction mixture containing, at
final concentrations, 100 mmol/l diethanolamine buffer (pH 7.4),
0.14 mmol/l NADPH, 2.27/1.14 mmol/l EDTA/MnCl2 (pH 7.0). The
oxidation of NADPH was started by the addition of 1 mmol/l
b-mercaptoethanol. Oxidation of NADPH was measured spectrophotometrically
at 340 nm. A standard curve for SOD activity was constructed
using SOD from bovine erythrocytes (Sigma, St Louis, MO, USA). One
unit of SOD activity was defined as the amount of the enzyme required to
inhibit the oxidation of NADPH by 50%. SOD activity was expressed in
units per gram of tissue or ml of serum.
2.8.3. Catalase (CAT)
CAT activity was estimated following the method of Aebi (1984). The
reaction was started by the addition of 20 ll of sample in 2 ml of 30 mmol/l
hydrogen peroxide (H2O2) in 50 mmol/l potassium phosphate buffer (pH
7.0). Enzyme units are expressed as mmol/l of consumed H2O2 per min g
or ml.
2.9. Isolated blood vessels and histopathology analysis
After 10 weeks of treatment, animals were euthanized using CO2
chamber and serum samples were taken through heart puncture for biochemical
parameters. The thoracic aorta was rapidly removed, rinsed in
0.9% saline, fixed in 10% buffered formalin, processed for dehydration,
and embedded in paraffin. Five-micron sections were cut transversally on a
glass slide, mounted, rehydrated and stained with Harris hematoxylin/
eosin (H&E).
2.10. Statistical analysis
The data was analyzed using GraphPad Prism V.4 (GraphPad Software,
Inc., San Diego, CA). The statistical significance of differences
between data means was determined by using analysis of variance one way
(ANOVA), followed by post hoc testing by Tukeys test. A value of
P < 0.05 was considered as statistically significant. Results are given as
means SD, n = 6. For correlation analysis, Carl Pearson Moment
correlation test was employed.
3. Results
3.1. Antioxidant activity of the T. indica L. extract in vitro
As an initial chemical characterization of the extract, an
HPLC-UV chromatogram was performed. An UV spectra
was obtained for each observed peak in the chromatogram.
All spectra showed a similar absorption pattern, with two
bands of maximum absorption around 230 and 290 nm,
with the exception of the peak at 12.15 min, which comprises
a single band of absorption at 282 nm. These data
suggest the predominance of compounds like flavonoids
that normally occurs as secondary metabolites in plants
by the characteristics absorptions of the A and B bands
(Fig. 1).
The crude tamarind extract was found to be rich in sugars
(70.25 8.56 mg/ml), polyphenols (34.02 2.11 nmol/
ml) and flavonoids (35.51 5.61 lg/ml), the later two
being well known antioxidant agents. The assays for quantification
of these components were all performed in triplicate
in three independent extract preparations.
DPPH and superoxide radicals scavenging activities are
present in the extract in a dose-dependent manner, with
Pearson correlation of 0.89 (Fig. 2). The results of
both scavenging activities of the extract were compared
with the reference compound BHT (not shown). BHT presented
61.65% 0.3 and 80.29% 0.2 of scavenging activities
against DPPH and superoxide, respectively, whereas
the extract, at the concentration of 1 mg/dl, presented
7.31% 0.5 and 42.01% 3.5 scavenging activities against
DPPH and superoxide, respectively (Fig. 2).
Fe2+/citrate-mediated lipid peroxidation in animal
serum, assessed as TBARS, was inhibited by about 50%
in the presence of 2.5% crude tamarind extract. In the presence
of 5% of the extract the inhibition was almost complete
(Fig. 3).
3.2. Dietary intake and growth rate of hamsters
Table 1 shows food and fluid consumption by the experimental
hamsters; no differences were observed between
groups, but it should be noted that the animals consumed

less tamarind extract than tap water. The animals were

weighed at the beginning and at the end of the treatment.
Similar increases in body weight were observed in all experimental
groups.
3.3. Biochemical parameters of hamsters
AST and ALT serum levels were performed to assess
liver function. As can be observed in Table 2, no alterations
are detected in the animal groups treated with the cholesterol
diet and/or tamarind extract, during 10 weeks, when
compared to the control group (Table 2).

The levels of fasting serum glucose for control animals

were within the normal range, but the glucose levels of
the group treated with 5% of tamarind extract (SC + E)
showed a significant decrease when compared to the
normal control (SC + W) (Table 2).
As expected, the serum total cholesterol clearly
increased in the group treated with the cholesterol-rich diet

for 10 weeks (CC + W), when compared to the normal

control (SC + W). Treatment of either the control
(SC + E) or the hypercholesterolemic groups (CC + E)
with tamarind extract, resulted in a significant decrease in
the serum total cholesterol levels, when compared with
their respective controls (SC + W and CC + W). A similar
response was also observed for serum non-HDL cholesterol
levels. Furthermore, the treatment of either group
with the tamarind extract (SC + E and CC + E) resulted
in a substantial increase in the serum HDL levels when
compared to their controls (SC + W and CC + W, respectively)
(Table 2).
Serum triglycerides also increased in the control group,
which was fed with the cholesterol rich diet (CC + W),
when compared to the normal control (SC + W). On the
other hand, both the control group (SC + E) and the
hypercholesterolemic group (CC + E) showed a significant
decrease of the triglycerides levels after treatment with the
tamarind extract (Table 2).
3.4. In vivo serum lipid peroxidation
The evaluation of lipid peroxidation products showed
that the levels of TBARS in the serum were significantly
increased in the control group submitted to a cholesterol
rich diet. In contrast, treatment of both control and hypercholesterolemic
groups with the tamarind extract led to a
significant decrease of TBARS (Fig. 4).
3.5. Antioxidant status in serum and liver of experimental
animals
The cholesterol-rich diet showed a significant decrease
in the activity of GPx in serum, but not in the liver.

Remarkably, treatment of either control or hypercholesterolemic

groups with the tamarind extract caused a significant
increase in GPx activity both in serum and liver
(Fig. 4).
CAT activity either in serum or liver was not significantly
affected by the cholesterol diet. However, treatment of the
control group with the tamarind extract, led to an increase
in CAT activity in both serum and liver (Fig. 4).
The activity of SOD both in serum and liver, decreased
in the control group submitted to the cholesterol rich diet.
The treatment with the tamarind extract resulted in an
increase of SOD activity both in the serum and liver of
the control group, but not in the hypercholesterolemic
one (Fig. 4).
3.6. Histologic analysis
Aortae of all animals treated with the cholesterol diet (6/
6) showed a well-developed and mature lesion with lipid
accumulation (Fig. 5Bblack arrow) and a deep invasion
of the intima vessel (Fig. 5Bwhite arrow). On the other
hand, all animals submitted to the cholesterol diet and
treated with the tamarind extract (6/6) showed a reduction
in lesioned area, with lower accumulation of lipid droplets
(Fig. 5Cblack arrow). No histological differences
between the aortae of the control group (SC + W,
Fig. 5A) and the group treated with the tamarind extract
(SC + E) were observed (not shown).
4. Discussion
It is well established that elevated blood lipids levels
constitute the major risk factor for atheroclerosis (Castelli
et al., 1986). The search for new drugs capable of reducing
and/or regulating serum cholesterol and triglycerides
levels has gained momentum over the years, resulting in
numerous reports on significant activities of natural agents
(Jahromi et al., 1993). Although plan extracts constitute
potential candidates, they very often contain highly complex
mixtures of many different compounds showing
distinct polarity, antioxidant and pro-oxidant properties
and sometimes presenting synergic actions concerning the
individual components (Kahkonen et al., 2001; Parejo
et al., 2002).
In view of previous evidence that many fruits extracts
are successful in the prevention of atherosclerosis, we evaluated
the effects of the pulp fruit extract of T. indica L. on
serum lipids of hypercholesterolemic hamsters, as well as
its antioxidant activities. Oxidative stress arises from
perturbations in the balance between the production of
reactive oxygen species (ROS) and the efficiency of the antioxidant
defense mechanisms (Halliwell and Gutteridge,
1999). Lipids are very susceptible to attack by free radicals,
and oxidized LDL species appear to contribute to the atherosclerosis
pathobiology within the artery wall. In recent
years, several compounds from vegetables and fruits i.e.,
food especially rich in natural antioxidants like polyphenols,
have been used to prevent oxLDL formation. An
inverse relationship between oxLDL formation and polyphenol/
flavonoid contents has been reported (Aviram and
Fuhrman, 1998). In view of the high content of these compounds
in a crude tamarind extracts, we evaluated their
antioxidant properties in vitro, and found both DPPH
and superoxide radicals scavenging activities. In addition,
tamarind extracts were able to reduce TBARS levels

in vitro, suggesting their potential in promoting protection

against lipid peroxidation. These results corroborate the
previously reported antioxidant property of the tamarind
seed, which was attributed to the ()epicatechin and two
b-diketone (Tsuda et al., 1994).
A hypercholesterolemic diet changes the in vivo antioxidant
status of blood by increasing the generation of
oxygen free radicals, which exert their cytotoxic effect by
causing lipid peroxidation (Prasad and Kalra, 1993), furthermore,
an increase of TBARS levels in animals fed with
a high cholesterol diet has been previously reported
(Prasad and Kalra, 1993; Dhuley, 1999; Shunkla et al.,
2004). Accordingly, we have also observed increased serum
TBARS levels in hypercholesterolemic hamsters.
Atherosclerosis is a progressive disease involving both
the large and medium-sized arteries. It is a common factor
in many cardiac complaints, but it can affect different
organs. Also, the occurrence of antioxidant effects is not
organ specific, so that we evaluated these effects on liver,
which is the most common experimental model for this
kind of study (see for example: Frei and Higdon, 2003;
Hatipoglu et al., 2004; Sun et al., 2004; Auger et al.,
2005). It was reported that during mild oxidative stress,
tissues respond by activating antioxidant mechanisms
(Halliwell, 1994) and that high oxidative stress levels, most
likely depress the antioxidant defenses. In agreement with
previous reports (Daniel et al., 1998; Mary et al., 2002),
we observed a decrease in the activities of the antioxidant
enzymes SOD, CAT and GPx in animals maintained on
high cholesterol diet, compared to those maintained on a
normal diet. Such decreases may be associated to the production
of a-, b-unsaturated aldehydes during lipid peroxidation.
These compounds have the ability to increase
oxidative stress by promoting the cellular consumption of

glutathione and by inactivating selenium-dependent glutathione

peroxidase (Kinter and Roberts, 1996). Moreover,
the reaction of these products with amino acid residues
of proteins may cause oxidative modification of antioxidant
enzymes (Bosch-Morell et al., 1999) and other products
resulting from polyunsaturated fatty acid damage
may cause protein breakdown (Esterbauer et al., 1991).
Recently it was demonstrated that the superoxide anion,
alkoxyl, peroxyl and other radicals could inactivate catalase,
reducing the effectiveness of cells to protect themselves
from damage by free radicals (Mayo et al., 2003). Thus,
although the administration of tamarind extract to hamsters
did not suppress the lipid peroxidation in liver, it
significantly increased the activity of antioxidant enzymes
in the normal-diet groups. These effects could be due to a
protective antioxidant effect, and/or a significant lipidlowering
effect of tamarind plant constituents present in
the extracts.
In serum the tamarind extract prevented the dietaryinduced
increase in indicators of lipid peroxidation
(TBARS) whereas the GPx activity was reverted by the
extract in the hypercholesterolemic group. In this context,
the ability of the tamarind extract to suppress lipid peroxidation
could be due to the antifree radical activities of its
phenolic components, know to act as free radical scavengers,
and/or to the increase in the activity of antioxidant
enzymes, and/or decrease of available lipid substrates.
Considering the endogenous stress-related markers
(SOD, GPx and CAT), our results suggest that the tamarind
extract could improve efficiency to the superoxide
radicals conversion to hydrogen peroxide to increased
SOD activity in the normal-diet group following deactivation
of hydrogen peroxide by glutathione peroxidase. The
observation that catalase activity was unchanged after

administration of the extract, may not indicate a discrepancy

with otherwise improved reactive species elimination
of the antioxidant status (Skottova et al., 2004). Increased
catalase activity is considered to indicate the excessive
hydrogen peroxide levels when the detoxification capacity
of glutathione system becomes insufficient.
In order to rule out a possible damage to the liver by
the hypercholesterolemic diet or by treatment with the
tamarind extract, we evaluated the serum transaminases
activity. AST and ALT levels remain the most useful tests
for the detection hepatic cell damage, because both are
present in high concentrations in hepatocytes. These
enzymes leak into the circulation when hepatocytes or their
cell membranes are damaged (Kew, 2000). It has been
reported that treatment with a high concentration of cholesterol
can cause liver damage (Bolkent et al., 2004). Our
results show that 1% cholesterol did not lead to a significant
increase neither in ALT nor in AST in the serum
levels. Furthermore, hamsters treated with the tamarind
extract did not present any alteration in serum AST and
ALT levels.
In addition to the antioxidant activity, the tamarind
extract exhibited a significant hypolipidemic activity,
decreasing total cholesterol, triglycerides and non-HDL
cholesterol in serum. A decrease in serum triglycerides
and cholesterol has been associated with the epicatechins
contents (Chan et al., 1999). Remarkably, serum HDL
levels increased by nearly 50% both in the normal and
hypercholesterolemic groups treated with the tamarind
extract. HDL cholesterol is an independent negative risk
factor for coronary artery disease, at present representing
the only protective factor against atherosclerosis. This
effect of HDL is largely attributed to its central function
in the reverse cholesterol transport, a process whereby
excess cell cholesterol is taken up and processed by HDL
particles for further delivery to the liver for metabolism
(Martinez et al., 2004).
To summarize, the tamarind extract was able to reduce
total cholesterol, triglycerides, non-HDL cholesterol and to
increase HDL cholesterol in serum. The extract also inhibits
atherogenesis in the aorta of animals fed a cholesterol
diet, apparently by improving the efficiency of the antioxidant
defense system. Therefore, these data suggest that the
tamarind extracts has the potential to control the risk of
atherosclerosis development in humans.
Acknowledgments
We thank A. Zanardo Filho and N.M.F. Rodrigues for
excellent technical assistance. We are grateful to Dr. Norberto
P. Lopes and PhD Student Leonardo Gobbo Neto
for HPLC Analysis. Flavia Martinello is a recipient of a
PhD fellowship from the Fundacao de Amparo a` Pesquisa
(FAPESP 02/03174-5). S.A. Uyemura is a fellow from the
Conselho de Desenvolvimento Cientfico e Tecnologico
(CNPq 475276/01-9).

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