INTRODUCTION:

Tamarindus indica occurs widely throughout the tropics of the Old World. a monotypic genus and belongs to the subfamily Caesalpinioideae of the family Leguminosae (Fabaceae), Tamarindus indica L., commonly known as Tamarind tree is one of the most important multipurpose tropical fruit tree species in the Indian subcontinent. Tamarind fruit was at first thought to be produced by an Indian palm, as the name Tamarind comes from a Persian word "Tamar-I-hind," meaning date of India. Its name "Amlika" in Sanskrit indicates its ancient presence in the country.

T. indica is used as traditional medicine in India, Africa, Pakistan, Bangladesh, Nigeria, and most of the tropical countries. It is used traditionally in abdominal pain, diarrhea and dysentery, helminthes infections, wound healing, malaria and fever, constipation, inflammation, cell cytotoxicity, gonorrhea, and eye diseases. It has numerous chemical values and is rich in phytochemicals, and hence the plant is reported to possess antidiabetic activity, antimicrobial activity, antivenomic activity, antioxidant activity, antimalarial activity, hepatoprotective activity, antiasthmatic activity, laxative activity, and anti-hyperlipidemic activity. Every part of the plant from root to leaf tips is useful for human needs. Thus the aim of the present review is to describe its morphology, and explore the phytochemical constituents, commercial utilization of the parts of the plant, and medicinal and pharmacologic activities so that T. indica's potential as multipurpose tree species can be understood. Medicines herbs are major constituents of Ayurvedic system of medicines. The role of free radical reactions in disease pathology is well established, suggesting that these reactions are necessary for normal metabolism but can be detrimental to health; the antioxidants protected against free radicals induced oxidative damage by antioxidant enzymes such as superoxide dismutase and catalase or antioxidant compounds(Kakoti BBet al).The liver is expected not only to perform physiological functions but also to protect against the hazards of harmful drugs and chemicals. Inspite of tremendous scientific advancement in the field of hepatology in recent years. Jaundice and hepatitis are two major hepatic disorders that account for a high death rate (Shahani S et al). Liver diseases are mainly caused by toxic chemicals, excess consumption of alcohol, infections and autoimmune disorders. Most of the hepatotoxic chemicals damage liver cells mainly by inducing lipid peroxidation and other oxidative damages (Dianzani MUet al). In spite of tremendous advances in modern medicine, there are no effective and reliable drugs available that can stimulate liver function, offer protection to the liver from damage or help to regenerate hepatic cells (Subramoniam A et al). However, there are a number of medicinal preparations in Ayurveda that are recommended for the treatment of liver disorders (Chatterjee TK. Et al, Espejo A, et al).

MATERIALS AND METHODS:

Plant material:

Fresh plants of Tamarindus indicaa were collected from Nalgonda AP plant specimen was aunthenticated by Prof. Dr. Raju, Kakatiya University, Warangal.

Animals:

The study was conducted in male Wistar strain albino rats, weighing about 180–225 g. They were housed in microlon boxes in a controlled environment (temperature 25 ± 2 °C) with standard laboratory diet and water ad libitum. The animals were acclimatized for a period of three days in the new environment before the initiation of experiment. The litter in the cages was renewed thrice a week to ensure hygiene and maximum comfort for animals. Ethical clearance for handling the animals was reviewed and approved by the University Animals Ethical Committee.

Acute toxicity studies:

Acute oral toxicity (ACT) of Tamarindus indica was determined using Swiss albino mice. The animals were fasted for 12 h before the experiment and were administered with single dose of extracts dissolved in 5% gum acacia and observed for mortality up to 48 h (short term toxicity). On the basis of short-term toxicity, the dose of next animal was determined as per CECD guideline 420. The limit test carried out first at 4 g/kg. b.w. All animals were observed for toxic symptoms and mortality for 72 h.

Preparation of plant extracts:

Hundred grams of the aerial parts were dried and powdered in a mechanical grinder. The powdered material was extracted by 500 ml of petroleum ether, ethylacetate, chloroform, methanol and water consecutively using a Soxhlet apparatus. These extracts were filtered and concentrated by a rotary vacuum evaporator and kept in a vacuum desiccator for the complete removal of solvent.

Percentage yield and physical appearance of extracts:

Sl. No.	Extract	% dry Weight	Colour	Consistency
1	Pet. Ether extract	10.8	Green	Resinus
2	Ethyl acetate	11.12	Green	Semi solid
3	Chloroform extract	16.17	Green	Solid
4	Methanol	8.8	Green	Powder
5	Aqueous	1.6	Brown	Powder

Antioxidant activity in vitro:

Inhibition of DPPH radical:

The free radical scavenging activity of the extract was analyzed by the DPPH (1,1-- diphenyl-- 2--picryl hydrazyl) assay.(Gupta M, et al). A total of 2 ml of the test extract, at concentrations ranging from 1 μg/ml to 100 μg/ml each, was mixed with 1 ml of 0.5 mM DPPH (in methanol). The absorbance at 517 nm was taken after 30 min of incubation in the dark at room temperature. The experiment was done in triplicate. The percentage antioxidant activity was calculated as follows:

%Antioxidant Activity [AA] = l00 --[ { (Abs_sample -- Abs_blank) X 100 }/Abs ml of methanol plus 2.0 ml of the extract was used as the blank while 1.0 ml of the 0.3 mM

DPPH solution plus 2.0 ml of methanol was used as the negative control. Ascorbic acid was used as the reference standard.

Inhibition of superoxide anion radical:

Measurement of superoxide anion scavenging activity of SM was performed based on the method described by Nishimiki (Starzynska AJ et.al.) and slightly modified. About 1 ml of nitroblue tetrazolium (NBT) solution containing 156 μM NBT which is dissolved in 1.0 ml of phosphate buffer (100 mM, pH 7.4), 1 ml of NADH solution containing 468 μM of NADH which is dissolved in 1 ml of phosphate buffer (100 mM, pH 7.4) and 0.1 ml of various concentrations of SM and the reference compounds (5, 10, 25, 50 and 100 μg) were mixed and the reaction started by adding l00 μl of phenazine methosulphate (PMS) solution containing 60 μM of PMS l00 μl of phosphate buffer (100 mM, pH 7.4). The reaction mixture was incubated at 25°C for 5 min and the absorbance at 560 nm was measured against the control samples. All the tests were performed in triplicate and the results were averaged. The percentage decrease in absorbance was calculated (Gupta M, et al) Quercetine was used as the standard.

Inhibition of nitric oxide radical:

Nitric oxide generated from sodium nitroprusside in aqueous solution at physiological pH interacts with oxygen to produce nitrite ions, which were measured by the Griess reaction (Hinneburg I. et. al., Tepe B. et.al.) The reaction mixture (3 ml) containing sodium nitroprusside (10 Mm) in phosphate buffered saline (PES) and SM and the reference compound in different concentrations (5, 10, 25, 50 and 100 μg) were incubated at 25°C for 150 min. In each 30 min, 0.5 ml of the incubated sample was removed and 0.5 ml of the Griess reagent (1% sulfanilamide, 0.1% naphthyl ethylene diamine dihydrochloride in 2% H₃PO₄) was added. The absorbance of the chromophore formed was measured at 546 nm. All the tests were performed in triplicate and the results were averaged. BHT was used as the reference compound. All the tests were performed in triplicate and the results were averaged. The percentage decrease in absorbance was calculated (Gupta M, et al). Quercetine was used as the standard.

Iron-chelating activity:

Chelating of iron (II) ions by extracts was carried out as described in the previous work (Zheng W. et.al.). Briefly, a given volume of extracts (0.1222 mg/ml), ascorbic acid (0.1564 mg/ml), or BHT (0.1890 mg/ml) was added to 50 μl of 2.0 mM aqueous FeSO₄ in a 5.0 ml test tube, then was added 1 ml of ethanol to complete 4.0 ml. After 5 min incubation, the reaction was initiated by 1.0 ml of 5.0 mM ferrozine. After 10 min of equilibrium, the absorbance at 562 nm was recorded. The controls contained all reaction reagents except extracts or positive control substance. Three experiments were performed and the average result was adopted. The iron-chelating activities were calculated from the absorbance of the control (Ac) and of the sample (As) using the following equation:

Inhibition (%) = Ac −As × 100 Ac

Hydroxyl radical scavenging assay:

The OH· scavenging ability was evaluated as the inhibition rate of deoxyribose oxidation by this radical as described by Hutadilok--Towatana.( Hutadilok TN et al) Tannic acid was used as the positive control. The capability to scavenge OH. was calculated based on the concentration of extract required to inhibit deoxyribose attack by 50% (IC₅₀).

Hepatoprotective activity:

Induction of in vivo carbon tetra chloride hepatotoxicity:

The animals were divided into control, carbon tetrachloride (CCl₄) and test groups (CCl₄ + extracts, silymarin and extracts) each containing six animals in all the sets of experiments. 50% v/v CCl₄ solution in olive oil was used for administration (Aly AAQ et al). Animals from the:

Group – I: Served as solvent control which received 1ml/kg of arachis oil p.o. for seven days

.Group – II: Served as toxic control and were given 3ml / kg of 50% v/v CCl₄in olive oil i.p. on the seventh day.

Group – III: The Animals from the test groups received single daily dose of the methanolic extracts (200, 400 y 600 mg/kg mg/kg i.p.) and silymarin (50 mg/kg i.p) for four days. The animals were also administered toxicant CCl₄ (2 ml/kg S.C.) 30 min after the administration of the test extracts.

All the rats were anaesthetized with ether anaesthesia 36 hours after administration of CCl_4.Then blood samples were collected from common carotid artery by carefully opening the neck region of the rat. After blood collection the blood samples were allowed to coagulate at room temperature for at least one hour. Serum was separated by centrifugation at 3000 rpm for 30 minutes at 50⁰C and then analysed for marker enzymes namely SGOT, (AST), SGPT (ALT), Alkaline phosphatase, (ALP), Albumin, bilirubin, lactate dehydrogenase (LDB) and triglycerides. The enzyme levels were assayed using standard kits obtained from Excel Diagnostics Pvt. Ltd., Hyderabad. The animals were sacrificed by cervical dislocation and liver samples were dissected out and washed immediately with ice cold saline to remove as much blood as possible. Liver homogenates (15% w/v) were prepared in cold 50mM potassium phosphate buffer (pH 7.4) using a Remi homogenizer and preserved in formalin solution (10% formaldehyde) for histopathological studies.

The hepatoprotective activity was calculated as:

[1-- (ALTdrug --ALTcontrol/ALT CCl₄ --ALTcontrol)] X 100

Statistical analysis:

Results of biochemical analysis are presented as mean values ±S.D. and % reduction was calculated by considering the difference between the control and the toxicant as 100% reduction. Statistical significance of the difference was analyzed through one way analysis of variance (ANOVA) by SPSS version 11.5 for Windows. Difference between the test group and the control was determined by least significant difference method at p<0.05 confidence levels.

RESULTS:

Acute toxicity studies:

For acute oral toxicity studies, the extract-treated animals were observed for mortality up to 72 h. On the basis of the results, it can be seen that the extract did not produce any mortality up to 4000 mg/kg body weight.

The DPPH radical scavenging abilities of the extracts (89.87%) were found to be less than those of ascorbic acid (97%)) at 100 μg/ml

Inhibition of nitric oxide scavenging and superoxide anion scavenging of the methanol extracts

Table 1, shows the dose--response results of nitric oxide scavenging and superoxide anion scavenging of the methanol extracts of the leaves of Tamarindus indica . The extract reduced the generation of nitric oxide radicals from sodium nitoprusside solution. This showed marked nitric oxide scavenging of the extract (69.79%). Also the extract showed significant superoxide scavenging activity (76.12 %) at 100 μg/ml.

TABLE:1 In vitro antioxidant effect of methanol extract of Tamarindus indica

Treatment mg/ml	DPPH scavenging	Nitric oxide scavenging	Superoxide anion scavenging
TI 20	27.45 ± 0.34^*	19.34 ± 1.23^*	20.98 ± 0.13^*
TI 40	38.16 ± 0.23^*	28.34 ± 2.56^*	36.45 ± 0.17^*
TI 60	53.27 ± 0.19^*	42.57 ± 3.15^*	48.79 ± 0.20^*
TI 80	68.98 ± 0.42^*	59.76 ± 1.98^*	54.36 ± 0.24^*
TI 100	89.87 ± 0.39^*	69.79 ± 4.17^*	66.12 ± 0.25^*
Ascorbic acid 100 (μg/ml)	97 ± 0.52^*	-	-

Effect of different concentrations of TI, ascorbic acid on DPPH free radical, nitric oxide and superoxide anion scavenging activities.

^*Data are mean representative of three experiments and the result are expressed as Mean ± S.E.M.

Iron-chelating activity:

In this study, the chelations of ferrous ions by extracts, ascorbic acid and BHT as controls were estimated. In the presence of chelating agents, the complex formation is disrupted, resulting in a decrease in the red color. The efficiencies of Fe²⁺ferrozine complex increase with the increasing concentration of the three antioxidants. In this assay, methanol extract showed iron-chelating activity [Table 2]. The median inhibitory concentration (IC₅₀) values for methanol extract, ascorbic acid and BHT were 66.5, 49.3 and 46.6 μg /ml, respectively.

Table-2: Ion-chelating and OH. Scavenging activities of methanol extract of Tamarindus indica

	Treatment	OH scavenging
Tamarindus indica	66.5	49.65
Ascorbic acid	49.3	-
BHT	46.6	-

Hydroxyl radical scavenging assay

In Table 2, methanol extract shows highest activity on OH· scavenging with IC₅₀ values of 49.65 μg/ml.

Hepatoprotective effect:

In addition to antioxidant, the ability of hepatoprotective action of SM was assessed by measuring the level of biochemical enzyme. As shown in Table 3, administration of CCl₄ significantly enhanced the biochemical markers like ALT, AST, SGPT, SGOT by three to four fold. ALP, total bilirubin, cholesterol and reduced levels of HDL are shown in Table 4. Pretreatment with AI (200, 400 and 600 mg/kg) reduced the elevated levels of all the above-mentioned biochemical indicators and increased the level of HDL. The groups treated with the hexane and chloroform extracts did not reduce the elevated biochemical parameters, indicating no protection.

DISCUSSION:

It has been already reported that phenolic compounds play an important role in scavenging of free radicals. The correlation between antioxidant activities and quantity of the flavonoids is still under discussion, a good linear relationship was observed in some published works (Starzynska AJ et al, Wangensteen H et al). However, Hinneburg (Hinneburg I et al ) found no linear relationship between them. The controversy might be contributed to the complexity of plant materials used by them. Results obtained in the present study revealed that the level of these phenolic compounds in the methanol extracts of the leaves and stem of Tamarindus indica was considerable. Polyphenolic compounds are known to have antioxidant activity and it is due to their redox properties, which play an important role in adsorbing and neutralizing free radicals, quenching singlet and triplet oxygen, or decomposing peroxides (Tepe B et al, Zheng W et al). In fact, many medicinal plants contain large amounts of antioxidants such as polyphenols, many of these phytochemicals possess significant antioxidant capacities that are associated with lower occurrence and lower mortality rates of several human diseases. The results strongly suggest that phenolics are important components of this plant, and some of its pharmacological effects could be attributed to the presence of these valuable constituents (Djeridane A et al). The effect of antioxidants on DPPH is considered to be due to their hydrogen donating ability (Yu L et al). The present study shows that the extracts have the proton-donating ability and could serve as free radical inhibitors or scavengers, acting possibly as primary antioxidants.

The methanol extract of SM shows significant superoxide anion, nitric oxide scavenging activities in a dose dependent manner. Simultaneous generation of NO and O₂^-- favors the production of a toxic reaction product, peroxynitrite (ONOO^--). The scavenging of the superoxide anion and nitric oxide indicate the possibility of preventing the formation of peroxynitrite in the cell. Reducing the nitric oxide generation in the digestive tract was found to be effective in preventing the reactions of nitrate with amines and amides to form carcinogenic nitrosamines and nitrosamides (Boone CW et al). Hence the NO scavenging activity of SM extract could play a preventive role against nitrosamine-mediated carcinogenesis.

Table 3: Effect of different doses of methanol extract from Tamarindus indica on ALT, AST, SGPT and SGOT on CCl₄-induced hepatotoxicity in rats

Group (mg/kg)	ALT (IU/L)	AST (IU/L)	SGPT (U/I)	SGOT (U/I)
Control	64.21 ± 1.47	61.21 ± 1.43	48.12 ± 1.86	100.98 ± 0.45
CCl₄	139.89 ± 1.76^**	141.89 ± 1.96^**	295.11 ± 1.72^**	421.41 ± 0.15^**
TI 200	104.60 ± 1.45^*	109.69 ± 1.65^*	145.70 ± 1.95^*	249.76 ± 0.34^*
TI 400	83.81 ± 2.55^*	89.21 ± 2.75^*	103.05 ± 1.96^*	166.59 ± 0.23^*
TI 600	70.53 ± 0.77^*	73.43 ± 0.97^*	78.76 ± 2.34^*	140.98 ± 0.19^*
Sylimarin 100	104.21 ± 1.37^*	105.61 ± 1.47^*	62.75 ± 2.06^*	132.62 ± 0.43^*

Each value represents the mean ± SEM, n = 6; ^*P<0.05 significantly different values from CCl₄ group.

^**P<0.01 indicate significantly values compared to control group.

Table 4: Effects of different doses of methanol extract from Tamarindus indica on ALP, total bilirubin cholesterol and HDL on CCl₄-induced hepatotoxicity in rats.

Group (mg/kg)	ALP	total bilirubin	Cholesterol	HDL
Control	135.67 ± 0.07	0.98 ± 2.45	105.47 ± 3.24	47.89 ± 1.29
CCl₄	257.38 ± 0.09^**	4.56 ± 2.03^**	175.36 ± 2.74^**	27.91 ± 1.56^**
TI200	148.90 ± 0.06^*	2.64 ± 2.87^*	141.32 ± 4.32^*	34.12 ± 1.70^*
TI 400	119.80 ± 0.04^*	1.89 ± 1.99^*	136.73 ± 4.56^*	38.61 ± 1.86^*
TI 600	95.99 ± 0.02^*	1.25 ± 2.65^*	124.50 ± 3.75^*	46.02 ± 1.73^*
Sylimarin 100	96.45 ± 0.03^*	1.28 ± 2.84^*	122.61 ± 3.56^*	47.54 ± 1.28^*

Each value represents the mean ± SEM, n = 6; ^*P<0.05 significantly different values from CCl₄ group.

^**P<0.01 indicate significantly values compared to control group

Iron-chelating capacity is important as it reduces the concentration of the catalyzing transition metal in lipid peroxidation via the fenton reaction. Ferrozine can quantitatively form complexes with Fe²⁺chelating agents, which form δ-bonds with a metal, that are effective as secondary antioxidants because they reduce the redox potential and then stabilize the oxidized form of the metal ion (Bhatia A et. al. ) OH· scavenging activities were determined based on the ability of the antioxidant components in the samples to inhibit deoxyribose oxidation by reactive OH· generated from Fenton's type reaction (Beckman JS et al). In this case, two anti oxidation mechanisms are involved. One is the suppression of the OH· generation from H₂O₂ by binding with metal ions and the other is a direct single electron transfer to the generated radical. Tamarindus indica is high in polyphenols that are known to be strong chelators of heavy metals, and are also believed to be related to such effective OH· scavenging ability. Apart from the phenolic compounds that are responsible for the antioxidant activity, there might be some other active compounds that also exert some effects.

This present study evaluated the hepatoprotective activities of Tamarindus indica in CCl_4- -induced liver toxicity. It is generally accepted that the hepatotoxicity of CCl₄ depends on the cleavage of the carbon--chlorine bond to generate tricloromethyl free radical (.CCl₃); this free radical reacts rapidly with oxygen to form a trichloromethyl peroxy radical (.CCl₃O₂). This metabolite may attack membrane polyunsaturated fatty acids and causes lipid peroxidation which plays a main role in the induction of liver injury (Gonzalez R et. al.) and further causes impairment of membrane function. CCl₄-induced hepatic injuries are commonly used as models for the screening of hepatoprotective plant extract and the extent of hepatic damage is assessed by the level of released cytosolic transaminases including ALT and AST in circulation (Agarwal M et al). When administrated prophylacticaly, methanol extract exhibited protection against CCl₄induced liver injuries as manifested by the reduction of toxin-mediated rise in serum enzymes in rats. Enzyme levels such as GOT and GPT are mainly determined. Necrosis or membrane damage releases the enzyme in to circulation; therefore, it can be measured in serum. A high level of GOT indicates liver damage such as that due to viral hepatitis as well as cardiac infarction and muscle injury. GPT catalyzes the conversion of alanine to pyruvate and glutamate, and is released in a similar manner. Therefore, GPT is more specific to the liver, and is thus a better parameter for detecting liver injury (Muriel P et al). Elevated levels of serum enzymes are indicative of cellular leakage and loss of functional integrity of cell membrane in liver. Prolonged destruction of the hepatic cells results in more hepatic releases to exacerbate hepatic dysfunction and causes an elevation in the serum levels of ALP, LDH, and bilirubin (Mitchell JR et al).

CONCLUSSION:

The antioxidant activity may be due to the inhibition of the formation of radicals or scavenging of the formed radical and the presence of the phenolic compounds. These results concluded that Tamarindus indica has promising antioxidant and hepatoprotective effects. The findings thus establish the potential medicinal value of the plant Tamarindus indica used in indigenous systems of medicines in Mexico and also initiate further detailed investigations on this plant in order to justify its use in polyherbal formulations prescribed in the treatment of liver disorders.

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Received on 08.07.2011 Accepted on 12.08.2011

Asian J. Pharm. Tech. 1(3): July-Sept. 2011; Page 73-78