Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 38, Num. 6, 2006, pp. 408-413

Indian Journal of Pharmacology, Vol. 38, No. 6, November-December, 2006, pp. 408-413

Research Paper

Antipyretic, antidiarrhoeal, hypoglycaemic and hepatoprotective activities of ethyl acetate extract of Acacia catechu Willd. in albino rats

Ray D, Kh. Sharatchandra, Thokchom IS

Department of Pharmacology, Agartala Govt. Medical College, Po. Kunjavan, Agartala-799 006, Tripura
Correspondence Address:Department of Pharmacology, Agartala Govt. Medical College, Po. Kunjavan, Agartala-799 006, Tripura Email: dr_raydebasis@yahoo.co.in

Date of Submission: 23-Jan-2006
Date of Decision: 06-Apr-2006
Date of Acceptance: 04-May-2006

Code Number: ph06116

Abstract

Keywords: Antidiabetic, cutch, pyrexia, liver

Introduction

The diverse culture of our country is a rich source of traditional medicines, many of which are of plant origin. Scientific data on such plant derivatives could be of clinical use.[1] Catechu or cutch (Katha in Hindi and Manipuri), the extract prepared from the hard wood of Acacia catechu, has been used for treating fever, diarrhoea, leucorrhoea, piles and erysipelas.[2] The juice of its fresh bark has been used in treat-ment of haemoptysis and gonorrhoea. Catechuic acid found in cavities of the wood of the Acacia catechu tree (Leguminosae) was valued for facilitating expectoration in chest infection.[3] Catechu contains catechuic acid, catechutannic acid (25%-33%), acacatechin (10%-12%), catechu red, quercetin, catechin (2%-12%), epicatechin, phlebotanin (25%-33%), gummy matter, quercitrin, quercitin and moisture. Quercitin is a phe-nolic flavonoid and catechu of acacia is a pseudotanin. Catechu and epicatechin usually accompany other flavonoids.[3],[4]

It is reported that Acacia catechu has hypoglycaemic activity[5] and catechu, a product of Acacia catechu , has hepatoprotective, antipyretic and digestive properties.[6],[7] Cyanidanol, an active principle of Acacia catechu , is claimed to be effective in treating liver diseases.[8] Catechu was used in the treatment of diarrhoea and throat infection[9] because the tannin and polyphenols present in it impart astringent activity. Considering the above facts, the present study was undertaken to evaluate the antipyretic, antidiarrhoeal, hepatoprotective and hypoglycaemic properties of Acacia catechu in experimental animal models.

Materials and Methods

Collection and preparation of extract

Catechu (250 g) was collected (from the local market during the month of October), identified and differentiated from Uncaria gambier by gambier fluorescin test.[10], [11]

The ethyl acetate extract of Acacia catechu was obtained by the procedure as described by Jayasekhar et al , [6] and Seikel.[12] The powdered catechu was defatted with a nonpolar organic solvent such as petroleum ether (40 ^o C-60 ^o C) to remove the phenolic and nonpolar substances from the dried material. After defatting, catechu was extracted with 95% ethanol. Then the dried ethanol extract was again extracted with ethyl acetate to concentrate the minute amounts of phenolic materials present. The yield at the end of extraction was 29.2%.

Phytochemical studies

Tests for presence of flavonoid compounds in the catechu were done according to the methods described by Kokate[11] and Seikel.[12] However, identification and estimation of different flavonoid compounds like catechin, quercetin and cyanidol were not done.

Animals

Colony bred, healthy, Wistar strain, albino rats of either sex weighing 150-200 g were used for the study. They were housed in standard polypropylene cages, under room temperature (24±2 ^o C); relative humidity (60%-70%) and exposed to 12:12 h light:dark cycle. The rats were fed with standard diet and water ad libitum . The ethics committee, Regional Institute of Medical Sciences, Imphal, approved the protocol of the present study.

Drugs

The following chemicals (analar/GR) and drugs were used: gum acacia, castor oil, tablet Lomotil (diphenoxylate 2.5 mg + atropine sulphate 0.025 mg per tablet), acetyl salicylic acid, dried yeast, carbon tetrachloride (Qualizen), olive oil, silymarin (Micro Lab), glibenclamide (Aventis) and alloxan monohydrate.

Antidiarrhoeal studies
Antidiarrhoeal activity was evaluated as described by Yegnanarayan and Shrotri[13] with slight modification to ensure evenness of dose of castor oil in rats according to body weight. Fifty albino rats were screened initially by administering 1 ml of castor oil orally and those (32) which developed diarrhoea were selected (consistency, i.e. loose stool, was the criterion for selection). The experimental set-up was as follows.

Group A (Control):received 3% aqueous gum acacia suspension 1 ml/200 g, p.o., at '0' hour and castor oil 1 ml/150 g, p.o., one hour later.

Group B (Test): received ethyl acetate extract of Acacia catechu 250 mg/kg as 5% suspension in 3% gum acacia in DW (1 ml/200 g, p.o.) at '0' hour and castor oil 1 ml/150 g, p.o., one hour later.

Group C (Standard): received diphenoxylate 10 mg/kg (0.2%) with atropine sulphate (0.002%) suspension in 3% gum acacia in distilled water at a dose of 1ml/ 200 g, p.o. and castor oil 1ml/150 g, p.o, one hour later.

Antipyretic activity

Antipyretic activity on albino rats was studied with fever induced by 20% Brewer's yeast as described.[14], [15] Albino rats (150-200 g) were fed uniformly till 24 h before giving drugs, when food was withdrawn. After measuring rectal temperature of the animals by introducing 1.5 cm of digital thermometer in rectum, pyrexia was induced by injecting, subcutaneously, 20% suspension of dried yeast in 2% gum acacia in normal saline at a dose of 20 ml/kg of body weight. After 18 h of yeast injection, rats which showed a rise in temperature of at least 1 ^o F (0.6°C) were taken for the study. Animals in the various groups were treated as follows.

Group A: 3% aqueous suspension of gum acacia (1 ml/200 g) as vehicle, orally.

Group B: Aqueous suspension of ethyl acetate extract of Acacia catechu 250 mg/kg (1 ml/200 g) with 3% gum acacia as 5% suspension, orally.

Group C: Aqueous suspension of ethyl acetate extract of Acacia catechu 500 mg/kg (1 ml/200 g) with 3% gum acacia as 10% suspension, orally.

Group D: Aqueous acetyl salicylic acid, 300 mg/kg (1 ml/200 g) with 3% gum acacia as 6% suspension, orally.

Rectal temperature was recorded every hour for four hours after administration of drugs.

Hepatoprotective activity

Hepatoprotective activity of Acacia catechu was studied using the method described by Jayasekhar et al .[6] Blood was collected from the orbital sinus of albino rats and allowed to coagulate for 30 min. Serum was separated by centrifugation at 2500 rpm for 20 min and aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were assayed. AST and ALT levels were estimated from blood by the widely used method of Reitman and Frankel.[27] The protocol for the study of hepatoprotective activity is shown in [Table - 1].

Study of hypoglycaemic activity of Acacia catechu

Hypoglycaemic activity of Acacia catechu was studied both in normal and alloxan induced diabetic rats.

Hypoglycaemic activity in normal rats

Twenty-four albino rats weighing 150-200 g were fasted for 18 h and were divided into four groups of six animals in each. The groups included i) vehicle control (5% gum acacia in normal saline, 1 ml/200 g rat); ii) test drug (250 mg/kg, p.o. 5% w/v, 1 ml/200 g rat); iii) test drug (500 mg/kg, p.o., 10% w/v, 1 ml/200 g rat) and iv) standard control, glibenclamide (0.5 mg/kg, p.o., 1% 1 ml/200 g rat). One millilitre of blood from the orbital sinus of each rat was collected at '0' hour. At two hours of treatment, blood samples were collected again from the treated animals and blood glucose was estimated by glucose oxidase method.[16]

Hypoglycaemic activity in diabetic rats

Albino rats (n = 44) were fasted for 48 h. Diabetes was induced by administering[5],[17],[18],[19],[25],[26] freshly prepared alloxan monohydrate 2.4% in normal saline subcutaneously at a dose of 120 mg/kg, body weight as single dose.[17] After 72 h of alloxan, 18 h fasting blood was collected from those that survived (n=34)[18] and blood sugar estimated by glucose oxidase method. Twenty-four diabetic rats with blood glucose level of 300-500 mg% were selected and were divided into four groups of six each. The selected groups were treated with the vehicle (5% gum acacia, 1 ml/200 g), test drug (250 mg/kg, p.o.), test drug (500 mg/kg, p.o.) and glibenclamide (0.5 mg/kg, p.o.), respectively, for seven days.[19] On the eighth day blood samples were collected after 18 h of fasting and blood glucose was estimated again.

Statistical analysis

The results were analysed for statistical significance using one-way ANOVA followed by Dunnett's test. Nonparametric data were analysed by Kruskal-Wallis one-way ANOVA. P values < 0.05 were considered significant.

Results

Antidiarrhoeal studies

The extract of Acacia catechu (250 mg/kg) markedly reduced the percentage of animals that had diarrhoea (50%). The latent period (5.25±0.31) was prolonged and average number of stool passed was significantly reduced (0.7±0.26), leading to very low purging index (16.28) compared to the control group. Both the standard and test drugs brought a highly significant ( P < 0.001) change in the latent period of onset of passing stool as well as in total number of stools passed. [Table - 2]

Antipyretic activity

[Table - 3] shows the effect of Acacia catechu in yeast induced pyretic rats. There was no significant difference in the basal temperature at '0' hour between the different groups. The ethyl acetate extract of Acacia catechu and aspirin significantly decreased the temperature of pyretic rats at 2nd, 3rd and 4th h after drug or extract treatment.

Hepatoprotective activity

Significant increase in the levels of AST and ALT were found in the toxicant group ( P < 0.001) after 24 h of administration of CCl ₄ orally. Pretreatment with Acacia catechu (250 mg/kg) and silymarin (25 mg/kg) in test group and standard group, respectively, daily for seven days showed highly significant ( P < 0.001) protective effect against CCl ₄ induced hepatotoxicity when compared to toxicant alone group. There was no significant difference ( P < 0.5) between the protective ability of the test drug and the standard drug silymarin, when compared. [Table - 4]

Hypoglycaemic activity

The hypoglycaemic activity of ethyl acetate extract of Acacia Catechu in normal (nondiabetic) and diabetic rats is shown in [Table - 4]. The test drug, at a dose of 500 mg/kg, p.o. significantly lowered the blood glucose level ( P < 0.01) as compared to the control group, at 2 h. However, the activity of the standard drug, glibenclamide (0.5 mg/kg/day), was more pronounced ( P < 0.001). In alloxan induced diabetic albino rats, Acacia catechu at a dose of 250 and 500 mg/kg/day and standard drug glibenclamide (0.5 mg/kg/day) for seven days was highly significant ( P < 0.001) in comparison with the control group. However, in diabetic rats the hypoglycaemic effect of the test drug at 250 mg/kg was significantly less than the standard drug glibenclamide.

Discussion

In the present study, the mean initial basal rectal temperature of rats before yeast injection corresponds with findings of other workers.[20],[21] In our study, the initial rise of temperature after 18 h of subcutaneous yeast injection was 1.71°F-2.1°F (0.95°C to 1.67°C), which corresponds with the findings of Hajare et al .[15] There was no significant difference between the initial mean basal temperature of the different groups and the mean temperature between the groups of pyrexia rats, after 18 h of yeast injection. Body temperatures of pyrexia rats were lowered significantly with the test drug. The antipyretic effect of the test drug may be due to presence of flavonoid compounds, as some flavonoids are predominant inhibitors of cyclooxygenase or lipooxygenase.[4], [22],[23]

Castor oil induced model of diarrhoea incorporates both secretory and motility diarrhoea as a result of prostaglandin release from the intestinal mucosa.[13] Acacia catechu significantly decreased the number of stool passed ( P < 0.001). The percentage of respondents and purging index were also significantly decreased. Highly significant ( P < 0.001) increase in the latent period of onset of diarrhea, as compared to the control group, was also produced. Parameters in the control group of animals agree with the findings of previous workers.[13] The result shows that the ethyl acetate extract of A cacia catechu (test drug) is as effective as the standard drug, diphenoxylate, in respect of total number of stool passed ( P >0.05). The antidiarrhoeal property of the ethyl acetate extract of Acacia catechu appears to be due to its tannin[9] content, which has astringent property.

Results of the present study shows that ethyl acetate extract of Acacia catechu (500 mg/kg) significantly decreases fasting glucose levels of normal rats ( P < 0.001). However, the reduction was found to be less effective than that of glibenclamide. [Table - 5] The blood glucose levels in normal and diabetic albino rats before administration of the drugs correspond well with the findings of previous workers.[25]

The test drug at doses of 250 and 500 mg/kg/day for seven days reduced the blood glucose level of diabetic rats significantly ( P < 0.001). Effect of the test drug at doses of 250 and 500 mg/kg, orally, on blood glucose level was comparable with that of the standard drug, glibenclamide. The hypoglycaemic effect of A. catechu may be due to presence of flavonoids which acts as insulin secretagogues.[18] Epicatechin, a flavonoid compound, is reported to promote regeneration of β cells of the Islets of Langerhans.[1],[5],[19],[23]

Carbon tetrachloride is a hepatotoxin commonly used for the production of experimental liver toxicity. The serum transaminase level is most widely used as a measure of hepatic injury, due to its ease of measurement and high degree of sensitivity. It is useful for the detection of early damage of hepatic tissue and requires less effort than that required for a histologic analysis, moreover without sacrifice of the animals. In the present study, AST and ALT levels in normal control group were in conformity with the findings of Kapoor et al .[28] Seven days pretreatment with the test drug (250 mg/kg) protected the animals significantly ( P < 0.001) from CCl ₄ induced hepatotoxicity as compared to toxic control, and reflects the hepatoprotective activity of ethyl acetate extract of A. catechu. There was no significant difference between the test drug group and standard drug group. The hepatoprotective activity of A. catechu could be due to the presence of bioflavonoids which have hepatoprotective and antioxidant properties.[4],[8],[23]

In the present study, preliminary phytochemical screening of Acacia catechu found the presence of flavonoids and reports substantiate the presence of catechutanic acid,[3] quercetin,[4] catechin,[4],[22] pseudotannin,[4] phlabotannin,[4] polyphenols,[9] epicatechin[22] and cyanidanol[8] compounds in A. catechu . The antidiarrhoeal and hepatoprotective properties of A. catechu could be attributed to the presence of tannins[9] and cyanidanol[8] and quercetin,[23] respectively. The antipyretic and hypoglycaemic properties of Acacia catechu may be ascribed to the presence of flavonoids, which have been shown to inhibit cyclooxygenases[23] and promote β-cell regeneration besides having insulin secretagogues and antioxidant properties.[1],[5],[18],[19],[23]

The results of the present study suggest that ethyl acetate extract of Acacia catechu in doses of 250 and 500 mg/kg, significantly reduced the temperature of pyretic rats, and also illustrate significant hypoglycaemic activity. The extract of A. catechu, 250 mg/kg, p.o., has also shown to possess significant antidiarrhoeal and hepatoprotective property.

References

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