Anti-Inflammatory Activity of Ethanolic and Acetone Extracts of Bauhnia variegate

D Sheshadri Shekar* C Velmurugan, G Ramakrishnan and B Vivek

Department of Pharmacology, Sri K .V College of Pharmacy, M.G.Road, Chickballapur-562101.

ABSTRACT

The ethanolic and acetone extracts of Bauhnia variegate bark was examined for anti-inflammatory in experimental animals. In this study both acute and sub-acute inflammation models were used to evaluate the anti-inflammatory activity of the Bauhnia variegate. In acute model carrageenan was used to induce inflammation in rat hind paw and in sub-acute inflammation, cotton pellet induced granuloma was performed. The bark of ethanolic and acetone extracts of bauhnia variegate at the doses of 400 mg/kg shows significant (p<0.001) inhibition of inflammation against both acute and sub-acute anti-inflammatory model. The percentage inhibition of ethanolic and acetone extract of Bauhnia variegate both acute and sub-acute anti-inflammatory model was 35.29%, 43.14% and 33.53%, 42.20% respectively. According to our results both the extracts of Bauhnia variegate in doses of 400 mg/kg reveals anti-inflammatory activity and supports tribal claim.

Keywords: Anti-inflammatory, Carrageenan, Cotton pellet granuloma and Bauhnia variegate.

INTRODUCTION:

Bauhinia variegate (Caesalpiniaceae) is a commonly found plant in muddy ground and open plantation. It is medium sized deciduous tree which are cultivated throughout India particularly in the District of Nilgris, Tamil Nadu where it is called as Mandarai (Tamil). The phytochemical characterization shows the presence of tannins, steroids, alkaloids, triterpens, flavonoids^1-3, sitosterol, lupeol, vitamin C, Kaempferol, flavonone and Quercentin⁴. The ethanolic stem bark extract of Bauhina variegate is used in ayurvedic treatment of liver tonic, blood impurities, leprosy, menorrhagia, wounds and in ulcers^1-3 and antitumour activity⁵. The buds are acrid, indigestible and used in piles, cough, eye disease, liver complaints; the root decoction is given for dyspepsia and flatulency. The root has been prescribed in combination with other drugs for the treatment of snake bite. Tribals of Nilgris has been using the bark for past several years to treat inflammatory conditions. But there is no sufficient scientific evidence for this plant having anti-inflammatory action, so the present study was performed in order to investigate its anti-inflammatory activity by acute and chronic inflammatory model.

MATERIALS AND METHODS:

Plant material:

The leaves of Bauhnia variegate were collected from Ootacamund. The taxonomical identification of plants was identified by Dr. Rajan, Department of Botany, J.S.S College of Pharmacy, Ootacamund.

Animal experiments:

Swiss albino rats weighed between 150-180gms of either sex were obtained from the colonies maintain at Central animal facility, Government Veterinary College, Bangalore. The rats were housed in the polypropylene cages with paddy husk as bedding and with stainless steel top grill having facilities for providing food and drinking water in polypropylene bottles with stainless steel sipper tube.

Table 1: THE ANTI INFLAMMATORY ACTIVITY OF BAUHNIA VARIEGATE BY CARRAGEENAN INDUCED PAW OEDEMA

S. No	Treatments	Dose (mg/kg)	Increase in paw volume (ml)	Percentage inhibition
Group I	Control	2ml	0.51±0.02	0
Group II	Ethanol	400	0.33±.01^*	35.29
Group III	Acetone	400	0.29±0.02^*	43.14
Group IV	Diclofenac sodium	50	0.25±0.01^*	50.98

Values are expressed as mean ± SEM (N=6), p<0.001* significant with respect to the control group.

TABLE 2: THE ANTI INFLAMMATORY ACTIVITY OF BAUHNIA VARIEGATE BY COTTON PELLET GRANULOMA MODEL

S.No	Treatments	Dose in mg/kg	Weight of cotton pellet (mg) (dry)	Percentage inhibition
Group I	Acetone	400	43±2.8^*	42.20
Group II	Ethanol	400	49.4±3.2^*	33.53
Group III	Diclofenac sodium	50	36.8±2.7^*	50.53
Group IV	Control	2 ml	74.4±4.2	0

Values are expressed as mean ± SEM (N=6), p<0.001* significant with respect to the control group.

The animals were housed at temperature of 24±2^o C and relative humidity of 30-70%. A 12:12 hr light and dark cycle was followed. The animal experimental protocol was approved by Institutional Animal Ethical Committee as per the guidance of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) 117/99/kvcp.

Preparation of extracts:

The stem park was collected using sickle during the month of August. The air dried bark was powdered and subjected to successive extractions with petroleum ether, acetone and ethanol (90%) for 24 to 36 hrs. These extracts were then concentrated at low pressure and dried at vacuum to evaporate the solvent.

Statistical analysis:

One way analysis of variance (ANOVA), unpaired ‘’t’’ test by parametric standard test was employed using Graphpad Instat 3.0 software for statistical analysis of the data. A probability value of < 0.001 was considered statistically significant. Values in the text and tables are represented as Mean ± SEM.

Acute toxicity:

The acute toxicity study was carried out as per Guidelines. Mortality in each group within 24 h was recorded. The animals were observed for a further 14 days for any signs for delayed toxicity. The alcoholic and acetone extracts of Bauhnia variegate has good margin of safety and did not shown the lethal effects on the animals up to the doses of 4000mg/kg. Hence LD₅₀ of Bauhnia variegate was considered as 4000mg/kg, studies were carried out with 1/10 of the LD₅₀ dose is 400mg/kg.

Pharmacological studies:

Carrageenan induced rat hind paw oedema model :

The rats were divided into four groups comprising six animals in each group. Group I served as control received refined ground nut oil (2 ml/kg) and groups II and III were treated orally with ethanolic and acetone extracts of Bauhnia variegate (400 mg/kg) respectively. Group IV received the standard drug Diclofenac sodium (50 mg/kg). The administration of test and standard drugs was 30min prior to injection of 0.1ml of 1% w/v of freshly prepared carrageenan suspension in normal saline at sub plantar region in the right hind paw of each rat⁶. The paw volume was measured by using plethysmometer at 0, 1^st, 2^nd, 3^rd and 4^th hours after the administration of Carrageenan. Third hour was taken as a measure of oedema formation. The percentage anti-inflammatory activity of Bauhnia variegate was calculated by following formula.

Anti-inflammatory activity (%) = Control-Test

Control

Effect of extract on cotton pellet granuloma method:

Male albino rats were weighed and divided in to four groups of six animals in each group. Under light ether anesthesia sterile cotton pellet weighing 10±1mg were implanted subcutaneously into the both sides of the groin region of each rat⁷. Group I and Group II received the acetone and ethanol extracts of Bauhnia variegate at dose of 400 mg / kg body weight for seven consecutive days from the day cotton pellet implantation. Group III received Diclofenac sodium 50 mg / kg body weight on the same day. Group IV served as solvent control and received the refined ground nut oil 1 ml / 500 gm. On 8^th day the animals were anaesthetized and the cotton pellets together with granuloma tissue were removed. The wet pellets were weighed and then dried in an oven at 60⁰ C for 24 hours. Then the dried pellets were weighed again. Increase in the dry weight of the pellet was taken as a measure of granuloma formation. The anti-proliferative effect of ethanolic and acetone extracts of Bauhnia variegate was compared with control. The percentage protection was calculated by following formula

% Protection = Control-Test

Control

RESULTS:

Effect of the extracts on carrageenan induced paw oedema:

The result shows (Table 1) that both the extracts of Bauhnia variegate at the dose of 400 mg / kg has significant reduction in the carrageenan induced paw oedema (p<0.001) when compared to control. But when comparing ethanolic extract, the acetone extract has high anti-inflammatory effect.

Effect of the extracts on the cotton pellet granuloma:

The effect of the extracts of the anti inflammatory activity using cotton pellet granuloma technique was studied. Our results showed a marked protection in granuloma by markedly reducing the dry weight of the cotton pellet in both extracts when compared to control (p<0.001) (Table 2). The acetone extract’s percentage protection was slightly higher than the ethanolic extract, which was also not weak when compared with that of the Diclofenac sodium (p<0.001).

DISCUSSION:

In living animal tissue, Inflammation caused by infection, auto immune stimuli. Several classes of compound such as plasma protein, tissue digestive enzyme and biologically derived oxidant are all associated with inflammation^{8, 9}. And also inflammatory process involve the release of several mediators, including prostaglandin, histamine, cytokines, proteinase, and so as well as that regulate adhesion of molecules and the process of cell migration, activation and degranulation¹². Varian form and model of anti-inflammatory reaction have been detected. Most of all investigators have reported that inhibition of carrageenan induced inflammation in rats is one of the most suitable acute model to screen anti-inflammatory agents¹⁰. The development of carrageenan induced oedema is bi- phasic, the first phase is attributed to the release of histamine, 5-hydroxy tryptamine and kinins, while, the second phase is release of prostaglandins¹¹. The inhibitory action observed after bauhnia variegate treatment on carrageenan induced paw oedema in rats may be mediated through either first phase alone or both first phase and second phase as per data shown in Table. 1. The above acute model result was indicate, the bauhnia variegate having anti inflammatory effect. The adoption of different anti inflammatory and anaelgesic experimental models for assessment of phytomedicine used in the health care system for the management of pain, asthma, arthritis, rheumatism and so on one considered desirable and justifiable¹³. Likewise, the granulomatous tissue formation is related to the chronic inflammatory process which is characterized by several phases¹⁴. The result of the cotton pellet implantation model for anti inflammatory activity further support the anti inflammatory activity of the extract of bauhnia variegate. Several flavonoids isolated from medicinal plants have been discovered to possess significant anti inflammatory activity¹⁵.

In the cotton pellet granuloma model inflammation and granulomas develops during the period of several days. It is model in an indication for the proliferate phases of inflammation. Inflammation involves proliferation of macrophages, neutrophils, and fibroblast, which are basic sources of granuloma formation. Hence the decrease in weight of granuloma indicates that the proliferate phase was effectively suppressed by the Bauhnia variegate extracts as shown in Table no. 2.

CONCLUSION:

In conclusion, this study has shown that the both the extracts of bauhnia variegate having significant anti-inflammatory effect at the dose of 400mg/kg in both acute and sub acute anti-inflammatory model. Our data obtained from the present study suggest that the bauhnia variegate useful for some inflammatory condition.

REFERENCES:

1. Gokhale A and Band Saraf MN. Recent developments in asthma therapy. Indian Drugs. 2002; 39(3): 121-132.

2. Kirtikar KR, and Basu BD. Indian Medicinal Plants. Allahabad. 1933: pp 314-315.

3. Rangari A. Text book pharmacognosy and phytochemistry. Carrier Publication, Allahabad. 2004: 1^st ed. pp 50-53.

4. Rajkapoor B, Jayakar B and Murugesh N. Antitumor activity of Bauhinia variegate Linn on Daltons Ascitic Lymphoma. Journal of Ethanopharmacology. 2003; 89: 107 -109.

5. Rajkapoor B. Jayakar B, Murugesh R and Anandan. Anti-ulcer effect of Bauhinia variegate Linn. in rats. Journal of Natural Remedies. 2003; 3(2): 215-217.

6. Winter CA and Porter CC. Effect of alteration in side chain upon anti-inflammatory and liver glycogen activities in hydrocortisone ester. J. Am. Pharmacol. Soc. 1957; 46: 515-519.

7. D’Arcy PF, Haward EM, Muggleton RW and Townsend SB. The anti inflammatory action of griseofulvin in experimental animals. J. Pharm. Pharmacol. 1960; 12: 659-665.

8. Larsen GL and Henson PM. Mediators Of inflammation. Ann rev immunol. 1983; 1: 335-9.

9. Brooks PM and Day RO. Non steroidal anti-inflammatory drugs: differences and similarities. N Engl J Med. 1991; 15: 452-5.

10. Todd PA and Sorkin EM. Diclofenac sodium, a re-appraisal of its pharmacodynamic and pharmacokinetic properties, and therapeutic efficiency. Drugs. 1988; 35: 244-85.

11. Winter CA, Risely EA and Nuss CW. Carrageenan Induced Paw Oedema in hind paw of the rats as an assay for anti inflammatory drugs. Proc soc exp biol med. 1962; 111: 544-7.

12. Hollander C, Nystrom M, Janciauskiene S and Westin U. Human mast cell decrease SLPI levels in type II like alveolar cell model in vitro. Can. Cell Int. 2003; 3: 14-22.

13. Omisore NOA, Adewunmi CO, Iwalewa EO, Ngadjui BT, Watchueng J, Abegaz BM and Ojewole JAO. Antinociceptive and anti-inflammatory effects of Dorstenia barteri (Moraceae) leaf and twig extracts in mice. J. Ethnopharmacol. 2004; 95: 7-12.

14. Swingle KF, Sanchez S, Rodriguez L, Pol O and Puig MM. Antinociceptive/antioedema effects of liposomal morphine during acute inflammation of the rat paw. Pharmacology. 2000; 60: 121-127.

15. Duke JA. Hand Book of Biologically Active Phytochemicals and Their Activities. CRC Press, Boca, Raton. 1992.

Received on 28.02.2009

Accepted on 10.09.2009

© A & V Publication all right reserved

Research Journal of Pharmaceutical Dosage Forms and Technology. 1(2): Sept.-Oct. 2009, 97-99