Prostaglandins are ubiquitous substances that indicate and modulate cell and tissue responses involved in inflammation. Most of the anti-inflammatory drugs now available are potential inhibitors of cyclooxygenase (COX) pathway of arachidonic acid metabolism which produces prostaglandins. Prostaglandins are hyperalgesic, potent vasodilators and also contribute to erythema, edema and pain. Hence for treating inflammatory diseases analgesic and anti-inflammatory agents are required

MATERIALS AND METHODS

The selected plant Rhynochsia beddomei is endemic to Southern parts India and mainly found in some parts of Cuddapah, Chittoor, Anantapur districts of Andhra Pradesh. Leaves of the plant are traditionally used in the treatment of all type of wounds as an antimicrobial agent. Leaves are also used as an abortifacient by Adivasi tribes in some districts of Andhra Pradesh. Plant parts are extensively used by traditional healers in India to treat a variety of bacterial diseases such as dysentery, diarrhoea and skin disorders. The tribals of Sikkim are using leaves of the plant in treatment of wounds, helminthes infections and as abortifacient. In Saurastra region of Gujarat leaves are utilized by aborigines for the treatment of asthma and piles.

Collection of plant materials:

For the present study the leaves of the Rhynchosia beddomei collected in the month of August from Arunachalam hills of Tirupati (Andhra Pradesh) as the active constituents are found to be maximum during these days. The fresh plant was identified, confirmed and authenticated by Dr. Madavachetty, Professor, Department Botany, Sri Venkateshwara University, Tirupati (A.P.). The powdered material was used for the extraction. Various extracts of the plant material were prepared by successive solvent extraction.

Animals

Healthy Wister rats and Swiss Albino mice of either sex, weighing between 150-200g and 20-30g respectively were procured from the animal house of Dayananda Sagar college of Pharmacy, Bangalore, India where the animals were kept in well ventilated spacious animal house with 12 ± 1 h day and night schedule. The animals were lodged in large and spacious hygienically maintained cages during the course of the experimental period. The room temperature was maintained at 25 ± 1°C. The animals were fed with standard rat feed (Brook Bond Lipton India Ltd., Bangalore.) and water ad libitum. The experiments were conducted as per the guidelines of CPCSEA, Chennai, India (approval no. 606/02/c/CPCSEA) and Institutional Ethical Committee clearance no. DSCP/ M. Pharm. Col/ 1AEC/62/11-12

Antioxidant activity

Evaluation of In vitro antioxidant activities of the leaf extracts of Rhynochsia beddomei.

Free-radical scavenging activity of aqueous ethanol extracts at different concentrations was tested in three in vitro models.

DPPH free radical scavenging activity

Free scavenging activity was measured by a decrease in absorbance at 516 nm of a methanol solution of colored DPPH brought about by the sample^[3][4][5].

Estimation of Superoxide Anion scavenging activity in the NADH/PMS/NBT System:

The superoxide anion scavenging activity of extracts were determined by the method described by^[6], slightly modified. Decreased absorbance of the reaction mixture indicated increased superoxide anion scavenging activity. All tests were performed in triplicate. The capability of scavenging the superoxide anion radicals was calculated using the following equation,

(A₀- A₁)

% scavenging effect =---------------------- X 100

A₀

Where, A₀ is the absorbance of the control (without test samples); A₁ is the absorbance of test samples.

Lipid per oxidation Assay

Lipid per oxidation was quantified by the method of determination of thiobarbituric acid-reactive substances (TBARS) described by^[7]using UV-Spectrophotometer(Shimadzu UV-2450). The experiment is performed in triplicate. The capability of scavenging the free radicals is calculated by using the following equation

[Control–Test]

% Scavenging effect = ----------------------- ×100

Test

Acute toxicity studies as per OECD Guideline 425.

Healthy Wister rats weighing between 150-180 g were used to carry out acute toxicity studies by the ‘staircase’ method. All successive extracts of Rhynchosia beddomei leaves were subjected for acute toxicity studies. The extracts were suspended in 0.5% tween 80 and administered orally by gavages in graduated doses to several groups of experimental animals under test, one dose being used per group. Subsequently, observations were made at 0,1,2,4 and 24 h. Observations include changes in skin and fur, mucous membranes and also muscle spasm, convulsion, motor activity and behavioral patterns and for any mortality.

Anti-inflammatory activity

Inflammation is an immune response to cellular/tissue injury or infection by pathogens. It is clinically characterized by features such as redness, warmth, swelling, and pain. The process itself is not considered a disease, but failure to contain it and successfully resolute it in a timely fashion results in exacerbation of tissue damage and modulation of cell signaling pathways^[8]. Inflammation involves a portfolio of cellular and molecular components collectively referred to as inflammation mediators.

Experimental model for testing anti-inflammatory activity:

Carrageenan-induced pedal inflammation

Acute inflammatory condition is produced in the animals by adopting the method of Carrageenan-induced pedal inflammation ^[9]. Paw volumes were measured using a Plethysmometer at different time intervals of 0, 30, 60, 120, 240 minutes. The reduction in the paw volume was calculated. The percentage inhibition of edema was calculated using the following formula:

% Inhibition of Edema = [1 – (V_t/V_c)] x 100

Where V_t is edema volume of the drug treated group and V_c is the edema volume of the control group.

Corton oil induced ear edema

The anti-inflammatory activities of leaf extracts of Rhynchosia beddomei were measured on mouse by using croton oil induced ear edema. The method of ^[10] was adopted for this assay. Mice were anesthetized with anesthetic ether and 20μl liter of an acetone solution containing 0.4 mg croton oil and appropriate amount of test samples(400 and 800 mg/kg) dissolved in acetone were applied to the inner surface of the each ear. The left ear remained untreated. Control animals received only the irritant while indomethacin 300mg/kg b.w. was served as the reference. The animals were sacrificed by cervical dislocation 5 hours later and a plug (7mm in diameter) was removed from the both the treated ear and the untreated ear. The difference in weight between two was taken as a measure of edematous response. The percentage response of protection was evaluated using following ratio

(Control mean – treated mean) x 100/ control mean.

RESULTS:

Herbal medicine is the oldest form of health care known to mankind. Herbs have been used by all civilizations through the history. It was an integral part of the development of modern civilization. Many drugs commonly used today are of herbal origin. Herbal medicines are major component in traditional medicine and a common element in ayurvedic, homeopathic, naturopathic, unani, sidda including allopathy. Substances derived from the plants remain the basis for large proportions of the commercial medicines used today for the treatment of heart diseases, high blood pressure, pain, asthma, antioxidant, analgesic, antiinflamatory and other problems. The major constituents of the plants extracts are alkaloids, glycosides, tannins, flavonoids, triterpenoids, resins, gums and carbohydrates^[11]. The characterization and estimation of these constituents can be analyzed by employing various phytochemical techniques.

Phytochemical studies

In the preliminary studies experiments were conducted to determine the pharmacognostic features of the leaves material of the selected plant Rhynchosia beddomei. In the present study the pharmacognostic features of all forms of extracts namely petroleum ether, chloroform, and ethanol and have been analyzed. The details of the quantity of the leaves powder taken for extraction and nature of the extracts are given in the table -1.

Qualitative phytochemical investigation

To screen the phytochemical constituents of the selected leaves, qualitative phytochemical investigations were carried out. All the extracts were subjected to appropriate preliminary qualitative chemical analysis where some of the important constituents were analysed such as, carbohydrates, triterpenoids, saponins, steroids; alkaloids, carbohydrates, triterpenoids, saponins, steroids, glycosides (Table -3 ). It is observed that the flavanoids, carbohydrates, alkaloids saponins and glycosides are the important active constituents found in ethanol and chloroform extracts. However, saponins are present are in all the extracts.

The ability of the antioxidant property of a plant extract can be determined by its total phenolic content. Hence, it is essential to determine the total phenolic contents of all the extracts of Rhynchosia beddomei leaves quantitatively by the method of Folin-ciocalteu. ^[11]. The total phenolic content is found to be very high in ethanol extract (23.24%) followed by chloroform (9.78%). However, the petroleum ether extracts showed very less quantities (4.21%).

From the above investigations it is clear that the pharmacologically active constituents are present mainly in the chloroform and ethanol extracts. The pet-ether extracts contain very less number of active constituents. Based on the presence of more active constituents in these extracts the chloroform and alcohol extracts have been selected for further studies.

Superoxide anion scavenging activity

The super oxide anion scavenging activity of the crude extracts of the leaves is expressed in terms of percentage inhibition. The ethanol extract is found to possess good scavenging activity on super oxide anion at all concentrations under test. Ethanol extract at concentrations range from 1-800µg/ml inhibited the production of super oxide anion radical by 27.43% to 88.75 Table-3. On the other hand the standard Butylated hydroxyl toluene showed significant scavenging activity in a dose dependent manner. The greatest scavenging activity was observed with BHT which effectively depressed the formation of super oxide anion. The maximum inhibition of super oxide anion was observed at 800µg/ml concentration and is 97.58% (Table-3,Fig-2). The super oxide scavenging activity is least with chloroform extract and is 15.65 and 48.54% respectively. The ethanol extract showed the maximum scavenging of super oxide free radicals of 88.75% at the dose of 800μg/ml.

Fig No: 2 Superoxide anion free radical scavenging activity Lipid per oxidation inhibition activities.

The two extracts of the leaves of Rhynchosia beddomei were subjected to lipid peroxidation inhibition activity against non-enzymatic in vitro lipid peroxidation in rat brain by the method of determination of thiobarbituric acid reactive substances (TBARS). The malondialdehyde formed as a result of lipid peroxidation induced by ferric chloride reacts with thiobarbituric acid releasing pink chromogen which indicates the extent of lipid peroxidation. Inhibition of pink chromogen formed indicates inhibition of lipid peroxidation. The ethanol extract under test showed the low absorbance values which indicates the highest level of antioxidant activity. It showed the ability of free radical inhibition activity in a dose dependent manner and maximum being 85.45% at 800µg/ml concentration. The absorbance is lesser than the chloroform extracts (Table-4 & Fig No-3). The inhibitory activity of chloroform and BHT are 54.02%, and 95.42% respectively. Interestingly, the activity exhibited by ethanol extract is almost very close to the standard BHT.

Table-4: Lipid peroxidation inhibition activity

Conc of extract/std mcg/mL	BHT	Chloroform extract	Ethanol extract
1	25.00±0.34	13.21±0.25	22.33±0.46
10	41.83±0.6	18.32±0.28	35.17±0.38
30	59.83±0.19	26.21±0.24	46.78±0.44
50	81.22±0.26	29.17±0.21	66.88±0.27
100	87.40±0.10	34.28±0.36	74.77±0.22
400	91.90±0.33	43.17±0.42	81.30±0.28
800	95.42±0.23	54.02±0.54	85.45±0.32

Fig No: 3 Lipid peroxidation inhibition activity Anti-inflammatory activity

Inflammation is caused by local release of prostaglandin, histamine, bradykinin. Carrageen induces the inflammation through the release of inflammatory mediators like prostaglandin, bradykinin and histamine^[12] where as 5-hydroxytrypamine additionally increases the permeability of blood vessels for various collagen. Phytochemical investigation of the present study indicates the presence of phenolic acids and triterpenoids in the extract of the leaves Rhynchosia beddomei, which are the potent antioxidant and anti-inflammatory compounds. Hence the anti-inflammatory parameter is taken for the present investigation. The ethanol extract showed anti-inflammatory activity on the inflammation induced by carrageen indicating that the ethanol extract inhibit all the endogenously released inflammatory mediators (such prostaglandin, bradykinin and histamine).

The animals are pre treated orally with the extracts of the leaves of Rhynchosia beddomei at the doses of 400mg/kg, 800mg/kg and a known standard - Nimesulide 50mg / kg b. w. respectively. Among two extracts ethanol extract showed maximum reduction in the paw volume induced by carrageen which is 1.00±0.025 when compared to the standard Nimesulide 0.97±0.04. Paw volume was maximum in the animals treated with, Chloroform extract showed a little reduction in the paw volume 1.08±0.25when compared to the control 1.16±0.032 (Table-5 and Fig No. 4-9).

Fig no: 4 Fig no: 5

Fig no: 6

Fig No: 7

Fig No: 8

Fig No: 9

Fig no: 4-9 Anti-inflammatory activity of the leaf extracts of Rhynchosia beddomei on Carrageenan (1%) Induced Rat Hind Paw Edema

Croton oil induced ear edema

The topical anti-inflammatory activities of Rhynchosia beddomei leaves were evaluated for chloroform and ethanol extract for both doses 400 and 800 mg/kg b.w. using inhibition of croton-oil-induced ear edema in mice. The percentage inhibition ear edema (Table-6) of extract under test is compared with the standard Indomethacin. The ethanol extract exhibited considerable anti-inflammatory activity at 800mg/kg b.w. which is 49.80% (Table-6, Fig:10S). In contrast, the chloroform fraction showed a mild effect of 32.48%, indomethacin appreciably inhibited the ear edema by 52.86%. The result demonstrates the topical anti-inflammatory properties of Rhynchosia beddomei leaves and justify the use of this plant extracts for the treatment of inflammatory diseases.

Table No: 6 Anti-inflammatory activity of the leaf extracts of

Rhynchosia beddomei on Croton oil induced ear edema

Treatment	Dose mg/kg	edema degree	Edema inhibition (%)
Control	_	7.85±0.86	_
Standard (Indomethacin)	300	3.7±0.76**	52.86
Chloroform	400	5.48±0.73*	30.19
Chloroform	800	5.30±0.46*	32.48
Ethanol	400	4.60±0.32*	41.40
Ethanol	800	3.94±0.65**	49.80

Note: N = 6 animals; ** P< 0.01 – when compared with control at respective time; *P < 0.05 – when compared with control at respective time.

Fig No: 10 Anti-inflammatory activity of the leaf extracts of Rhynchosia beddomei on Croton oil induced ear edema

DISCUSSION:

Free radical scavenging activity:

The free radical scavenging activity of various extracts is expressed in terms of percentage inhibition. The decrease in the percentage inhibition shows increased absorbance of DPPH, hence the compound is said to be having less anti-oxidant potency. The scavenging properties of anti-oxidants are often associated with their ability to form stable radicals. DPPH has long been recognized as a convenient reagent to qualify anti-oxidants in complex biological systems and has been widely used for this purpose ^[13][14] .

The free radical scavenging activity of ethanol extract is maximum when compared to chloroform extract which may be due to the presence of high concentration of flavonoids, triterpenoids and phenolic acids in the alcohol extract. Earlier reports also indicate the presence of phenolic acids, gallic acid and flavonoids such as quercetin, rutin, luteolin and hesperidin in the alcohol extract^[15][16] .

The compounds which contain hydroxyl groups may donate hydrogen to free radical to reduce the DPPH radical. So many hydroxyl containing compounds such as flavonoides, glycosides, etc have been isolated from Rhynchosia species^[17][18], so these compounds also be present in Rhynchosia beddomei as per the reports phytochemical investigations as shown in table no:3.

To confirm the free radical scavenging activity, the superoxide anion scavenging activity of the crude extracts of Rhynchosia beddomei were determined by NBT system. The ethanol extract showed the significant scavenging scavenging activity and is almost equal to that of standard drug BHT the reason may be due to the presence of flavonoids, triterpinoids and phenolic compounds in the ethanol extract i.e., quercetin, rutin, luteolin and gallic acids as reported by earlier workers.

Lipid peroxidation is another method of determining the free radical inhibition activity. Lipid peroxides, derived from poly unsaturated fatty acids, are unstable and decompose to form a complex series of compounds. These include reactive carbonyl compounds. The most abundant among them is malondialdehyde (MDA) and is one of the important secondary metabolite of lipid peroxidation. The concentration of MDA indicates the measure of lipid peroxidation. The ethanol extracts showed significant inhibition of lipid peroxidation which indicates the less formation of TBARS and hence shows less absorbance. But, the anti-oxidant properties of chloroform extract is less as they may contain less quantity of phenolic compounds and other active constituents as compared to ethanol extract. These reports confirm the occurrence of potent antioxidants in the extracts of Rhynchosia beddomei.

The results of antioxidant evaluation by employing three important protocols such as, DPPH free radicals scavenging, superoxide anion scavenging and lipid peroxide inhibition have clearly indicated the strong occurrence of active principles effectively involved in the removal of hazardous free radicals in the in vitro conditions.

Anti-inflammatory activity:

Inflammation is caused by the release of local hormones like prostaglandin, histamine and bradykinin. Carrageen induces inflammation through the release of prostaglandin, bradykinin and histamine^[28]where as hydroxytryptamine additionally increases the permeability of total blood vessels for various collagen. The most widely used primary test to screen new anti-inflammatory agents is to measure the ability of a compound to reduce local edema induced in the rat hind paw by injection of an irritant substance ^[10]Edema is due to the exudation of fluids and plasma proteins and the migration of leucocytes, most notably neutrophils and macrophages into the injured area ^[19]. Carrageenan induced edema has been commonly used as an experimental model for the determination of acute inflammation. The early phase (1-2 hr) of inflammation in the carrageenan model is mainly attributed to the release of histamine, serotonin and increased synthesis of Prostaglandins into the surrounding area of damaged tissue. The late phase is an accelerated phase of swelling, due to sustain release of prostaglandins and other mediators of inflammation like bradykinin, protease, leukotrines and infiltration of PMNS (Polymarphonuclear nutrophils) and macrophages ^[20][21]. It has been reported that the second phase of edema is sensitive to both steroidal and non-steroidal anti-inflammatory drugs, which is generally used to access the edematous effect of natural products^[22][23] . Prostaglandins play a major role in the development of the secondary phase of reaction, which is measured at around 3 hr time ^[37]. Edema and pain are the characteristic signs of an inflammatory response where the role of prostaglandins and histamine is well established^[24].

Cyclooxygenase (COX) is a key enzyme in the biosynthesis of prostaglandin from arachidonic acid and has two iso-types. COX-1 is responsible for producing the basal levels of prostaglandin needed for gastrointestinal tract homeostasis, where as COX-2 is an inducible enzyme which is involved in inflammatory events. Well known non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen and naproxen inhibit COX-2^[25]. The carrageen induced paw edema in rats is known to be sensitive to cyclooxygenase (COX-2) and has been used to evaluate the effect of anti-inflammatory agent against PGE-2 production and on COX-2 protein and mRNA expression.

The development of carrageenan induced inflammatory reactions in rats results from the activation of Kinin system, the accumulation of leukocytes and release of several inflammatory mediators such as prostaglandins and cytokines^[26] . Pro-inflammatory cytokines, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) or sequentially released in the pleural exudates induced by carrageenan in rats^[27] . These cytokines cause chemotaxis to attract granulocytes and monocytes at the site of inflammation. The migrated leukocytes in turn produce cytokines such as TNF-α and IL-1β and other pro-inflammatory mediators^[27] . IL-6 has been proposed as a crucial mediator for the development of carrageenan induced edema and further accumulation of leukocytes at the inflammatory sites.

In the present study ethanol extract of the leaves Rhynchosia beddomei exhibits maximum protection against carrageen induced paw edema and is almost nearing to the value of the volume when compared to control. The other extracts chloroform show moderate reduction in the paw volume. The ethanol extract 800mg/kg b.w. significantly reduces the inflammation due to the inhibition of the enzyme cyclooxygenase which is a basic substance for the synthesis of prostaglandin. From the result, it is observed that there is a reduction in the paw volume at the second stage i.e. after 60min of administration of carrageenan. ^[28]claimed that the inhibitory effects of inflammatory agents that act on the first stage of carrageenan induced hind paw inflammation are attributed to inhibition of the release of chemical mediators such as histamine and serotonin. They also claimed that the second stage of the hind paw edema may be related to arachidonic acid metabolites since it is inhibited by aspirin and other arachidonate cyclooxygenase inhibitors . Therefore, it is predicted that the ethanol and chloroform extracts may reduce the carrageenan induced paw volume largely in the later phase, which may be due to the inhibition of enzyme arachidonate cyclooxygenase. In this study, the Phytochemical investigation has already shown the presence of flavonoides, triterpenoids and phenolic acids in the ethanol and chloroform extracts which can be attributed to the maximum reduction in the paw volume . As these compounds are potent anti-inflammatory agents which may act in a similar way as that of the standard non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen and naproxen^[29] .

CONCLUSION:

In the present study preliminary phytochemical screening of the extract of leaves of Rhynchosia beddomei reveled the presence of flavonoids, alkaloids, triterpenoids, saponins and glycosides in alcohol extract and that of chloroform are alkaloids, triterpenoids, steroids, saponins and glycosides.

Ethanol extract at the concentration of 800mg/kg b.w. exhibits maximum protection against carrageen induced paw edema and is almost nearing to the value of the standard indomethacin. The other extracts chloroform show moderate reduction in the paw volume. Phytochemical investigation has already shown the presence of flavonoides, triterpenoids and phenolic acids in the ethanol and chloroform extracts which can be attributed to the maximum reduction in the paw volume. As these compounds are potent anti-inflammatory agents which may act in a similar way as that of the standard non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen and naproxen^[30].

The antioxidant and anti- inflammatory activity of the extracts may be due to the presence of these phytochemical constituents. The significant reduction in the paw volume of the plant extracts might be due to the presence of active principles which inhibits the production of secondary metabolites like prostaglandins.

In conclusion the active constituents present in ethanol and chloroform extract of the leaves of the plant Rhynchosia beddomei is capable of inhibiting free radicals with antioxidant and anti inflammatory activities.

Though the ethanol extract showed significant activity against the inflammation. It is necessary to determine the exact compound responsible for this activity. Further work is needed to determine the mechanism of antioxidant and anti- inflammatory activity of the drug.

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Received on 04.07.2012

Modified on 15.07.2012

Accepted on 27.07.2012

Research J. Pharmacology and Pharmacodynamics. 4(5): September –October, 2012, 319-327

Extracts	Quantity used for extraction		Nature of the extract	Yield %
Extracts	Powder(g)	Solvent(ml)
Petroleum ether (40-60^oC)	100	250	Slightly yellowish sticky mass	12.25%
Chloroform	100	250	Dark brown semisolid	18.75%
Ethanol	100	250	Dark brown sticky solid	25.75%

Conc of extract/std mcg/mL	Ascorbic acid	Chloroform extract	Ethanol extract
1	24.95±0.27	14.47±0.35	20.0±0.31
10	41.17±0.36	25.27±0.29	39.50±0.16
30	61.27±0.45	53.77.±0.42	53.35±0.45
50	80.35±0.78	77.53 ±0.47	84..80±0.23
100	90.25±0.38	80.12±0.22	87.04.±0.25
400	93.67±0.30	66.30±0.38	76.24.±0.29
800	96.68 +056	38.22±0.34	61.12+047

Conc of extract/std mcg/mL	BHT	Chloroform extract	Ethanol extract
1	24.98±0.12	15.65±0.25	27.43±0.28
10	40.08±0.18	17.55±0.29	38.85±0.37
30	61.32±0.23	25.68±0.32	55.43±0.49
50	83.05±0.15	29.25±0.73	69.87±0.11
100	92.18±0.09	39.07±0.33	83.00±0.45
400	94.57±0.15	43.20±0.25	86.53±0.31
800	97.58±0.32	48.54±0.32	88.75±0.42

Groups	Dose (mg/kg oral)	Difference in Paw Oedema Volume (Mean ± SEM)
Groups	Dose (mg/kg oral)	0 Min. 30 Min. 60 Min. 120 Min. 240 Min.					360 min
Control	0.5% Tween 80	1.15 ± 0.02	1.27 ± 0.025	1.35 ± 0.028	1.25 ± 0.029	1.20 ± 0.025	1.16±0.032
Standard Nimesulide	50	0.97 ± 0.04**	1.10 ± 0.06**	1.14 ± .04**	1.12 ± 0.03**	0.96 ± 0.05**	0.93±0.023**
Chloroform extract	400	1.13±0.03*	1.25±0.032*	1.33±0.032*	1.24±0.032*	1.20±0.03*	1.12±0.042*
Chloroform extract	800	1.09 ± 0.05*	1.24± 0.04*	1.32 ± 0.04	1.24 ± 0.03*	1.21± 0.04*	1.08±0.023*
Ethanol extract	400	1.07±0.014*	1.18±0.02*	1.24±0.02*	1.22±0.21*	1.20±0.025*	1..08±0.032*
Ethanol extract	800	1.04±0.032*	1.14±0.023*	1.20±0.24*	1.15±0.24*	1.14±0.012*	1.00±0.025**

Extraction and Screening of Antioxidant, Anti-inflammatory and Analgesic Activity of Rhynchosia beddomel Leaves