Phytochemical screening and evaluation of analgesic and antiinflammatory activities of Phaseolus vulgaris linn . , seeds in rodents

M. R. Pradeepkumar , Shrinivas D. Joshi, V. H. Kulkarni, Chetan Savant Centre for Research and Development, Prist University, Thanjavur, Tamil Nadu 613 403, India. Novel Drug Design and Discovery Laboratory, Department of Pharmaceutical Chemistry, S.E.T’s College of Pharmacy, S. R. Nagar, Dharwad, Karnataka 580 002, India. Department of Pharmacology, S.E.T’s College of Pharmacy, S. R. Nagar, Dharwad, Karnataka 580 002, India.


INTRODUCTION
Inflammation is a body's defense mechanism in response to various infectious or metabolic stimuli.It is a body defence reaction in order to terminate or limit the spread of injurious agent as well as to remove consequent necrosed cells and tissues.Suppression of inflammation still continues to be a challenge to the scientists despite the availability of number of NSAID's.This is because NSAID's not only exhibit antiinflammatory and analgesic activity but also cause gastrointestinal complications ranging from dyspepsia to upper GI tract bleeding and perforation.Efforts to improve adverse effect profile of current NSAID's have been focused on developing pro-drugs or modification of marketed formulations (Schenone et al., 2006).
A recent approach is development of selective cyclooxygenase inhibitors (COX-2).Currently available NSAID's inhibit both COX-1 and COX-2.Inhibition of COX-1 reduces production of cytoprotective prostaglandins PGE2 and PGI2 and hence causes ulceration, while inhibition of COX-2 inhibits inflammation.Complete inhibition of COX-1 is therefore not preferred and drugs that selectively inhibit COX-2 are better antiinflammatory agents.Another field of greater concern is synthesis of more effective analgesic agents.Pain is in fact a very serious problem associated in 90% of diseases.NSAID'S, which are used to treat pain, have several side effects.There is a great need to develop some natural agents (phytoconstituent), which are capable of treating both acute and chronic pain.Plants have payed a remarkable role in health care since the ancient times.Traditional plant based medicines still exert a great deal of importance to people living in developing countries and also lead to discovery of new drug candidates (Orhan et al., 2007).Phaseolus vulgaris (Linn) seeds belong to the family of Fabaceae (Romero-Arenas et al., 2013).
Phaseolus vulgaris (Linn) seeds have an important place in the folk medicine of the world and in the traditions of many cultures because of their pharmacotherapeutic effects (Mishra al., 2010).However, Phaseolus vulgaris (Linn) has not been investigated for analgesic and antiinflammatory activities.Hence, this study was carried out to evaluate the potent bioactive constituents for analgesic and antiinflammatory activities in Phaseolus vulgaris (Linn) seeds.The Seeds of Phaseolus vulgaris (Linn) were shade dried and finely powdered to particle size (#) 40.About 300 g of dried powder was subjected to continuous hot soxhlet exhaustive extraction with petroleum ether, chloroform and ethanol (95%).Aqueous extract was also obtained by cold maceration of the drug (300 g) with 2% chloroform water.After the extraction, the extracts were filtered and concentrated under reduced pressure using a rota evaporator.The yield of petroleum ether, chloroform, ethanol and aqueous extract was found to be 8.36 g (3.12 % w/w), 6.285 g (1.95 % w/w), 24 g (7 % w/w) and 11 g (4.01 % w/w), respectively.All the extracts were kept in a dessicator for drying.

Pharmacological activity
The pharmacological activities of synthesized compounds were investigated in albino rats and mice.Rats were used for acute anti-inflammatory and chronic anti-inflammatory activities.Mice were used for analgesic activity.All animal experiments were approved by institutional animal ethical committee.All the animals were stabilized to laboratory conditions before starting the experiments.

Acute toxicity
The acute toxicity test was carried out according to the Organization for Economic Co operation and Development (OECD) guidelines (OECD/ OCDC, 2000) to establish the effective dose of all the synthesized and extracted compounds.

Anti-inflammatory activity
Anti-inflammatory activity was evaluated using the well known Carageenan induced rat paw oedema model (Winter et al., 1962) using groups of six animals each.A freshly prepared aqueous suspension of carrageenan (1% w/v, 0.1 ml) was injected in the sub planter region of right hind paw of each rat.One group was kept as control and the animals of the other group were pretreated with the test extract at a dose of 200 mg/ kg body weight of rat, 1 h before the carageenan treatment.The volume was measured before and after carageenan treatment at the 30 min.interval with the help of digital plethysmometer.Standard drug used was diclofenac at a dose of 200 mg/kg body weight of rat.

Analgesic activity
Twenty four hours prior to actual testing a large number of mice (20-25 gm) received intraperitoneally (i.p) 10 ml/kg of 0.6 % glacial acetic acid.Animals were observed for writhing movements.Only those showing one or other type of writhing movements (positive responders) were chosen for the test on the next day.On the test day the responders received compounds half an hour prior to glacial acetic acid challenge (Koster et al., 1959).Extracts were given at a dose of 200 mg/kg orally.Standard drug used was aspirin at a dose of 30 mg/kg body weight of mice.

Phytochemical screening
Phytochemical screening revealed the presence of steroids, flavonoids (Milan, 2011) in the petroleum ether extract of seeds of Phaseolus vulgaris (Linn).The results of the physicochemical parameters and preliminary phytochemical screening of Phaseolus vulgaris Linn seeds is given in Table 1 and Table 2 respectively.Hence, the presence of flavonoids and steroids in petroleum extract could be attributed for observed significant analgesic (Jain et al., 2011;Parveen et al., 2007) and antiinflammatory activities (Sakat et al., 2010;Nagore et al., 2010).However, research work is under progress to confirm the exact mechanism of action and to elucidate the structure of bioactive principle for the claimed analgesic and antiinflammatory activities.

CONCLUSION
The present study provides an evidence for the analgesic and antiinflammatory activities of Phaseolus vulgaris (Linn) seeds.Aspirin used as standard drug for screening the analgesic activity which act by obtunding of peripheral pain receptors and prevention of PG-mediated sensitization of nerve endings (Tripathi, 2013) and the diclofenac which is used as standard drug for screening the antiinflammatory activity that acts by inhibiting the prostaglandin synthesis and specially it is COX-2 selective (Tripathi, 2013).As the activity results (petroleum ether extract) are closer to those of diclofenac and aspirin, the bioactive principles present in the extract may be having the mechanism of action similar to that of the tested standard drugs.However research is under progress to confirm the exact mechanism of action and to elucidate the structure of bioactive principles for the claimed analgesic and antiinflammatory activities.The present study may form the basis for the selection of plant species for further investigation in potent bioactive compounds for analgesic and antiinflammatory activities.
Seeds of Phaseolus vulgaris (Linn) were collected from the local areas of Dharwad in Karnataka and were authenticated by Dr. S. S. Hebbar, Department of Botany, Government Pre-university College Dharwad.A voucher specimen (No-SETCPD/Pharmacog/Herb/2011/12) has been deposited in the Herbarium of Department of Pharmacognosy, S.E.T.'s College of Pharmacy, Dharwad, Karnataka.

Table 2 :
Preliminary phytochemical analysis of various extracts of Phaseolus vulgaris Linn seeds.

Table 3 :
Antiinflammatory activity screening of Phaseolus vulgaris Linn seeds.

Table 4 :
Analgesic activity screening of Phaseolus vulgaris Linn seeds.