Research Article

Analgesic and anti-nociceptive activity of hydroethanolic extract of Drymaria cordata Willd

Chandana Choudhury Barua, Jayanti Datta Roy, Bhaben Buragohain, Acheenta Gohain Barua1, Prabodh Borah2, Mangala Lahkar3

ABSTRACT

Objectives: To study analgesic and anti-nociceptive activity of hydroethanolic extract of Drymaria cordata Willd. Materials and Methods: Wistar rats and Swiss albino mice were used for studying analgesic Departments of Pharmacology and anti-nociceptive activity of Drymaria cordata hydroethanolic extract (DCHE) at doses and Toxicology, 1Veterinary Public Health, 2Microbiology, College 50, 100 and 200 mg/kg p.. Various models viz. acetic acid induced writhing model of Veterinary Science, Assam (female mice), Eddy’s hot plate (mice) and tail flick model (rat) for analgesic study and Agricultural University, Khanapara, formalin-induced paw licking model (mice) were used for anti-nociceptive study. 3Department of Pharmacology, Results: In acetic acid induced writhing model, effect of DCHE was better than the standard Guwahati Medical College, drug- indomethacin 10 mg/kg (p.o.). In the hot plate model, the maximum effect was Bhangagarh, Guwahati, observed at 60 min at dose of 200 mg/kg p.o., which was higher than the standard drug Assam, India morphine sulfate (1.5 mg/kg .p.), whereas in the tail flick model, effect was comparable with morphine sulfate. In formalin-induced paw licking model, administration of DCHE Received: 30-09-2009 completely abolished the early phase at 100 and 200 mg/kg p.o. and in the late phase, Revised: 21-11-2010 Accepted: 31-12-2010 the effect of DCHE (200 mg/kg p.o.) was higher than indomethacin (10 mg/kg p.o.). Conclusion: DCHE was effective in both non-narcotic and narcotic models of nociception, Correspondence to: suggesting its possible action via peripheral and central mechanism. It also abolished the Prof. Chandana Choudhury Barua, early phase in formalin-induced paw licking model, suggesting complete inactivation of -mail: [email protected] C-fiber at higher dose. The activity can attributed to the phyto-constituents viz tannins, diterpenes, triterpenes and steroids present in the DCHE extract. In conclusion, DCHE can be developed as a potent analgesic and anti-nociceptive agent in future.

KEY WORDS: Analgesic, acetic acid, Drymaria cordata, Eddy’s hot plate, hydroethanol extract, tail flick

Introduction novel drugs.[1] Many medicines of plant origin with analgesic and anti-nociceptive activity had been used since long time The use of plant products is increasing in many segments without any adverse effect. North East India is considered as of the population. According to an estimate, 80% of the world’s one of the “hotspots” for biodiversity in India, since out of the population relies upon plants for their medication. Most of 1500 species of medicinal plants available in India, almost 350 the synthetic drugs used at present for analgesic and anti- species belong to Assam and many of these traditionally used nociceptive effect cause many side and toxic effects. Plants plants have not yet been studied scientifically which can be still represent a large untapped source of structurally novel developed as a potential drug after scientific validation. compounds that might serve as lead for the development of Drymaria cordata Willd (Caryophyllaceae) locally known as “Laijabori” is one of the traditionally used plants of North East Access this article online India for its analgesic, wound healing, anti-inflammatory activity Quick Response Code: Website: www.ijp-online.com and is also used as an antidote, appetizer, depurative, emollient, DOI: 10.4103/0253-7613.77337 febrifuge, laxative and stimulant in both human and animals. It is a sprawling herb with procumbent and more or less ascending branched stems, often rooting at the lower nodes, quadrangular, glabrous or papillose especially in the upper internodes, which are slender, generally 2-6 cm long. The pounded leaf is applied to

121 Indian Journal of Pharmacology | February 2011 | Vol 43 | Issue 1 | 6-12 Indian Journal of Pharmacology | April 2011 | Vol 43 | Issue 2 121 Barua, et al.: Analgesic activity of D. cordata snake bites.[2] Studies on Drymaria cordata exhibited significant Initially a particular dose, on the basis of the above study, was anti-tussive,[3] anti-bacterial[4] and anti-inflammatory[5] property. administered to single female rat and the rat was observed Hydroethanolic extracts of Drymaria cordata have been shown for 48 h with close surveillance up to initial 4 h (same as in to possess significant anxiolytic activity.[6] have also studied case of first rat) after 48 h (of the second administration), its anti-inflammatory, adaptogenic, anti-convulsant and anti- same dose was administered in two more female rats and the depressant activity (unpublished reports). observation was done same as for the previous rat. The rats In our previous study, we have reported analgesic activity were observed for 14 days and no adverse observation was of the plant using methanol extract.[7] The present study aims found morphologically. The weight of the animals was recorded to select the active extract responsible for analgesic activity on 7th and 14th day. using hydroethanolic extract and thereafter to isolate the Animals were divided into five groups of six animals each. phytoconstituent from the active fraction. The first group (Group I) served as a control group. The second (Group II) was used as the reference standard. Three groups Materials and Methods (Group III, IV and V) received DCHE at three different doses (50, Collection of Plant material 100 and 200 mg/kg p.o.). The doses were selected on the basis The leaves of Drymaria cordata Willd were collected during of our preliminary screening. The research was conducted in the month of July to September, 2008 from the medicinal accordance with the ethical rules on animal experimentation, garden of the Department of Pharmacology and Toxicology, approved by ethical committee, Guwahati Medical College, C.V.Sc. Khanapara, Guwahati and were identified by Botanical Guwahati (Registration numbers- 351). Survey of India, Shillong, Meghalaya. A voucher specimen (No Drugs AAU/CVSC/PHT/ 07-08/ 03) was deposited in the Herbarium Indomethacin (Jagsonpal Pvt Ltd) and morphine sulfate of Botanical Survey of India, Shillong, Meghalaya. (Drugs India Pvt. Ltd. Dispur, Guwahati-5) were used for the Preparation of extract of Drymaria cordata study. Distilled water was used as vehicle. All the chemicals Fresh leaves of Drymaria cordata were cleaned and dried and solvents were of analytical grade. under shade in clean dust free environment, grinded and Analgesic and anti-nociceptive activity stored in an air-tight container. They were (250 g) soaked in Acetic acid induced writhing model in mice 1000 ml of hydroethanol (50:50) for 72 h in separate beakers. It was stirred every 18 h using a sterile glass rod. The solvent In this model, the animals were pretreated with drugs 30 was filtered every third day using muslin cloth and Whatman’s min before induction of writhing. The Group I animals received filter paper No 1. The filtrate obtained was concentrated in vehicle and Group II animals received the reference standard rotary evaporator (Equitron, Roteva) at 50-60°C under reduced drug indomethacin (10 mg/kg p.o.). Analgesic activity of DCHE pressure leaving a dark brown residue. The Drymaria cordata at the doses 50, 100 and 200 mg/kg p.o. (Group –III, IV and hydroethanolic extract (DCHE), thus obtained, was transferred V) was assessed by counting the number of writhes induced to a Petri dish and kept over water bath (50˚C­) until the solvent by 0.7% acetic acid. The number of writhes per animal were [10] gets completely evaporated. It was stored at 4˚C for future use. counted for 20 min. Percent reduction in writhing syndrome Recovery was 18.06% (w/w). was calculated and compared with the standard drug. Percent reduction indicates the percentage protection against abdominal Phytochemical screening constriction which was taken as an index of analgesia. Freshly prepared DCHE was subjected to standard It was calculated as: phytochemical screening tests for various constituents by {(W –W ) × 100}/W standard methods.[8] c t ) c where, Wc = number of writhing of the control Animals and treatment regimes group Swiss albino mice (20-30 g) and Wistar rats (120-130 g) W = number of writhing of the treated group were used for the study. The animals had free access to food and t Eddy’s hot plate model in mice water. They were fasted overnight before the experiment. They In this model, the animals received vehicle (Group I) were housed in animal room, with alternating light-dark cycle and standard drug indomethacin (10 mg/kg p.o.) (Group II). of 12 h each. The animals were acclimatized to the laboratory conditions for at least five days prior to the experiments. All Analgesic activity of DCHE at doses 50, 100 and 200 mg/kg experiments were conducted between 0900 h and 1800 h. p.o. (Group III, IV and V) was assessed by observing the reaction Acute toxicity study was carried out according to the time in the treated groups. The reaction time was noted at 0, 30, [11] Organization of Economic Corporation Development (OECD) 60, 90 and 120 min following the administration of drugs. A guidelines No. 425. DCHE was administered orally in doses cut-off time of 20 sec was considered. of 100, 200, 400, 800, 1000 and 2000 mg/kg to the group of Tail flick model in rat mice (n=3) and the percentage mortality was recorded for a In this model, the animals received vehicle (Group I) period of 24 h. During the first 1 h after the drug administration, and standard drug indomethacin (10 mg/kg p.o.) (Group II). the mice were observed for any gross behavioral change Analgesic activity of DCHE at doses 50, 100 and 200 mg/kg p.o. and the parameters observed were hyperactivity, grooming, (Group III, IV and V) was assessed by observing the reaction convulsions, sedation and loss of righting reflex, respiration, time in the treated groups.[12] Following the administration salivation, urination and defecation[9] of drugs, the reaction time was noted at 0, 30, 60, 90 and Based on the above toxicity study, direct limit test was done. 120 min.

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Formalin- induced paw licking model in mice mortality was observed and the extract was found to be safe In this model, the animals received vehicle (Group-I) and at the given dose. standard drug indomethacin (10 mg/kg p.o.) (Group II). Anti- Analgesic and anti-nociceptive activity nociceptive activity of DCHE at doses 50, 100 and 200 mg/kg, Acetic acid induced writhing model in mice p.o. (Group III, IV and V) was assessed by observing the reaction The DCHE produced significant (P<0.01) reduction in the time in the treated groups. Fifteen minutes after treatment, 20 μl number of writhing in mice in dose dependent manner. At 100 of 1% formalin was injected subcutaneously under dorsal surface and 200 mg/kg oral dose, the percent reduction of writhing was of the hind paw and the time spent for licking the paw injected 88.89% and 92.14% respectively, as compared to the control with formalin was counted for 30 min post formalin injection and group, whereas the standard drug indomethacin (10 mg/kg p.o.) considered as indicative of the pain stimuli.[13] The formalin test showed a reduction of 86.45% [Table 1]. has two distinctive phases possibly reflecting different types of pain. The first phase peaked at 5 min and the second phase at Eddy’s hot plate model in mice 20–30 min after formalin injection. This represented neurogenic In this model, the reaction time in DCHE treated group and inflammatory responses, respectively. increased significantly (P<0.01) in comparison to the control group. The maximum effect was observed at the highest dose Statistical analysis viz. 200 mg/kg p.o. at 60 min which showed a reaction time of The statistical analysis of data was done using one-way 18.2 sec, whereas the standard drug morphine (1.5 mg/kg i.p.) analysis of variance by using the SPSS software (version 11.5). showed a reaction time of 12.3 sec. The extract also showed P< 0.01 was considered as highly significant. dose and time dependent activity [Table 2]. Results Tail flick model in rat In the tail flick test, increase in the reaction time was Phytochemical screening significant (P<0.01) as compared to the control group. The phytochemical screening of DCHE showed the presence Maximum effect was 8.94 sec at 60 min post treatment of diterpenes, tannins by ferric chloride and gelatin test; with 200mg/kg p.o. of DCHE, whereas in the vehicle treated triterpenes by Salkowski’s and Liberman Buchardt’s test and control group the reaction time was 5.88 sec at 60 min, clearly steroids by Salkowski’s and Liberman Buchardt’s test. indicating the analgesic property of the plant extract [Table 3]. Acute toxicity studies Formalin- induced paw licking model in mice Oral administration of DCHE up to 2 g/kg did not produce Administration of DCHE at 50 mg/kg p.o. caused reduction any toxic effects in the normal behavior of the mice. No in duration of paw-licking (20.4 sec) as compared to the control group (58.4 sec). Higher doses of DCHE at 100 and 200 mg/kg Table 1 p.o. showed complete abolishment of the early phase indicated Analgesic activity of DCHE in female albino mice in acetic acid by absence of paw licking after the formalin injection. However, induced writhing method the standard drug- indomethacin (10 mg/kg p.o.) exhibited a reduction of paw licking time of 22.9 sec only in the early Groups Drugs Dose mg/kg No. of Percent phase. In late phase, the administration of DCHE decreased (p.o.) writhing reduction (%) the duration of paw licking dose dependently from 51.81 sec at I Control - 77 ± 1.67 0.00 50 mg/kg p.o. to 25.73 sec at 200 mg/kg p.o. in the late phase. II Indomethacin 10 8.33 ± 0.63* 86.45 On the other hand, the standard drug- indomethacin (10 mg/ III DCHE 50 11.8 ± 0.79* 80.76 kg p.o.) exhibited a reduction of paw licking time of 35.53 sec IV 100 6.83 ± 0.83* 88.89 in the late phase. The effect of DCHE at 200 mg/kg p.o. was V 200 4.83 ± 0.47* 92.14 better than the standard drug [Table 4]. Values are expressed in terms of mean ± SEM, n = 6 in each group, *P <0.01 statistically highly significant as compared with control group. DCHE = Drymaria Discussion cordata hydroethanolic extract Any injury or tissue damage is associated with pain and

Table 2

Analgesic activity of DCHE in Eddy’s hot plate model in mice

Groups Drugs Dose Reaction time in seconds mg/kg 0 min 30 min 60 min 90 min 120 min

I Control - 4.99 ± 0.25 5.03 ± 0.09 5.70 ± 0.15 5.31 ± 0.35 4.69 ± 0.20 II Morphine (i.p.) 1.5 8.55 ± 0.25* 10.1 ± 0.09* 12.3 ± 0.15* 13.8 ± 0.35* 14.9 ± 0.20* III DCHE 50 8.80 ± 0.89* 11.7 ± 0.65* 13.7 ± 0.63* 14.4 ± 0.85* 16.5 ± 0.88* IV (p.o.) 100 12.0 ± 0.60* 14.4 ± 0.58* 15.9 ± 0.33* 17.6 ± 0.74* 15.4 ± 0.45* V 200 13.6 ± 0.52* 15.6 ± 0.81* 18.2 ± 0.64* 15.2 ± 0.48* 14.6 ± 0.53* Values are expressed in terms of mean ± SEM, n = 6 in each group, *P <0.01 statistically highly significant as compared with control group. DCHE= Drymaria cordata hydroethanolic extract

Indian Journal of Pharmacology | April 2011 | Vol 43 | Issue 2 123 Barua, et al.: Analgesic activity of D. cordata

Table 3

Analgesic activity of DCHE in Wistar rat by tail flick method

Groups Drugs Dose Reaction time in seconds mg/kg 0 min 30 min 60 min 90 min 120 min

I Control - 5.22 ± 0.16 5.56 ± 0.22 5.88 ± 0.17 5.71 ± 0.22 5.81 ± 0.20 II Morphine (i.p.) 1.5 3.31 ± 0.22* 6.10 ± 0.23* 16.2 ± 0.71* 17.9 ± 0.48* 19.9 ± 0.34* III DCHE 50 6.17 ± 0.03* 6.32 ± 0.02* 6.46 ± 0.01* 6.56 ± 0.01* 6.78 ± 0.01* IV (p.o.) 100 7.07 ± 0.02* 7.26 ± 0.01* 7.37 ± 0.01* 7.84 ± 0.01* 7.55 ± 0.01* V 200 8.18 ± 0.01* 8.34 ± 0.01* 8.94 ± 0.01* 8.55 ± 0.01* 8.44 ± 0.01* Values are expressed in terms of mean ± SEM, n = 6 in each group, *P <0.01 statistically highly significant as compared with control group. DCHE = Drymaria cordata hydroethanolic extract.

Table 4 The formalin-induced paw licking model comprises of early phase and late phase. The early phase (immediately after Anti-nociceptive activity of DCHE in albino mice in formalin induced paw-licking test injection) seems to be caused by C-fiber activation due to the peripheral stimulus. The late phase (starting approximately Groups Drugs Dose Early phase Late phase 20 min after formalin injection) appears to depend on the (mg/ (duration of (duration of combination of an inflammatory reaction, activation of NMDA kg) paw licking paw licking [18] p.o. in seconds) in seconds) and non-NMDA receptors and NO cascade in the peripheral I Control - 58.4 ± 1.02 142.48 ± 1.14 tissue and the functional changes in the dorsal horn of the [19] II Indomethacin 10 22.9 ± 1.44* 35.53 ± 2.62* spinal cord. In our study, DCHE completely abolishes the early III DCHE 50 20.4 ± 1.01* 51.81 ± 1.27* phase at the dose 100 and 200 mg/kg p.o. suggesting complete IV 100 0 35.76 ± 1.57* inactivation of C- fiber in the early phase. DCHE decreased the V 200 0 25.73 ± 1.11* reaction time in dose dependent manner in the late phase also Values are expressed in terms of mean ± SEM, n = 6 in each group, *P <0.01 which might suggest that DCHE causes partial inactivation of statistically highly significant as compared with control group. DCHE= Drymaria NMDA and non-NMDA receptors. cordata hydroethanolic extract. Our previous studies have shown anxiolytic[6] activity in the hydroethanol extract and analgesic activity in the methanol [7] inflammation. Analgesics can act on peripheral or central extract. Analgesic activity of hydroethanol extract of Drymaria nervous system. Peripherally acting analgesics act by blocking cordata in the present study showed better effect in both the generation of impulses at chemoreceptors site of pain, while narcotic and non narcotic models, whereas in the methanol centrally acting analgesics not only raise the threshold for pain, extract, the activity was more prominent in non narcotic but also alter the physiological response to pain and suppress models (acetic acid induced writhing) only. The methanol extract the patient’s anxiety and apprehension. Pain and inflammation contains flavonoids, steroids, triterpenes, diterpenes, alkaloids are an essential prelude to the repair process.[14] and tannins whereas the hydroethanol extract contains steroids, Acetic acid induced writhing response in mice is not only triterpenes, diterpenes and tannins. Hence, significant analgesic simple and reliable but also affords rapid evaluation of peripheral activity in hydroethanolic extract might be attributed to the type of analgesic action. Acetic acid causes inflammatory pain presence of these bioactive principles. by inducing capillary permeability and liberating endogenous In conclusion, it can be interpreted that DCHE possesses substances that excite pain nerve ending. Acetic acid is also promising analgesic and anti-nociceptive properties, which are [15] probably peripherally mediated via inhibition of prostaglandin known to increase PGE1 and PGE2 peripherally. NSAIDs can inhibit COX in peripheral tissues and therefore interfere with the synthesis as well as central inhibitory mechanism and may be mechanism of transduction of primary afferent nociceptors.[16] of potential benefit for management of pain. Further studies on The mechanism of analgesic activity of DCHE could be probably isolation and fractionation of the active components from the due to the blockade of the effect or the release of endogenous leaf of Drymaria cordata and study on its mechanism of action substances that excite pain nerve endings similar to that of to ascertain their analgesic and anti-nociceptive properties will indomethacin and NSAIDs. Thus, the reduction in the number throw light on mode of action. of writhing indicates that DCHE might exert anti-nociceptive Acknowledgements activity by inhibition of prostaglandin synthesis or action of prostaglandin. The authors are grateful to National Medicinal Plant Board, Govt. of India, New Delhi for providing financial assistance to carry out this In the hot plate model, nociceptive reaction toward thermal work. Physical facility provided by the Director of Research (Vety), stimuli in mice is a well-validated model for detection of opiate C.V.Sc is also gratefully acknowledged. analgesics as well as several types of analgesics drugs from spinal origin.[17] The tail flick model is also used to evaluate References analgesic agents acting through central nervous system. DCHE 1. Ahmad F, Khan RA, Rasheed S. Study of analgesic and anti-inflammatory activity was found to be effective in both the models. from plant extracts of Lactuca scarliola and Artemsia absinthium. J Int Acad Sci

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