INTRODUCTION:

The natural products are used in the traditional medicine in India and in other countries. Many medicinal plants provide relief of symptoms comparable to that obtained from allopathic medicines. The majority of clinically important medicines in allopathy has various and severe side effects. Herbal medicine contains hundreds of chemicals and which are potentially active. Herbs and herbal medicine can cause toxic adverse effects has been reported¹.There fore agents of natural origin with little side effects are required to as substitute for chemical therapeutic agents. The alternative medicine for treatment of various diseases is getting more popular.

Leucas aspera (Wild.) Link belonging to the family Lamiaceae. A coarse erect diffusely branched annual herb with white flowers , the stems hispid or scab rid².The plants is widely distributed in most plains districts of India upto 3000, ft., as a weed in cultivated fields, waste lands and road sides^3,4. The plant is being used as the drug source in ayurveda system of medicine in kerala and in other traditional system of medicine in India. The plant is well known and the flowers of this plant are used in the onam festival in kerala. The whole plant is used medicinally⁴.The use full parts of the plant are wholeplant, leaves and flowers ^3,5,6. The photochemical studies reveal the presence of sterol, αsitosterol, βsitosterol, oleanolicacid, urosolicacid, essentialoil, alkaloids, diterpenes, leucasperones, leucasperols, asperphenamate, maslinicacid, isololioli, linifolioside, nectandrin, mesodihydroguaiareticacid, maceligna, acecentin, chrysoeriol, apigenin, myristargenolB, andmachilin C.

Leucas aspera is used in traditional system of medicine for various disorders^3-7,9,12-16. In previous studies, the diterpines from Leucas aspera inhibited prostaglandin induced contraction in guinea pig ileum ¹¹, the methanolic extract of Leucas aspera flowers showed antimicrobial activity ¹⁷, the ethanolic extracts of Leucas aspera roots showed antinociceptive, antioxidant, cytotoxic activities¹⁸,even though there is an experimental evidences for the analgesic activity with respect to roots of Leucas aspera, but to the best of our knowledge there is no scientific report on the antinociceptive activity of whole plant of Leucas aspera. There fore the present study an attempt has been made to investigate the antinociceptive activity of whole plant aqueous extract of Leucas aspera using acetic acid induced visceral nociceptive responses (classical chemical and thermal model of nociception) in mice.

The traditional uses and the prostaglandin inhibitory activity of this plant suggest that the plant might possess antinociceptive activity and this stimulated our interest to study the effect of whole plant aqueous extract of Leucas aspera in acetic acid induced visceral nociception responses and thermal induced nociception in experimental mice.

MATERIALS AND METHODS:

Plant material:

Leucas aspera (Wild.) Link was collected from cultivated fields, waste lands in local areas of Kancheepuram district, Tamil Nadu , India. It was identified by Prof. P. Jayaraman, Director, Plant Anatomy Research Center (PARC), Pharmacognosy Institute, West Tambaram, Chennai, Tamil Nadu, India a voucher specimen (NO. PARC/07/SRM/32) was deposited in the herbarium of the institute.

Preparation of aqueous extract:

The plant material was washed well with water, dried under shade and powdered to a fine grade by using laboratory scale mill. A batch of 500gm of the whole plant powder was suspended in 5 Liters of the distilled water and the mixture was boiled for 30 minutes. The decoction was centrifuged and filtered by using filter paper. The filtrate was evaporated in vacuum to give a residue. The yield of the product was approximately 21.6% w/w of the whole plant of Leucas aspera. The final product was stored in vacuum desiccators at room temperature until use.

Animals:

Male Swiss albino mice weighing 20-30gm were procured from the inbred stock of the Tamil Nadu Veterinary and Animal Sciences University (TANUVAS), Madhavaram, Chennai Tamil Nadu, India was used for the study. They were housed in well ventilated polypropylene cages with a 12h light / 12h dark cycle, received a standard pellet diet (Hindustan lever limited, Bangalore, India.) and water ad libitum. The mice were acclimated to laboratory condition for 7days. Animals were kept under fasting for over night, but allowed free access of water before commencement of experiment. The experiments were conducted according to the ethical norms approved by Ministry of social justices and empowerment, Govt. of India and the study was got approval from the Institutional Animal Ethical Committee (IAEC) of Committee For The Purpose of Control and Supervision of Experiments on Animal (CPCSEA).

Drugs and Chemicals

The following drugs and chemicals were used for the study: Acetyl Salicylic Acid (USV limited, Mumbai.), Pentazocine (Ranbaxy Fine Chemicals, Mumbai), Acetic acid (Rankem , Mumbai. ), Sodium chloride ( Rankem, Mumbai.).

Acute toxicity studies:

Acute toxicity studies were carried out using acute toxic class limit test dose guidelines 425 of Organization for Economic Co-operation and Development (OECD). Acute toxicity of the plant extract was carried out using groups of three Swiss albino mice by administering a dose of 2000 mg kg ^-1body weight,p.o., while control group received normal saline. The toxicological effects were assessed on the basis of mortality and behavioral changes during 48 h (19).

ANALGESIC ACTIVITY:

Writhing test:

This test in mice was carried out using the method of Koster with slight modifications (20). Animal were injected intra-peritoneally (i.p.) 0.6 % acetic acid solution of 10 ml kg ^-1 body weight and the test group were pretreated with Leucas aspera extract 200 and 400 mg kg ^-1body weight, p.o. and the positive control group with Acetyl Salicylic Acid (ASA) 200 mg kg ^-1body weight, i.m., 30 minutes prior to the peritoneal irritation. Control group received 10 ml kg ^-1body weight of 0.9 % of sodium chloride solution , i.p. The resulting writhes and stretching were observed and counted of for 60 minutes after acetic acid injection.

Hot plate test:

The method originally developed by Woolfe and Mac Donald²¹. In this test, animals were placed on the Eddy’s hot plate temperature maintained at 55 ± 1⁰C. The paws of mice are very sensitive to heat at temperature, which are not damaging the skin. The latency of nociceptive response such as licking or shaking of one paw or withdrawal of the paw or jumping was recorded as reaction time. Those showing a reaction time below 15 seconds were places again on the hot plate. The test was terminated at 30 seconds in the absence of response. The latency was recorded at 30, 60, 90 and 120 minutes after administration of the test compounds. Control group received 0.9 % Normal saline 10 ml kg ^-1body weight,p.o., Pentazocine was used as positive control 10 mg kg ^-1body weight ,i.m. Test group received Leucas aspera extract 200 and 400 mg kg ^-1body weight,p.o.The latency was recorded of the above timings.

STATISTICAL ANALYSIS:

The statistical analysis of all the result was carried out using one- way ANOVA followed by Dunnett’s multiple comparison using graph pad instat 3 software and all the results obtained in the study were compared with the control group. P value <0.05 were considered statistically significant.

RESULTS:

Acute toxicity:

The behavior of the treated mice appears to be normal. No toxic effect was seen up to 10 times the effective dose of the aqueous extract and there was no mortality during 48 h. Therefore an LD₅₀> 2000 mg kg ^-1body weight may be assumed.

Analgesic activity:

Writhing test:

As shown in Table 1, our results showed that the number of acetic acid induced writhes was significantly reduced by aqueous extract administered orally at 200 and 400 mg kg ^-1 body weight in dose related manner with 52.63 and 64.35 % of inhibition respectively. The results were statically significance (p<0.01) and similar to that of standard drug ASA with significant ( p<0.01) of inhibition at dose of 200 mg kg ^-1body weight.

Hot plate test:

In hot plate test Table 2, the Leucas aspera aqueous extract at doses of 200 and 400 mg kg ^-1 body weight showed a significant increase in the latency time in a dose dependent manner. The result was found to be statically significant ( p<0.01) in comparison to the control and similar to that of Pentazocine treated at a dose of 10 mg kg ^-1 body weight.

Table 1 : Effect of aqueous extract of Leucas aspera on acetic acid induced writhing response in mice.

Treatments

Dose mg kg ^-1Body weight

Number of Writhes^*during 60 min

Percentage of writhes inhibition

Control

110.83 ± 1.990

Aqueous extract

ASA

200

400

200

52.50 ± 0.9916*

39.50 ± 0.6708*

30.33 ± 1.990*

52.63

64.35

72.63

^*Values are expressed in Mean ± S.E.M. (n = 6); Difference between groups were statistically analyzed by one-way ANOVA;*p<0.01, Dunnett’s test as compared to control which received normal saline.

Table 2: Effect of aqueous extract of Leucas aspera on the latency of mice exposed to hot plate.

Treatments

Dose mg kg ^-1Body weight

^bReaction time(s)

Basal

30 min

60 min

90 min

120 min

Control

Aqueous extract

Pentazocine

200

400

3.20 ± 0.032

3.48 ± 0.094*

3.72 ± 0.063**

3.90 ± 0.056**

3.30 ± 0.2192

5.22 ± 0.1929**

6.35 ± 0.6184**

7.60 ± 0.1291**

3.36 ± 0.0767

4.82 ± 0.0687**

6.09 ± 0.0945**

6.58 ± 0.0850**

3.41 ± 0.0570

4.14 ± 0.0733**

5.42 ± 0.0675**

5.98 ± 0.0467**

3.28 ± 0.0746

3.62 ± 0.0982*

3.53 ± 0.0774^NS

3.88 ± 0.0377**

^bValues are expressed in Mean ± S.E.M.(n = 6); Difference between groups were statistically analyzed by one-way ANOVA; *p<0.05, **p<0.01, Dunnett’s test as compared to control which received normal saline.

DISCUSSION:

The results showed that the Leucas aspera aqueous extract administered orally to mice produced significant antinociceptive action when assessed in acetic acid induced and thermal induced nociception models in experimental mice.

In acute toxicity study a high dose of 2000 mg kg ^-1 body weight of PCEE administered orally caused neither any sign of mortality nor any observable negative symptoms over a period of 48 h. No death even with 10 times of the effective dose indicating the high margin of safety and there fore an LD₅₀> 2000 mg kg ^-1body weight may be assumed.

The acetic acid induced visceral nociceptive response in mice is regarded as a model of inflammation pain and used as screening tool for evaluation of analgesic or inflammatory agents. Intaperitoneal injection of acetic acid produced pain through activation of chemosensitive nociceptors ²². It has been suggested that acetic acid acts by releasing endogenous inflammatory mediators or irritation of the visceral surface, which lead to the liberation of histamine, kinins, prostanoids, serotonin and substance p. It is a sensitive procedure to evaluate peripherally and centrally acting analgesics ^23-27.The major transmission pathway for inflammatory pain has been documented as that comparing polymodal receptors, such as vanilloid, bradykinin, prostaglandin and tachykinin receptor, among others around small vessels that signal to the central nervous system (CNS) via sensory afferent C- fibers entering the dorsal horn ^27,28. The nociceptive activity of acetic acid may be due to cytokine release, such as TNF-α , inteleukin-1β and interleukin – 8, by resident peritoneal macrophages and mast cells ²⁹.The intraperitoneal injection of acetic acid induced an increase in the concentration of glutamate and aspartate in the cerebrospinal fluid³⁰.In writhing test the quantification of the prostaglandins by radio immunoassay in the peritoneal exudates of rats, obtained after intraperitoneal injection of acetic acid. High levels of prostaglandins PGE_2α and PGF_2αduring the first 30 min after acetic acid injection were reported³¹.We have reported that the Leucas aspera aqueous extract was effective in inhibiting the writhing in mice by 52.63 and 64.35 % respectively, in a dose dependent manner, in comparison to both the negative control group and the positive control group treated with ASA, which inhibit the writhing by 72.63 %. There fore the aqueous extract may involve in the peripheral analgesic activity.

In this work , we measured the nociceptive reactivity to thermal stimuli in mice using the hot plate test , which is sensitive acute pain test for detecting opiate analgesia as well as several types of hyperalgesia reactions from spinal origin the hotplate test could be a simple and sensitive procedure to evaluate analgesics and hyperalgesics reactions in mice . This method is considered to be selective for opioid like compounds in animals ³². The results indicate that the oral administration of aqueous extract of Leucas aspera significantly attenuated the hot plate thermal stimulation. Hot plate is normally used to study the central analgesics effects of drugs. There fore it is probable that Leucas aspera could be producing its effects centrally. These shows the extract increased the stress tolerances capacity of the animals and the possible involvement in higher centre. Although the underlying mechanism is unknown the observed activity can be attributed to the over all effects of the plant constituents or the compounds having similar NSAIDs or opioids.

In conclusion, the results of the present work clearly demonstrated the antinociceptive activity of Leucas aspera aqueous extract and also we conclude that the aqueous extract have peripheral and central analgesic property. The crude extract has been reported to have interaction with opioid receptors ³³.The antinociceptive effect of this extract may be a result of inhibition or reduction of proinflammatory mediators³⁴. The analgesic activity of Leucas aspera aqueous extract can be due to the presence of sterols or β-sitosterol or linifolioside.Analgesic activities of some sterols have already been shown on the models of pain induced by acetic acid and formalin ^35,36. In acetic acid induced writhing test showed that β-sitosterol decreased the number of squirms induced by acetic acid and the hot plate method conformed its analgesic activity³⁷, linifolioside showed inhibition of prostaglandin induced contraction contractions in guinea pig¹¹. Fact that this class of compounds from Leucas aspera may justify their antinociceptive activity. Further studies are required to isolate, characterize and finding the mechanism of action of the active compounds in aqueous extract responsible for peripheral and central analgesic activity.

REFERENCES:

1. Nagasamy Venkatesh, D., R. Adhiyaman and B. Sanat kumar,2007.Potential adverse drugs reactions of herbal drugs and its safety issues,Antiseptic,104(8):431-433.

2. Gamble,J.S.,1921.Flora of the presidency of madras,Published under the authority of the secretary of the india in council,Vol. 11.,pp:1150.

3. Vasudevan Nair,R., 2001.Indian medicinal plants a compendium of 5000 species, Orient Longman ltd,Vol. 3.,pp:316-318.

4. Sivarajan,V.V and I.Balachandran,2002.Ayurvedic Drugs and Their Plant Sources, Oxford & IBH Publishing co. Pvt. Ltd., New Delhi, pp: 271-272.

5. Chopra,R.N.,S.L.Nayar andI.C.Chopra,1996.Glossary of Indian medicinal plants, National institute of science communication, New delhi,1^st Edn.,pp:153.

6. Joshi,S.G.,2000.Medicnal Plants, Oxford & IBH Publishing co. Pvt. Ltd., New Delhi,pp:224.

7. Rastogi,R.Pand.B.N.Mehrotra,1993.Compendiumof Indian Medicinal Plants,Publications and Information Directorate, CSIR, New Delhi, Vol.2., pp : 414.

8. Rastogi,R.Pand.B.N.Mehrotra,1995.Compendium of Indian Medicinal Plants,Publications and Information Directorate, CSIR, New Delhi, Vol.1., pp : 245.

9. Nadkarani,K.M,1976.IndianMeteriaMedica,PopularPrakashanPrivateLimited,Mumba i,3^rdEdn.,pp :739.

10.     Sadhu, S.K., E.Okuyama,H.Fujimoto,M.Ishibashi,2003.Separation of Leucas aspera, a medicinal plant of Bangladesh, guided byprostaglandin inhibitory and antioxidant activities. Chem Pharm Bull, 51(5):595-598.

11. Sadhu,S.K.,E.Okuyama,H.Fujimoto and M.Ishibashi,2006.Diterpines from Leucas aspera inhibiting prostaglandin induced contractions.J.Nat prod.,69(7):988-994.

12. Chopra,R.N.,I.C.Chopra and B.S.Varma,1980.Supplement to glossary of Indian medicinal plants, Publications and Information Directorate, CSIR, New Delhi,pp:55.

13. Chopra,R.N.,S.L.Nayar andI.C.Chopra,1996.Glossary of Indian medicinal plants, National institute of science communication, New delhi,1^st Edn.,pp:153.

14. Kiritikar,K.R., and B.D.Basu,1996.Indian Medicinal Plants, International book distributors nook sellers and publishers, Dehra dun,Vol.3,2^nd Edn.,pp:2019-2020.

15. Ambasta.S.P,1992.The useful plants of india, Publications and Information Directorate, CSIR, New Delhi,pp:326.

16. Anonymous,1995.The Wealth of India. A Dictionary of Indian Raw Materials and Industrial Products, Publications and Information Directorate, CSIR, New Delhi,Vol. 6., pp : 7a.

17. Mangathayaru,K.,J.Lakshmikant,N.ShyamSundar,R.Swapna,X.F.GraceandJ.Vasantha,2005. Antimicrobial activity of Leucas aspera flowers.Fitoterapia,76(7-8):752-754.

18.     Rahman,M.S.,S.K.Sadhu,C.M.Hasan,2007.Preliminaryantinociceptive,antioxdant and  cytotoxic activities of Leucas aspera root. Fitoterapia. Jul 4(available online).

19. OECD Test Guideline 425, 2001. Guidelines for Testing of Chemicals, Guidelines 425, Acute Oral Toxicity – Up-and-Down Procedure,( www.oecd.org).

20. Koster,R.,M.Anderson and E.J.Beer,1959. acetic acid for analgesic screening.Fed. Proc. 18:412

21. Woolfee,G. and A.D.MacDonald,1944.The evaluation of the analgesic action of pethidine hydrochloride (DEMROL).J.Pharmacol.Exp.Ther.,80:300-307.

22. Stai,H.Y.,Y.F.Chen and T.S.Wu,1995.Anti-inflammatory and analgesic activites of extract from roots of Angellica pubescens.Planta med.,61:1-8.

23. Murray, C.W.,F.Porreca and A.Cowan,1988.Methodolgical refinement in the mouse paw fomalin test: an animal model of tonic pain .J.Pharmacol. Method.,20:175-186.

24. Tjosen,A.,D.G.Berg,S.Hunskaar,J.H.Rosland,K.Hole,1992.The formalin test : an evaluation of the methods.Pain,51:5-17.

25. Gaertner,M.,L.Muller,J.F.Roos,G.Cani,A.R.S.Santos,R.Niero,N.BCalixto,R.AYunes,F.DelleManacheandV.CechinelFilho,1999.Analgesictriterpenesfromsebastiania schottiana roots.Phytomed., 6:41-44.

26. Collier,H.O.J.,J.C.Dinneen,C.A.Johnson and C.Schneider,1968. The addominal constriction response and its suppression by analgesic drugs in mouse.Br.J.Pharmacol.Chemother.,32:295-310.

27. Ikeda,Y.,A.Ueno,H.Naraba and S.Oh-ishi,2001.Involment of vanilloid receptor VR1and prostanoids in the acid –induced writhes responses of mice.Life Sci.,69:2911-2919.

28. Kumazawa,T.,K.Mizumura,H.Koda and H.Fukusako,1996.EP receptor subtypes implicated in the PGE2 Induced sensitive of polymodal receptors in esponse to bradykinin and heat , J.Neuro. physiol.,75:2361-2368.

29. Ribeiro,R.A.,M.L.Vale,S.M.Thomazzi,A.B.P.Paschoalato,S.Poole,S.H.FerreiraandF.Q.Cunha,2000.Involvement of resident macrophages and mast cells in the writhing nociceptiveresponse induced by zymosan and acetic acid in mice.Eur.J.Pharmacol.,387:111-118.

30. Feng,Y.,M.Cui andW.Willis,2003.Gabapentin markedly reduces acetic acid induced visceral nociception.Anesthesiology,98:729-733.

31. Deraetdt,R.,S.Jouquey,F.Delevallee and M.Flahaut,1980.Release of prostoglandinsE and F in an Algogenic reaction and its inhibition.Eur. J.Pharmacol.,61:17-24.

32. Janseen, P.,C.Niemegeers and J.Dony, 1963. The inhibitory effects of phentannyl (R- 4263) and other morphine –like analgesics on the warm water induced tail withdrawal reflex in rats. Arzeinmittel Foursching,13:502-507.

33. Awe,E.O., J.M.Makinde, O.A. Olajide and O.K. Wahkeel,2004b. Evaluation of antiinflammatory and analgesic activity of Russelia Equisetiformis. Inflammoparmacol,12:399- 405.

34. Fermandez M.A.,B.DE Las Haras , M.D.Gacia, M.T.Saen and A.Villar ,2001.New insights into the mechanism of action of anti inflammatory triterpenes lupeol. J.pharm.pharmacol., 53:1533-9.

35. Cechinel Filho,V.,A.R.S.Santos,R.O.P.De Campos, O.G.Miguel, R.A.Yunes, F.Ferrai, I.Messana, J.B.Calixto, 1996. Chemical and pharmacological studies of hyllanthus carolinienses in mice.J.Phar.Pharmacol.,48:1231-1236.

36. Santos,A.R.S.,R.Neiro,V.CechinelFilho,R.A.Yunes,M.G.Pizzolatti,F.DelleMonache, J.B.Calixto,1995.Antinociceptive properties of steroids isolated fromphyllanthus corcovadensis.Planta Med.,61:329-331.

37. Villasenor,I.M.,J.Angelada,A.P.Canlas, and D.Echegoyen,2002. Bioactivity studies of beta sitosterol and its glucoside ,Phytother. Res.,16:417-421.

Received on 25.06.2009 Modified on 23.08.2009

Research J. Pharm. and Tech. 3(1): Jan.-Mar. 2010; Page 95-98