INTRODUCTION ^[1-6]

Cyathocline lyrata is annual herb, growing to 20-25 cm high, branched grooved stem has soft hair covering it. Whole Plant is strongly aromatic. Alternatively arrange stalkless leaves are toothed covered with soft hair and flowers occurs in corymbs at the end of branched in purple color.

Cyathocline lyrata widely spread in Himalyas range, Assam, India and also available in Local area of Betul M.P. Cyathocline lyrata is well known drug in Indigeneous system of medicine for its various used as a bitter tonic. It acts as germicide and appetizer. The essential oil of aerial part of Cyathocline lyrata had show fairly pharmacological activity. It also shows anthelmatic, insect repellant and anti-microbial activity.

Pain is defined as an “unpleasant sensation in animals that is caused by actual or perceived injury to body tissues and produces physical and emotional reactions”.

Presumably, pain sensation has evolved to protect our bodies from harm by causing us to perform certain actions and avoid others. Pain might be called a protector, a predictor, or simply a hassle. Pain arising from the skin and from the deep structures like muscles, bones, and joints is also termed as somatic pain. It is usually well defined and is generally caused by inflammatory reaction in the tissues; it may be accompanied by contraction of the surrounding skeletal muscles as in patients with rheumatoid arthritis. Inflammation is the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells or irritants. Non steroidal anti-inflammatory drugs (NSAID‟s) are widely used in the treatment of pain, fever and inflammation. However these drugs have no side effects especially on the gastro intestinal tract. Therefore there is a need to search for novel analgesic agents from natural source, which could be used in medicine and as additives to Nutraceuticals. Many natural products have been reported to have analgesic action. An attempt is made during this research work to evaluate the chloroform extracts of whole part of Cyathocline lyrata for analgesic activity.

MATERIAL AND METHOD:

Plant material collection and authentication:

The entire plant of Cyathocline lyrata Cass were collected from local area of Betual (M.P.) in the month of June, 2012. The plant specimen was conﬁrmed by Dr. Madhuri Modak, Professor, Dept. of Botany, Motilal Vigyan Mahavidyalaya, Bhopal (M.P). A voucher specimen (Her/Bot/1212.90-374) is deposited at the herbarium of MJPR University,Jaipur(RJ).

Evaluation of In vivo analgesic activity:

Procurement of Experimental Animals:

Male albino rats (100-150 g) of approximate same age were used in the present studies was procured from VNS College of pharmacy, Bhopal India. The animals were fed with standard pellet diet and water ad libitum. All the animals were housed in polypropylene cages. The animals were kept under alternate cycle of 12 hours of darkness and light. The animals were acclimatized to the laboratory condition for 1 week before starting the experiment. The animals were fasted for at least 12 hours before the onset of each activity. The animals received the drug treatments by oral gavage tube. After preparation of chloroform extract the next step was to formulate a suspension of extracts of Cyathocline lyrata Cass which was subjected to animal studies. Suspension of the extract was made by suspending in 0.5% CMC.

Acute toxicity studies:

This study is needful before pharmacological screening on animals. The acute oral toxicity study of Cyathocline lyrata chloroform extracts was carried out according to OECD 423 guideline (Organization for Economic Cooperation and Development) which is based on a stepwise procedure with the use of a minimum number of animals per step. All animals were received respective dose for seven days and observed mortality on 7th day.The animals Male albino rats were treated drug of Chloroform extract of Cyathocline lyrata.

Screening of Analgesic Activity:

Tail Flick Method / Analgesiometer Method^[^7]:

Analgesia is defined as a state of reduced awareness to pain, and analgesics are substances, which decrease pain sensation (pain - killers) by increasing threshold of painful stimuli. The commonly used analgesics are Diclofanac potassium, Aspirin, Paracetamol (non - narcotic type) and Morphine (narcotic type). Painful reaction in experimental animals can be produced by applying noxious (unpleasant) stimuli such as (i) thermal (radiant heat as a source of pain), (ii) chemical (irritants such as acetic acid and bradykinin) and (iii) physical pressure (tail compression).

Materials used:

Animal : Rats (100 – 150 g)

Drugs: Diclofenac Potassium (10 mg / kg, body weight)

Wama Pharmaceutical Ltd. (Wadi) Nagpur

Extract: Chloroform extract of Cyathocline lyrata (100, 200, 400, mg/ kg body weight)

Six groups of six animals were taken. Tail flick response was evoked by placing rat tail over a wire heated electrically. The analgesiometer adjusted in 2 amp. The intensity of heat was adjusted so that the baseline tail flick latency averaged 3-6 seconds in all the animals. Cut of period of 10 seconds was observed to prevent the damage to tail. The initial reaction time of each rat was recorded. The animals were treated with the test drug and post treatment reaction time of each animal was determined at 30 minutes interval for 120 minutes.

MPE (Maximum possible effect) = 100(TL▬CL) / 10▬CL

CL = Control latency

TL = Test latency

Hot Plate Method^[^8]:

Experimental animals of either sex were randomly selected and divided into four groups designated as group-I, group-II, group-III and group-IV consisting of six rats in each group for control, positive control and test group respectively. Each group received a particular treatment i.e. control (1% Tween-80 solution in water, 10ml/kg, and p.o.), positive control (Diclofenac potassium 10 mg/kg, p.o.) and the test (chloroform extract of 100 mg/kg, p.o.200 & 400, mg/kg, p.o. respectively). The animals were positioned on Eddy’s hot plate kept at a temperature of 55±0.5 0C. A cut off period of 15 s was observed to avoid damage to the paw. Reaction time was recorded when animals licked their fore or hind paws, or jumped prior to and 0, 30, 60, 90 & 180 min after oral administration of the samples.

RESULT AND DISCUSSION:

Acute toxicity study:

Acute toxicity studies for chloroform extract of Cyathocline lyrata was conducted as per OECD guidelines 423 using albino rats. Each animal was administered extracts by oral route. There was no change in normal behavioral pattern of animals and no sign and symptoms of toxicity were observed during the observations which was done continuously for the first two hours and then observed up to twenty four hours and then for seven days for mortality.

Screening of Analgesic Activity:

Tail Flick method/Analgesiometer Method:

Analgesic activity of of chloroform extract of Cyathocline lyrata cass was assayed by using analgesiometer and results are presented in Table 1.

Table 1: Effect of Chloroform Extract of Cyathocline lyrata using Tail Flick Method

Treatment	Time in minute after drug administration
Treatment	0 min	30 min	60 min	90 min	180 min
Control	3.32±0.25	3.82±0.29	3.12±0.26	3.10±0.22	2.49±0.17
DP (10 mg/kg)	3.23±0.24	3.3±0.22	3.29±0.28	3.49±0.30*	3.21±0.28*
CECL (100 mg/kg)	3.21±0.25	3.29±0.22	3.2±0.19	3.35±0.23	3.32±0.22*
CECL (200 mg/kg)	3.19±0.12	3.17±0.24	3.46±0.21	3.55±0.13*	3.98±0.18**
CECL (400 mg/kg)	3.32±0.1	3.31±0.23	3.11±0.1	3.62±0.12**	3.45±0.15**

All values are expressed as a Mean ± S.E.M, n=6. Results were analyzed using one way ANOV followed by Dennett’s comparison multiple test. DP- Diclofenac Potassium, CECL- chloroform extract of Cyathocline lyrata. Asterisks (*) represent significance level with * indicating P<0.05 and ** indicating <0.01 as compared to the control.

Table 2: Effect of Chloroform Extract of Cyathocline lyrata using Hot Plate Test

Treatment	Paw licking or jumping in seconds
Treatment	0 min	30 min	60 min	90 min	180 min
Control	3.32±0.13	3.33±0.13	3.33±0.11	3.32±0.11	3.03±0.08
DP (10 mg/kg)	3.32±0.17	3.41±0.16	3.98±0.13*	4.10±0.13**	4.10±0.17**
CECL (100 mg/kg)	3.31±0.15	3.33±0.13	3.42±0.11	3.59±0.1	3.52±0.09
CECL (200 mg/kg)	3.32±0.13	3.34±0.11	3.62±0.15	3.71±0.17*	3.84±0.13**
CECL (400 mg/kg)	3.33±0.1	3.43±0.12	3.82±0.14*	4.22±0.17**	4.14±0.15**

All values are expressed as a Mean ± S.E.M, n=6. Results were analyzed using one way ANOVA followed by Dennett’s comparison multiple test. DP- Diclofenac Potassium, CECL- chloroform extract of Cyathocline lyrata. *p<0.05 and **P<0.01 indicate statistical significance when compare to control.

CONCLUSION:

The results obtained in different pharmacological evaluation of cyathocline lyrata cass has analgesic effect. The results have been obtained in carefully controlled experiments with laboratory animals where psychological factors can presumably be ruled out. In all the tests the responses have been assessed by actual measurement and not by subjective comparisons which may be influenced by the observer. Therefore the statistical validity of the findings has been proved and they provide a scientific foundation for the use of the biologically active ingredients of cyathocline lyrata in pain conditions and explain the clinical effectiveness of the plant. In our study we have made an attempt to prove its efficacy in experimental animals. Further study can be done in human subjects.

ACKNOWLEDGEMENTS:

Researchers are very much thankful to the Dept. of Botany, Motilal Vigyan Mahavidyalaya, Bhopal (M.P), Wama Pharmaceutical Ltd. (Wadi) Nagpur, MJRP College of Heath Care and Allied Sciences, MJRP University Jaipur, Sharad Pawar College of Pharmacy, Nagpur for providing necessary facilities.

REFERENCES:

1. Shrivastava R, Anthelmintic properties of essential oil of Cyathocline lyrata Cass., Indian journal of Pharmaceutical Sciences 1979;41:228-229.

2. Tripathi KD. Essentials of Medical Pharmacology. 5th Edn, Jaypee Brothers Medical Publishers (P) Ltd.; 2003,p.453

3. Rang HP, Dale MM, Ritter JM, Moore PK. Pharmacology. 5th Edn, Elservier; 2006,p. 246.

4. Sosa S, Balicet MJ, Arvigo R, Esposito RG, Pizza C, Altinier GA. Screening of the topical anti-inflammatory activity of some central American plants. J Ethanopharmacol 2002; 8:211–215.

5. Michel YB, Dubois, Christopher G, Allen HL. Chronic pain management. In: Healy TEJ, Knight PR, eds. Wylie and Churchil-Davidson’s A practice of Anaesthesia .7th edition. London, 2003, p.1235–1139.

6. Pilotto A, Franceschi M, Leandro G. The risk of upper gastrointestinal bleeding in elderly users of aspirin and other non-steroidal anti-inflammatory drugs: the role of gastroprotective drugs. Aging Clin Exp Res 2003; 15(6):494–499.

7. D’Amour FE, Smith DL. A method for determining loss of pain sensation. J Pharmacol Exp Ther 1941; 72: 74–79.

8. Eddy NB, Liembach D. Synthetic analgesics II: Dithienylbuttenyl and dithiennylbulyl-amines.J Pharmacol Exp Ther 1957; 107: 385–393.

Received on 14.04.2013 Accepted on 18.05.2013

Asian J. Pharm. Res. 3(2): April- June 2013; Page 82-85