INTRODUCTION:

Michelia champaca Linn. known as champaca is belonging to the family of Magnoliaceae.[1] It consists of 12 genera and 220 species of evergreen trees and shrubs, native to tropical and subtropical South and Southeast Asia (Indomalaya), including southern China. It’s commonly referred as yellow champaca. There are three species of Michelia available in Malaysia. They are Michelia Alba (white chempaka), Michelia champaca (orange chempaka) and Michelia figo (dwarf chempaka) with Michelia champaca and Michelia Alba being the most popular species within the family. [2] In recent times there are several reports of medical specialty roles and activities of Michelia champaca and its active principals on the circulatory system, Michelia, known by the scientific name Michelia champaca, is a very tall tree that grows up to 30m tall. The young branches are covered with grey hairs.

The leaves are ovate in shape and are up to 30.5cm long and 10.2cm wide narrowing to a fine point at the apex. Stem bark of Michelia champaca Linn. belonging to the family Magnoliaceae is commonly used by many traditional healers in most of the herbal preparations for diabetes[3] and kidney diseases.[4] Traditionally, it is being used in fever, colic, leprosy, post partum protection[5] and in eye disorders.[6] It has been reported to possess antipyretic, anti- inflammatory,[7] insecticidal,[8] antimicrobial[5], and leishmanicidal activities.[9] The active constituents reported in this plant are alkaloids, saponins, tannins, sterols, flavonoids and triterpenoids.[5] From the above reference this study was conducted to evaluate the extract of Michelia champaca stem bark for central analgesic activity along with anti-inflammatory activity in rats.

MATERIALS AND METHODS:

Plant Material:

Stem bark of M. champaca were collected in May 2016 from Tirupati, India. The taxonomical identification of the plant was done by internet source. (https://en.wikipedia.org/ wiki/ Magnolia_champaca)

Preparation of extracts:

Dried and powdered Stem bark material of Michelia champaca (500 g) was successively Soxhlet extracted with petroleum ether (60-80°), chloroform, acetone, ethanol and water for 48 h each. Crude aqueous extract of this plant was prepared separately by boiling the plant material (25 g) with 200 ml of water for 15 min. The obtained extracts were evaporated in vacuum to give residues and their percentage yields were determined.

Animals and Treatment:

Healthy Wistar rats of either sex (150-180 g), with no prior drug treatment, were used for the present studies. The animals were fed with commercial pellet diet and water ad libitum. The animals were acclimatized to laboratory hygienic conditions for 10 days before starting the experiment. Animal study was performed in the Division of Pharmacology,

Acute toxicity studies:

The acute toxicity test of the extracts was determined according to the OECD (Organization for Economic Co-operation and development) guidelines No. 420. Female Wistar rats (150-180 g) were used for this study. After the sighting study, starting dose of 2000 mg/kg (p.o.) of the test samples was given to various groups containing five animals in each group. The treated animals were monitored for 14 days, for mortality and general behaviour. No death was observed till the end of the study. The test samples were found to be safe up to the dose of 2000 mg/kg, and, from the results, 400 mg/kg dose was chosen as the maximum dose for further experimentation.

Analgesic Study:

Analgesic effects was evaluated using three different models: the writhing test, tail flick test and formalin test.

Writhing Test:

Male Swiss rats (180-200 g) were used according to the method described previously by.[10] The total number of writhings, following intraperitoneal (i.p.) administration of 0.6% acetic acid , was recorded for 20 min. starting 10 min. after injection. The animals were pretreated with hydroalcoholic extracts (HAEs) from Stem bark of M. champaca 100, 150, 200 mg/kg b.wt respectively.

Analgesic activity:

Table 1: writhing test:

Tail-Fick Test:

The basal reaction time of each mouse was determined using tail-withdrawal response when one-third of the tail was immersed in water bath at 51˙C.[11] The cutoff time for immersion was 180 s. The reaction time was evaluated 30, 60, 90, 120 and 240 min after oral administration of extracts, distilled water or acetylsalicylic acid.

Formalin Test:

The method used in our study was similar to that described previously.[12] Twenty microliter of 5% formalin was injected subcutaneously into the right hind paw of mice. The time (in seconds) spent in licking and biting responses of the injected paw was taken as an indicator of pain response. Responses were measured for 5 min after formalin injection (early phase) and 20–30 min after formalin injection (late phase). Michelia champaca stem bark M extracts (0.5 and 1.0 g/kg, i.p.) were administered 60 min before formalin injection. Indomethacin (10 mg/kg, i.p.) was administered 30 min before formalin injection. Control group received the same volume of saline by oral administration.

Anti-inflammatory activity:

Carrageenan induced hind paw edema in rats Paw edema was produced in rats by carrageenan following the methods of Winter et al. (1962) respectively.[13] Male rats eighing 100–120 g were divided into groups of six animals. A volume of 0.05 ml of 1% carrageenan in normal saline solution (NSS) in 0.2M carbonate buffer was injected intradermally into the plantar side of the right hind paw of the rat. Test drugs and vehicle were given 1 h prior to carrageenan injection. Paw volumes were measured using a plethysmometer (model 7150, Ugo Basile, Italy) before as well as 1, 3 and 5 h after carrageenan, injection. Results obtained were compared with those obtained from there.

Analgesic activity

Table 1: writhing test

Group	Treatment	N	Route of administration	Dose mg/kg	No.fo writches	Inhibition writhing response
1	Comtrol	6		-	49.06±4.08
2	Aspirin	6	300	i.p	12.05±2.14	90
3	Stem bark of M. champaca	6	50	i.p	20.65±2.10	40
4	Stem bark of M. champaca	6	100	i.p	18.85±1.78	65
5	Stem bark of M. champaca	6	150	i.p	8.68±1.14	85

Mean = S.E.M. of 6 animals. ** = P≤0.001= highly significant. Group II, III , IV, and V compared with Group I.

Anti-inflammatory activity:

Table2 Carrageenan –induced paw edema method.

Group	n	Dose (mg/kg)	Paw volume increase .(ml)			Inhibition (%)
Group	n	Dose (mg/kg)	1hr	3hr	5hr	1hr	3hr	5hr
Control	6		0.36±0.07	0.69±0.05	0.82±0.03
Aspirin	6	300	0.10±0.02**	0.21±0.02**	0.27±0.03**	72	70	67
Stem bark of M. Champaca ME	6	100	0.22±0.04*	0.47±0.01*	0.51±0.02*	39	32	38
Stem bark of M. Champaca ME	6	150	0.18±0.03*	0.39±0.01*	0.42±0.04*	50	43	49
Stem bark of M. Champaca ME	6	200	0.07±0.01**	0.24±0.02**	0.35±0.02**	81	65	57

n = 6 animals in each group. * = p≤0.01 (significant).** = p≤0.01(highly significant) control groups, which received vehicle only

RESULT:

Analgesic activity of administration of Michelia champaca stem bark ethanolic extract at the dose level of 100, 150 and 200 mg./kg b. wt. to the rats produced weak effect on the writhing induced by the injection of 0.6% acetic acid when compared with the aspirin (300mg/kg) by 79% while the treated group with Michelia champaca stem bark ethanolic extracts inhibited the writhing by 40%, 65%, 85% respectively(table 1). The ethanolic extract of Michelia champaca stem bark (100- 200 mg/kg) produced inhibition of formalin induce biphasic pain response (neurogenic and inflammatory pain) in rats. The analgesic effect of this fraction occurred predominately during the II phase; 200 mg dose level was more efficient in the late phase. Anti-inflammatory activity of Michelia champaca stem bark, The inhibitory activity on carrageenan induced rat hind paw edema, caused by the subplanatar administration of Michelia champaca stem bark ethanolic extract, at various assessment times after carrageenan injection are shown in table 2. aspirin, a cyclooxygenase inhibitor, at the dose of 300mg/kg body weight exhibited significant (p≤0.01) edema inhibition. Michelia champaca stem bark ethanolic extract at doses of 50,100,150 mg/kg boy weight also possessed significant (p≤0.001) inhibitory effect on carrageenan induced paw edema at all recorded times. This increase was observed at 1 hr. and was maximum at 5hr. after administration of carrageenan in the vehicle group.

DISCUSSION:

The inflammatory effect, analgesic properties of Michelia champaca stem bark ethanolic extract ethanolic extracts were investigated in the present study The writhing test allows us to identify central and peripheral analgesic compound.[15] The tail formalin test is recent algesiometric assay in which only behaviour suggestive of pain is the licking of tail. The lack of 2 distinct phases after the administration of formalin in the tail may be due to a different pattern of the release of the chemical pain mediators at both the spinal and peripheral levels and this method mainly identifies peripheral analgesic.[16] The thermal model of the tail flick test is considered to be spinal reflex, but could also involved higher neural structures at this method identify mainly central analgesic.[17] The extracts derived from Michelia champaca stem bark ethanolic extract exhibited analgesic activity in albino rats by inhibiting acetic acid induce writhing, which is a model of visceral pain.[18] Acetic acid induced writhing is a highly sensitive and useful test for analgesic drug development , but not a selective pain test as it gives false positives with sedatives , muscles relaxants and other pharmacological activities.[19] The analgesic property of Michelia champaca stem bark ethanolic extract can also probably be to the blockade of the effects or the synthesis and /or release of PGs and /or other endogenous substances that excite pain nerve endings.[20] Although the writhing response test is very sensitive it has poor specificity as in analgesic screening test , therefore the formalin test and tail flick test were conducted to confirm and study the possible analgesic mechanism of the tested plants .The formalin test consist of 2 distinct phases that possibly reflecting different types of pain mechanisms. The I phase immediately after injection of formalin and last about 5 minutes this is due to result chemical, peripheral stimulation of nococeptors.[21] The II phase starts approximately 15-20 min after formalin injection and lasts for 20- 40 min. the formalin test is sensitive to nonsteroidal anti- inflammatory drugs and other mild analgesics. In the study aspirin inhibited analgesic behavior during both the early and late phases. The carrageenan test was selected because of its sentivity in detecting orally active inflammatory agents particularly in the acute phase of inflammation. [22] The intraplatar injection the carrageenan in rats leads to paw edema. Its first phase that is 1 hour after infection results from the concomitant release of mediators, histamine, serotine and kinins on the vascular permeability. The IInd phase is correlated with elevated production of prostaglandins, oxygen derive free radicals and production of inducible cyclooxygenase. [23] Oral administration of the Michelia champaca stem bark M extract extracts suppressed that edematous response 1 hr after carrageenan injection and this effect continues up to 5 hr It is well known that most of the anti-inflammatory analgesic activity Michelia champaca stem bark ethanolic extract. In general , anti- inflammatory drugs produce their antipyretic action through inhibition of prostaglandin synthesis within the hypothalamus.[24] Although there is no direct evidence of Michelia champaca stem bark ethanolic extract to interfere with prostaglandin synthesis in hypothalamus but it can be supported by a related study in which D. Odorifera extract was found to inhibit prostaglandin by synthesis[25] From these results is concluded that the extract from Michelia champaca stem bark ethanolic extract possess both peripheral and central analgesic activity along with marked anti-inflammatory activity in rats and also the present study provoke the traditional use of Michelia champaca stem bark ethanolic extract for the purpose various ailments like analgesic and anti- inflammatory.

CONCLUSION:

The ethanol extract of Michelia champaca stem bark has moderate and safe oral both peripheral and central analgesic activity along with marked anti- inflammatory activity in rats.

ACKNOWLEDGEMENT:

The authors thanks to Principal, Management Hindu College of Pharmacy, Guntur for place of research and providing financial support and encouragement for completion of the research work.

REFERENCES:

1. Rout, P. K., Naik, S. N., and Rao, Y. R. Composition of the concrete, absolute, headspace and essential oil of the flowers of Michelia champaca Linn. Flavour and Fragrance Journal, 2006; 21(6): 906-911.

2. Ibrahim, R., Salahbiah, A. M., Khoo, C. K., Azhar, M., Ashanul, K. A. W., Rasol, A., and Muse, R. Development of embryogenic culture system for the production of essential oils using bioreactor technology from Michelia alba. P-INCOBB- 18. 2005.

3. Rajagopalan PM. Siddha medicine. Madurai: Siddha Maruthuva Gurukulam., 2000.

4. Nadkarni M. Indian Materia Medica. 1st ed. Mumbai: Popular Book Depot, 1954.

5. Khan MR, Kihara M, Omoloso AD. Antimicrobial activity of Michelia champaca. Fitoterapia., 2002; 73:744–8.

6. Sobhagini N, Soumit KB, Malaya KM. Ethno- medico-botanical survey of Kalahandi district of Orissa. Indian J Trad Knowledge. 2004; 3: 72–9.

7. Vimala R, Nagarajan S, Alam M, Susan T, Joy S. Antiinflammatory and antipyretic activity of Michelia champaca Linn., (white variety), Ixora brachiata Roxb. and Rhynchosia cana (Willd.) D.C. flower extract. Indian J Exp Biol. 1997; 35: 1310–4.

8. Ulla J, Vijaya K, Shantini S. Sesquiterpene lactones from Michelia champaca. Photochemistry, 1995; 39:839–43.

9. Takahashi M, Fuchino H, Satake M, Agatsuma Y, Sekita S. In vitro screening of leishmanicidal activity in myanmar timber extracts. Biol Pharm Bull., 2004; 27: 921–5.

10. Koster R, Anderson M, De Beer J. Acetic acid for from Dalbergia odorifera. Chem Pharma Bull., analgesic screening. Federal Proceedings. 1959; 18: 1985; 33: 5606-9. 412-417.

11. Jansen PAJ, Niemergeers CJE, Dony JGH. The inhibitory effect of fentanyl and other morphine–like analgesics on the worm induced tail withdraw reflex in rats rzneimittel forschung., 1963; 13: 502-507.

12. Shibata M, Ohkubo T, Takahashi H, Inoki R. Modified formalin test: characteristic biphasic pain response. Pain, 1989; 38: 347-352.

13. Winter CA, Risley EA, Nuss GW. Carrageenin- induced edema in hind paw of the rat as an assay for anti-inflammatory drug. In: Proceedings of the Society for Experimental Biology and Medicine, 1962; 11: 544–547.

14. Yesilada E, Ku¨peli E. Berberis crataegina DC. Root exhibits potent antiinflammatory, analgesic and febrifuge effects in mice and rats. Journal of Ethno pharmacology, 2002; 79: 237–248.

15. Le Bars D, Gozariu M, Cadden S. Animal models of nociception. Pharmacological Reviews. 2001; 53: 628–651.

16. Koleniskov Y, Cristea M, Oksman G, Torosjan A, Wilson R. Evaluation of tail formalin test in mice as a new model to assess local analgesic effects. Brain Research 2004; 1029: 217–233.

17. Jensen TS, Yaksh TL. Comparison of antinociceptive action of morphine in the periaqueductal gray, medial and paramedial in rat. Brain Research. 1986; 363: 99-113.

18. Vykicky L. Techniques for the study of pain in animals. IN: Bonica, J.J., Liebeskind, J.C. Albe- Fessard, D.G. (Eds.), Advances in pain research and therapy. Raven Press, New York, 1978; 727- 745.

19. Elisbetsky E, Amdor TA, Albuquerque RR, Nunes DS, Carvalno ACT. Analgesic activity of Psychotria colorata (Wild ex R and S) Muell-Arg. Alkaloids. Journal of Ethno pharmacology. 1995; 48: 77-83.

20. Panthong A, Supraditaporn W, Kanjanapothi D, Tae sotikul, T, Reutrakul V. Analgesic, anti - inflammatory and ventonic effects of Cissus quadrangularis Linn. Journal of Ethno pharmacology. 2007.

21. Heapy CG, Jamieson A, Russell NJW. Afferent Cfiber and A-delta activity in models of inflammation. British Journal of Pharmacology., 1987; 90: 164.

22. Dirosa M. Biological properties of carrageenan, Journal of Pharmacy and Pharmacology. 1972; 24; 89-102.

23. Pathong A, Kanjanapothi D, Taesotikul T, Phankummoon A, Panthong K, Reutrakul V. Anti- inflammatory activity of ethanolic extracts of Ventilago harmandiana Pierre . Journal of Ethan pharmacology. 2004; 91: 237-242.

24. Clarke WO, Cumby HR. The antipyretic effect of indomethacin. Journal of Physicol., 1975; 248: 625-38.

25. Goda Y, Katayama M, Ichikawa W, Shibuya M, Kiuchi F. Inhibition of prostaglandin biosynthesis from Dalbergia odorifera. Chem. Pharma. Bull., 1985; 33: 5606-9.

Received on 08.04.2017 Accepted on 20.05.2017

Asian J. Pharm. Res. 2017; 7(2):94-97.

DOI: 10.5958/2231-5691.2017.00016.8