Continental J. Pharmacology and Toxicology Research 4 (1): 11 - 17, 2011 ISSN: 2141 4238 Wilolud Journals, 2011 http://www.wiloludjournal.

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THE ANTINOCICEPTIVE EFFECT OF THE AQUEOUS STEM BARK EXTRACT OF Amblygonocarpus andogensis IN ALBINO RATS H .Ighodaro and S.O Bello Department of Clinical Pharmacy,UsmanuDanfodiyo University,Sokoto. 2Department of Pharmacology and Toxicology,Usmanu Danfodiyo University,Sokoto. ABSTRACT Amblygonocarpus andogensis is a perennial plant commonly used in Nigeria traditional medicine for the treatment of pain, particularly body and joint pains. However, little scientific evidence exists in literature on the antinociceptive property of this plant.Furthermore; current analgesics being used in the treatment of pain have numerous undesirable side effects. There is therefore the need for further research for new analgesics acting on new pain receptors. This study was therefore undertaken to investigate the antinociceptive activity of the aqeous stem bark extract of Amblygonocarpus andogensis in Albino rats. The acetic acid induced abdominal constriction test and the Formalin-induced paw licking test methods were used for the pain evaluation. In the acetic acid induced abdominal constriction test, the method used was that described by Koster et al (1959) as modified by Amos et al 2002. A total of 20 rats were divided into two sets of two groups of rats with one control group; with n=4. The first set was pre-treated with the extract at 100 and 200 mg/kg p.o; with pre-treatment time of 30 min. The second set was similarly treated but with a pre-treatment time of 60 min. Each group was administered 10ml/kg intra peritoneal(i.p) of an aqueous solution of acetic acid (0.7%). The rats were then held upward and the number of abdominal constriction for each rat counted for 10 min immediately after treatment with acetic acid. The observer of the abdominal constriction was blinded to the exact treatment the animal received. The control group was given normal saline for pre-treatment and compared with the extract treated groups. The % inhibitions of abdominal constrictions for the extract treated groups were calculated. The Formalin test used was similar to that described by Dubusson and Dennis (1977) and modified by Tjolsen et al (1992). Three groups of rats weighing between 100-160g consisting of 4 rats per group were pre-treated as follows: Group one normal saline (acted as control) Group two was given 100mg/kg of extract Group three was given 200mg/kg of extract Thirty minutes after this treatment, they were administered 50l of a 2.5% solution of formalin subcutaneously under the plantar surface of the left hind-paw. They were then placed in an observation chamber and monitored for 1 hour, and the severity of pain was recorded based on the following pain score; (0) Rat walked or stood firmly on infected paw. (1) The infected paw was favoured or practically elevated. (2) The infected paw was clearly lifted off the floor. (3) The rat licked, chewed or shook the infected paw. The observer was blinded to the exact treatment the animal received. Antinociceptive effect was determined in two phases. (i) The early phase been recorded during the first five minutes, while the late phase (ii) Was recorded during the last 45 minutes with a 10min lag period in between both phases. The aqeous extract(200 and100mg/Kg) significantly and in a dose dependent manner reduced the nociception induced by the acetic acid and in both the early and late phases of Formalin test (P<0.05). Acetic acid induced writhing is a model of visceral pain and is a highly sensitive and useful test for analgesic drug development but not a selective pain test.Formalin test however is sensitive to non steroidal anti-inflammatory drugs and other mild analgesics.The extract of Amblygonocarpus andogensis produced significant analgesic effect in both phases of the Formalin pain test. This probably indicates that the analgesic effect of the extract was mediated by both neurogenic and inflammatory mechanisms.

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H .Ighodaro and S.O Bello: Continental J. Pharmacology and Toxicology Research 4 (1): 11 - 17, 2011

KEY WORDS:Antinociception,acetic acid,formalin,Amblygonocarpus andogensis. INTRODUCTION The problem of pain sensations has been with man and has given cause for concern from time immemorial. Pain itself is a difficult Science to study and the need to study it remains as long as there are pains, which we do not understand, and which are inadequately treated. These pains are indications of our ignorance about pain mechanisms and therapy. To achieve adequate understanding and treatment, there is need for further research into the phenomenon of pain. Furthermore, current analgesics being used in the treatment of pain have numerous undesirable side effects. There is therefore the need for further research for new analgesics acting on new pain receptors such as PAR-2 (protease activated receptor) which is found in the skin, in joints and in the digestive system. AC 264613 peptide is an agonist of protease activated receptor, while F SLLRY is an antagonist. Several herbal agents have been known to exhibit analgesic properties. Such includes Alstonia boonie, whose stem bark is used in traditional medicine for treating painful micturition and rheumatic conditions. The plant Erythrina senegalenses was reported by Etkin (1997) to have some significant analgesic activity against the acetic acid induced abdominal constriction in mice. In line with this understanding, the plant Amblygonocarp andogensis was assessed as a way of sourcing for possible useful and better analgesic agents. The word Pain is frequently used especially in research, to refer to a class of behaviours, which operate to protect the organism from harm or to enlist aid in effecting relief. The behaviour may be a reflex withdrawal as in pulling ones hand away from heat or it may be any of a number of physiological processes which accompany the presumed experience of pain and are used as objective measures of it, such as changes in cardiac or blood pressure, histamine production, or catecholamine levels. Such changes are considered to be operational definitions of pain for experimental purposes, or for purposes of objective clinical evaluation and are referred to as pain response (Patrick, 1989). Another definition of pain is that given by the International Association for the study of pain (IASP) which defines it as an unpleasant sensory and emotional experience association with actual or potential tissue damage, or described in terms of such damage. Most pain researchers and therapists have accepted this definition, which is now used widely to qualify the meaning of the word pain (IASP 1986). The World Health Organization in recognition of the immense value of herbal medicine to primary health care has advocated for the proper identification, sustainable exploitation, scientific development and appropriate utilization of herbal medicine which provides safe and effective remedies in Medicare. (Wambebe, 1998). Preliminary enquiry through local traditional herbal practitioners shows that the plant does have medicinal analgesic properties worth investigating. Paul et al, (2000); (Patrick, 2001) described Andongensis as a tree with clear potential values. Rogger (2000) also described this plant as an economic tree. But above all, this plant has a long standing history of analgesic as well as antipsychotic claim among the leading traditional medical practioners. Despite these claims; there is no documentation on the scientific validation of the plant. This is because traditional medicine in Africa is not codified but verbally passed unto apprentices as folks medicine (Ohaeri,1989). It was also discovered that the plant despite its useful medicinal values have been under investigated and very scanty information about it exist in literature. The present study was therefore intended to fill the highlighted vacuum . MATERIALS AND METHODS ACETIC ACID-INDUCED ABDOMINAL CONSTRICTIONS IN ALBINO RATS. The method used was that described by Koster et al (1959) as modified by Amos et al 2002. A total of 20 rats were divided into two sets of two groups of rats with one control group; with n=4. The first set was pre-treated with the extract at 100 and 200 mg/kg p.o; with pre-treatment time of 30 min. The second set was similarly treated but with a pre-treatment time of 60 min. Each group was administered 10ml/kg intra peritoneal(i.p.) of an aqueous solution of acetic acid (0.7%). The rats were then held upward and the number of abdominal constriction for each rat counted for 10 min immediately after treatment with acetic acid. The observer of the abdominal constriction was blinded to the exact treatment the animal received. The control group was given normal saline for pre-treatment and compared with the extract treated groups. The % inhibitions of abdominal constrictions for the extract treated groups were calculated.

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H .Ighodaro and S.O Bello: Continental J. Pharmacology and Toxicology Research 4 (1): 11 - 17, 2011

FORMALIN TEST IN RATS The method used was similar to that described by Dubusson and Dennis (1977) and modified by Tjolsen et al (1992). Three groups of rats weighing between 100-160g consisting of 4 rats per group were pre-treated as follows: Group one normal saline (acted as control) Group two as given 100mg/kg of extract Group three was given 200mg/kg of extract Thirty minutes after this treatment, they were administered 50l of a 2.5% solution of formalin subcutaneously under the plantar surface of the left hind-paw. They were then placed in an observation chamber and monitored for 1 hour, and the severity of pain was recorded based on the following pain score; (4) Rat walked or stood firmly on infected paw. (5) The infected paw was favoured or practically elevated. (6) The infected paw was clearly lifted off the floor. (7) The rat licked, chewed or shook the infected paw. This method of scoring allows a graded determination of responses thus showing finer degrees of antinociception as opposed to the method in which only the time the animal spent licking the infected paw was recorded. The observer was blinded to the exact treatment the animal received. Antinociceptive effect was determined in two phases. (iii) The early phase been recorded during the first five minutes, while the late phase (iv) Was recorded during the last 45 minutes with a 10min lag period in between both phases. RESULTS AND DISCUSSION TABLE 1: ACETIC ACID INDUCED PAIN TEST IN ALBINO RATS ______________________________________________________________________________ GROUP DOSE NUMBER OF CONSTRICTIONS DURING; mg/kg 30min pretrt %inhibition 60minpretrt %inhibition ______________________________________________________________________________ Control 112.25 8.47 112.25 8.47 A. Andogensis 100 35.50 4.27* 68.40 27.25 2.56* 75.78 A. Andogensis 200 14.50 1.26* 87.11 15.50 3.30* 86.22 ______________________________________________________________________________ * P < 0.05 TABLE 2: GROUP Control A. FORMALIN INDUCED PAIN TEST IN ALBINO RATS DOSE PAIN SCORE Mg/kg ---3 Andogensis 100 200 1* 0*

3 0* 0*

A. Andogensis

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H .Ighodaro and S.O Bello: Continental J. Pharmacology and Toxicology Research 4 (1): 11 - 17, 2011

ACETIC ACID INDUCED ABDOMINAL CONSTRICTIONS IN ALBINO RATS

140 120
NUMBERS OF CONSTRICTIONS

100 80 60 40 20 0 1 2 3 4 5 6

PRE-TREATMENT GROUPS 1 30 mins control 4 60 mins control 2 30 mins A.A. 100 5 60 mins A.A. 100 3 30 mins A.A. 200 6 60 mins A.A. 200

Fig : Antinociceptive effect of the aqueous stem bark extract of A.A. Acetic acid induced nociception. Data are means with vertical error bar indicating S.E.M; n=4; ANOVA, with student t test, *P <0.05 (significant compared to control). ANTINOCICEPTIVE ACTIVITY The antinociceptive activity of A.Andogensis was investigated using the acetic acid writhing test and the formalin pain test. The result of both studies strongly suggests that the aqueous extract of A.Andogensis has remarkable antinociceptive effect. The result of the acetic acid induced abdominal constrictions showed that the extract produced a dose dependent antinociceptive activity but was not significantly affected by the pretreatment time. Thus at the dose range of 100-200mg/kg, the extract significantly protected the animals against acetic acid induced writhing. Acetic acid induced writhing is a model of visceral pain (vyklicky, 1979). Writhing induced by chemical substances injected ip are due to sensitizations of nociceptors by protaglandins. This test is useful for evaluation of mild analgesic non-steroidal anti-inflammatory compounds (Ferreira and Vane 1974; Berken Kopf and Weichman, 1988). Acetic acid induced constrictions are a highly sensitive and useful test for analgesic drug development but not a selective pain test. It gives false positives with sedatives, muscle relaxants and other pharmacological activities (Elisabetsky et al., 1995) The formalin test however is sensitivie to non steroidal anti-inflammatory drugs and other mild analgesics. The test possesses two distinct phases; the earlier phase reflects inflammatory pain (Hunskaar and Hole 1997; Elisabetsky et al. 1995) The extract of A.Andogensis produced significant analgesic effect in both phases. this probably indicates that the analgesic effect of the extract was mediated by both neurogenic and inflammatory mechanisms. CONCLUSION From the results obtained in this study it can be concluded that A. andogensis does not possess any significant anti-inflammatory and antipyretic properties in this study. Also it can be said that the extract is safe at dose tested in oral formulations.

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H .Ighodaro and S.O Bello: Continental J. Pharmacology and Toxicology Research 4 (1): 11 - 17, 2011

RECOMMENDATIONS Based on the shortcomings/findings of this research, it will be pertinent to make the following recommendations i. The phytochemical analysis of the extract needs to be carried out so that the various classes of natural constituent in the extract can be evaluated. ii. The active principles responsible for the antinoceiceptive effect of A.andogensis needs to be investigated so that the mechanism of action can be elucidated. REFERENCES Akil, Shiom, H., and Mathews, J. 1985. Induction of the intermediate Pituitary by stress: synthesis and release of a non Opioid form beta-endorphin. Science, 227:424-426. Asuzu, I.U., Anaga, A.O., 1991. Pharmacological screening of the aqeous extract of Alstonia boonie stem bark. Fitoterapia 63, 411-417. Amos, S., Chindo, B., Edomond, I., Akah, P., Wambebe, C., Gamaniel, K., 2002. Anti-inflammatory and antinociceptive effects of Ficus Platyphylla in rats and mice. Journal of Herbs, Spices and Medicinal Plants 9, 47 53. Baiyewu, O (2001) Tradtional Medicine and Healthcare; Nigeria Journal of Psychaiatry Vol. 1 No. 5 Pp 284 288. Borda, I.T., and Koff, R.S., eds NSAIDS: A profile of adverse effects. Hanley and Belfus, Philadelphia, 1992. Cervero, F. and Laird, J.M.A. (1991) one pain or many pains? : a new look at pain mechanisms. NIPS 6, 268273. Cervero F. and Laird MA (1996): The neurophysiology of Pain; in International Practice of Anaesthesia, Vol: 1 pg 3/4/5 Dalziel, J.M., (1937), the useful plants of West tropical Africa. The Crown Agents for the Colonies. Royal Botanical Garden, Kew, p. 242. Dubuisson, D., Dennis, S.G., (1977). The formalin test: a quantitative study of the analgesic effect of morphine, meperidine and brain stem stimulation in rat and cats. Pain 4, 161-174. Eltkin, L.N., (1997). Antimalarial plants used by the Hausas in Northern Nigeria. Tropical Doctor supplement 1, 1-5. Exell and Torre, (1989), Notes on A. andogensis: in Savanna plants, An illustrated Guide by Shashina G. Elisabetsky, E., Amador, T.A., Albuquerque, R.R., Nunes, D.S Carvalho, A.C.T., (1995). Analgesic activity of Psychotria colorata (Wild ex R. & S.) Muell Arg. Alkaloids. Journal of Ethnopharmacology 48, 77-83. Fernando C. and Laird M.A. (1996): The neurophysiology of pain in international practice of Anaesthesia vol. Pg 1/2/5 Farnsworth, N.R., and Saejarto, D.D. , (1988). Global importance of Medicinal plants. In conservation of medicinal plants, ed Olayiwola Akerele, Pp 25 40. Gabriel, S.E., Jaakkimaine L., and Bombardier, C. Risk for serious gastrointestinal complication related to use of non steroidal anti-inflammatory drugs. A meta-analysis. Ann. Intern. Med., 1991, 115: 787-796. Goodman and Gilman (2001) The pharmacological basis of therapeutics, 10th ed. McGraw Hill, pp 687-714. Graham, D.Y. , White, R.H; Moreland, L.W. , Schubert, T.T. , Katz, R; Jaszzewski, R. , Tindall, E; Tri adafilopoylos, G. , Stromatt, S. C; and Teoh, L.S. Duodenal and gastric ulcer prevention with misoprostol in arthritis patients taking NSAIDS. Misoprostol study group. Ann. Intern. Med. , 1993, 119: 257 262.

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Wambebe C (1998) Development and production of standardized Phytomedicine. West African Journal of Pharmacology Vol. 12. No. 2: 12-18 Zagon, I.S., Goodman, S.R., and McLaughlin, P. J. characterization of Zeta: a new opioid receptor involved in growth. Brain Res. , 1989, 428: 297 305. Received for Publication: 18/05/2011 Accepted for Publication: 24/07/2011 Corresponding Author H .Ighodaro Department of Clinical Pharmacy, Usmanu Danfodiyo University,Sokoto. E.MAIL: harrisonighodaro@yahoo.com

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