Asian Journal of Medical Sciences 2(3): 121-126, 2010 ISSN: 2040-8773 M axwell Scientific Organization, 2010 Submitted Date:

: April 09, 2010 Accepted Date: April 22, 2010

Published Date: June 25, 2010

Reversal of Coumarin-Induced Toxicity by the Extracts and Fractions of Ageratum conyzoides

Peter A. Akah, Chinyelu C. Osigwe and C hukwuemeka S. N woru Departm ent of Pharmacology and Toxicology , Facu lty of Pharmaceutical Sciences, University of Nigeria, Nsukka, 410001 , Enugu State, Nigeria
Abstract: This study examined the antidotal effects of the methanol extract (MeOH ) and fractions of Ageratum conyzoides L. (Asteraceae) on couma rin-indu ced toxicity in ra ts. The effects o f these e xtracts o n cou marin acute toxicity (LD 5 0 ) and mortality rate were assessed. The LD 5 0 of coumarin was increased by as much as 487.2 and 238.1% when co-administered with the MeO H extract (1 g/kg) and the methanol fraction, (MF) (1 g/kg) respectively. The MeOH extract and MF significantly (p # 0.05) reduc ed co umarin-indu ced m ortality to 0 and 40%, respectively when compared to the 100% mortality recorded for the control group. Vitamin K and activated charcoal, which were used as standard antidotes, also produced similar reductions in percentage mortality, but MeOH extract and M F performed better. The Hexane Fraction (HF) and the ethyl acetate fraction, (EF) did not significantly (p>0.05) affect coumarin-induced mortality. Maximal antidotal effect was recorded when MeO H extract was given within 3 h of coumarin administration. The MeO H extra ct a nd the MF demonstrated antidotal activities against acute poisoning b y cou marin in rodents. On their own, the extracts did not produce any gross pathological change in the animals, and could therefore be harnessed as use ful antidote in coumarin poisoning. Local activities of the phytoconstituents of A. conyzoides on the GIT which limits absorption and h aem orrhagic damag es are believed to be responsible for the reversal of the coumarin toxicity. Key w ords: Ageratum conyzoides, antidote, coumarin, coumarin- toxicity, poisoning INTRODUCTION Couma rin is a toxic chemical compound (benzopyrone) found in many plants, notably in high concentration in the tonka bean, woodruff, mullein, and sweet clover (Majerus and T ollefsen , 2006 ). Toxicity from coumarins was first observed in animals. Livestock that were fed moldy spoiled sweet clover hay died from a p r e v i o u s l y u n k n o w n h e m o r r h a g i c d i s o rd e r . Bishydroxycoumarin, the active ingredient responsible for this hemorrhagic disorder, was discovered by Campbell and Link (1941). Bishydroxycoumarin is formed when fungi in moldy sweet clover oxidize coumarin to 4hydroxycoumarin, an anticoagulant. Bishydroxycoumarin was synthesized and used clinically one year later as an oral anticoagulant under the American trade name dicumarol (Campbell and Link, 1941 ). Altho ugh only somewhat dangerous to humans, coumarin is a potent rodenticide. Rats and o ther rod ents larg ely metabo lize it to 3, 4-coumarin epoxide, a toxic compound that can cause internal hemorrhage and death (Cain et al., 1997). Humans largely metabolize it to 7-hydroxycouma rin, a compound of lower toxicity (Kaminsky and Zhang, 1997). Ageratum conyzoides L. (Asterace ae), comm only called tropical whiteweed, Billy goat weed, or floss flower and also synonymo usly known as A. latifolium L., A. cordifolium L., A. album L.and A. odoratum L. is a common tropical annual herbaceous weed which grows up to a height of 2.5 feet with little white flowers and opposite leaves (Dalziel, 1958). The stem is often reddish and has long white hairs. The weak aromatic unpleasant smelling leaves are also covered with fine hair. A. conyzoides is thought to be native to South America, but ha s sprea d to the tropical and subtropical regions. A. conyzoides is used in folk medicine for various ailments in Africa (Almagboul et al., 1985), Asia, and South America (Ekundayo et al., 1988). The common medicinal uses of A. conyzo ides in West Africa is for wound healing, especially burns (Watt and BreyerBrandwijk, 1962). Ageratum is also used as an antiepileptic, antihelminthic, and as an insect repellent (Adodo, 2004). A. conyzoides has also been reported in the treatment of Human Immunodeficiency Virus (HIV) and acquired immunodeficiency syndrome among the Igede community of Benue State, Nige ria (Igoli et al., 2005). Some pharmacological properties have also been associate with A. conyzoides such as haem ostatic (Akah, 1988), muscle relaxant (Achola et al., 1994), antinflamm atory (M aglhaes et al., 1997), antimicrobial (Almagboul et al. , 1985 ; Akinyem i et al., 2005;

Corresponding Author: Peter A. Akah, Department of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka, 410001, Enugu State, Nigeria 121

Asian J. Med. Sci., 2(3): 121-126, 2010 Amadi et al., 2007), glucose lowering effect (Nyunai et al., 2006 ), and gastroprotective activity (Shirwaikar et al., 2003). A variety of phytochemical compounds have been identified in the tropical whiteweed. These include alkaloids, coumarins, essential oils, flavonoids and tannins (Horiet et al., 1993; Adeleke et al., 2002). The essential oil of A. conyzoides is composed of phenols, phe nolic esters, c oum arin, $ -cariophylene and ageratochrome (Oliver-Bever, 1986; Vera, 1993). The African species of A. conyzoides contain eugenol as the major essential oil (Ekundayo et al., 1988). In Igboukw u Comm unity of South Eastern Nigeria, the plant is popularly used to arrest bleeding from fresh wounds and to stop epitasis. Also victims of various poisons are made to chew copious quantity of the fresh leaves of A. conyzoides, a practice that has been reputed to result in high degree of antidotal efficacy. This study is aimed at evaluating the antidotal effects of the leaf extracts of A. conyzoides on co uma rin-indu ced toxicity in rodents. MATERIALS AND METHODS Anim als: Adult albino rats (100-200 g) and adult albino mice (21-25 g) of both sexes obtained from the Laboratory Animal Facility C entre of the Department of Pharmacology and T oxico logy, U niversity of Nigeria, Nsukka were used in the study. The animals were housed under standard conditions (252C and 12 h light/dark cycle). The rats and mice we re maintained on standard pellets (Livestock feed ), and allowe d unrestricted access to drinking water. The use and ca re of laboratory animals were conducted in accordance w ith the internation ally accepted best practices as co ntained in the E uropean Comm unity guidelines (EEC D irective of 1986; 86/609/E EC). These stud ies were carried ou t in 2007 in the Department of Pharmacology and Toxicology, University of Nigeria, Nsukka (UNN ), Nigeria. Preparation of plan t ma terial: The fresh leaves of A. conyzoides were co llected in Igbouk wu, So uth Eastern Nigeria in the month of July, 2005 and were authenticated by Mr. J.M.C. Ekekwe, of the Department of Botany, University of Nigeria, Nsuk ka, wh ere a voucher specimen has been deposited. The leaves were air-dried under shade and then pulverized with a laboratory mill. The leaf powder (3 kg) was divided into two portions. The first portion (1 kg) was extracted with methanol in a soxhlet extractor to obtain the methanol extract (MeOH ) and the second portion (2 kg) was successively extracted with nhexane (HF), ethylacetate (EF) and methanol (MF) using soxhlet extractor to obtain three solvent fractions. Preliminary phytoche mical studies were carried out on the leaf powder and on the extracts using standard procedures (Harbo urne, 1984). Acu te toxicity (LD 5 0 ) test of the extracts and fractions: The acute toxicity profile of the extracts and fractions of A. conyzoides was estimated in mice using method described by Lorke (1983). Briefly, the tests involved two phases. The first phase is the d etermination of the toxic range. The mice were placed in three groups of three mice each. The groups were administered the ex tracts intraperitoneally (ip) at the doses of 10, 100, and 1000 mg/kg respectively. The extracts were solubilized in a solution of 3% Tw een 8 0 in water. The treated mice were observed for 24 h for the number of deaths. The death pattern in the first phase determined the doses used for the seco nd ph ase ac cording to Lorke (1983) estim ation. In this phase, four groups of one mouse each w ere used for each extract tested. Each group received suitable doses of the extract i.p. as predetermined in the first phase and the animals observed for lethality or signs of acute intoxication for 24 h. The LD 5 0 was calculated as the geometric mean of the highest non-lethal dose and the least toxic dose (Lorke, 19 83). Antidotal tests: Letha lity of coumarin: The acute toxicity of cou marin was assessed by determining its LD 5 0 as described in the previous section (Lorke, 1983). Effect of the extracts on the lethality (LD 5 0 ) of couma rin in mice: The LD 5 0 of coumarin was reevaluated after the animals were pretreated orally w ith graded doses (100, 250, 500, 1000, and 5000 mg/kg) of the extracts. Effect of the extracts on the letha lity of coum arin in rats: The effect of A. conyzoides extracts on coumarin toxicity was also studied in eight groups of rats (n/gp = 6). The animals were fasted for 12 h prior to the ex perim ent, but were allowed access to drinking water. The minimum lethal dose of coumarin (600 mg/kg) as previously determined was administered orally to the animals in the groups. Five groups were immediately given graded doses of the extracts (100-5000 mg/kg) orally while three groups were treated orally (in two divided doses) w ith activated charcoal (6000 mg/kg), Vitamin K 1 (5 mg/kg), vehicle (3% Tween 80, 1 ml/rat) respectively. The animals were then observed for 48 h and the number of deaths recorded as percentage mortality. Time-dependent effect of the methanol extract on couma rin toxicity: The MeO H extract, which was found most active in the previou s procedures, was use d in this study. Six groups (n/gp = 6) of albino rats were employed. The rats were administered orally once with 600 mg/kg of coumarin followed by 1000 mg/kg of the MeO H extract at 0, 1, 3, 6, 12, and 24 h to the different groups respectively. The animals were observed for 48 h for deaths and the percentage mortality recorded.

122

Asian J. Med. Sci., 2(3): 121-126, 2010

Table 1: Phy tochemical constituents and ex tractive yield of A. conyzoides Extract Yield (g) # Ph ytoc ons tituen ts MeOH 123 (12.3) Alkaloids, saponins, tannins, glycosides, flavonoids, resins, protein, carbohyd rate, reducing sugars, steroids, and terpenoids HF 70 (3.5) Alk aloid s, flav ono ids, res ins, ca rbo hyd rate EF 86 (4.3) Alk aloid s, flav ono ids, res ins, ca rbo hyd rate MF 134 (6.7) Alka loids, sapo nins, tann ins, glyco sides, flavo noids , resins, protein, ca rboh ydrate, redu cing su gars # : Perc enta ge y ield in pare nthe sis Table 2: Effect of the ex tract and fractions of A. conyzoides on the LD 5 0 of coumarin Dose (mg/kg) of extract LD50 of coumarin (mg/kg) ------------------------------------------------------------------------------------------------------------------------------MeOH HF EF MF 100 367.42 (26.8) 282.84 (-2.4 ) 289.83 (0.0) 288.53 (-0.5) 250 471.12 (62.6) 289.83 (0.0) 288.53 (-0.5) 400.00 (38.0) 500 1264.91 (336.4) 113.14 (-61.0) 227.60 (-21.5) 565.69 (95.2) 1000 1319.09 (355.1) 80.0 (-72.4) 200.0 (-31.0) 979.80 (238.1) Percentage increases in the LD 5 0 of co um arin (2 89.8 3 m g/kg ) are sh ow n in p aren thesis

Statistical analysis: Results obtained were analyzed using one way analysis of variance (ANO VA) followed by Fischer LSD post hoc test and w ere expressed as mean standard error of the mean (SEM). Differences between paired mean observations were regarded as significant at p # 0.05. The SPSS statistical software (version 11.0) was used. RESULTS Phytochemical test: Phytochemical studies showed that the leaves of A. conyzoides contain alkaloids, tannins, flavonoids, saponins, resins, carbohydrate, protein, fats and oil. The occurrence of these metabolites in the different extracts and fractions are shown in Table 1. Acu te toxicity (LD 5 0 ) of the extracts of A. conyzoides: The intraperitoneal (ip) LD 5 0 were estimated as 1265, 2150, and 3 810 mg/kg for HF, EF, and MF, respectively. The MeO H extract administered up to a dose of 5 g/kg did not cause any m ortality in the mice. Letha lity (LD 5 0 ) of coumarin: The intraperitoneal (ip) LD 5 0 of coumarin was estimated to be 289.83 m g/kg. The animals that died within 24 h of the experiment showed signs of coumarin anticoagulation toxicity. These signs include fatigue, anorexia, haematuria, bruises, melena, reduced activity and eventual death. Effect of the extract and fractions on the LD 5 0 of coumarin: There was a dose-related increase in the LD 5 0 of coumarin when co-administered with the MeOH extract and MF (Table 2). The LD 5 0 of coum arin was increased for as much as 487.2 and 238.1% when the animals were co-administered with the methanol extract (5 g/kg) and the methanol fraction (1 g/kg) respectively. Generally, the HF caused a dose-dependent decrease in the LD 5 0 of coumarin at doses above 500 mg/kg. The EF did not affect the LD 5 0 of coumarin at doses below

Tab le 3: Effect of the extract and fractions of A . conyzoides on cou mar in-ind uce d m ortality Treatment Dose (mg/kg) M o rta lity (% ) MeOH 100 60 250 60 500 20 1000 0 5000 0 HF 100 100 250 80 500 100 1000 NT 5000 NT EF 100 100 250 100 500 80 1000 100 5000 NT MF 100 80 250 60 500 40 1000 40 5000 NT Activated charcoal 6000 60 Vitamin K 5 mg/kg 40 Tween 80 1 ml 100 N T represents the doses that were not tested considering the LD 5 0 of the respective extract

1000 mg/kg, but caused significant decreases at doses above 1000 m g/kg (Tab le 2). Effect of the extract and fractions on coum arininduced mortality: The study showed that only MeOH extract and MF exhibited significant (p # 0.05) antidotal effect as indicated by the decrease in percentag e mo rtality recorded when the rats were pretreated with these extracts (Table 3). Vitamin K and activated charcoal, which were used as standard antidotes in the study, produced similar decreases in percentage mortality, but MeOH extract and MF performed better. The HF and EF did not affect couma rin-indu ced m ortality significantly when compared to the negative control. The MeOH extract performed better than all other extracts tested (Table 3).

123

Asian J. Med. Sci., 2(3): 121-126, 2010

Tab le 4: T ime- dep end ent e ffects M eO H e xtrac t of A . con yzo ides o n co um arin to xicity M o rta lity (% ) -------------------------------------------------------------------------------------------------------------------------Treatment Dose (mg/kg) 0h 1h 3h 6h 12 h 24 h MeOH 1000 0 0 0 60 100 50 Activated charcoal 6000 40 40 60 80 100 100 Vitamin K 5 20 20 40 20 40 40

Time-dependent effect of MeO H extract on co um arin toxicity: The maximal antidotal effect was recorded when the MeO H extract was given within 3 h of coum arin administration, thereafter there was a gradual loss of the protection as shown by increased mortality rate (Table 4). DISCUSSION In this study, the methanol extract and the methanol fractions of Ageratum conyzoides exhibited antidotal properties against coumarin-induced toxicity since they caused significant (p # 0.05) increase in the LD 5 0 of coumarin when co-adm inistered. Althoug h both sha re some similarities in phytochemical constituents, the methanol extract show ed hig her an tidotal activities than the methanol fraction. Higher levels of active constituents, which are responsible for the antidotal pro perties, could explain this observation. It could also be explained by the better tolerability of the MeOH extract by the animals as compared to MF . In our tox icity experiment, the methanol extract could be considered safe, since the mice tolerated dose up to 5 g/kg b ody weight w ithout m ortality or signs of acute intoxication (Lorke, 1983). It is interesting to observe that the antidotal effect of the methanol extract of A. conyzoides was quite com parab le to that shown by activated charcoal, a loc ally acting adsorbent-antidote com mon ly used clinica lly in acute substance-intoxication. We also performed a timeof-intervention study to assess the antidotal effect over different time of administration. In the study, amelioration of coumarin toxicity by the extract of A. conyzoides was only remarkable when it was given not later than 3 h of coumarin administration. Early adm inistration before the third hour of cou marin adm inistration resulted in zero mortality rates in the methanol extract treated group. The mortality rate incre ased with increase in the elapsed time before intervention. This result suggests that the extract may have a local antidotal action, acting early to prevent absorption or to protect gastric mucosa against hem orrhagic lesions. Coumarin is known to be absorbed slowly, which could take up to the 3 h (Hoult and Paya, 1996). Administration of the extract at 6 h after coumarin did not produce significant antidotal protection. This also supports our earlier assertion on a possible local action even though more studies is needed to rule out possible systemic effects. Unlike the methanol extract of A. conyzoides, the antidotal effectiveness of Vitamin K was more pronounced w hen given 6 h after coum arin

administration. Couma rins inhibit hepa tic synth esis of the Vitam in K-dependent coagulation factors II, VII, IX, and X and the anticoagulant proteins C and S (Colman et al., 2005). Vitam in K is a cofactor in the synthesis of these clotting factors. The Vitamin Kdependent step inv olves carbo xylation of glutamic acid residues and requires regeneration of Vitamin K to its reduced form. C oum arins an d related com pounds inhibit Vitam in K 1 -2, 3 epoxide reductase, preventing Vitamin K from being reduced to its active form (Colman et al., 2005). The result of the stud y with Vitam in K in the reversal of cou marin toxicity is therefore systemic. The administration of the methanol extract and the methanol fraction of A. conyzoides increased the LD 5 0 of coum arin significantly (p # 0.05), this dem onstrates the potent antidotal effect of the extracts in coumarin poisoning. We have estimated the oral LD 5 0 of cou marin to be 289.83 mg/kg, quite comparable to an earlier estima te of 290 mg/kg (Hazleton et al. , 1956).Resin-drug complexes have been used extensively in drug delivery systems to delay absorption and achieve sustained release characteristics (Woodw orth et al., 1992; Wen et al., 1997; Um amah eshw ari et al., 2003). It is possible that the abundance of resinous substances in the methanol extract and fraction could be responsible for the antidotal activity since resins are capable of complex formation with wide range of molecu les which could d elay absorp tion. The presence of resins may not fully explain the reversal of coumarin toxicity by the extracts since the hexane and ethyl acetate fractions which also contain resins did not show remarkable antidotal effects. Coumarin is a plant glycoside and tann ins are k now n to precip itate glycosidal drugs and poisons (Hoult and Paya, 1996). It is therefore possible that the presence of tannins in the methanol extract and fraction could also partly or wholly explain the observed antidotal activity. The major site of anticoagulation in couma rin is the gastrointestinal tract leading to haemorrhagic lesions. Antioxidants have been show n to be gastroprotective and to alleviate such haemorrhagic damages (Ligumsky et al. , 1995 ; Mahmood et al. , 2005). Flavonoidal compounds isolated from A. conyzoides and the aqueous extract from the plant have been show n to possess significant antioxidant properties in a previous study (Gonzalez et al., 1991). This effect could also have contributed to the reduced mortality and increased LD 5 0 caused by A. conyzoides in coumarin -treated rats.

124

Asian J. Med. Sci., 2(3): 121-126, 2010 In an earlier study (Akah, 198 8), the aqueous extract of A. conyzoides was reported to have h aem ostatic activity by two mechanisms. F irstly, the precipitation of blood constituents as a result of its high tannin content, which results in the formatio n of an artificial clot to arrests bleeding. Secondly, the vasoconstrictive action of the extracts of A. conyzoides was also identified. These same mechanisms may also occur locally in the gastrointestinal tract to protect the animals. Our preliminary study had identified the gastrointestinal tract as the major site of coumarin haem orrhagic toxicity. The methanol extract and the methanol fraction of the leaves of A. conyzoides demonstrated antidotal activities against acute poisoning b y cou marin in roden ts and could therefore be harnessed as a useful antidote in coumarin poisoning. A combination of several mechanisms which are believed to be localized in the GIT may have contributed in the reversal of the couma rin toxicity. T his study could offer explanation to the successes claimed by herbalists in their use of Ageratum conyzoides extracts as a general antidote in the treatment of different kinds of poisoning. CONCLUSION The results of this study revealed that the extracts of Ageratum conyzoides demonstrated potent antidotal activity against acute coum arin po isoning. Local activities of the second ary metabolites p resent in the ex tract, which limits absorption, and hemorrhagic damage of the gastrointestinal tract may be responsible for the reversal of cou marin toxicity. REFERENCES Achola, K.J., R .W . Munenge and A .M. Mwaura, 1994. Pharmacological properties of root and aerial parts extracts of Ageratum conyzoides on isolated ileum and heart. Fitoterapia, 65: 322-325. Adeleke, A.K ., P. W interhalter, M.A. Adew ale, K. Holger and B. Bernd, 2002. Chromenes in Ageratum conyzoides. Flav. Frag J., 17 : 247-2 50. Adodo, A., 20 04. N ature P ower-a C hristian A pproach to Herbal Med icine. 3rd Edn., Lagos Generation press Ltd., pp: 139-143. Akah, P.A., 1988. Haemostatic activity of aqueous leaf extract of Ageratum conyzo ides L. Int. J. Crude Drug Res., 26: 97-101. Akinyem i, K.O., O. Oladapo, C.E. Okwara, C.C. Ibe and K.A. Fasure, 2005. Scre ening of crud e extracts of six medicinal plants used in south-west Nigerian unorthodox med icine for anti-methicillin resistant Staphylococcus aureus activity. BMC Compl. Altern. Med., 5: 6. Almagboul, A.Z ., A.A . Farroq and B .B. Tyagi, 1985. Antimicrobial activity of certain Sudanese plants used in folkloric medicine: Screening of antibacterial activity Part II. Fitoterapia, 56: 103-109. Am adi, E.S., C.A. Oyeka, R.A. Onyeagba, O.C. Ugbogu and I. Okoli, I 2007. Antimicrobial screening of Breynia nivosus and Ageratum conyzoides. J. Biol. Sci., 7: 354-358. Cain, D ., S.M . Hutson and R . Wallin, 199 7. Assem bly of the warfarin-sensitive vitamin K 2, 3- epoxide reductase enzy me comp lex in the endo plasm ic reticulum membrane. J. Biol. Chem., 272: 29068-29075. Campb ell, H.A . and K .P. Link, 1941. Studies on the hemorrhagic sweet clover disease. IV. The isolation and crystallization of the hemorrhagic agent. J. Biol. Chem., 138: 21-33. Dalziel, J.M. and J. Hutchinson, 1958. Flora of West Tropical Africa. Vo l. 1, Part II, 2nd Edn., Crown A g e n t s f o r O v e r s e e s G o v e r n m e n t a nd Administration, London, pp: 728. Colman, R.W ., 2005. Anticoagulant Therapy for Major Arterial and Venous Thromboembolism. In: Colman, R.W., V.J. Marder, A.W. Clowes, J.N. George and S.Z. Goldhaber (Eds.), Hemostasis and Thrombosis: Basic Principles an d Clinical Practice. Lippincott Williams and Wilkins, pp: 1676-1684. Ekundayo, O., S. Sharama and E.V. Roa, 1988. Essential oil of Ageratum conyzoides. Planta Med., 54: 55-57. Gonzalez, A.G., G. Thomas and P. Ram, 1991. C h r o m e n e s f ro m A g e r a t u m c o n y z o i d e s . Phytochemistry, 30: 1137-1139. Harbourne, J.B., 1984. Phytochemical Methods. A Guide to Modern Technique of Plant Analysis. 2n d Edn., Chapman and Hall, London, pp: 282. Hazleton, L.W., T.W. Tusing, B.R. Zeitlin, R. Thiessen Jr and H.R . Murer, 19 56. Toxicity of cou marin . J. Pharmacol. Exp. Therap., 118: 348-358. Horiet, T., H. Tominaga and Y. Kawamura, 1993. Revised structure of a natural flavone from Ageratum conyzoides. Phytochemistry, 32: 1076-1077. Hoult, J.R. and M. Paya, 1996. Pharmacological and biochemical actions of simple coumarin: Natural products with therapeutic potential. Gen . Pharm acol., 27: 713-722. Igoli, J.O., O.G. O gaj, T.A . Tor-A nyiin and N .P. Igoli, 2005. Traditional Medicine Practice Amongst the Igede People of Nigeria Pa rt II. Afr. J. T rad. C omp l. Altern. Med., 2: 134-152. Kaminsky, L.S. and Z.Y. Zhang 1997. Human P450 metabolism of warfarin. Pharmacol. Ther., 73: 6774 . Ligumsky, M., M. S estieri, E. O kon and I. G insburg, 1995. Antioxidant inhibits ethanol indu ced g astric injury in the rat. Role of manganese, glycine and carotene. Scand. J. Gastroenterol., 30: 854-860.

125

Asian J. Med. Sci., 2(3): 121-126, 2010 Lorke, D., 19 83. A new appro ach to practical acute toxicity testing. Arch. Toxicol., 54: 275-287. Mag lhaes, J.F.G., C.F.G . Viana, A .G.M .A. Junior, V.G. Moraes, R.A . Ribeiro and M.R. V ales, 1997. Analgesic and a ntiinflammtory activities of Ageratum conyzoides in rats. Phytother. Res., 11: 183-188. Mahm ood, A., K .K. Sidik, I. Salmah, K.A.R.Suzainur and K. Philip, 2005. Antiulcerogenic activity of Ageratum conyzoides leaf extract against ethanolinduced gastric ulcer in rats as animal model. Int. J. Mol. Adv. Sci., 1: 402-405. Majerus, P.W. and D.M. Tollefsen, 2006. Anticoagulant, Thro mbo lytic and Antiplatelet Drugs. In: Brunton, L.L., J.S. Lazo and K.L. Pa rker (Eds.), Goodman and Gilman's The Pharmacological Basis of Therapeutics. 11th Edn., Mc Graw-H ill, New Y ork, pp: 1467-1488. Nyunai, N., N. Njikam, C. Mounier and P. Pastoureau, 2006. Blood glucose lowering effect of aqueous leaf extracts of Ageratum conyzoides. Afr. J. Trad. Compl. Altern. Med., 3: 76-79. Oliver-Bever, B., 1986. Medicinal Plants in Tropical W est Africa. Cambridge University Press, Cambridge, pp: 132. Shirwa ikar, A., P.M. Bhilegaononka, S. Malini and K.J. Sharath, 2003. The gastroprotective activity of the ethanol extract of Ageratum conyzoides. J. Ethnopharmacol., 86: 117-121. Um ama hesh wari, R.B., S. Jain and N.K. Jain, 2003. Anew approach in gastroretentive drug delivery System using cholestyramime. Drug Delivery, 10: 151-160. Vera, E.J., 1993. Chemical composition of essential oil of Ageratum conyzoides L. from Reunion. Flav Frag J., 8: 256-260. W att, J.M. and M.G. Breyer-Brandwijk, 1962. The M edicinal Plants of South and Eastern Africa. 2nd Edn., E & S Livingstone Publisher, London, pp: 197-198. W en, B., M.P. Ramsay, H . Scheurer, V. Dokuzovic and V. Lam, 1997. An titussive Drugs Delivered by Ion Exchange Resins. Retrieved from: http//www. pharmacast.com/patents. (Accessed date: September 09, 2007 ). W oodworth, J.R., T .M . Ludden, L.K. Ludden, A .M .M . Shepherd and K.S. Rotenberg, 1992. Comparative bioavailability of a sustained release ion-exchange hydralazine product with a potassium cation exchange. J. Pharm. Sci., 81: 541-548.

126