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1 Serial No University of Nigeria Virtual Library Author 1 Author 2 Author 3 Title Keywords Description I. U. ASUZU U. O.OKORO A. O. ANAGA THE EFFECTS OF THE METHANOLIC LEAF EXTRACT OF TRICILISIA SUBCIRDATA IKUVE ON SMOTH MUSCLES.NTIHISTAMINE, CONTRACTIONS, EZIZE, H 2 - RECEPTORS, MEDICINAL PLANT, OGWU-AJU, SMOOTH MUSCLES, TIETIE, UTERINE Category Publisher VETERINARY PHYSIOLOGY AND PHARMACOLOGY THE HAWORTH PRESS, INC. Publication Date 1994 Signature

2 The Effects of the Methanolic Leaf Extract. of Triclisia subcordata Oliv. on Smooth Muscles I. U. Asuzu U. 0. Okoro A. 0. Anaga ABSTRACT. The methanolic leaf extract of Triclisia subcordata obtained by macerating dried and pulverized leaves in 50 percent methanol f a 48 hour produced relaxant effects in the rabbit jejunum and rat fundus (stomach), but produced contractile effects in the rat uterus. The effective concentration (ECso 0.07 mg/ml) of the extract in the rat jejunum was determined from a plot of the percent relaxation of the tissue against the log concentration of the extract. No observable effects in the guinea pig ileum were produced by the extract. Effects of extract on jejunum, rat fundus, and uterus were antagonized by histamine. The effects of the extract appeared to be mediated through H2- receptor blocking activity. KEYWORDS. Antihistamine, contractions, Ezize, H2-rece-, nal plant, Ogwu-aju, smooth muscles, Tielie, uterine. medici- I. U. A&u, U. 0. Okoro, and A. 0. Anaga are affiliated with the Department of Veterinary Physiology and Pharmacology, University of Nigeria, Nsukka, Nigeria The authors wish to thank Dr. E. N. Muogbo, who gave the plant sample used for the present study and members of the Department of Veterinary Physiology and Pharmacology for technical assistance. Journal of Herbs, Spices & Medicinal Plants, Vol. 2(4) 1994 O 1994 by The Haworth Press, Inc. All rights reserved. I1

3 12 JOURNAL OF HERBS. SPICES & MEDICINAL PLANTS INTRODUCTION Triclisia subcorduta (Menispermaceae), a medicinal plant commonly known as Tietie in Nigeria, grows in tropical and subtropical areas of West Africa and in a few temperate regions (14). Because of the fibrous climbing stem, the plant, which is also known in Nsukka as Ogwu-aju (meaning antidizziness drug), in Ohafia as Ezize (meaning benign skin growth), and in Umuoji, in the eastern part of Nigeria, as Ike Mbekwu (meaning, from the shape of the leaf, like the anal region of the turtle), frequently serves as rope used for tying purposes. The importance of the species, however, rests with the medicinal applications (4). including the use of the root extract for treatment of snake bite, ulcer, diarrhea, malaria, pyorrhea, swelling of extremities, anemia, joint pains, cancer, rheumatic pains, and hypertension in Nigeria (7). In Abeokuta and Akure regions in western Nigeria, Ihe plant extract is used for the treatment of strangulated hernia, gastric fever, and various mensmal irregularities (12). Extract of the root is also used as an arrow poison (7). The bisbenzylisoquinoline alkaloid fricordatine and the alkaloid tetrandrine have been isolated from Triclisia subcordata (5, 13). Other compounds in the plant with known pharmacological properties include fangchinoline which has anticancer and curare-like activity and cosculine rnagnoflaine and palmitine which have antibacterial adivity (8,9). Tetrandrine has been reported to have hypotensive effects that are mediated through a parasympathetic mechanism in the cat (3.6). The present study investigated the pharmacological effects of the methanolic leaf extract of Triclisia subcordata on various smooth muscles to establish a phmacological basis of folklore medicinal applications. MATERIALS AND METHODS Plant material. The leaves of Triclisia subcorduta (Oliv.) were collected during November, 1991, in Nsukka Town, Enugu State, Nigeria, from a plant identified by Mr. A.O. Ozioko of the Botany Department, University of Nigeria, Nsukka. The leaf material was subsequently dried in the sun and then pulverized with a mortar and pestle. Extraction. Pulverized leaf tissue, 50 g, was macerated in 200 ml of 50 percent methanol and left for 48 h with intermittent, vigorous shaking. The mixture was then filtered through No. 1 Whatmann filter paper and the filtrate concentrated at 40 C. 210 millibars using a vacuum rotary evaporator. Total solids were recovered and compared with the weight of the starting material.

4 Asuzu, Okoro, and Anaga 13 Test animals. Male rabbits ( g), guinea pigs of both sexes ( g), and maiden inbred albino rats ( g) were used for the experiments. The animals, purchased from the laboratory animal units of Veterinary Medicine and Pharmaceutical Sciences, University of Nigeria, Nsukka, were kept in clean wire mesh cages and, except for fasting times, fed ad lib with standard animal ration. Chemicals and -solutions. Acetylcholine (Dumex), histamine (Fisher), dieth ylstilbestrol (May & Baker), ox ytocin (Scanpharm), methanol (Biomec Chemical Industries, Ltd) and olive oil (BDH), were used in the experiments. Isolated tissues were bathed in Tyrode, De-jalon or Krebs solution as appropriate. Tissue preparation. A segment of guinea pig ileum (2-3 cm long), taken from stunned animals which had been fasted for 24 h, was suspended in an organ bath containing Tyrode solution maintained at 32 C and aerated with air generated by an air pump (2). The tissue in the bath was allowed to stabilize for 15 min before introduction of any drug or plant extract. Acetylcholine (10 Uml) and various concentrations (0.05, 0.1, 0.2, 0.4 mglml) of the extract were tested on the tissue with responses amplified (2.5 x ) through an isotonic T2 transducer and recorded with a Harvard oscillograph. Rabbit jejunum was taken from animals fasted for 24 h and the ileum was isolated and prepared following the procedures described for the guinea pig ileum. The ileum was aerated in solution with 95 percent O2 and 5 percent CO2 at 32 C. Following a 30 min stabilization period, the tissue response to various concentrations of the extract (same as above), histamine (10 g/ml), and acetylcholine (10 g/ml) were determined according to the same procedure used for the guinea pig ileum. The effect of various concentrations ( mg/ml) of histamine on the ileum response to a fixed concentration (0.4 mg/ml) of the extract was also investigated. Rat uterus was taken from animals primed with diethylstilbesterol in olive oil (0.75 ml of 0.05 g/ml, s.c.) 24 h before testing of the tissue response. The rats were killed by stunning and the uterine horns were isolated quickly and transferred into a Petri plate containing De-jalon solution where the 2 horns were separated and freed from fat. Each horn was aerated with 95% 02 and 5% C02 and suspended in an organ bath at 30 to 32 C by means of threads attached to both ends. The free end of the thread was attached to an isotonic T2 transducer and the responses were recorded on an oscillograph. The tested concentrations of extract and acetylcholine were the same as those used above. In addition, the effect of histamine (3 mg~l) and oxytocin (2.5 IUIml) on the tissue were evaluated.

5 14 JOURNAL OF HERBS, SPICES & MEDICINAL PLATS The induced responses were recorded as described above. The concentration of histamine was later varied (3-6 mglrnl) while the concentration of the extract was kept constant. In each test, 3 readings were made. Rat fundic strip was isolated following a standard procedure (10). Female rats, fasted for 24 h, were killed after stunning and the fundus of the stomach was identified, excised, cut into ships (4-5 cm long) by transverse cuts, and placed in a Petri plate containing Krebs' solution. The preparations were suspended using threads attached at both ends in an organ bath containing Krebs' solution aerated with 95 percent 0 2 and 5 percent C02 and maintained at 37 C. The free end of the thread was attached to an isotonic T2 transducer and, after a 30 min stabilization period, respbnses to drugs and extracts were recaded on a physiograph as previously described. Tested concentrations of the extract and acetylcholine were the same as used with the guinea pig ileum. In addition, the effect of histamine (1.0 mgfml) on the tissue was tested. In each test, 3 readings were made. Statistical analysis. All results were subjected to one-way analysis of variance (ANOVA) and means were separated using Duncan's New Multiple Range Test (DNMRT). RESULTS The methanolic extract removed 10.4 percent of the initial sample, was a deepyellow to dark-amber color, and possessed a characteristic repugnant odor. Shaking the extract produced little to moderate frothing, suggesting the presence of only limited amounts of saponin. The extract neither induced relaxation nor contraction in the guinea pig ileum. When 0.2 mglml of histamine and Triclisia subcordata were added to the tissue bath, no significant increase or decrease in the contractile response due to histamine was observed (data not presented). A simcant relaxation of the rabbit jejunum was noted when the tissue was treated with extract (Table I). The relaxation increased with increasing concentration of the extract and when the percentage relaxation was plotted against the log of the extract concentration a straight line regression was obtained from which an ECso of 0.07 mglrnl was calculated The extract seemed to potentiate the contractile response of the tissue to acetylcholine when both extract and drug were added to the bath simultaneously. The extract also significantly inhibited the contractile effects of 0.05 and 0.08 mghnl histamine by 90 percent and 75 percent, respectively. Increasing the concentration of Triclisia subcorduta extract on rat uterus induced increased uterine contractions as compared with conlrac-

6 Asuzu, Okoro, and Anaga 15 tions induced by oxytocin at 1.25 x IU/rnl (Figure 1). Histamine at 0.03 mg/d completely blocked the contractile effect of 0.4 mglrnl of extract on the rat uterus (Figure 2). The extract produced a concentration dependent relaxation effect on the rat stomach (Figure 3). Contractile effects in the stomach induced by 0.01 mg/rnl of histamine were completely blocked by the extract (Figure 4). DISCUSSION In each of the tissues (stomach, jejunum and uterus) where the extract produced an effect, whether contraction (uterus) or relaxation (stomach and jejunum), the action of histamine was antagonized by various concentrations of the extract. While all the tested tissues possess HI and H2 receptors to various degrees, the guinea pig ileum, which did not respond, has primarily the HI receptor as opposed to the predominate H2 receptors in the other tissues. Such differences in action within tissues with different numbers of HI and H2 receptors may explain differences in tissue response to extract. The uterus, in particular, is very rich in H2 receptors and serves as a good model for identifying these receptors (1). The extract of Triclisia subcordata could apparently be accessing the Hz receptors in tissue in a manner and location similar to that of histamine. After inhibition by histamine, the activity of the extract could be reestablished by washing the tissue. In the uterus, particularly, contractile responses due to extract following a complete block by histamine were reestablished soon after washing the tissue. The blocking effect of the extract on histamine receptors was also reversible. One of the folklore uses of Triclisia subcordata extract is the treatment of stomach ulcers, a use supported by the results obtained in the present TABLE 1. The relaxant effect of TricIisia subcordata extract in rabbit jejunum. Extract Mean Concentration Relaxation P value Relaxation

7 16 JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS FIGURE 1. The effect of Tridisia subcordata extract on the rat uterus. Wash-out I Wash-out 4 Wash-out Wash-out t t t t Oxytocin Extract Extract Extract 1.25 x 1u2 IU/d 0.4 mg/d 0.2 mg/d 0.1 mg/d 7 = Addltion of drug or extract, l. = Washout of drug or extract. FIGURE 2. The effect of histamine on rat uterine contractions induced by Tnclisia subwrdata extract. 'Hresh-out b Wash-out t t t t Histamine Ace lcholine Extract 0.3 mg/d Extract 0.d: pglml 0.4 mglml mglml Extract 0.4 mgld L 7 = Addition of drug or extract, l. = Wash-out of dnrg or extract. study. The extract could produce positive healing effects by blocking H2 receptors in the stomach, one of the mechanisms associated with antihistamines used for the treatment of stomach ulcers which inhibit histamine release and subsequent hydrochloric acid release (I I). The effect of the extract at 0.4 mglml was comparable with that of oxytocin and suggests that like oxytocin it could serve as a useful ecbolic.

8 Asuzu, Okoro, and Anaga 15 tions induced by oxytocin at 1.25 X IU/ml (Figure 1). Histamine at 0.03 mglml completely blocked the contractile effect of 0.4 mglml of extract on the rat uterus (Figure 2). The extract produced a concentration dependent relaxation effect on the rat stomach (Figure 3). Contractile effects in the stomach induced by 0.01 mgtrnl of histamine were completely blocked by the extract (Figure 4). DISCUSSION In each of the tissues (stomach, jejunum and uterus) where the extract produced an effect, whether contraction (uterus) or relaxation (stomach and jejunum), the action of histamine was antagonized by various concentrations of the extract. While all the tested tissues possess H1 and Hz receptors to various degrees, the guinea pig ileum, which did not respond, has primarily the H1 receptor as opposed to the predominate H2 receptors in the other tissues. Such differences in action within tissues with different numbers of H1 and H2 receptors may explain differences in tissue response to extract. The uterus, in particular, is very rich in H2 receptors and serves as a good model for identifying these receptors (1). The extract of Triclisia subcordara could apparently be accessing the HZ receptors in tissue in a manner and location similar to that of histamine, After inhibition by histamine, the activity of the extract could be reestablished by washing the tissue. In the uterus, particularly, contractile responses due to extract following a complete block by histamine were reestablished soon after washing the tissue. The blocking effect of the extract on histamine receptors was also reversible. One of the folklore uses of Triclisia subcordara extract is the treatment of stomach ulcers, a use supported by the results obtained in the present TABLE 1. The relaxant effect of Tricisia subcordataextract in rabbit jejunum. Extract Mean Concentration Relaxation P value Relaxation

9 Asuzu. Okoro, and Anaga 17 FIGURE 3. The relaxant effect of Tlclisia subcordata extract on rat stomach muscle. Wash-out Wash-out Wash-out Wash-out T.5-- "t\ J Extract Extract Extract Extract mglml 0.05 mglml 0.1 mglml 0.2 mg!ml t = Additbn of drug or extract, 1 = Wash-ouf of drug or extract. FIGURE 4. The inhibiiorv action of Tdclliia subcordata extract on histamine induced contraction of rat stomach muscle. twash-out C I t Extract Histamine Extract 0.8 mglml 0.01 mglml 0.8 mglml? = Additkn of drug or exlract, 4 = Wash-out of drug or extract. REFERENCES 1. Ash, A.S.F. and H.O. Schild Receptors in smooth muscles. British J. Pharmacol. pp. 27, Asuzu, LU. and A. Undie Some observations on the toxic effects of the seed extract of Sphenotylis stenocarpa (Hochst ex. A. Rich) harms on testinal muscle. Qualitas Plantarum 36: Chang, TM, K.C. Chao, EH. La. 1%8. The mechanism of the hypotensive effect of tetrandrine and dernethylyetrandrine. Yao Hsuch Hsuch Pao 6: Dalziel. J.M Useful Plants of West Tropical Africa. Secretary of State for the Colonies, Crown Agents for the Colonies, London, 15 p. 5. Dwuma-Badu, D., J.S.K. Ayim, A.N. Tackie, J.E. Knapp, DJ. Slatkin, and RL. Schiff Additional alkaloids of Triclisia patens and Triclisia subcordata. Phytochemistry 14:

10 18 JOURNAL OF HERBS, SPICES & MEDICINAL PLANTS 6. Gralla, E.J., G.L. Coleman, and A.M. Jonas Toxicological studies with tetrandrine, a plant alkaloid with vascular and lymphotoxic effects in dogs and monkeys. Cancer Chemotherapy 3: Irvine, F.R. 1%1. Woody Plants of Ghana. Oxford University Press, London. pp Kuroda, H., S. Nakazawa, K. Katagiri, 0. Shirori, M. Kozuka, K. Fujitan, M. Tornita Antiturnour effect of benzylisoquinoline alkaloid Chemistry Pharm. Bull (Tokyo) Mitscher, L.A., W.N. Wu, and J.L. Bear Antibiotics from higher plants. Mycobacterium smegmatics. Experentia Perry, W.LM Pharmacological Experiments on intact Preparations. Churchill Livingston, London. pp Siegbert, H. 1%8. Gashic secretion and its role in the etiology of gastric and duodenal ulcers. Ann. Rev. Phannacol. 8: Tackie, A.N., D. Wumba-Badu, and J.S.K. Ayim The potential of Menispermaceous plants of West Africa as tumour inhibitors, muscle relaxants, antitussives and analgesics. Report of the Faculty of Pharmacy, University of Technology, Kumasi, Ghana. 13. Tackie, A.N., D. Dwuma-Buda, T.U. Okarter, J.E. Knapp, D.J. Slatkin, and P.L. Schi ff Two new bisbenzylisoquinoline alkaloids from Triclisia spp. Phytochemistry 12: Trease, G.E. and W.C. Evans. Pharamacognosy, 12th ed. Balliere T il, London, 6 12 p.