Acute toxicity studies of butanol fraction of leaves of Moringa stenopetala in rats

Transcription

1 Acute toxicity studies of butanol fraction of leaves of Moringa stenopetala in rats Abdu Hassen Musa 1*, Prabhanjan Kumar Vata 2, Asfaw Debella 3 *1 College of Health Sciences and Medicine, Dilla University, P.O. Box 419 Dilla, Ethiopia 2 College of Health Sciences and Medicine, Dilla University, P.O. Box 419 Dilla, Ethiopia 3 Ethiopian Public Health Institute, P.O. Box 1242/5654, Addis Ababa, Ethiopia ABSTRACT The leaves of Moringa stenopetala are traditionally used for the treatment of various ailments such as malaria, hypertension, asthma, and diabetes. This study was carried out to evaluate the acute toxic effect of butanol fraction of the leaves of M. stenopetala in experimental rats. For this study the leaves were collected in Arba-Minich, Southern Ethiopia. The leaves were dried, crushed to powder and extracted with ethanol and fractionated with butanol. Fifteen female albino rats were randomly divided into four experimental groups (which received 500, 1000, 2000, and 5000mg/kg respectively of the fraction) and one control group, which received distilled water by oral gavage. In the acute toxicity study rats treated with up to dose of 5000mg/kg showed no toxic signs on behavior, gross pathology, and body weight, as compared with the controls. These results showed that butanol fraction of M. stenopetala did not produce adverse effects in treated rats after acute treatment. Although, further detailed studies should be carried out to recommend its therapeutic use. Keywords: acute toxicity, behavioral, body weight changes, LD 50, Moringa stenopetala. Introduction M. stenopetala belongs to the family Moringaceae; it is a fast growing tree with 6-12 m tall, white to pale gray smooth bark, alternate multi-pinnate leaves and long taproots with few lateral roots. It is widely distributed in the Southern region of Ethiopia and is known by different vernacular names such as Shiferaw (Amh), Halako (Gamo & Wollayita), Shelchada (Konso), and Cabbage tree (Eng)[1-3] Many primary & secondary chemical compounds have been isolated from the fresh leaves of M. stenopetala such as: proteins, fats, carbohydrates, minerals (K, Fe, Ca, P, and Zn, in significant concentrations), alkaloids, flavonoids, glycosides, polyphenols, saponins etc[4-6]. In southern Ethiopia, around Arba-Minch, Konso and Wollayta the fresh leaves are eaten as vegetables and also used as herbal medicine its alcholic extract & fractions has shown significant changes on blood glucose level and body weight in experimental mice[6-11]. *Correspondence Abdu Hassen Musa College of Health Sciences and Medicine, Dilla University, P.O. Box 419 Dilla, Ethiopia. E Mail: ahdu97@yahoo.com Despite its nutritional & medicinal values, toxicity studies of M. stenopetala leaves are limited. Therefore, toxicological testing is needed in stenopetala leaves. This study was aimed to investigate the acute toxic effects on behavior, general body weight, gross pathology of the liver and kidneys and to determine median lethal dose (LD 50 ) of butanol fraction of the leaves of M.stenopetala in experimental rats. Materials and methods Preparation of Plant Materials The present study was conducted in the Ethiopian public health Institute (EPHI), traditional and modern drug research laboratories, in March The leaves were collected in Arba-Minich, Southern Ethiopia, about 502 kilometers south of Addis Ababa. The leaves were identified and authenticated by a taxonomist, cleansed from extraneous materials and dried under shade and powdered. The powdered leaves were extracted with 70% ethanol using percolator and the resulting ethanol extract was filtered using Whatmann no.1 filter paper and concentrated using a rotary evaporator (BUCHI Rota-vapor type R-205, Switzerland) under reduced pressure at a temperature of C. Then the residue was dried by steam bath at 40 0 C. The dried ethanol extract was dissolved in 160

2 warm distilled water and filtered with Whatmann no.1 Acute toxicity evaluation of the butanol fraction filter paper. The dissolved extract was partitioned in a separatory funnel with n-hexane (3x50), After administration they were kept under close dichloromethane (3x50) and n-butanol (5x50) observation continuously for 1 hour and intermittently successively until the extracting solvent became for 4 hours to determine the LD 50 and to assess the colorless in each case. After completing the separation effect on behavior. Thereafter clinical observations were process, the butanol fraction was collected and made once every 24 hours for the next 14 days, for concentrated using a rotary evaporator. The residue mortality, behavioral, body weight and gross physical was then dried by steam bath at 40 0 C. Finally, the dried changes. Finally, on the 15 th day, their final weights n-butanol fraction was kept in the refrigerator for the were measured and then they were anesthetized, experiments. The yield of the extract was 20% and that sacrificed and dissected to examine gross pathological of the butanol fraction was 7% relative to the weight of changes on the liver and kidneys. the powdered leaves (500gm). The procedure for plant material preparation was adopted from Debella (2002), Statistical analysis Ranjan and Reeba (2002) with some modification[13-14].. Preparation of experimental animals and treatment For this study fifteen healthy adult female rats (weighing gm, with age of weeks old) were obtained from animal house of EPHI. They were kept under standard conditions (at a temperature of C, with 12 hours light/ 12 hours dark cycle) and provided with free access to standard pellet laboratory diets and drink tap water ad-libitum. Before the experiment they were grouped into 5 groups (3 female rats per group, one control and four test groups) and then kept in their cages for 5 days to allow acclimatization to the laboratory conditions. After acclimatization all groups were fasted overnight. Following the fasting period, they were weighed and the doses and the vehicle were calculated based on their body weight (2 ml/100g body weight). The butanol fraction was then administered orally, using oral gavage, at a single dose of 500, 1000, 2000, and 5000mg/kg body weight of rats in the test groups II, III, IV, & V respectively. The control group (I) received distilled water. This acute toxicity study was conducted based on the Organization for Economic Co-Operation and Development (OECD 423) guideline[15]. The toxicity results were expressed as mean + standard error of mean (SEM). The data obtained was analyzed by a one- way analysis of variance (ANOVA) using the SPSS version 20 program followed by a Dunnet s t- test. P-values < 0.05 were considered statistically significant. Results Acute toxic effects of fraction of M.stenopetala In the present toxicity study the fraction of M.stenopetala leaves up to the highest dose of 5000mg/kg did not show any significant changes on behavior (such as (alertness, aggressiveness, irritability), gross physical appearance (condition of fur, general cleanliness) compared with the controls. No death was observed, at any of the doses employed, including the dose 5000mg/kg. The gross pathological examination on the liver and kidneys of treated rats showed no significant changes in color, size, shape, and texture compared with the rats in the control group. Moreover, as summarized in table-1, the mean absolute weights of the liver & kidneys increased in a dose - dependent manner, though, were not statistically (p>0.05) significant as compared with the control groups. Table: 1. Comparison of organ weights of rats treated with different doses of the butanol fraction Doses Liver weight (g) kidney weight (g) Control (DW) mg/kg mg/kg mg/kg mg/kg *Values are expressed as Mean ± SEM, n= 3/group There was a gradual increase in the body weight of both the treated and control rats. At the end of the experiment (after 14 days) the mean body weight gain for rats treated with 500 mg/kg, 1000 mg/kg, 161

3 2000mg/kg, and 5000mg/kg, were g (6.9%), the mean body weight gains for rats treated at different g (7.19%), g (11.68%), and doses of the fraction weree not statistically (p>0.05) g (10.93%) respectively, while the control rats was significant as compared to that of the control groups g (4.4%). As summarized in table-2 and graph-1, Table: 2 Comparison of the effect of fraction of the leaves of M. stenopetala on the body weight Doses Initial body weight (g) Final body weight (g) Mean weight increment (g) Control (DW) mg/kg mg/kg mg/kg mg/kg Values are expressed as Mean ± SEM, n= 3/group BUTANOL FRACTION OF THE LEAVES OF M.stenopetalaON THE BODY WEIGHT. % of weight gain Initial body weight (g) Final body weight (g) Fig.1: Comparison of the effect of fraction of the leaves of M. stenopetala on the body weight Discussion Because of the widespread use of M. stenopetala as a cleanliness) and behavioral profile (such as alertness, food and medicinal plant in the southern Ethiopia[3]. It aggressiveness, irritability), as compared to the controls. was felt necessary to investigate the potential toxicity Moreover, no death was observed in the treated groups the leaves of M. stenopetala in experimental animals. up to the dose of 5000mg/kg during the 14 days of the The present study was, therefore, aimed at evaluating experimental period. This indicates that the LD50 of the the effects of the butanol fraction of the leaves of M. fraction could be greater than 5000mg/kg body weight. stenopetala on behavior, general appearance, body According to Clarke and Clarke (1977), any compound weight, gross pathology of the liver and kidney in or drug with the oral LD 50 estimate greater than 1000 laboratory-bred rats. The acute toxicity study was mg/kg could be consideredd low toxic and safe. The conducted at the doses of 500, 1000, 2000, and butanol fraction may, therefore, be considered relatively 5000mg/kg body weight. Administration of the fraction safe on acute exposure. Similar results were reported by of M. stenopetala, up to the dose of 5000 mg/kg body other workers and it was suggested that, no behavioral weight, did not produce significant changes on the changes and death were noted following administration general appearance (condition of fur, general of the fraction of leaves of M. stenopetala up to dose of 162

4 5000mg/kg body weight in mice[11-12]. (Nardos et al., Ethiopian public health institute for sponsoring this 2011; Toma et al., 2012) in both normal and diabetic research study. mice. Liver and kidneys of rats are used by many researchers to assess the safety or toxicity of drugs or plant materials[16-17]. In the present acute toxicity References study, gross pathological examination of the liver and kidneys did not show any significant difference/change in size, shape, color and texture upon treatment with the fraction. Though not significant, the absolute liver and kidney weight of treated rats appeared to increase as compared with the control group (Table-1) perhaps due to presence of active compounds in the butanol fraction of the leaves of M. stenopetala. According to Mekonnen and Dräger, (2003) rats that fed with diets containing glucosinolates produce small increase in the weights of the liver and kidneys. This is in-line with the previous studies carried out on the same plant by Ghebreselassie et al., (2011).[10,18]. Body weight change is an important index for assessment of toxicity[19]. In the two weeks acute toxicity study, there was a gradual increase in the mean body weight of the treated rats though the differences were not statistically significant (p>0.05) as compared with the control group (Table-2). The higher mean body weight gain observed with the fraction, suggests that the fraction might not have harmful effect on body growth patterns and the plant might rather have essential nutritional values, which supports the traditional use of the leaves of M. stenopetala for its food value. The increase in body weight was in agreement with the findings of Ghebreselassie et al., (2011), Toma et al., (2012) with the leaf extract & fraction in mice[10,12]. Conclusion In the current acute toxicity study, the butanol fraction of M. stenopetala was tolerated in rats up to an oral dose of 5000mg/kg. This suggested that the butanol fraction of the leaves is safe on acute exposure in rats and justifies the use of the leaves of the plant as a source of food by some localities in the southern parts of Ethiopia. It is recommended that, further subchronic and chronic toxicity studies should be carried out on blood parameters, histopathological structures of kidney, liver and other vital organs in experimental animals. Acknowledgment We would like to express over sincere gratitude to dr. Asfaw Debella for his constructive and stimulating advice, consistent effort and valuable suggestions during this study. I would also like to thank the 1. Clarke, E. and Clarke, M. (1977). Lander s Veterinary Toxicology. London: Bailliére Tindall. 8; Mark, E.O. (1998). Research on applied uses of M. stenopetala. FAO Technical Bulletin No.4. pp: Mekonnen, Y. The multi purpose Moringa tree: Ethiopia. Examples of the development of pharmaceutical products from medicinal plants. UN Development Program, New York, 2005;(10): Abuye, C., Urga, K., Knapp. H., Selmar, D., Omwega, A.M., Imungi, J.K. and Winterhalter, P. A compositional study of Moringa stenopetala leaves. East African Medical Journal, 2003;80 (5): Mengistu, M. (2007). Hypotensive effects of aqueous extract of Moringa stenopetala in both in vivo and in vitro animal models. MSc thesis, Addis Ababa University, unpublished. 6. Sileshi, T. (2010). Antihyperglycemic effect of solvent fractions of total hydroalcholic extract and column chromatographic fractions in alloxan induced diabetic mice, (MSc thesis, Addis Ababa University, unpublished). 7. Mekonnen, Y. and Gessesse, A. Documentation on the uses of Moringa stenopetala and its possible antileishmanial and antifertility effects. SINET: Ethiopian Journal of Science, 1998;21: Jiru, D., Sonder, K., Alemayehu, L., Mekonnen, Y., and Anjulo, A. (2006). Leaf yield and nutritive value of Moringa stenopetala and Moringa oleifera accessions: Its potential role in food security in constrained dry farming agroforestry system. Moringa and other highly nutritious plant resources: Strategies, standards and markets for a better impact on nutrition in Africa. Accra, Ghana. Pp: Mussa, A., Makonnen, E. and Urga, K. Effects of the crude aqueous extract and isolated fraction of Moringa stenopetala leaves in normal and diabetic mice. Pharmacology Online 2008;3: Ghebreselassie, D., Mekonnen, Y., Gebru, G., Ergete, W., and Huruy, H. The effects of Moringa stenopetala on blood parameters and histopathology of liver and kidney in mice. Ethiopian Journal of Health Development, 2011;25 (1):

5 11. Nardos, A., Makonnen, E., and Debella, A. Effects 15. OECD. (2001). OECD Guideline for Testing of of crude extracts and fractions of Moringa Chemicals. Test No Acute Oral Toxicity- Acute stenopetala (Baker f) Cufodontis leaves in Toxic Class Method. pp: normoglycemic and alloxan-induced diabetic mice. 16. Graaf, K.V.D. (1995). Human Anatomy (4th ed), African Journal of Pharmacy and Pharmacology, Wm.c.Brown Communications, Inc. U.S.A; pp: ;5(20): Toma, A., Makonnen, E., Debella, A., and Tesfaye, 17. Satyapal, S.U., Kadam, V.J. and Ghosh, R. B. Antihyperglycemic effect on chronic Hepatoprotective activity of Liveobond, a polyherbal administration of butanol fraction of ethanol extract formulation against CCl4 induced hepatotoxicity in of Moringa stenopetala leaves in alloxan induced rats. International Journal of Pharmacolog, 2008;4: diabetic mice. Asian Pacific Journal of Tropical Biomedicine, 2012;S1606-S Mekonnen, Y. and Dräger, B. Glucosinolates in 13. Debella, A. (2002). Manual for photochemical Moringa stenopetala. Planta Medica, 2003; 69:380- screening of medicinal plants. Department Drug 382. Research, EHNRI, Addis Ababa, Ethiopia. pp: Vahalia, M.K., Thakur, K.S., Nadkarni, S., and 15. Sangle, V.D. Chronic toxicity study for Tamra 14. Ranjan, C. and Reeba, K. Antidiabetic and bhasma (A generic Ayurvedic mineral formulation) hypolipidemic activity of Helicteres isora in animal in laboratory animals. Recent Research in Science models; Journal of Ethnopharmacology, 2002;81: and Technology, 2011;3(11): Source of Support: Nil Conflict of Interest: None 164