OSADEBE, P.O. AND UZOCHUKWU, I.C.*

Size: px

Start display at page:

Download "OSADEBE, P.O. AND UZOCHUKWU, I.C.*"

Charlotte Anderson
6 years ago
Views:

1 Journal of Pharmaceutical and Allied Sciences 3 (1) (2006) Journal of Pharmaceutical and Allied Sciences CHROMATOGRAPHIC AND ANTI-MOTILITY STUDIES ON EXTRACTS OF LORANTHUS MICRANTHUS LINN. OSADEBE, P.O. AND UZOCHUKWU, I.C.* DEPARTMENT OF PHARMACEUTICAL CHEMISTRY, UNIVERSITY OF NIGERIA, NSUKKA, ENUGU STATE, NIGERIA ABSTRACT The anti-motility properties of the leaves of African mistletoe, Loranthus micranthus (Linn), Loranthaceae harvested from Kola acuminata host tree was studied by the charcoal meal test in mice. The intraperitoneal LD 50 of the methanol extract was determined in mice by the Locke s method. The phytochemical constituents of the leaf extract were also determined. An attempt was also made to resolve the extracts into its components using thin layer chromatography (TLC). The leaves of L. micranthus were found to contain alkaloids, cyanogenetic glycosides, saponins, flavonoids, tannins, proteins and resins. The intraperitoneal LD 50 of the methanol extract of the leaves in mice was calculated to be 5916 mg/kg. Among the chromatographic solvent systems tested, toluene: diethylamine (19:1) gave the best resolution in all the extracts. The highest number of spots was obtained with the ethanol extract. Result of the charcoal meal test revealed that the methanol extract had a significant dosedependent anti-motility effect. At a dose of 200 mg/kg, the methanol extract produced a decrease in gastric transit time, which was significantly (P< 0.05) higher than that of atropine (10 mg/kg). Keywords: African mistletoe, anti-motility activity, phytochemical tests, chromatographic studies. INTRODUCTION The need for more potent, safe and affordable drugs has led to intensified research into herbal drugs, the result of which is the introduction of new herbal preparations for therapeutic uses. African mistletoe, Loranthus micranthus (Linn), is an obligate semi-parasitic plant (1). Some common host trees of African mistletoe are Kola acuminata, Baphia nitida, Citrus limon, Jatrova curcas, Pentaclethra macrophylla, Azadirachta indica and Persea americana. There are several similar plants such as the *Correspondence author Journal of Pharmaceutical and Allied Sciences Vol 3 No 1 (2006) ISSN: Website: European mistletoe (Viscum album Linn), Australian mistletoe (Ligaria cuneiflia) and Korean mistletoe (Viscum album Coloratum). The chemical constituents and biological activities of these plants depend to a large extent on the host plant and the season of harvest (2, 3). Information on the European, Australian and Korean mistletoes are widely documented (4, 5, 6). The African mistletoe may be regarded as the natural substitute of the widely studied Viscum album, but differences in the metabolic pathway, characteristic protein pattern, and in the immunogenic capacity between the Australian and the European species have been documented (7). Such differences may also exist between the European and the African mistletoe because of obvious climatic and host tree variations. Lectins, viscotoxins, viscumin 263

2 and alkaloids have been isolated from the Viscum album (8, 9). Other compounds isolated from Viscum album include flavonoids, glycosides, aglycones, triterpenes, saponins, β-amyrins, lupeol, oleanolic acid, ursolic acid, β-sitosterol, stimasterol, sugars and palmitic acid (10). African mistletoe is used folkloric medicine in the treatment of epilepsy, hypertension, headache, infertility, cancer, menopausal syndrome and rheumatism (11). The antidiabetic and antimicrobial activities of L. micranthus have also been reported (12, 13). It is equally used in many parts of West Africa as an anti-microbial and antispasmodic agent (14). Lectins from Viscum album exhibit antitumor activity (14) while Loranthus begwensis (a species of mistletoe found in Northern Nigeria) has been shown to possess antidiabetic activity (15). Due to the extensive work already done on the European and the Asian mistletoes, several commercial preparations of the plant are now available for treatment of various ailments. This work is aimed at evaluating the anti-motility properties of L. micranthus and detecting the classes of constituents present in the leaves. METHODOLOGY Plant material The leaves of L. micranthus were harvested from K. acuminata located in Nsukka, South Eastern Nigeria and authenticated by Mr. J. Ekekwe, a taxonomist in the Department of Botany, University of Nigeria, Nsukka. A voucher specimen was deposited in the Department of Botany Herbarium (Herbarium No.U.N.HB214D). Extraction procedure The leaves were dried under shade at 25 0 C for seven days and pulverized using pestle and mortar. For the purpose of chromatographic studies, different 10 g portions of the powder were extracted with methanol, ethanol, chloroform and petroleum ether (50 ml each). Each 10 g portion was soaked for 24 h (with intermittent shaking) and filtered to obtain the extract. The extracts were concentrated to dryness in a water bath and weighed. The methanol extract was used for the anti-motility study while all the extracts were used for the chromatographic study. Animals used White albino mice (20-28 g) of either sex were used. All animals were conditioned in standard metal cages. They were allowed access to both food and water and were kept in 12/12 h light/dark cycle. Chemicals and Reagents The following analytical grade chemicals and reagents were used in the study: chloroform, ethanol, methanol, and petroleum ether (BDH, England); diethyl amine, benzene, ethyl acetate, ammonia solution, and toluene (May and Baker, England); Silica gel G60 (Merck, Germany). Phytochemical tests Standard procedures (16) were followed in the detection of the different classes of constituents present in the dried leaves of the plant. Classes of the constituents tested for were carbohydrates, alkaloids, glycosides, saponins, tannins, flavonoids, resins, proteins, oils and steroids. 264

3 Acute toxicity study The acute toxicity test (LD 50 ) of the crude methanol extract was determined according to the Locke s method (17). Three groups of mice (n=3) were given doses of 10, 100, and 1000 mg/kg of the methanol extract suspended in Tween 20 solution respectively by the intraperitoneal route and observed for 24 hours. Based on the result of the first range finding stage, where no death of the mice was observed even with 1000 mg/kg, the second step was carried out in which doses of 5000, 7000 and mg/kg were administered to three mice respectively. LD 50 was calculated as the geometric mean of the lowest dose showing death and the highest dose showing no death. Effect of the methanol extract on small intestinal transit Twenty albino mice of either sex (20-35 g) were randomly divided into five groups (n=4). The animals were fasted for 24 hours prior to the experiments, but were allowed free access to water. The negative control group received 20 % Tween 20 solution (10 ml/kg) and the positive control group received 10 mg/kg of atropine in 20 % Tween 20 solution. The remaining three groups received three different doses of the methanol extract (100, 200 and 300 mg/kg) respectively suspended in 20 % v/v Tween 20 solution. The administrations were by oral route. Five minutes after drug administration, 0.5 ml of a 5 % charcoal suspension in 5 % aqueous solution of tragacanth was administered to each animal orally. The animals were sacrificed 30 minutes later and the abdomen opened. The percentage distance on the small intestine (from the pylorus to the caecum) traveled by the charcoal plug in the treatment groups were determined. Determination of TLC solvent system for extracts of L. micranthus Thin-layer chromatography was used to resolve the extracts following a standard procedure (18). Clean glass plates (20 cm by 20 cm) were coated with silica gel G60 to a thickness of 0.25 cm using Kenso CJK 520 spreader. The coated plates were air-dried and activated in an oven for 1 h at C. The plates were then cooled at room temperature. While the methanol and ethanol extracts were each dissolved in methanol, the chloroform and petroleum ether extracts were each dissolved in chloroform. The dissolved extracts were spotted at different points on a plate, maintaining the same distance from one edge to another. The plates were allowed to dry at room temperature. The spotted plates were developed in different solvent systems: - Chloroform: methanol (1:1), benzene: ethylacetate (6:4), benzene: ethyl acetate (7:3), benzene: ethyl acetate (8:2), toluene: diethylamine (19:1) and chloroform: methanol (9:1). The solvent system was allowed to travel a predetermined distance of 15 cm from the origin. The plates were removed from the chamber, dried and the spots located with the aid of iodine. The number of spots eluted was counted and their R f values calculated. Statistical analysis The results were presented where necessary as mean + standard deviation. Significances of the 265

4 difference in activity between control and the test groups were determined using Student s t-test (19). RESULTS AND DISCUSSION The yields of the methanol, ethanol, chloroform and petroleum ether extracts were 15.6, 12.0, 8.0 and 5.5 % respectively. From the phytochemical study, the presence of alkaloids, cyanogenetic glycosides, saponins, flavonoids, tannins, proteins, and resins were detected. An intraperitoneal LD 50 of 5516 mg/kg was obtained for the crude methanol extract. The result of the anti-motility screening is given in Table 1. There was a significant difference (P<0.05) in the percentage distance traveled by chromatographic study is shown in Table 2. Toluene: diethylamine (19:1) afforded a good separation of the spots and also gave the highest number of spots in all the extracts. However, it resolved particularly well the ethanol extract, giving a total of eight spots. With an LD 50 above 5916 mg/kg, methanol extract of the leaves of L. micranthus is relatively safe based on the criteria proposed by Loomis (20). Following the toxicity test, three doses (100, 200, 300 mg/kg) of the methanol extract were chosen for the evaluation of anti-motility activity. They were doses that were safe enough to give 100 % survivors of the animals without any changes in the behavioural, neurological and autonomical response for 14 days. The better resolution of the ethanol extract into eight spots by TLC suggests Table 1: Effect of Loranthus micranthus extract on gastrointestinal transit-time in mice Group Test drug received Dose Percentage distance traveled by the charcoal I Tween ml/kg II Atropine 10 mg/kg *** III IV V Methanol extract Methanol extract Methanol extract 100 mg/kg 200 mg/kg 300 mg/kg *** *** a *** a *** Significant (P<0.05) compared to negative control, Tween 20 solution. a Significant (P<0.05) compared to the positive control, atropine the charcoal in the presence of L. micranthus (100 mg/kg) when compared with the negative control (20 % Tween 20). The result of the that extraction with polar solvent such as ethanol will improve the phytochemical constituents of the decoctions used in traditional medicine. 266

5 The methanol extract (100 mg/kg) exhibited anti-motility effect, which was comparable with the effect of 10 mg/kg of atropine. On the other hand, 200 mg/kg of the methanol extract produced a substantiated the folkloric use of African mistletoe as an antispasmodic agent. CONCLUSION The methanol extract of L. micranthus Table 2: R f values of the TLC spots of extracts in various solvent systems. Extracts R f values in different solvent systems A B C D E F Methanol Ethanol Chloroform Petroleum ether Key: A = Benzene/ ethyl acetate (8:2) D= Toluene/diethylamine (9:1) B = Benzene/ ethyl acetate (7:3) E = Toluene/diethylamine (19:1) C = Benzene/ethyl acetate (6:4) F = Chloroform/ethanol (1:1) statistically significant reduction in percentage distance traveled by the charcoal plug, which was significantly higher than the effect of atropine. The anti-motility effect of the extract was found to be dose-dependent. The result Linn (200 mg/kg) harvested from K. acuminata showed anti-motility activity, which was significantly higher than that of atropine (10 mg/kg). 267

6 ACKNOWLEDGEMENT We are grateful to Onwutalu A. U. and Okorie J. for their contributions in the realization of some of the experimental data. REFERENCES 1. Griggs, P. (1991) Mistletoe, myth, magic and medicine, The Biochemist, 13: Mortan, J.S. (1977) Major Medicinal Plants, University of California Press, Los Angeles, p Tuquet, C. and Salle, G. I. (1996) Characteristics of chloroplast isolated from two plants originating from temperate and tropical areas, J. Plant Physiol. and Biochem, 34: Kuttan, G., Vasudevan, D. M. and Kuttan, R. (1990) Effect of a preparation from Viscum album on tumour development in-vitro and in mice. J. of Ethnopharmacol. 29 (1): Fernandez, T., Marcelo, L. W., Beatriz, G. V., Rafeal, A. R., Silvia, E. H., Alberto, A. G. and Elida, A. (1998) Study of an Argentine mistletoe, the hemiparasite, Ligaria cuneifolia (R. et. P) tiegh. (Loranthaceae). J. of Ethnopharmacol. 62(1): Yoon, T. J., Yung, C. Y., Tae, B. K., Young, J. B., Chul, S. H., Seong, K. S., Kwan, H. L., Ichiro, A. and Jong, B. K. (1998) Prophylactic effect of Korean Mistletoe (Viscum album coloratum) extract on tumour metastasis is mediated by enhancement of NK cell activity. Int. J. of Immunopharmacol. 20 (4-5): Joller, U., Parnham, M. J., Weyhenmeyer, R. and Lentzen, H. (1996) Stimulation of Cytokine Production via a special standardized mistletoe preparation in an in-vitro human skin bioassay, J. Carmeittel Forschung, 46, 6: Fisher, S., Scheffler, A. and Kabelitz, D. (1996) Oligoclonal in-vitro response of CD 4 T- cells to vesicles of mistletoe extracts in mistletoe-treated cancer patients, J. Cancer Ther. and Immunother. 44, 3: Evans, C. W. (1992) Textbook of Pharmacognosy, Bailiere Tindall, London, p Neamtu, M.A. and Bodea, C. (1970) Chemotaxonomic studies on higher plants: III Carotenoids of Viscum plant, Plant Biochem., 73: Nwude, N and Ibrahim, M.A. (1980) Plants used in traditional veterinary medicine practice in Nigeria, J. Vet. Pharmacol. Therap., 3: Osadebe, P. O., Okide, G. B. and Akabogu, I. C. (2004) Study on the Anti-diabetic activity of crude methanolic extracts of Loranthus micranthus (Linn.) sourced from five different host trees, J. Ethnopharmacol., 95: Osadebe, P.O. and Ukwueze, S.E. (2004) Comparative study of the antimicrobial and phytochemical properties of mistletoe leaves sourced from six host trees, J. of Biolog. Res. and Biotech., 2, 1: Oliver-Bever, B. (1986) Medicinal plants of tropical West Africa, Cambridge University, London, p Obatomi, A. K., Bikomo, P.O. and Temple, V.J. (1994) Anti-diabetic properties of the African mistletoe in streptozotocin-induced diabetic rats, J. Ethnopharmacol., 43: Harborne, J. B. C. (1973) Phytochemical methods, Chapman and Hall, London, p Lorke, D. (1983) A new approach to practical acute toxicity testing, Arch. Toxicol., 54: Touchstone, J. C. (1992) Practice of thin layer chromatography, John Wiley and Sons, Ave, New York, p Woodson, R. F. (1987) Statistical methods for the analysis of biochemical data, Probability and Mathematical Statistics, Wiley, Chichester, pp Loomis, I. (1980) Essentials of Toxicology, ed 3, Lea and Febiger, Philadelphia, USA, pp

7 . 269

Antidiabetic principles of Loranthus micranthus Linn. parasitic on Persea americana

Antidiabetic principles of Loranthus micranthus Linn. parasitic on Persea americana Asian Pacific Journal of Tropical Medicine (2010)619-623 619 Contents lists available at ScienceDirect Asian Pacific Journal of Tropical Medicine journal homepage:www.elsevier.com/locate/apjtm Document