Trypanocidal and Cytotoxic Effects of 30 Ethiopian Medicinal Plants
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Trypanocidal and Cytotoxic Effects of 30 Ethiopian Medicinal Plants Endalkachew Nibreta,b and Michael Winka,* a Institut für Pharmazie und Molekulare Biotechnologie, Universität Heidelberg, Im Neuenheimer Feld 364, D-69120, Heidelberg, Germany. Fax: +49 6221 544884. E-mail: [email protected] b College of Science, Bahir Dar University, 79 Bahir Dar, Ethiopia * Author for correspondence and reprint requests Z. Naturforsch. 66 c, 541 – 546 (2011); received March 1/September 15, 2011 Trypanocidal and cytotoxic effects of traditionally used medicinal plants of Ethiopia were evaluated. A total of 60 crude plant extracts were prepared from 30 plant species using CH2Cl2 and MeOH. Effect upon cell proliferation by the extracts, for both bloodstream forms of Trypanosoma brucei brucei and human leukaemia HL-60 cells, was assessed using resazurin as vital stain. Of all CH2Cl2 and MeOH extracts evaluated against the trypano- somes, the CH2Cl2 extracts from fi ve plants showed trypanocidal activity with an IC50 value below 20 μg/mL: Dovyalis abyssinica (Flacourtiaceae), IC50 = 1.4 μg/mL; Albizia schimpe- riana (Fabaceae), IC50 = 7.2 μg/mL; Ocimum urticifolium (Lamiaceae), IC50 = 14.0 μg/mL; Acokanthera schimperi (Apocynaceae), IC50 = 16.6 μg/mL; and Chenopodium ambrosioides (Chenopodiaceae), IC50 = 17.1 μg/mL. A pronounced and selective killing of trypanosomes with minimal toxic effect on human cells was exhibited by Dovyalis abyssinica (CH2Cl2 ex- tract, SI = 125.0; MeOH extract, SI = 57.7) followed by Albizia schimperiana (CH2Cl2 extract, SI = 31.3) and Ocimum urticifolium (MeOH extract, SI = 16.0). In conclusion, the screen- ing of 30 Ethiopian medicinal plants identifi ed three species with good antitrypanosomal activities and low toxicity towards human cells. Dovyalis abyssinica might be a promising candidate for phytotherapy of trypanosomiasis. Key words: In vitro Trypanocidal Activity, Trypanosoma brucei brucei, HL-60 Cells, Ethio- pian Medicinal Plants Introduction disease. Nevertheless, there are times when a complex mixture of plant secondary metabolites Ethiopia is characterized by great physiogeo- is preferred to a single compound especially when graphic variation that accentuated the diversity of therapeutic effect is additive or synergistic (Wink, plant and animal life (Abebe and Ayehu, 1993). 2008). The Ethiopian fl ora comprises more than 7000 The African trypanosomes are protozoan blood species of higher plants, of which about 12% are parasites and mainly transmitted by tsetse fl ies endemic (Gebre Egziabher, 1991); about 715 spe- of the genus Glossina. The diseases caused by cies of plants have been documented to be used these pathogenic protozoans are fatal and are as medicinal drugs (Abebe et al., 2003). Medici- called sleeping sickness in humans and nagana nal plants are quite often employed in cultural in domestic livestock (Brun et al., 2010). Human and health care systems of many Ethiopians. sleeping sickness is caused by two subspecies of Therefore, plants remain indispensable sources Trypanosoma brucei namely: T. b. gambiense and of preventive and curative traditional medicines T. b. rhodesiense (Brun et al., 2010). African ani- for both human beings and livestock. Most of the mal trypanosomiasis is a disease complex caused time, medicinal plants comprise 87% of tradition- by T. congolense, T. vivax, or T. brucei brucei, or a al medical preparations (Abebe and Ayehu, 1993; simultaneous infection with one or more of these Abebe et al., 2003). Indeed, many controlled clini- trypanosomes. Both, human and animal trypano- cal studies have shown the therapeutic values of somiases, negatively affect the whole economy plant extracts used in phytotherapy (Wink, 2008). of Africa by weakening both the health of hu- In modern medicine, however, a single compound mans and their domestic animals. Currently only is usually preferred for treatment of a particular seven trypanocidal drugs are available for the © 2011 Verlag der Zeitschrift für Naturforschung, Tübingen · http://znaturforsch.com 542 E. Nibret and M. Wink · Trypanocidal and Cytotoxic Effects of Ethiopian Plants treatment of both types of diseases. Except the Material and Methods recently (1990) introduced drug efl ornithine (di- Reagents fl uoromethylornithine), the other six drugs have been in use for more than 50 years. The appear- Fetal bovine serum, MEM, and RPMI 1640 me- dia were purchased from Invitrogen (Karlsruhe, ance of drug-resistant trypanosomes, the toxicity Germany). Resazurin and diminazene aceturate of trypanocidal drugs to patients, unaffordability were purchased from Sigma-Aldrich (Steinheim, of the drugs, and lack of either their sustainable Germany). production or development of new trypanocidal drugs by pharmaceutical companies call for an Plant materials urgent search for new, less or non-toxic, and af- The plants were collected from different parts fordable drugs. of Ethiopia at different times between Decem- In Africa and elsewhere in the world, natural ber 20, 2007 and February 5, 2008 by one of us products of plant origin are culturally accepted as (E. N.) from their natural habitats. Plant samples alternative medicine and/or complementary med- were identifi ed by Mr. Melaku Wondafrash (Ad- icine to the modern drugs. However, the problem dis Ababa University, Ethiopia) and deposited with this type of traditional medicine in Africa at National Herbarium, Addis Ababa University, and particularly in Ethiopia is that the traditional Ethiopia and at Department of Biology, Institute healers (herbalists) do not tell the composition or of Pharmacy and Molecular Biotechnology, Hei- even the dose of the putative therapeutic prepa- delberg, Germany for further reference. ration. Moreover, they pass their knowledge se- Preparation of plant crude extracts cretively to the most trusted and knowledgeable person of their own families. It is highly likely that The parts of each plant investigated in this study were ground and macerated in MeOH and indigenous knowledge of folk medicine might be CH Cl , respectively, and left on a shaker for two lost. To mitigate the aforementioned problem, 2 2 consecutive days. Extracts were then fi ltered and over the last three decades various scholars have evaporated to dryness under reduced pressure documented the traditional medicinal uses of using a rotary evaporator (Büchi, Labortechnik, Ethiopian plants. But, only a few comprehensive Essen, Germany) at 45 °C. pharmacological or biological studies have been carried out to validate the putative medicinal uses Cell cultures of these traditional medicines. Sometimes, the ap- The human myeloid cell line HL-60 was ob- plication of traditional medicinal plants involves a tained from Deutsche Sammlung von Mikroor- magico-religious approach which is not totally re- ganismen und Zellkulturen GmbH (DSMZ, lated to the causative agent or specifi c symptoms Braunschweig, Germany). Trypanosoma brucei of diseases like trypanosomiasis. There is also lit- brucei TC221 bloodstream form was initially ob- tle or no information on the toxicity of medici- tained from Prof. Peter Overath (Max-Planck In- nal plants being used in the traditional medical stitut für Biologie, Tübingen, Germany) and was practices. continuously maintained in our laboratory. The present study was thus designed with the Bloodstream forms of T. b. brucei TC221 cells primary objective to identify and validate 30 me- were grown in Baltz medium (Baltz et al., 1985) dicinal plants of Ethiopia as potential candidates supplemented with 20% inactivated fetal bovine for the treatment of trypanosomiasis. In addition, serum and 1% penicillin-streptomycin where- the present study was carried out to document the as HL-60 cells (human myeloid cell line) were grown in RPMI 1640 medium supplemented with cytotoxicity of these plants against human leukae- 0.2 mM L-glutamine, 1% penicillin-streptomycin, mia (HL-60) cells as new anticancer substances and 10% heat-inactivated fetal bovine serum. are also in need. A comparison of IC values of 50 Both cell types were incubated in a humifi ed at- extracts on T. b. brucei with respect to their cor- mosphere containing 5% CO at 37 °C. responding cytotoxicity to HL-60 cells can serve 2 as a guide to the initial selection of extracts for Trypanocidal and cytotoxicity assays early drug discovery or phytotherapy of trypano- The extracts and compounds were dissolved in somiasis. dimethyl sulfoxide (DMSO). The extracts were E. Nibret and M. Wink · Trypanocidal and Cytotoxic Effects of Ethiopian Plants 543 further serially diluted with the medium in a tia ferruginea (IC50 = 87.5 μg/mL), Rosa abyssinica two-fold fashion into seven different concentra- (IC50 = 58.7 μg/mL), Vernonia amygdalina (IC50 = tions so as to attain fi nal concentrations ranging 22.4 μg/mL), Leonotis ocymifolia (IC50 = 61.0 μg/ from 250 to 3.91 μg/mL in 96-well plates. Each mL), Hagenia abyssinica (IC50 = 32.3 μg/mL), and concentration of the drug was tested in triplicate, Solanum incanum (IC50 = 82.0 μg/mL). The selec- each test was repeated twice. The solvent DMSO tivity index (SI) which is the ratio of cytotoxicity did not exceed 1.25% in the medium that con- in HL-60 (IC50) to that of T. b. brucei (IC50) cells tained the highest concentration of extract tested. is also shown in Table I. Wells containing DMSO as well as wells without DMSO were included in the experiment. Discussion Both T. b. brucei and HL-60 cells were seed- In Ethiopia a number of plants are used for ed into 96 wells at a density of 1 · 104 cells per the treatment