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A Comparative Study of in Vitro Susceptibility of Madurella

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A Comparative Study of in Vitro Susceptibility of Madurella Original Article A comparative Study of In vitro Susceptibility of Madurella mycetomatis to Anogeissus leiocarpous Leaves, Roots and Stem Barks Extracts Ikram Mohamed Eltayeb*1, Abdel Khalig Muddathir2, Hiba Abdel Rahman Ali3 and Saad Mohamed Hussein Ayoub1 1Department of Pharmacognosy, Faculty of Pharmacy, University of Medical Sciences and Technology, P. O. Box 12810, Khartoum, Sudan 2Department of Pharmacognosy, Faculty of Pharmacy, University of Khartoum, Khartoum, Sudan 3Commission of Biotechnology and Genetic Engineering, National Center for Research, Khartoum, Sudan ABSTRACT Objective: Anogeissus leiocarpus leaves, roots and stem bark are broadly utilized as a part of African traditional medicine against numerous pathogenic microorganisms for treating skin diseases and infections. Mycetoma disease is a fungal and/ or bacterial skin infection, mainly caused by filamentous Madurella mycetomatis fungus. The objective of this study is to investigate and compare the antifungal activity of A. leiocarpus leaves, roots and stem bark against the isolated mycetoma pathogen, M. mycetomatis fungus. Methods: The alcoholic crude extracts, and their petroleum ether, chloroform and ethyl acetate fractions of A. leiocarpus leaves, roots and stem bark were prepared and their antifungal activity against the isolated M. mycetomatis fungus were assayed according to the Address for NCCLS antifungal modified method and MTT assay compared to the Correspondence Ketoconazole, standard antifungal drug. The most bioactive fractions were subjected to chemical analysis using LC-MS/MS Department of chromatographic analytical method. Pharmacognosy, Results: The results demonstrated the potent antifungal activity of A. Faculty of Pharmacy, leiocarpus extracts against the isolated pathogenic M. mycetomatis University of Medical compared to the negative and positive controls. The chloroform Sciences and fractions showed higher antifungal activity among the other extracts, Technology, P. O. Box while the bark chloroform fraction was found to be the highest one. 12810, Khartoum, The chromatographic analysis of the chloroform fractions showed the Sudan presence of important bioactive compounds such as ellagic and flavellagic acids derivatives, known for their antifungal activity and E-mail: kramela_07 toxicity to the filamentous fungi, steilbenoid compounds known as @yahoo.com phytoalexins secondary metabolites with potent antifungal activities American Journal of Phytomedicine and Clinical Therapeutics www.ajpct.org Mohamed Eltayeb et al_______________________________________ ISSN 2321 – 2748 and the antimicrobial agents, flavonoids Conclusion: These studies present that the A. leiocarpus extracts posse’s potent antifungal activity against mycetoma causing pathogen compared to the ketoconazole standard drug and the highest activity was found to be in the stem bark of the plant. Keywords: In vitro, Susceptibility, Madurella, mycetomatis, Anogeissus, leiocarpus, Leaves, Roots, Stem barks, extracts. INTRODUCTION Combretaceae is a family of require amputation leading to loss of the flowering plants, widely distributed in the infected limbs33, 23, 34. tropical climates of Africa, Asia and South In Sudan mycetoma is a serious America. It incorporates 20 genera and common disease leading to the loss of around 600 species of shrubs, trees numerous limbs. The rate of mycetoma (evergreen or deciduous) or woody lianas1-3. infections in Sudan has not changed and The family is an important resource in around 400 new cases are found in clinics traditional medical practices for many and outpatient centers every year35, 33, 23, 34. human diseases3-8, many of these indications There is no potent and effective drug are related to treating infections7. The for treating mycetoma infection. efficacy of the plants may be due to the Ketoconazole is the favored antifungal presence of different classes of antimicrobial medication utilized for mycetoma secondary metabolites4, 5, 9. treatment36,23,34. Adequate treatment requires Anogeissus leiocarpus, is an African a prolonged antifungal drug combined with evergreen tree10 of genus Anogeissus extensive surgical treatment33, 23, 34. Meager belonging to this family with different uses data is available for susceptibility of M. in traditional medicine8,11-19. It mainly used mycetomatis to the plants secondary in treating skin diseases and infections, metabolites37. wounds infections, sore feet, boils, cysts, The present paper reported the syphilitic and diabetic ulcers13,20-23. The results of comparative study and activity plant was found to exhibited potent assessment of alcoholic leaves, barks and antibacterial and antifungal activity against roots extracts of the plant and their several pathogenic microorganisms24-30,19, chloroform fractions and ethyl acetate 31, 22, 32. fractions against M. mycetomatis. Emphasis Mycetoma is a chronic has been laid on the fungal susceptibility to granulomatous subcutaneous and deep the different metabolites occurring in tissues skin disease or a number of skin different morphological parts of the same infections caused by numerous fungi plant used in traditional therapy for the (eumycetoma) primarily Madurella infections treatment. mycetomatis fungus, or by bacteria (actinomycetoma). Progressive destruction EXPERIMENTAL of tissues leads to loss of function and impaired the affected site. Serious cases Plant Material The leaves, roots and stem barks of A. leiocarpus were collected from El AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al_______________________________________ ISSN 2321 – 2748 Damazeine region in Sudan; their botanical hyphae which were harvested in identities were authenticated in the mycological peptone (BDH) water broth siliviculture department, Faculty of Forestry, medium with chloroamphenicol. The University of Khartoum, Sudan. The harvested mycelia or hyphae was washed voucher specimen was deposited at two to three times with RPMI 1640 with L- Department of Biochemistry, Commission glutamine medium, and then incubated for of Biotechnology and Genetic Engineering, 24 hours. The harvested mycelia, was National Centre for Research, Sudan. Barks sonicated for two minutes till homogenous and roots were chipped using saw mill, and suspension of mycelia was obtained. the plant materials were air dried under shade at room temperature, ground to a Anti Fungal Procedure coarse powder using electric grinder. While the leaves were ground into powder utilizing NCCLS Antifungal Modified Assay mortar and pestle. One ml of RPMI medium containing serially diluted extracts (10-0.31mg/ml) Preparations of the Extracts were placed in sterile test tubes, then 1ml of Plant powdered materials were prepared suspension was added. Tow set of extracted by maceration over night in 80% control tubes were used in the experiment, alcohol. Alcoholic extracts were fractionated one is growth control tubes(-ve) contained using solvents with gradually increasing 1ml of RPMI medium without any treatment polarities; petroleum, chloroform and ethyl and 1ml of prepared suspension, and the acetate). The obtained fractions were other one was standard drug (+ve) control concentrated by evaporation of the solvents tubes contained 1ml of RPMI medium with under reduced pressure using a rotary serially diluted ketoconazole (5-0.31mg/ml). vacuum evaporator. The optical density of the prepared growth control suspension was measured prior Madurella mycetomatis Collection incubation using a spectrophotometer at 680 M. mycetomatis Isolated fungus was nm red filter and reported as initial reading. collected from mycetoma research center at Then all test tubes were incubated at 37°C Soba hospital, Sudan. The black grains were for a week then, the optical density was exuded from open sinuses and surgical measured at 680 nm38, 39 biopsy from the lesion, freed from tissues MIC value is the least concentration and carried by forceps in sterile container before the spectrophotometer transmission with saline. reading is the same as or more than the initial reading37. It the least concentration, Culture and Preparation of Fungal when, there is no any growth of inoculated Suspension tested organisms had been seen40. The isolated grains were washed several time with saline solution and were MTT Assay firstly cultured in blood agar media,and then The assay is a quick sensitive sub--cultured in sabouraud dextrose agar colorimetric method; utilize tetrazolium salt and incubated at 37°C for 8 days. as indicator of microbial metabolism for The isolated strains were sub - evaluation of cell death41. This method is cultured again to maintain pure isolate of actually the reduction of yellow MTT salt hyphae. The subculture of hyphae was [tetrazolium salt (3-{4, 5-dimethylthiazole- repeated for two weeks to maintain pure 2-yl}-2, 5-diphenyl tetrazolium bromide)] AJPCT[4][05][2016] 135-164 Mohamed Eltayeb et al_______________________________________ ISSN 2321 – 2748 into the green blue or violet blue formazan addition to the stem bark chloroform by the mitochondrial dehydrogenase, show fraction was found to be the most active just in the living cells and henceforth fraction. The results were compatible with discharged into the supernatant. The color the results of other related Anogeissus spp intensity is directly proportional to the living (Anogeissus latifolia) against skin disease cell numbers in the culture. One drop of the organisms44. indicator was added to all tested tubes after The leaves extract and fractions measuring the final optical
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