Phytochemical investigation of zanthoxylum holstzianum for antimicrobial principles
As a response to the worldwide alarm of increased resistance of microbes to readily available antibiotics, the stem bark of Zanthoxylum holstzianum (Rutaceae) with no prior phytochemical report was investigated so as to isolate, and elucidate secondary metabolites with likely antimicrobial activities. The plant material was collected from Diani Veminant forest (coastal province of Kenya), dried at room temperature under shade, pulverised and extracted using acetone. The crude extract was subjected to fractionation and purification using a range of separation techniques including, partitioning, Column Chromatography (CC), Preparative Thin Layer Chromatography (PTLC) and crystallization. The structure of the isolated compounds was determined using a combination of spectroscopic techniques such as UV, MS, and NMR. In total seven compounds were isolated, of these, three were benzophenanthridine alkaloids [dihydrochelerythrine (2), 8-acetonyldihydrochelerythrine (5) and 8oxochelerythrine (7)], one canthin-6-one alkaloid [N-methylflindersine (3)], a flavanone [hesperidine (1)], a fatty acid [hexadecanoic acid (6)], and an amide (2E,4E)-Nisobutyltetradeca-2,4-dienamide (4)]. This is the first report of the occurrence of (2E,4E)N-isobutyltetradeca-2,4-dienamide (4), hexadecanoic acid (6) and hesperidin (1) from the genus Zanthoxylum. A summary of the isolated compounds is shown in figure 1. The crude extract and isolated compounds were screened against four microbial pathogens, namely: Escherichia coli NC 35218 (Bacterium), Staphylococcus aureus ATCC 259213 (Bacterium), Candida albicans SC 5314 (Yeast fungus), and Aspergillus niger ATCC 16404 (Filamentous fungus) using the disc diffusion technique as recommended by the Clinical and Laboratory Standards Institute (CLSI, 2012). Dihydrochelerythrine (2), N-methylflindersine (3), (2E, 4E)-N-isobutyltetradeca-2,4dienamide (5) and the crude extract, each had minimum inhibition concentration (MIC) of 6.25 μg/disc against Staphylococcus aureus. The crude had MIC of 62.5 μg/disc against Candida albicans and the essential oils showed a MIC of 12.5 μg/disc against both Staphylococcus aureus and Candida albicans.