Development of chromatographic fingerprinting and other quality evaluation methods for warburgia ugandensis sprague herbal materials
Onyambu, Meshack Ondora
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Warbugia ugandensis Sprague is among the ten most utilized herbal medicines in Kenya with both scientific and ethnobotanical evidence of antimalarial, antileishmanial and antibacterial activity. The contribution of this plant to primary healthcare of many Kenyans is evident from the sale of its products in commercial outlets. The plant is currently endangered and therefore, there is fear of adulteration and/or substitution because there are no standardized procedures to assure quality and authenticity of its products in the market. The objective of this study was to investigate the macroscopic, microscopic, microbiological, physicochemical, phytochemical and chromatographic parameters for use to control the quality of Warbugia ugandensis leaf and stem-bark products. Plant samples from Kenyatta University Medicinal Plant Research Garden were used as reference during method development while twelve plant samples from six geographical zones and ten commercial ones were analyzed for similarity and quality respectively using the developed methods. All the samples were obtained by random purposive sampling technique. The samples used for method development and similarity evaluation were harvested, processed and stored using WHO recommended methods on good agricultural, collection and processing practices for medicinal plants. Macroscopic and microscopic studies of the whole and powdered leaf and stem-bark were done based on a modified method from the American herbal pharmacopoeia while physico-chemical, microbiological and phytochemical studies were done based on modified WHO methods. Chromatographic methods including thin layer chromatography, high performance liquid chromatography and gas chromatography-mass spectrometry were developed and used for quality analysis. The study revealed over 5 major organoleptic characteristics of W. ugandensis leaf and stem-bark including strong aromatic odor and bitter taste. Major microscopic characteristics of the leaf included anomocytic stomata and epidermal cells with anticlinal primary walls. Microscopy of stem-bark revealed brownish lignified parenchyma and schlerenchyma cells, xylary fibers in addition to clusters of simple starch granules. Microbial purity showed less than 30 colony forming units (cfu) for both the leaf and stem-bark in addition to absence of Escherichia coli, Salmonella typhi and Pseudomonas aeruginosa. Physicochemical studies showed ethyl acetate extractable matter of 8.55% and 9.18%, for the stem-bark and leaf, respectively. Phytochemical evaluation of both stem-bark and leaf powders showed relatively high concentrations of cardiac glycosides, tannins and saponins. Normal phase silica TLC method visualized sixteen spots for the stem-bark and eighteen for the leaf. Reversed-phase HPLC method showed four to six well separated peaks while the GC-MS method separated more than 100 compounds. However, 22 and 38 compounds from stem-bark and leaf respectively were selected as fingerprints for quality evaluation. The phytochemical, physicochemical, TLC and GC fingerprinting methods showed that there were some similarities and differences in the chemical patterns of Warbugia ugandensis stem-bark and leaf from each of the six geographical zones in Kenya. Analysis of commercial products by TLC confirmed that they contain Warbugia ugandensis as per the claim. Four samples passed with 80-100% consistency with the reference GC-MS fingerprint. The study therefore concluded that the quality of Warbugia ugandensis leaf and stem-bark can be evaluated by macroscopic, microscopic, microbiological, physicochemical, phytochemical and chromatographic techniques. It is therefore recommended that these techniques should be used to verify purity and authenticity of herbal materials based on W. ugandensis.