Biological and phytochemical analysis of Chungtia medicinal plants of Nagaland, India

The MPhil study presented in this thesis was an extension of a collaborative research partnership between the Indigenous Bioresources Research Group (IBRG) of Macquarie University and Chungtia village (Chungtia Senso Mokokchung Town, CSMT), Nagaland, for documentation of ethnobotanical knowledge of Chungtia village Elders and healers as well as phytochemical and biological activity investigation and isolation of bioactive constituents from Nagaland medicinal plants. The project was initiated by Meyanungsang Kichu, a Nagaland person, who conducted an ethnobotanical study of medicinal plants used by Chungtia villagers and documented 135 plants for their various ethnomedicinal and ethnobotanical applications. This MPhil study completed an up to date literature review of the 135 medicinal plants, then investigated the antimicrobial potential of those plants used by Chungtia villagers for skin conditions, conducted antimicrobial screening of a selection of these, and finally investigated in detail one plant for its antimicrobial activity and bioactive constituents. A comprehensive literature review covering traditional usages of all 135 plants by other Indigenous traditional healers’ worldwide and phytochemical and biological properties of these plants was conducted. This revealed that the traditional usages by the Chungtia community of 93 of their medicinal plants are in agreement with the uses of other Indigenous communities. Thirteen species were found to have no reports on their traditional uses, other than our first-hand accounts of the Chungtia community. Out of 93 species that were found to be used in a similar way by other communities, 80 had traditional uses that were consistent with pharmacological studies that have been reported in the literature and 55 of these plants had also had phytochemical studies conducted that showed bioactive compounds that aligned with their traditional uses by the Chungtia villagers. A detailed literature review was conducted on the antimicrobial properties and relevant phytoconstituents of 35 plants used by the Chungtia villagers for skin related conditions of a possible microbial origin. This highlighted twelve species with either no antimicrobial properties reported and/or no antimicrobial compounds identified. Out of these, seven species (Dendrocnide sinuata, Duabanga grandiflora, Erythrina stricta, Eurya acuminata, Holboellia latifolia, Maesa indica and Prunus persica) that were available for collection were selected for antimicrobial screening. The antimicrobial screening of the 70% aqueous ethanolic extracts of the plants (D. sinuate stem, D. grandiflora stem bark, E. stricta stem, E. acuminata leaves, H. latifolia leaves, M. indica leaves and P. persica roots) was performed using disc diffusion and MTT microdilution assays against the human pathogenic microorganisms Staphylococcus aureus (susceptible S. aureus), methicillin resistant S. aureus (MRSA) and multi drug resistant S. aureus (MDRSA), susceptible beta-lactamase negative Escherichia coli (β- E. coli), β- lactamase positive (antibiotic resistant) E. coli (β+ E. coli), Pseudomonas aeruginosa, Streptococcus pyogenes, Salmonella typhimurium and Candida albicans. The highest inhibitory activities were exhibited by the P. persica root extract, with MIC values of 156 μg/mL for all tested S. aureus strains. Based on the antibacterial screening results, P. persica was selected for further biological and chemical investigations for its antibacterial constituents. The 70% aqueous ethanolic P. persica roots extract was subjected to partitioning with different polarity solvents (n-hexane, dichloromethane, ethyl acetate). The most potent inhibitory activity was observed for the n-hexane and ethyl acetate partitions against susceptible and resistant strains of S. aureus. The GS-MS analysis of the n-hexane partition revealed the presence of eight constituents, out of which three were reported in the literature as antibacterial against S. aureus. TLC bioautographic methods reported in the literature were trialled with the aim to develop the most appropriate technique for the bioautography guided isolation process. The overlay method was found to be the most effective for the purpose of this study. TLC bioautography guided isolation by normal phase chromatography, size exclusion chromatography and preparative TLC led to the isolation of β-sitosterol (5.1) from the n-hexane partition and afzelechin (5.2) and ent-epiafzelechin-(2α→O→7’,4α→8’)-(-)-ent-afzelechin (5.3) from the ethyl acetate partition. The structures of these three compounds were determined based on various spectroscopic methods, including mass spectrometry, nuclear magnetic resonance spectroscopy, Infrared spectroscopy and circular dichroism. β-Sitosterol was found to be moderately active (MIC 1250 μg/mL) against P. aeruginosa as well as weakly active (MIC 2500 μg/mL) against susceptible strains of S. aureus, E. coli and S. typhimurium. ent-Epiafzelechin-(2α→O→7’,4α→8’)-(-)-ent-afzelechin showed good antibacterial activity against all the tested strains of S. aureus (MIC 156 μg/mL for susceptible and 312 μg/mL for resistant) as well as weak activity against the susceptible strains of E. coli, P. aeruginosa and S. typhimurium (MIC 2500 µg/mL, for all bacteria). This is the first report of this compound possessing antibacterial activity. The antimicrobial properties of afzelechin were not tested due to the small quantity of sample.