Isolation and characterisation of antimycobacterial compounds from schkuhria pinnata (lam.) duntse ex thell against mycobacterium smegmatis

Abstract

Schkuhria pinnata was selected for this study based on its use in traditional medicine. This study was aimed at isolating and charactserising antimycobacterial compounds from S. pinnata. Different extraction procedures coupled with solvents of varying polarities were used in extraction of the plant materials. Solvents of intermediate polarity had the highest mass of the extracts and serial exhaustive extraction was the best extraction procedure which extracted high amounts of plant material obtained with dichloromethane solvent. The chromatograms were developed in three solvent systems (BEA, CEF and EMW) and sprayed with vanillin-sulphuric acid reagent for colour development. Different colours on the chromatograms indicated various phytochemical constituents. Standard chemical tests confirmed the presence of tannins, flavonoids, alkaloids, phlabotannins, terpenes, steroids, cardiac glycosides and saponins. It was discovered that S. pinnata possesses high phenolic and tannin content which could be behind the antioxidant and anti-inflammatory activities observed. Antioxidant activity was analysed using 2, 2–diphenyl-1-picrylhydrazyl (DPPH) qualitative and quantitative experiments. Chromatograms were sprayed with 0.2% DPPH solution, yellow bands or spot against the purple background indicated the presence of antioxidant compounds. On quantitative analysis methanol extracts had a good scavenging activity at various concentrations. Ferric ion reducing power of antioxidants from plant extracts was determined using FRAP assay. S. pinnata extracts had high ferric reducing power which was in a concentration-dependent manner. Antimycobacterial activity was evaluated using Bioautography and broth microdilution assays. Plant extracts indicated antimycobacterial activity observed on bioautograms with low MIC values ranging from 0.27 mg/ml to 2.5 mg/ml. African green monkey Vero kidney cells were used to evaluate the toxicity of crude extracts. The plant extract had cytotoxic value of 25 µg/ml with a selectivity index of 0.02 SI. It was observed that S. pinnata had anti-inflammatory activity on LPS-induced Raw 246.7 macrophage cells in a concentration-dependent manner. Bioassay guided fractionation on column chromatography managed to isolate two compounds which were characterised using nuclear magnetic resonance techniques. The compounds were elucidated to be helaingolide and eucannabinolide sesquiterpene lactones. Biological assays indicated that the compounds were active against Mycobacterium smegmatis. The compounds were toxic to Vero monkey kidney cells with less than 30 µg/ml LC50 value and <1 selectivity index. These compounds had a good anti- inflammatory activity on LPS-induced Raw 246.7 macrophage cells which was in a concentration dependent manner. The compounds can be used as new leads in the development of anti-inflammatory and antimycobacterial drugs. The crude extracts and the isolated compounds from S. pinnata should be evaluated for their cytotoxicity and anti-inflammatory effects in in vivo experiments.