Antimicrobial And Antioxidant Activity Of Crude Extracts And Fractions From The Stems And Leaves Of Tephrosia Lupinifolia

ABSTRACT

Tephrosia lupinifolia is used traditionally for the treatment of malaria, diarrhea, toothache and tuberculosis. Two flavonoids, lupinifolin and lupinifolinol were previously isolated from the hexane root extract of T. lupinifolia, but there are no reports on phytochemical and pharmacological studies conducted on the stems and leaves of the plant. The purpose of this study was to prepare and fractionate crude extracts from the leaves and stems of T. lupinifolia, and to evaluate their antioxidant as well as antimicrobial activities. Crude extracts were prepared by suspending the powdered plant material in an equivolume mixture of dichloromethane and methanol at room temperature for 48 hours on an orbital shaker. The extracts were then subjected to a combination of trituration, column chromatography and preparative TLC, which yielded nine partially purified fractions as revealed by Gas Chromatography-Mass Spectrometry (GC-MS) analysis. The fractions and crude extracts were tested for antimicrobial activity against one gram-positive bacterium (Staphylococcus aureus), two gram-negative bacteria (Klebsiella pneumonia and Escherichia coli) and one fungal strain (Candida albicans), using the agar disc-diffusion method. The best activity, albeit moderate, was recorded for the crude stem extract with zones of inhibition of 14 and 12 mm against K. pneumonia and C. albicans, respectively. The crude leaf extract displayed equipotent activity against E. coli and C. albicans with zones of inhibition of 10 mm. According to the GC-MS analysis fraction L5, which was obtained by subjecting the crude leaf extract to column chromatography, consisted of 10 compounds and the major compound was tentatively identified as 4-tetradecane. Fraction L6 comprised of four compounds of which two major compounds were tentatively identified as hexadecane and cyclohexanone with the aid of mass spectral libraries. Only fractions S1, S2, S3, L1,

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L5 and L6 were tested for antioxidant activity using the 2, 2-diphenyl-1-picrylhydrazyl radical (DPPH) assay. The test revealed that S3 showed the highest IC50 value of 23.50 ± 3.28 mg/mL whereas the lowest, and thus the best, antioxidant activity was recorded for fraction L6 with an IC50 of 0.80 + 0.01 mg/mL.

Quantitative determination of the total phenolic and total flavonoid contents of the methanolic leaf, root and stem extracts was done using the Folin Ciocalteu method and aluminum chloride complex forming assays, with the results expressed in mg of gallic acid equivalents (GAE)/g of dry weight (DW) and mg of quercetin equivalents (QE)/g DW, respectively. Total phenolic content was recorded as 45.3 mg GAE/g DW for the leaves, 58.3 mg GAE/g DW for the roots and 250.5 mg GAE/g DW for the stems. Flavonoid contents were obtained as 31.8 ±0.032 mg QE/ g DW for the leaves, 13.2 ±0.003 mg of QE/g DW for the roots and 20.3 ±0.013 mg of QE/g DW for the stems. The methanolic stems extract showed the highest total phenolic content whereas the highest total flavonoid content was shown from the methanol leaves extract.

Keywords: Antimicrobial activity, antioxidant activity, ethnomedicine, phytochemical screening, Tephrosia lupinifolia.