Evaluation of In vitro Antimicrobial Potential in the Sub-fractions of the Ethanolic Extract of Leucas biflora and Their Application in Herbal Soap Formulation.

Abstract

Background: Medicinal plants show promising activity for antimicrobial solutions worldwide. Leucas biflora plant is used for conjunctivitis, skin diseases and cleaning wounds for ethnomedicinal purposes. Objectives: To conduct bioassay-guided fractionation to assess the antimicrobial activity of ethanolic extracts derived from the entire plant of Leucas biflora, and to develop an antimicrobial soap formulation Methods: The whole plant was authenticated, dried (200 g), then refluxed and freeze-dried. Four concentrations (1.5, 3, 6, and 12 mg/mL) of crude extract underwent phytochemical analysis (PA) and Agar well-diffusion (AWD) against Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922) and Candida species (Clinical isolate). Ethanolic extracts were fractionated and four fractions (hexane, dichloromethane, ethyl acetate and residual) underwent PA and AWD. Ciprofloxacin and fluconazole were used as positive controls (PC) while dimethyl sulfoxide was used as negative control (NC). Crude extracts were incorporated (0.2, 0.4, and 0.8 g) into soap formulations and evaluated for antimicrobial activity (commercial antimicrobial soap-PC, distilled water-NC), Total Fatty Matter (TFM) and pH. Results: The crude extract indicated presence of phenols, flavonoids, saponins, carbohydrates, reducing sugars, terpenoids and triterpenoids. Crude extract (12 mg/mL) showed the highest activity of 13.92±0.02 mm zone of inhibition (ZOI) against E. Coli. The residual fraction exhibited the most potent inhibition, 13.03±0.26, 12.75±0.37, and 7.99±0.21 mm for E. coli, S. aureus, and Candida species, respectively. The highest ZOI showed for ciprofloxacin against E. Coli. (45.37±1.58 mm) and fluconazole against Candida species (17.89±1.13 mm). The highest ZOI showed for 0.8 g soap formulation against E. coli, S. aureus, and Candida species as 14.37±0.18, 21.83±1.13, and 16.15±0.65 mm, respectively even better than PC 11.54±0.51, 8.60±0.50 and 0.00 mm, respectively. Developed soaps were yellowish solids with a pleasant odor with TFM of 70.993% and pH of 9.15. Conclusions: Ethanolic crude extracts of the L. biflora whole plant and all fractions possess antimicrobial activity against chosen bacterial strains and fungi. Formulated herbal soaps were of expected quality and antimicrobial efficacy against tested pathogens.