Bioactivity-Guided Studies on the Crude Extract Andfractions of Hibiscus Asper Hook F. (Malvaceae) Leaf

Abstract

The antibacterial and anti-inflammatory activities of the leaf extract of Hibiscus asper Hook F. (Malvaceae) were investigated. Solvent extraction of the leaves yielded the crude methanol extract (CME), while solvent-guided fractionation of CME yielded a n-hexane fraction (HF), chloroform fraction (CF), ethyl acetate fraction (EF), acetone fraction (AF), butanol fraction (BF), methanol fraction (MF) and water fraction (WF). TheCME and the solvent fractions were subjected to phytochemical analysis using standard procedures. The acute toxicity test (LD50) of oral doses of CME was conducted using mice. CME and the solvent fractions were screened for antibacterial activity using agar well diffusion method against Gram-positive bacteria: Staphylococcus aureus, Bacillus subtilis and Gram-negative bacteria Pseudomonas aeruginosa, Escherichia coli and Klebsiellapneumoniae (clinical strains). The crude extract, CME was also screened for anti-inflammatory activities using egg albumen-induced rat paw oedema as a model for acute inflammation, while formaldehyde-induced rat paw oedema was used as a model for chronic inflammatory states. Results of the phytochemical analysis of CME revealed the presence of various substances including alkaloids, tannins, flavonoids, saponins, sterols, terpenoids, carotenoids and glycosides.The solvent fraction HF contains mainly steroids, terpenoids and carotenoids, CF contains alkaloids, carotenoids, flavonoids, terpenoids and glycosides, EF contains flavonoids and glycosides, AF contains tannins and glycosides,while BF, MF and WF contains saponins and glycosides. The acute toxicity test for CME revealed that it has a high safety profile (LD50>5000 mg/kg) as the extract was well tolerated by the animals. Result of the antibacterial screening showed that CME and the solvent fraction EF inhibited the growth of Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa and Escherichia coli, with their minimum inhibition concentrations (MIC) determined to be 11.34, 10.11, 6.78 and 7.61 mg/mL for CME and 5.75,3.05,9.54 and 3.37 mg/mL, for EF respectively. Klebsiellapneumoniae was not susceptible to CME and EF. The five test organisms were prominently susceptible to MF with MIC values as 6.25, 9.59,10.84,9.63 and 5.90 mg/mL for Staph. aureus, B. subtilis, E. coli,P. aeruginosa and K. pneumoniaerespectively. Result of the anti-inflammatory studies showed that the crude extract, CME elicited a dose-related inhibition of egg albumen-induced (acute) oedema in rats. At the dose level 200 mg/kg (i.p.), CME showed moderate inhibition of rat paw oedema (% oedema inhibition = 54.10), while 300 mg/kg (i.p.) showed high activity (% oedema inhibition = 73.77) at 4 h (P<0.05 and P<0.01). The oedema inhibition by CME at doses of 200 and 100 mg/kg were significant (P<0.05) at 1 h and 4 h after treatment respectively. The crude extract, CME also ameliorated chronic inflammation (arthritis) induced by formaldehyde in rats. At 100 mg/kg (i.p.), CME showed small but significant (P<0.05) inhibition of rat paw oedema (% inhibition of arthritis = 14.79). At dose levels of 200 and 300 mg/kg (i.p.), CME showed fair and moderate activities (% inhibition of arthritis = 26.06 and 40.14 respectively). This work indicates that Hibiscus asper leaf has both antibacterial and anti-inflammatory--activities.