Phytochemical and Bioactivity-Guided Evaluation of the Antibacterial Constituents of Psidium Guajava (Linn.) and Loranthus Micranthus (Linn.) Leaves

ABSTRACT Antibiotics have remained the mainstay of drug therapy of infectious diseases worldwide. Their use is, however, limited by their numerous adverse effects and rapid development of microbial resistance. Identification of natural products from plants that may serve as valuable sources of antimicrobial agents for medicinal or agricultural uses seems to be a viable alternative to the conventional antibiotics. To achieve this goal, biological assays should be carried out in order to identify promising plant extracts, 7 guide the separation and isolation, and to evaluate "lead" compounds. Psidium guajava Linn. and Loranthus micranthus Linn. have been employed traditionally in Nigeria and other parts of the globe for the treatment of various human ailments such as wounds, gastrointestinal tract disorders and other forms of infective and non-infective disorders. The main objective of this study was to identify and isolate the antibacterial compounds from the leaves of Psidium guajava and Loranthus micranthus L. The specific objectives were to: (i) carry out phytochemical evaluation and isolation of antibacterial constituents of the plants using standard methods, (ii) elucidate the structures of the isolated secondary metabolites, and (iii) carry out antibacterial assay of the isolated compounds. Fresh leaves of Loranthus micranthus (Linn.) parasitic on the stem of Persea americana were collected at Nsukka while those of Psidium guajava were collected from the bioresource area of the University of Port Harcourt in June 2010. The leaves were then cleaned, air-dried for 14 days and milled to coarse powder. The powdered materials (800 g each) were defatted with n-hexane (5 L) and extracted in a soxhlet extractor with 90.0 % methanol. The methanol extract was further fractionated to yield the chloroform, ethyl acetate, acetone and methanol soluble fractions. Each of the fractions was screened for antibacterial activity using Agar-well diffusion method. Phytochemical tests were carried out using standard procedures. The fractions that had the best antibacterial activity were subjected to column chromatographic separation and monitored by analytical thin layer chromatography (TLC). The ethyl acetate fraction (PsG-EF) from P. guajava that gave satisfactory bioassay result was subjected to further Sephadex-LH 20 chromatographic fractionation and purification to afford ten fractions (PsG-EF1 to PsG-EF10) which were pooled. Fractions PsG-EF4, PsG-EF5 and PsG-EF7 that had good antibacterial activity were subjected to semi-preparative reverse phase high pressure liquid chromatography (HPLC) purification to isolate the phenolic compounds; I-V. The structures of these compounds were elucidated by analytical and spectral techniques which included: ultra violet (UV), proton nuclear magnetic resonance (1H-NMR), carbon-13 nuclear magnetic resonance (13C-NMR), distortionless enhancement by polarization transfer (DEPT), proton-proton correlation spectroscopy (1H-1HCOSY), heteronuclear multiple quantum correlation (HMQC), heteronuclear multiple bond correlation (HMBC) and electron spray ionization-mass spectroscopy (ESI-MS) analyses. The isolated compounds were screened against standard strains of Staphylococus aureus (ATCC 25923) and Escherichia coli (ATCC 35219) using broth dilution assay method, and the MIC values determined and compared with ceftriaxone. All data obtained were analyzed by GraphPad Prism® 5 using differences in mean by two-way ANOVA and further subjected to Bonferroni post-tests to compare replicate 8 means. The results were presented as mean ± SEM. Differences between means were considered significant at P< 0.05) antibacterial activities while for Loranthus micranthus L., its various fractions showed significantly lower values (p > 0.001) when compared with the control (ceftriaxone) suggestive of a generally weak or negligible antibacterial action. All the isolated compounds from P. guajava were also found to have moderate antibacterial activities against E. coli and S. aureus in comparison with ceftriaxone whlie I and IV showed lower MICs than those of the other isolates against the test organisms.