Isolation and Purification of Beta Lactamase Inhibitor from Herbal Extracts to Enhance the Drug Susceptibility of Uropathogens

Abstract

The study is to isolate and purify beta-lactamase inhibitor/s from herbal extracts to enhance the drug susceptibility in uropathogens. For isolation of beta-lactamase inhibitior/s from medicinal plants following ten plants were screened Syzygium aromaticum (bud), Phyllanthus emblica (fruit), Datura metel (leaves), Citrus sinensis (fruit peel), Picrorhiza kurroa (roots), Lawsonia inermis (leaves), Punica granatum (flower), Terminalia chebula (fruit), Terminalia bellirica (fruit) and Ocimum tenuiflorum (leaves). Extracts of these plants were prepared with 6 solvents of different polarity. Beta-lactamase inhibition study was performed by micro- idometric assay and agar cup bioassay using uropathogens. Multidrug-resistant clinical strains of Escherichia coli ND-1, ND-2; Hospital Isolates-2, 7 and 13 (HI-2, HI-7 and HI-13); standard strain MTCC-729 (Escherichia coli acquired from MTCC; produces beta-lactamase) and DJ1.2 (DH5and#945; transformed with pJET1.2 plasmid containing Ampr gene; positive control for beta-lactamase) were used in this study. Results from agar cup bioassay, as well as micro-iodometric assay for enzyme inhibition, confirmed the presence of beta-lactamase inhibitor in Syzygium aromaticum, Citrus sinensis, Ocimum tenuiflorum and Datura metel and they were selected for purification. It was found that 4-allyl-2-methoxyphenol from S. aromaticum proved to be better beta-lactamase inhibitor when combined with ethanolic extract of D. metel (as natural antibiotic). Efficacy of HPLC purified fractions of beta-lactamase inhibitors from D. metel (D2), C. sinensis (C4) and O. tenuiflorum (O3). D2 and C4 proved to be better beta-lactamase inhibitor when compared with clavulanic acid whereas O3 exhibited lower beta-lactamase inhibition activity when compared with clavulanic acid. Results clearly demonstrate that 4-allyl-2-methoxyphenol is better than the commercial beta-lactamase inhibitor, it work in combination with existing beta- lactam antibiotics and extracts of medicinal plants such as D. metel and M. micrantha.