Detection of antibacterial compound from kesum leaves (Polygonum minus Huds) by TLC-bioautography method and the effect to escherichia coli and staphylococcus aureus membrane.

Abstract

Kesum (Polygonum minus Huds) is the one of biodiversities from West Borneo and commonly used as flavouring ingredient for local culinary. Kesum leaves contain some bioactive compounds i.e high phenolic compounds, flavonoids, alkaloids, tannins and terpenoids which are potential as natural antibacterial agent. Detection of antimicrobial fractions of compounds from natural extracts could be done by TLC-bioautography. It is a combining method of chemical (thin layer cromatography/TLC) and microbology technique (antimicrobial activity assay) which allowed the localization of active compound. Generally, the kesum leaves extracts have been obtained by conventional maceration technique. This technique is often time consuming and requires relatively large amounts of solvent and sometimes diminishes the active compounds. Ultrasound assisted extraction (UAE) is one of the emerging technologies being developed in order to minimize the length of the extraction time, to reduce the cost, and to maximize the extract quality. The aims of this study are: 1) comparing extraction of kesum leaves maceration technique and UAE using some solvent with different polarity by multilevel extraction, 2) screening of extract based on antibacterial activity of kesum leaves extracts against E. coli and S. aureus, 3) fractination of kesum leaves extracts with the highest antibacterial activity by TLC, 4) to detect the active fraction of kesum leaves extracts with TLC-bioautography, and 5) to find out the mechanism inhibition of kesum leaves extracts against E. coli and S. aureus cell membrane. The experiment was conducted in four stages. The first stage covers extraction was done by steam distillation to obtain essential oils, followed multilevel extraction by maceration and UAE to obtain different extracts, i.e. polar, semipolar and nonpolar. The second stage covers qualitative analysis of phytochemical extracts, preparation of bacterial test and test the bacterial activity of extracts using disc – diffusion method and Minimum Inhibitory Concentration (MIC) using macrodilution method. The third stage was fractionation of extract with the highest antibacterial activity by TLC method. The fourth stage was detection the active fraction of kesum leaves extracts with TLC-bioautography, followed by TLC-bioautography. The last stage was study of mechanism inhibition of kesum leaves extracts against cell membrane by DNA probe staining using fluorescen microscopy and detection leakage of the cytoplasmic membrane has been analysed by spectrophotometer UV – Vis. This study exhibited that UAE provided higher extraction yields and shortened the extraction time, in comparison to the conventional maceration technique. Phytochemical analysis showed kesum leaves extract contain phenolics, alkaloids, flavonoids, triterpenoids, tannins and saponins. The ethanolic extract of kesum leaves were potential as source of antibacterial agent. The MIC of the ethanolic extract, was 25 mg/ml for E. coli and 30 mg/ml for S. aureus. Fractination by TLC showed six spots under UV light at λ 254 nm. TLC-bioautography with agar overlay technique effectively detected that fractions of ethanolic extract of kesum leaves with Rf4 = 0.30 and Rf5 = 0.37 demonstrated the most noticeable antibacterial activity for both E. coli and S. aureus. Inhibitory mechanism of kesum leaves extract against E. coli and S. aureus is the disruption cytoplasmic membrane permeability that causes leakage of cell materials.