Antibacterial activity and chemical compositions OF Apis mellifera propolis extracts from nan province, Thailand

Abstract

Propolis is sticky and dark brown. It is derived from plant resins, tree buds, sap flows, and other botanical sources collected by honeybees. It is mixed with wax and used to construct and repair the nest. It possesses many bioactivities. In this research, propolis from Apis mellifera from Nan province was tested for the antibacterial activity against Staphylococcus aureus (Gram + bacteria), Paenibacillus larvae larvae (Gram + bacteria), and Escherichia coli (Gram – bacteria). Before all of three crude extracts were tested for antibacterial activity, the correct types of three selected bacteria were confirmed by two methods which were Gram staining and partial sequence of 16S rDNA analysis. Propolis was extracted by methanol, dichloromethane, and hexane. Thereafter, the antibacterial activity of these crude extracts was determined by agar well diffusion and microbroth dilution assay. In addition, streptomycin was used as a positive control. Crude methanol extract of propolis (CME) showed the best antibacterial activity with the minimum inhibition concentration or MIC value of 5 mg/ml and was effective to inhibit both S. aureus and E. coli. In addition, CME was effective to inhibit P. larvae larvae with MIC value of 6.25 mg/ml. Thus, CME was used for further purification by quick column chromatography. Only three active fractions were able to inhibit the growth of both S. aureus and E. coli with the MIC values of 6.25 and 31.25 µg/ml, respectively. Later, the active fraction was further purified by adsorption chromatography. The purified fraction A1A could present the IC50 values of 0.175 µg/ml for E. coli and of 0.683 µg/ml for P. larvae larvae. After fraction A1A was further analysed for chemical structure by using Nuclear Magnetic Resonance, it was in a cardanol group. The morphology change and time-killed assay by cardanol on E. coli was observed by scanning and transmission electron microscopy. The anti-E. coli activity was observed in the early period of treatment which was before 4 h. For treated cells, unusual shape, especially in dividing cells, damaged and dead cells could be distinguished.