Evaluation of the effect of Humulus lupulus alcoholic extract on rifampin-sensitive and resistant isolates of Mycobacterium tuberculosis

Abstract

The increasing incidence of Multi Drug Resistance Tuberculosis (MDR-TB) and Extensively Drug Resistance TB (XDR-TB) worldwide highlight the urgent need to search for newer anti-tuberculosis compounds. It has been determined that pharmaceutical plant, hops (Humulus lupulus), possesses some antibacterial effect. In this study, the antimycobacterial effect of this plant on rifampin sensitive and resistant strains of Mycobacterium tuberculosis were examined. Sensitivity and resistance of 37 Iranian isolates of M. tuberculosis to rifampin was determined by proportion method. Ethanolic extract of hops was prepared using maceration method. PCR-SSCP and direct sequencing were used for confirming existence of mutations in 193-bp rpoB amplicons related to the rifampin resistance in Mycobacterium tuberculosis isolates. Two different concentrations of hops alcoholic extract (4 and 8 mg/ml) were prepared and its effects against 21 resistant and 15 sensitive isolates was determinate using proportion method. Six different mutations in the 193-bp amplified rpoB gene fragments and seven distinguishable PCR-SSCP patterns in 21 Iranian rifampin resistant isolates were recognized. This study showed that the percentage of resistance and the type of mutations were correlated with the PCR-SSCP patterns and the type of mutations in rpoB gene (P<0.05). The results of hops antimycobacterial effect showed that different concentrations of hops ethanolic extract (4 and 8 mg/ml) had a remarkable inhibitory effect on rifampin sensitive and resistant isolates of Mycobacterium tuberculosis. Identification of the effective fraction of hops against Mycobacterium tuberculosis is a further step to be studied.

Keywords: Antimycobacterial effect; Ethanolic extract; Humulus lupulus; Mycobacterium tuberculosis.

Fig. 1

PCR-SSCP patterns of 3 sensitive and 10 rifampin-resistant M. tuberculosis isolates. From left, Lane: 1: ladder 50, lane 2 is H37Rv, lanes 3, 4, and 5 are sensitive strains and their patterns are indistinguishable from that of M. tuberculosis H37Rv (lane 2). Lanes 6 to 15 are rifampin-resistant strains and their patterns are distinguishable from that of M. tuberculosis H37Rv. Lane 6 is pattern 1 rifampin-resistant strains with 4 bands, Lanes 7 and 8, pattern 2 rifampinresistant with 5 bands, Lane 9, pattern 3 rifampin-resistant with 5 bands different from 7 and 8, Lane 10, pattern 4 rifampin-resistant with 4 bands different from Lane 6 and 11 to 15, pattern 5 rifampin resistant with 4 similar bands. All strains show distinguishable patterns from that of M. tuberculosis H37Rv pattern.