Antimicrobial activity of torilin isolated from Torilis japonica fruit against Bacillus subtilis

Torilis japonica fruit has been used in therapeutic antimicrobial treatments in Korea and China since ancient times, but there is still little information on the mechanism underlying this activity. We found that the ethanol extract of T. japonica fruit showed excellent antimicrobial activity against Bacillus subtilis ATCC 6633 spores and vegetative cells. The crude ethanol extract (75%) reduced the spore concentration by 3 log cycles and the vegetative cell concentration to lower than the detection level (reduction in spore concentration by more than 6 orders of magnitude) at a concentration of 1% (w/v). The ethanol extract of T. japonica fruit was fractionated into n-hexane (H) and a water layer. The active antimicrobial compound was isolated and purified from the hexane layer, and identified as torilin (5-[1-(acetyloxy)-1- methylethyl]-3,8-dimethyl-2-oxo-1,2,4,5,6,7,8,8a-octa-hydroazulen-6-yl(2E) -2-methylbut-2-enoate; C₂₂H₃₂O₅). Torilin immediately reduced vegetative cells counts by 5 to 6 orders of magnitude, and reduced spores counts by 1 order of magnitude. The integrity of structures such as the inner, middle, and outer layers of the coat and the cortex, protoplast membrane, and core are vital to spores. Torilin functions as a surfactant with hydrophilic and hydrophobic properties related to denaturalization of various proteins. The distortion of coat proteins due to direct binding polar groups of spore coats with hydrophilic groups of torilin may be responsible for the observed rapid inactivation of bacterial spores.