| Summary: | Industrial hemp leaves have raised much interest in nutraceuticals and functional foods areas. To expand its application ranges, the antibacterial activities of industrial hemp leaf extract on <i>Escherichia coli</i>, <i>Staphylococcus aureus</i>, and <i>Bacillus cereus</i> were evaluated and the active components were screened. As a result, the industrial hemp leaf extract was found to have strong bacteriostatic effects on <i>E. coli</i> and <i>S. aureus</i>. Bioassay-guided fractionation and isolation from fractions active against <i>E. coli</i> were conducted. Two compounds, cannabidivarinic acid and cannabidiolic acid, were firstly recognized by analytical HPLC by comparing the retention times and UV spectra with standards and later isolated using preparative HPLC. Moreover, the antibacterial mechanisms of cannabidivarinic acid and cannabidiolic acid were investigated by testing the alkaline phosphatase activity, β-galactosidase activity, conductivity, proteins leakage, nucleic acid leakage, and scanning electron microscope observation. The results demonstrated that cannabidivarinic acid and cannabidiolic acid could destroy the cell wall and membrane of <i>E. coli</i>, resulting in the inhibition of enzyme activity and leakage of contents. They could damage the bacteria cell envelope as well. Presented results pointed out cannabidivarinic acid and cannabidiolic acid as promising natural bacteriostatic agents for the food, pharmaceutical, and cosmetic industry.
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