Study On The Anti-HBV Active Constituents Of Miso

Abstract:Hepatitis B virus (HBV) pose a lethal threat upon human being’s health. Nucleoside analog are the major drugs for anti-HBV treatment as they can effectively inhibit HBV replication, but fail to completely eradicate the virus and significant inevitable drug resistance have been noted. Therefore, to find the candidate compounds with anti-HBV activity from the natural products and further develop into a novel medicine carry substantial significance.Anti-HBV activity of the miso extract has been found for the first time and then the n-butanol fraction was enriched from miso under the guidance of the anti-HBV assay in vitro. In this dissertation, the chemical constituents of the active fraction from miso were deeply and systematically studied under the guidance of activity. Those isolated compounds were further studied for their anti-HBV activities, and their structure-activity relationships were initially discussed.Miso is a traditional Japanese fermented soybean serviced as a seasoning in Asia with time honored history.In this dissertation, different column chromatography including silic gel, Sephadex LH-20, reversed phase silic gel and high performance liquid chromatography were applied in the chemical constituents study of the active fraction from miso. Thirty-three compounds were isolated from this fraction, and twenty-seven of them were elucidated by spectroscopic methods as follows:cyclo-(Pro-Gly)(FSE-1), cyclo-(Phe-Tyr)(FSE-2), cyclo-(Leu-Tyr)(FSE-3),4’,7,8-trihydroxy-6-methoxy isoflavone (FSE-4),4’,5,7,8-tetrahydroxy isoflavone (FSE-5),4’,7,8-trihydroxy isoflavone (FSE-6), glycitein (FSE-7), genistein (FSE-8), daidzein (FSE-9), isoleucine (FSE-10), valine (FSE-11), leucine (FSE-12), L-Pyroglutamic acid methyl ester (FSE-13), tyrosine (FSE-14), N-methyl leucine (FSE-15), phenylalanine (FSE-16) phenylalanine methyl ester (FSE-17), N-acetylphenylalanine (FSE-18), glutamic acid (FSE-19), glycine (FSE-20), aspartic acid (FSE-21), uracil (FSE-22), thymine (FSE-23), behenic acid (FSE-24), palmitic acid (FSE-25), pyrocatechol (FSE-26) and daucosterol (FSE-27). From the perspective of chemical structures, twenty-seven compounds include three cyclopeptides, six isoflavones, twelve amino acids and six other structural types. To the best of our knowledge, twenty-four compounds have not yet been isolated from unfermented soybean or its related products before this study, and eighteen compounds were reconstructed by bacterium.Anti-HBV activity of these compounds were evaluated on vitro model and five active compounds (FSE-5、FSE-6、FSE-13、FSE-19and FSE-23) were reported for the first time. FSE-13inhibited both the secretion of HBsAg and HBeAg by22.9%and42.7%(500.0μg/mL or3.50μM); FSE-19specifically inhibited the secretion of HBeAg by37.6%(500μg/mL or3.40μM); FSE-23inhibited both the secretion of HBsAg and HBeAg by50.2%and41.3%(250.0μg/mL or1.98μM); both FSE-5and FSE-6shown inhibition activities on the secretion of HBsAg and HBeAg,19.9%and17.1(100μg/mL or0.35μM),15.4%and24.6%(5μg/mL or18.5nM), respectively.Initial analysis of structure-activity relationships of those active isoflavones indicated that the absence of6-methoxy group may be favorable to its activities (FSE-6> FSE-4), while the presence of8-hydroxy group may contribute to its activities (FSE-5> FSE-8and FSE-6> FSE-9).Those findings in this study shed light on anti-HBV active constituents of miso. Investigating the reconstructed compounds generated during fermentation and discovering their structure-activity relationships will not only provide candidates for anti-HBV medicine development, but also lay a consolidate foundation for further development of fermented soybean.