Fast Screening For Human Aromatase Inhibitors From Mulberry (Mori Alba L.) Leaves And The Selectivities Of P450 Enzymes To Flavonoids

Aromatase, a cytochrome P450 (CYP19) enzyme, catalyzes the rate-limiting aromatization step for the conversion of androgens to estrogens and is an effective target for inhibition of estrogen biosynthesis to treat hormone-sensitive breast cancer in postmenopausal women. Naturally occurring products, especially the flavonoids, has been proved to be potential therapeutic agent for breast cancer by inhibiting the activity of aromatase and is one of the main active constituents of mulberry leaves. In this study, we provide a new method that can be used for fast screening of potential inhibitors of aromatase. Furtherly, this paper determined the binding capacities of twenty-one flavonoids on aromatase, and their binding characteristics and the structure requirement for all classes of flavonoids to binding aromatase were discussed. The selectivity of the twenty-one flavonoids was evaluated by binding assay with human cytochrome P450 enzymes (CYP19 and CYP1A1). The major contents of this dissertation are described as follows:1.3,5,7,3',4'-pentahydroxyflavone 3-glucosylglucoside/3,5,7,3',4'-pentahydroxyflavo-ne 3,7-diglucoside; moracin-M 6,3'-diglucoside (mulberroside F); 3,5,7,3',4'-penta-hydroxyflavone 3-(6"-malonyl)-glucoside; 3,5,7,4'-tetrahydroxyflavone 3-(6"-malo-nyl)-glucoside; 3,5,7,4'-tetrahydroxyflavone 3-(6"-acetyl)-glucoside; 3,5,7,3',4'-pentahydroxyflavone 3-rutinoside (rutin); 3,5,7,3',4'-pentahydroxyflavone 3-glu-coside/3,5,7,2',4'-pentahydroxyflavone 3-glucoside; kuwanon G; 5,7,3',4'-tetra-hydroxyflavone 7-rutinoside/3,5,7,4'-tetrahydroxyflavone 3-rutinoside; 3,5,7,4'-tetrahydroxyflavone 3-glucoside (astragalin); 3,5,7,3',4'-pentahydroxyflavone-3-(6"-acetyl)-glucoside; compounds 12 and 13; chalcomoracin/mongolicin F; 9,12,15-octadecatrienoic acid (linolenic acid), were tentatively identified by HPLC-DAD-ESI-MS/MS method.2. The present work successfully prospected for seven chemical constituents from Mulberry (Morus alba L.) leaf simultaneously using LC-MS, and their binding capability for aromatase was evaluated by measuring reduce of HPLC peak areas of the detected chemicals in samples treated with CYP19 compared to the control analyses (treated without CYP19). A model system by in vitro bio-assay was conducted to verify our findings. The seven compounds are,3,5,7,3',4'-pentahydroxyflavone 3-rutinoside (rutin),3,5,7,3',4'-pentahydroxyflavone 3-gluco-side/3,5,7,2',4'-pentahydroxyflavone 3-glucoside, chalcomoracin/mongolicin F, compounds 12 and 13,3,5,7,4'-tetrahydroxyflavone 3-glucoside(astragalin) and 9,12,15-octadecatrienoic acid (linolenic acid).3. The binding capacities of twenty-one flavonoids to aromatase and CYP1A1 were determined. Meanwhile, the aromatase inhibiting activities of these flavonoids were evaluated by in vitro bio-assay. Twenty-one flavonoids'selectivity for binding to aromatase or CYP1A1 was discussed. Generally, aromatase have tight selectivity in reacting with flavonoids, and the selectivity of flavonoids for interaction with CYP1A1 is less high compared with CYP19 (aromatase). Flavane nearly have no binding activity to aromatase and have moderate potency to CYP1A1, flavones have higher enzymes binding activity than flavanone, isoflavone and isoflavanone.