Discovery Of Aromatase Ligands From The Extract Of Glycyrrhiza Uralensis And Their Aromatase Inhibitory Activities

The chemical constituents from dichlormethane fraction of 95% ethanol extract of Glycyrrhiza uralensis were studied. Thirty compounds were separated and identified, twenty-six of which were flavonoids. Aromatase, a member of the cytochrome P450 superfamily, catalyzes the final and key steps of C19 steroids conversion to estrogens. Its inhibitors are widely used in breast cancer therapy. One of the mechanisms of aromatase inhibitors is to bind to the aromatase active-site. The offen used screening methods of aromatase inhibitors are TLC and 3H2O. The operations of the two methods are complicated and the expense is also too high.The aim of our work is to find a simple, fast and effective method to discover small molecure aromatase ligands and to screen aromatase inhibitors.It has been reported that some flavonoids (flavones, flavanones, and isoflavones) inhibit aromatase activity. G. uralensis, a traditional Chinese medicine enriched in flavonoids, was selected for the screening of aromatase ligands in this work. The peak areas of twenty-one compounds from CH2Cl2 extract of G. uralensis were significantly reduced or disappeared after treatment with aromatase. Eleven of the binding compounds (4,7,8,10,11,13,15,18,19,20, 23) were isolated from the G. uralensis in our work. Other ten (A-J) compouds were not isolated, structures of compounds A,B,D～F were speculated on their MS data with referring to literatures. They are isoflavones, isoflavanone, and isopentenyl substituted flavane. The isopentenyl may be facilitated for the affinity of this compound to aromatase. The aromatase used in this article was a recombination protein which can induce false positive result. In order to further confirm the results, the method of IP coupled with HPLC-MS/MS was designed to test the compounds, which can bind to aromatase in MCF-7 cells. Seven compounds (7,15,18,19,23,D,E)were detected, they also have strong binding ability to aromatase. An active compounds screening system was conducted to verify if the method of IP coupled with HPLC-MS/MS to discover aromatase ligands is efficient or not. Ten compouds (4,7,8,10,11,13,15,18,19,20) isolated from the G. uralensis were choosed to test the inhibitory activity on aromatase. The results are consistent with the method of IP coupled with HPLC-MS/MS. Thus, the method of IP coupled with HPLC-MS/MS can be used as a method to discover aromatase inhibitors initially.