AUTHOR=Qiang Yining , Bai Lu , Tian Shuran , Ma Yi , Xu Pingxiang , Cheng Mingchang , Wu Yi , Li Xiaorong , Xue Ming , Zhou Xuelin 

TITLE=Daidzein is the in vivo active compound of Puerariae Lobatae Radix water extract for muscarinic receptor-3 inhibition against overactive bladder

JOURNAL=Frontiers in Pharmacology

VOLUME=13

YEAR=2022

URL=https://www.frontiersin.org/journals/pharmacology/articles/10.3389/fphar.2022.924251

DOI=10.3389/fphar.2022.924251

ISSN=1663-9812

ABSTRACT=<p><bold>Background:</bold> In the previous study, Puerariae Lobatae Radix (named Gegen in Chinese) water extract attenuated M3 receptor agonist carbachol-induced detrusor contraction after 3-week oral administration in a hypertension-associated OAB (overactive bladder) model. This research aimed to investigate the active ingredients from Gegen water extract against OAB.</p><p><bold>Methods:</bold> Bioassay-guided fractionation was performed by using preparative HPLC for fast isolation of fractions followed by screening their <italic>ex vivo</italic> activity through carbachol-induced bladder strip contraction assay. Chemicals in each active fraction were analyzed by HPLC-UV. Urine metabolites were quantified by LC-MS/MS after sub-acute administration. Thermal shift assay with the recombinant human M3 receptor protein was performed, and molecular docking analysis was used for molecular modelling of M3 receptor inhibition.</p><p><bold>Results:</bold> Bioassay-guided fractionation results for isolating M3 receptor inhibitors indicated that four compounds were identified as active ingredients of Gegen water extract, and their inhibition potency on carbachol-induced detrusor contraction was ranked in descending order according to their inhibition concentrations as follows: genistein &gt; daidzein &gt; biochanin A &gt;&gt; puerarin. Daidzein in urine reached an <italic>ex vivo</italic> effective concentration to inhibit detrusor contraction, but others did not. Daidzein concentration-dependently increased the melt temperature (<italic>T</italic>m) of recombinant human M3 receptor protein with a positive binding (Δ<italic>T</italic>m = 2.12 °C at 100 μg/ml). Molecular docking analysis showed that daidzein can potently bind to the ligand binding pocket of the M3 receptor via hydrogen bonding.</p><p><bold>Conclusion:</bold> Puerarin and its derivatives were pro-drugs, and daidzein was their <italic>in vivo</italic> active form via M3 receptor inhibition for treating OAB.</p>