Study On The Material Basis And Mechanism Of Phytoestrogenic Effects In Selaginella Moellendorffii Hieron

Selaginella moellendorffii Hieron.,which belongs to the family Selaginellaceae.It contains flavonoids,phenylpropanoids and other components,which have the effect of clearing heat and detoxifying,activating blood circulation and reducing swelling.It can be used for the treatment of metabolic diseases and so on.The group’s preliminary research found that S.moellendorffii has estrogen-like activity,but its pharmacological basis and mechanism of action are still unclear.Therefore,in this paper,we intend to investigate the pharmacological basis and mechanism of action of S.moellendorffii at the cellular and animal levels.Objective: In this study,we will screen the effective parts and active ingredients of S.moellendorffii for their estrogenic activity,clarify their pharmacological basis,and investigate the mechanism of estrogenic activity of S.moellendorffii in vitro and in vivo.Methods:（1）A transgenic recombinant yeast model was constructed,and the hormonal activity of five Selaginella species was evaluated by detecting the relative activity of b-galactosidase;HPLC and LC-MS were used to analyze the active components of estrogenic active parts of S.moellendorffii.（2）Using E-screening method,breast cancer cells with different types of estrogen receptor expression（MCF-7,T-47-D,ZR-75-1,SK-BR-3,MDA-MB-231）,and different estrogen receptor antagonists ICI182,780,MPP and PHTPP were selected to observe the effects of S.moellendorffii on the proliferation of different cells;combined with Western Blot assay,the effects of S.moellendorffii on the expression of ERa,ERb,and estrogen signaling pathway PI3K/AKT and MAPK/ERK and other related proteins were investigated.（3）Sexually immature mice were selected for uterine weight gain experiments to study the effects of S.moellendorffii on uterine coefficient,uterine tissue morphology,endometrial thickness and serum estrogen content,as well as to observe the mean fluorescence intensity and protein expression of uterine tissue ERa,and to evaluate the toxic effects of S.moellendorffii in terms of liver and kidney function.Results:（1）The results of transgenic recombinant yeast showed that the aqueous extracts of S.tamariscina and S.moellendorffii possessed both estrogen agonistic and antagonistic activities.The n-butanol site and the components of apigenin and apigenin-6,8-di-C-b-D-glucopyranoside also showed weak estrogenic agonistic activity,and it is assumed that the pharmacokinetic substance basis for estrogenic activity in S.moellendorffii may be the flavonoid glycoside components.The HPLC and LC-MS results of different extracts of Cyperus rotundus showed that more than 10 components,including 5-carboxymethyl-4’-hydroxyflavone-7-O-b-D-glucopyranoside,neoselaginellic acid and Moellenoside A,were analyzed and identified in the aqueous and n-butanol parts of S.moellendorffii that exerted estrogenic activity.（2）Both aqueous extracts and n-butanol sites of S.moellendorffii in the concentration range of 6.25-100 μg/m L promoted the proliferation of ER（+）cells（MCF-7 and T-47-D cells）within 48 and 72 h,while no significant proliferative effects were observed on ZR-75-1 cells and ER（-）cells（SK-BR-3and MDA-MB-231 cells）.The proliferative effect was antagonized by the estrogen receptor antagonist ICI182,780 and estrogen receptor a antagonist MPP,and the estrogen receptor b antagonist PHTPP had no significant effect on them.This indicates that S.moellendorffii has estrogenic activity and has a higher affinity for ERa compared to ERb.In addition,the flavonoid glycoside components and apigenin all promoted the proliferation of MCF-7 cells and exhibited some estrogenic activity.The effect of aqueous extract of S.moellendorffii on MCF-7 cells resulted in elevated ERa protein expression and a non-significant increase in ERbprotein expression.PI3 K,p-AKT/AKT,p-m TOR/ m TOR,p-ERK/ERK and pP38/P38 protein expression were all elevated to different degrees.100 n M of ICI182,780 and 100 n M of MPP both inhibited the expression of related proteins.（3）Uterus weight gain experiment showed that: the n-butanol site of S.moellendorffii increased the uterine coefficient of sexually immature mice,increased endometrial thickness,increased serum estrogen content,increased mean fluorescence intensity of ERa and ERa protein expression in uterine tissues,which were consistent with the results of in vitro cellular experiments,while ethyl acetate site of S.moellendorffii did not have this phenomenon.The results were consistent with the in vitro cellular assay,but not with the ethyl acetate site.Both n-butanol and ethyl acetate parts of S.moellendorffii had no significant effects on the liver and kidney functions of mice,while ethyl acetate part caused a decrease in MDA level and an increase in SOD level in the liver of mice.Conclusion: The aqueous extracts and n-butanol parts of S.moellendorffii have estrogen-like effects,and the material basis for the estrogen-like activity may be flavonoid glycoside components,and the mechanism of action is mainly manifested by higher affinity with ER,and can promote the expression of estrogen-related pathway proteins such as PI3K/AKT and MAPK/ERK.At the same time,S.moellendorffii could promote the growth of mouse uterus and had no obvious side effects on liver and kidney function.