Screening Of Active Ingredients From Mountain Cultivated Ginseng And Cultivated Ginseng On Delaying Ovarian Aging In Drosophila And Studying Its Mechanism

Objective: The aim of this study was to compare the efficacy of mountain cultivated ginseng(MCG)and cultivated ginseng(CG)in relieving ovarian aging,to investigate in depth the differences in the composition of secondary metabolites between MCG and CG,to screen for active ingredients with the ability to reduce ovarian aging,and to reveal their mechanisms of action in relieving ovarian aging.Methods:(1)MCG and CG were applied to naturally aging female Drosophila models,and their effects on fertility,longevity,locomotion,sleep quality,ovarian size,and steroid hormones were examined by behavioral monitoring,Drosophila sleep monitoring system,microphotography,and enzyme-linked immunosorbent assay to compare their efficacy in reducing ovarian aging.(2)The non-target metabolomics of traditional Chinese medicine was used to analyze the secondary metabolites in MCG and CG and to clarify the differential composition and distribution of secondary metabolites.The extractions and purifications of the different secondary metabolites were carried out by solvent leaching and chromatography,membrane filtration and other preparative techniques,and their contents were examined.Their effects on the fertility and ovarian aging-related symptoms of aged female Drosophila were analyzed,and the active fractions with the ability to alleviate ovarian aging were screened.(3)The expression of the screened differential fractions on ecdysteroid receptor(ECR),early transcription factor E74,Broad(Br),juvenile hormone reductase(HMGR),juvenile hormone transferase(JHAMT),juvenile hormone receptor Met and effector gene Kr-h1 was examined using real-time quantitative PCR to evaluate their effects on steroid signaling.(4)The above efficacy components were quantified by liquid-phase high-resolution mass spectrometry to detect their effects on fertility and steroid hormone levels,and to screen for active monomeric components that alleviate ovarian aging.(5)The binding of this monomeric component to ECR was analyzed using the molecular docking technique.(6)Real-time quantitative PCR,enzyme-linked immunosorbent assay,protein immunoblotting and immunofluorescence staining were used to detect the effects of the active ingredients on the number of steroid hormones,calmodulin(E-cadherin),pro-differentiation factor Bam,stemness maintenance factor Nos and germinal stem cells in the ovarian microenvironment and to determine their regulatory effects on the ovarian microenvironment.(7)The effects of ECR,Br,E74 and chromatin remodeling factor ISWI,and Dpp,Smad4 and p Smad1/5/8,which are related to the bone morphogenetic protein(BMP)signaling pathway,on the regulation of ovarian microenvironment,were detected by real-time quantitative PCR and protein immunoblotting to elucidate the mechanisms of active ingredients on the regulation of ovarian microenvironment.(8)Based on the ECR mutant female Drosophila model to detect fertility,ovarian volume,steroid hormone levels,expression of E-cadherin,Bam,Nos,and the effects of steroid signaling and BMP signaling pathways in the ovarian microenvironment to verify their effects on mitigating ovarian aging.Results:(1)The effects of MCG and CG on the reproduction and development of old female Drosophila offspring were significantly improved,resulting in a 16% and 28%increase in reproductive capacity,inhibition of aging ovarian atrophy,restoration of steroid hormone levels,improvement of sleep quality,and improvement of body motility to delay aging,and the effects of the MCG were superior to those of the CG.(2)A total of 71 significantly different secondary metabolites were identified by comparing the composition and relative contents of the secondary metabolites in MCG and CG.Among them,53 were significantly up-regulated and 18 were significantly down-regulated in MCG,and the differentially enriched secondary metabolites in MCG were mainly concentrated in triterpenoid saponins.The total ginsenosides of MCG and CG were extracted,isolated and purified from MCG and CG,both of which were >80% pure.Both total ginsenosides were found to alleviate the symptoms of ovarian aging when fed to aged female Drosophila,while the effect of the total ginsenosides from the MCG was more significant than that of the total ginsenosides from the CG.(3)All the total ginsenosides of MCG and CG increased the expression levels of ECR,E74 and Br,promoted the expression of HMGR and JHAMT genes,enhanced the expression of the effector gene Kr-h1 gene and regulated ovarian function in the ovaries of aged female Drosophila.(6)We detected 17 ginsenoside monomers with different contents in total ginsenosides of MCG and CG using liquid-phase high-resolution mass spectrometry,among which Rg1,Rd,Rc,Rb1,F2,F1,Rg3,Rh1,Rb3,Rf,Rg5,Rb2,PPT,Re,R0,Rh2 and CK were higher in total ginsenosides of MCG than in total ginsenosides of CG.The effects of 17 ginsenoside monomers on the reproductive capacity and steroid hormones of aged female Drosophila were subsequently examined,and it was found that ginsenosides Rg1,Rb1 and Re enhanced ovarian function in aged females,while the other 14 ginsenoside monomers had no significant effects.(5)Ginsenosides Rg1,Rb1,and Re were docked to the active site of ECR,and it was found that ginsenosides Rg1,Rb1,and Re could bind to the functional structural domain LBD of ECR,and Rg1 had the strongest binding ability to ECR with an affinity energy of-5.9 Kcal/mol.(6)Ginsenoside Rg1 promoted the number and development of offspring of aged female Drosophila and was dose-dependent.1 mg/m L Rg1 increased the reproductive capacity of senescent Drosophila by 17.72%,inhibited the atrophy of senescent ovaries and restored steroid hormone levels.In addition,ginsenoside Rg1 could extend the lifespan of senescent female Drosophila and restore their muscle capacity to a youthful state.(7)Ginsenoside Rg1 inhibited the decrease of anchoring protein E-cadherin,self-renewal maintenance factor nanos(Nos)and pro-differentiation factor Bam caused by aging and reconstructed the microenvironment of aging ovaries.Ginsenoside Rg1 activated the expression of ECR,E74 and Br,triggered the steroid signaling cascade,and enhanced the expression of ISWI,promoting the secretion of BMP into the microenvironment.(8)The targeting effect of ginsenoside Rg1 was verified based on ECR mutants,and it was found that the regulatory effects of ginsenoside Rg1 on ovarian function,microenvironmental cytokine expression and the regulation of steroid signaling and BMP signaling pathways in the ovarian microenvironment of female Drosophila mutants were dependent on its binding to the receptor ECR,which clarified the targeting regulatory effect of ginsenoside Rg1.Conclusion: In this study,we found that MCG and CG have efficacy in alleviating ovarian aging,but the effect of MCG is better.The differences in the composition of the secondary metabolites of MCG and CG were investigated in depth,mainly focusing on the triterpene saponins.Systematic screening of the main active components of total ginsenosides from MCG and CG to alleviate ovarian aging revealed that ginsenoside Rg1 had the most significant effect,could bind to the LBD region of the hormone receptor ECR,upregulated the expression of chromatin remodeling factor ISWI through the target ECR,activated microenvironmental BMP signaling pathway to maintain germinal stem cell stemness,enhanced ovarian function and delayed ovarian aging,with potential estrogen-like effects.