Preliminary Study On The Active Ingredients Of Epimedium Koreanum Nakai For The Treatment Of Male Infertility

Objective:This study intends to investigate the molecular mechanism of Epimrdii Herba in treating male infertility based on network pharmacology and molecular docking technology,and to predict the active ingredients of Epimedium koreanum Nakai in the treatment of male infertility by investigating the Quantity-effect relationship between the active ingrediedients of Epimedium koreanum Nakai and the improvent of male infertility outcomes in mice,and to put forward a theoretical support for the judgment of the quality of Epimedium koreanum Nakai and its clinical application in male infertility.Methods:(1)Network pharmacology: The active ingredients of Epimrdii Herba were screened by TCMSP database and the targets of male infertility were obtained by Gene Cards and OMIM databases,and the common targets were taken to set up the active ingredient-target network and protein interactions network by Cytoscape 3.7.1 software.GO and KEGG enrichment analysis of the common targets was executed with the help of the Metascape database.Molecular docking validation of active ingredients and key targets was implemented with the help of software such as Auto Dock Vina 1.1.2.(2)Determination of active ingredient content: Single factor test and orthogonal experiment were conducted by HPLC to grope for the optimal extraction conditions for determining the content of Quercetin,Luteolin,and Kaempferol in Epimedium koreanum Nakai,and to grope for the conditions and methods for determining the content of icariin,Epimedin A,Epimedin B,Luteolin,Epimedin C,Quercetin,and Kaempferol in Epimedium koreanum Nakai.Methodological investigation and content determination were conducted respectively.(3)Animal experiments: Rando mly select 13 of 86 KM adult male mice as the normal group,and the rest were injected intraperitoneally with cyclophosphamide 53 mg/kg/d for5 d to build a male infertility model;the successfully modeled mice were stochasticly separated into the model group,normal group,Naozhi group,Hekou group,Liangshui group,Hongya group,Kuandian group,and Yaoyuan group,with 10 individuals in each group.From the 6th day,each group of Epimedium koreanum Nakai was administered by gavage for 40 d.The testicular index,epididymal index and sperm parameters of mice were measured.The morphological changes of testicular tissue were detected by HE staining,the LH,FSH and T concentrations in serum were determined by ELISA,and the expression levels of caspase-3,caspase-9,Bcl-2 and Bax proteins in testes were detected by Western Blot.Results: 1.We screened 23 active ingredients of Epimrdii Herba with 293 targets,such as among which the key ingredients were Quercetin,Luteolin,Kaempferol,and Icariin etc.There are 1146 male infertility targets and 111 targets intersected with Epimrdii Herba,and the key targets were TPT53,AKT1,VEGFA,JUN,ESR1,etc.In GO enrichment analysis,it mainly involves hormone response,protein kinase activity,kinase regulatory activity,cell cycle protein-dependent protein kinase holoenzyme complex,etc.In KEGG enrichment analysis,it mainly involves prostate cancer,PI3K-Akt signaling pathway,breast cancer,chemical carcinogenesis-reactive oxygen species,cellular senescence,etc.The key targets TP53,AKT1,VEGFA,JUN,and ESR1 showed good binding to the main active ingredients Quercetin,Luteolin,Kaempferol,and Icariin.2.The best results were obtained when the content of Quercetin,Kaempferol,and Luteolin in Epimedium koreanum Nakai was determined at a solid-liquid ratio of 0.2:8(g/ml),an ethanol-15% hydrochloric acid ratio of 3.5:1,an extraction temperature of 75 ℃,and an extraction time of 3 hours.The analysis of the results of content determination revealed differences in the content of each active ingrediengt in different Epimedium koreanum Nakai samples,among which icariin were higher and Luteolin was the lowest in each sample;the total content of Quercetin,Luteolin,Kaempferol and icariin were markedly higher in the Kuandian and Yaoyuan groups than in the other groups,predicting that the Kuandian and Yaoyuan groups may have more advantages in the treatment of male infertility.3.The analysis of animal experimental results showed that the body weight of the mice was reduced,and the reduction was more obvious in the model group;the testicular index and epididymal index of the mice in each drug administration group were improved.The sperm parameters of mice were descended,while the sperm density and sperm viability rate of each dosing group were markedly increased unlike those of the model group(P<0.01).Spermatocytes,secondary spermatocytes,germ cells,and more newborn spermatozoa in the official cavity were clearly visible in the testes of mice in each administration group;however,there was no significant difference in testicular tissue structure between the administration groups.unlike the normal group,the LH and FSH levels were markedly higher and T levels were markedly lower in the model group(P<0.01),and the LH levels in the Hongya and Kuandian groups were close to the normal group;unlike the model group,the LH levels were lower in the Naozhi,Hekou,Hongya and Yaoyuan groups((P<0.05),and markedly lower in the Kuandian groups(P<0.01),and the FSH levels were markedly lower in each drug administration group(P<0.01),and T levels were markedly raised(P<0.01).Unlike the normal group,Bax,Caspase-3 and Caspase-9 protein expression levels were markedly upregulated in the model group(P<0.01),and Bcl-2 protein expression levels and Bcl-2/Bax ratio were markedly descended in the model group(P<0.01);unlike the model group,Bax and Caspase-9 expression levels were markedly descended in each administration group(P<0.01),except for the Hekou group the expression level of Caspase-3 was markedly lower(P<0.01),while the expression level of Bcl-2 protein and the Bcl-2/Bax ratio were markedly higher(P<0.01)in each administration group.Correlation analysis of the active ingredients with the results of ameliorating male infertility mice showed that Luteolin and Epimedin C were correlated but not markedly correlated,Epimedin A was markedly correlated(P<0.01);Quercetin,Kaempferol,Epimedin B,and Icariin were closely correlated(P<0.05).unlike the normal group,the LH and FSH levels were markedly higher and T levels were markedly lower in the model group(P<0.01),and the LH levels in the Hongya and Kuandian groups adjacence the normal group;unlike the model group,the LH levels were lower in the Haoxi,Hekou and Yayuan groups((P<0.05),and markedly lower in the Hongya and Kuandian groups(P<0.01),and the FSH levels were markedly lower in each drug administration group(P<0.01),and T levels were markedly increased(P<0.01).unlike the normal group,Bax,Caspase-3 and Caspase-9 protein expression levels were markedly upregulated in the model group(P<0.01),and Bcl-2protein expression levels and Bcl-2/Bax ratio were markedly descended in the model group(P<0.01);unlike the model group,Bax and Caspase-9 expression levels were markedly descended in each administration group(P<0.01),except for the Hekou group the expression level of Caspase-3 was markedly lower(P<0.01),while the expression level of Bcl-2 protein and the Bcl-2/Bax ratio were markedly higher(P<0.01)in each administration group.Correlation analysis of the active ingredients with the results of ameliorating male infertility mice showed that Luteolin was correlated but not markedly correlated with Epimedin C,and Epimedin A was markedly correlated(P<0.01);Quercetin,Kaempferol,Epimedin B,and Icariin were closely correlated(P<0.05).Conclusion: Epimedium koreanum Nakai improves testicular function in cyclophosphamide-induced male infertility and has a facilitative effect in the treatment of male infertility.Initially,Icariin,Quercetin,Kaempferol,Epimedin A and Epimedin B were identified as the active ingredients of Epimedium koreanum Nakai for the treatment of male infertility.