Studies On The Active Constituent And Mechanism Of Cuyun Recipe In Improving Embryo Implantation

Although many fertility disorders have been overcome by a variety of assistedreproductive technologies (ART), especially in vitro fertilization-embryotransplantation (IVF-ET) which has experienced important technological progress inrecent years, implantation remains the rate-limiting step for the success of the IVF-ET.Accordingly, the pregnancy success rate remains disappointingly due to high numbersof transferred embryos fail to implant. Therefore, the objective of screening effectivedrugs to improve implantation rate and explore factors that responsible forimplantation dysfunction in pregnancies resulting from controlled ovarian stimulation(COS) has been a hot research topic. This study is aimed to investigate the activeingredients, and mechanism of improving implantation of Cuyun recipe. Moreover, apreliminary study on quality standards was proceeding based on the effect of itsactivre ingredients.Part1: Screening of Different active fractions from Cuyun recipe of improvingembryo implantationThe gradient polarity system and chromatography were employed to screen theimproving implantation acitivity of different fractionations and ingredient in theCuyun recipe. The embryo implantation dysfunction (EID) mice model induced bycontrol ovarian stimulation (COS) was established and all mice were divided intocontrol, model and treated groups with6in each. Pregnancy rate and number ofimplantation blastocysts were observed on8pregnancy day (PD8). Compared withthe control group, pregnancy rate and mean implanted blastocysts in the model group were remarkably reduced. Compared with the model group, pregnancy rate and meanimplanted blastocysts in the ethyl acetate parts group and DS147group weresignificantly improved (P<0.05, P<0.01). It was concluded that DS147couldimprove mice pregnancy more remarkably than ethyl acetate part, and no adverseeffects on normal pregnancy were found.Part2: Study on the mechanism of the active constituent DS147in improvingembryo implantationAll female mice were treated with COS, mice in DS147groups received a gastricperfusion of DS147decoction per day at the responding doses. On PD5and PD6,uteri and ovaries samples were collected to observe the endometrial decidualizationand ovarian development. Further, expression of MMP-2, TIMP-2, ANG and CD34inthe DS147group was detected by immunohistochemistry. The results suggested thatDS147could raise the expression level of MMP-2/TIMP-2ratio as well as CD34andANG to promote extracellular matrix degradation and angiogenesis. Meanwhile,improve the condition including insufficient development of corpus luteum, impairedendometrial decidualization under COS treatment. It is concluded that DS147couldimprove embryo implantation under COS treatment.Part3: Study on the spectrophotometry method and quality standard for DS147The object was to establish a spectrophotometry method and quality standard forDS147. The content of total flavonoids were determined withNaNO2-Al(NO3)3-NaOH colorimetric method at505nm wavelengths, total saponinswere measured with5%vanillin-glacial acetic acid solution and perchloric acid ascolor developing reagents and detection wavelength of540nm, and phenol-sulfuriccolorimetric method was used to determine the content of polysaccharides at490nmwavelengths. Radix Astragali Mongolici, Radix Scutellariae Baicalensis and RadixDipsaci in this prescription were identified by TLC; and the content of AstragalosidteⅣ in Cuyun mixture was determined by HPLC. The contents of flavnoids, saponins and polysaccharides of DS147were33.78%,40.42%and9.36%respectively, andRSD were1.36%,2.35%,0.69%respectively. TLC spots were clear with strongspecificity and easy identification. While the content of Astragalosidte Ⅳ was0.331%,RSD=1.01%. The UV method is simple, accurate and can be used for the contentdetermination. TLC spots were clear with strong specificity and easy identification,and the HPLC-ELSD method is accurate and reproducible. It can be used effectivelyfor the quality control of DS147.