Preparation Of Ginsenoside CK And Its Derivative And The Mechanism On Hepatocellular Carcinoma

Primary liver cancer has been reported to be the fifth commonly cancer and the second leading cause of cander death in China according to the global cancer data in 2020.Existing studies have shown that ginsenoside CK has multiple biological activity.In our research,ginsenoside CK with high purity was obtained from Panax notoginseng root through extraction,transformation and separation.The inhibition effect of ginsenoside CK on hypoxia-induced EMT of HCC was studied through in vitro induction of hypoxia model of HCC cells and xenograft model.In addition,a molecular modification of ginsenoside CK was carried out,and the effect and mechanism of the new product on HCC was investigatedm,so as to provide a theoretical basis for the development of drugs based on ginsenoside CK on HCC.The main research contents of this paper are as follows:（1）Preparation of ginsenoside CKIn our experiment,the panax notoginseng root was used for the preperation of ginsenosides,which was ground into powder first and the total ginsenosides were extracted by organic solvent extraction.After optimizing the type of extraction solvent,extraction ratio,extraction temperature and time,it is finally determined to adopt more safe and environmentally friendly ethanol as the extraction solvent,solvent volume（m L）:substrate mass（g）is 6:1,after 40 min extraction at 45℃and repeated extraction for three times,the content of total ginsenosides in the extract was 41.58%,and the extraction rate of total ginsenosides was83.29%.After four different macroporous adsorption resins were used for the separation of total ginsenosides in the extract,D101 resin was finally determined to achieve a better separation efficiency of diol type and triol type ginsenosides.After that,the snail enzyme was used to transforme protopanaxadiol saponins into rare ginsenosides.The optimal transformation conditions were confirmed as follows:substrate concentration 1.4 mg/m L,enzyme concentration 1.6 mg/m L,reaction temperature 50℃,reaction time 36 h,buffer p H5.5.The transformation product with high content of ginsenoside CK（41.31%）was obtained.Next,the transformed product was repeatedly extracted by ethanol to remove insoluble impurities,and then separated by preparative HPLC to obtain high purity ginsenoside CK.The purity of ginsenoside CK could reach more than 98%by analytical HPLC.（2）The effect and mechanism of ginsenoside CK on hypoxia-induced EMT in HCC The cytotoxicity of ginsenoside CK on different tumor cells were detected by MTT method,and HCC cells were selected for subsequent experiments as the higher sensitive reactivity to CK.Co Cl₂ and TNFαwere used to simulate hypoxia and activate the NF-κB signaling pathway in HCC cells,respectively.The results of western blotting and RT-q PCR demostrated the crosstalk between HIF-1αand NF-κB signaling pathway and their effects on EMT in HCC cells.The results of migration and invasion test showed that ginsenoside CK could inhibit the metastasis capacity of HCC cells enhanced by hypoxia.Mechanistically,ginsenoside CK suppressed HIF-1α/NF-κB signaling and expression level of EMT-related proteins and cytokines in hypoxia-induced or TNFα-stimulated HCC cell lines.An in vivo study revealed that the oral delivery of ginsenoside CK could inhibit the growth of xenograft tumors and block HIF-1αand NF-κB signaling as well as EMT marker expression.Therefore,we speculated that ginsenoside CK could reduce the potential of HCC metastasis by targeting HIF-1α/NF-κB crosstalk,which is a potential treatment for HCC.（3）We performed an acetylation modification of ginsenoside CK（CK-3）and investigated the effects of the CK-3 in vitro and in vivo.The cytotoxicity analysis revealed that CK-3could inhibit the proliferation of multiple tumor cell lines with a lower concentration compared with CK.Treating of HCC cells with CK-3 induced cell cycle arrest and cell apoptosis through flow cytometry,AO/EB staining and TUNEL analysis.Meanwhile,CK-3significantly suppressed the tumor growth in vivo with no side effect on the function of main organs.Mechanistically,the whole transcriptome analysis revealed that the antitumor effect of CK-3 was involved in Hippo signaling pathway.The immunoblotting and immunofluorescence results illustrated that CK-3 directly facilitated the phosphorylation of YAP1 and decrease the expression of the main trancription factor TEAD2 in HCC cells and tumor tissue sections.Collectively,our results illuminated the formation of a new derivative of ginsenoside CK and its regulatory mechanism to HCC,it could activate Hippo-YAP1-TEAD2 signaling pathway to regulate HCC progression.This research could provide some new mentality for traditional Chinese medicine on the therapeutic of tumor.