Research On The Anti-hepatocellular Carcinoma Effect Of B.Simaoensis Triterpenes And Its Active Ingredient Lupeol

Balanophora simaoensis S.Y.Chang belongs to the genus Balanophora of family Balanophoraceae,mainly distributed in Yunnan.Balanophora is the folk medicine in our country,according to the Chinese Materia Medica,it has the effect of tonifying liver and kidney,promoting blood circulation,swelling,hemostasis effect.The research group previously evaluated the anti-tumor effect of triterpenes of B.simaoensis,and screened some monomers,founding that lupeol had a strong anti-tumor effect.Therefore,in this study,the total triterpenes of B.simaoensis and its active ingredient lupeol were used as research objects to explore the anti-hepatocellular carcinoma effects and potential mechanisms.The main experimental contents are as follows:1.Determination of total triterpenoids content from B.simaoensis and Optimization of Macroporous Resin Separation and Purification Process:In this study,the method of colorimetric determination of total triterpenoids of B.simaoensis was firstly determined by single factor optimization.It was found that the optimal dosage of colorimetric agent was 0.3 mL 5%vanillin glacial acetic acid solution and 1 mL perchloric acid solution,the optimal colorimetric temperature was 60℃,and the optimal detection wavelength was 550 nm.Total triterpenoids were extracted by reflux extraction with 80%ethanol.According to the above method,the average content of total triterpenoids in the alcohol extract of B.simaoensis was 1.76%based on oleanolic acid.The effective fractions of total triterpenoids were enriched and purified by macroporous resin after condition investigation and optimization.Static adsorption and desorption experiments show that the adsorption capacity of different polar resins on triterpenoids was significantly different.The adsorption rate of weak polar resin XAD7HP was 87.61%,and the desorption rate was 74.88%,which was the best purification effect.Therefore,the macroporous resin XAD7HP was selected to purify the total triterpenoids of B.simaoensis,and the average content of total triterpenoids was 42.70%based on oleanolic acid after purification.2.The anti-hepatocellular carcinoma effect of total triterpenoids(10 g·kg-1)of B.simaoensis in vivo was evaluated by the establishment of H22 xenograft mice model.The results showed that compared with the model group,total triterpenoids(10 g·kg-1)of B.simaoensis significantly inhibited tumor growth of H22 xenograft mice(inhibitory rate:51.48%),but had no significant effect on body weight,thymus index and spleen index of mice.The results showed that the total triterpenoids of B.simaoensis had a small immunosuppressive effect on liver cancer in vivo and high safety.3.Anti-hepatocellular carcinoma of lupeol in vitro and in vivo:First,UHPLC-Q-TOF-MS/MS was used to identify lupeol in triterpenoids of B.simaoensis;then MTT assay,Annexin V/PI double-staining assay,Transwell assay and Western blot assay were used to evaluate the anti-HCC effect of lupeol in vitro;Immunohistochemical assay and HE staining assay were used to evaluate the anti-liver cancer effect of lupeol in vivo by establishing H22 xenograft mice model and HepG2 xenograft mice model.The results showed that in vitro lupeol inhibited the proliferation and invasion of HCC cells and promoted the apoptosis.In vivo,lupeol(40 mg·kg-1)significantly inhibited the tumor growth of HepG2 xenograft mice(inhibition rate:41.79%).The replacement of E-acdherin by N-cadherin in mice tumor tissues will lead to the increase of EMT and promote the invasion of tumor cells,while lupeol can reduce the expression of N-cadherin and up-regulate the expression of E-cadherin,which may be its anti-tumor mechanism.Lupeol also significantly inhibited tumor growth in H22 xenograft mice(inhibition rate:49.01%),but had no significant effects on body weight,thymus index and spleen index of mice.Therefore,lupeol has good anti-hepatocellular carcinoma effects in vitro and in vivo.4.RNA-seq was used to investigate the mechanism of lupeol inhibiting proliferation and migration and inducing apoptosis of HCC cells,and RT-qPCR was used to verify the experimental results.The RNA of lupeol-treated HepG2 cells and control cells were sequenced by RNA-seq,GO and Kyoto KEGG analysis of differentially expressed genes showed that lupeol down-regulated the expression of tumor cell proliferation and migration related genes VEGFA,EGLN3,SCARB1,TXNIP,EFNA1,PFKP,and up-regulated the expression of tumor cell apoptosis related gene HERPUD1.These genes are located in the PI3K-Akt signaling pathway,Fructose and Mannose metabolism,AMPK signaling pathway,Phagosome,MicroRNAs in cancer pathways.The results of RT-qPCR were consistent with the results of transcriptome sequencing,suggesting that lupeol may play an anti-hepatocellular carcinoma role by regulating the expression of genes related to proliferation,migration and apoptosis of tumor cells.In conclusion,the total triterpenes of B.simaoensis and its active ingredient,lupeol,have anti-hepatocellular carcinoma effect.The anti-hepatocellular carcinoma effect of lupeol may be related to the inhibition of EMT and the regulation of tumor cell proliferation,migration and apoptosis related gene expression.