Study On Preparation Artwork And Anti-Cervical Cancer Activity Of Heat-Processed Hydrolysate Of Ginsenosides Rb1

Ginseng has been used as a valuable medicinal material in Chinese traditional medicine.It has many pharmacological functions such as regulating anti-inflammator effect,tranquilizing sleep and anti-diabetes.At first,the researchers found that the glycosides of ginsenosides were hydrolyzed during the process of processing fresh ginseng into red ginseng by high temperature,dehydration and isomerization occurred at C-20,and a large number of active small molecules were formed: secondary ginsenosides.Secondary ginsenosides,also known as rare ginsenosides,can only be formed from the original ginsenosides,and they have significant biological activities in terms of cell biology and pharmacological effects.The secondary ginsenosides formed in the heat process are also called "heat-processed hydrolysate of ginsenosides".Based on the theory that red ginseng pyrolyses when heated,in order to obtain more secondary saponins than red ginseng,researchers obtained black ginseng by repeated high temperature and high pressure cooking,namely "nine steaming and nine exposure",to obtain rare saponins.In this paper,based on the scientific theory and experimental basis of ginseng pyrolysis to produce secondary ginsenoside,secondary ginsenoside was directly extracted from ginsenoside by high temperature pyrolysis method.Rare ginsenosides with high activity and high concentration were successfully developed and prepared by the extraction method.Objective: To prepare heat-processed hydrolysate of method,and to optimize the optimum technological conditions for the production of rare ginsenosides with high concentration.Ginsenoside RK1,one of the main pyrolysis products of ginsenoside Rb1,was studied in vitro for anti-tumor drug experiments and further analysis of the mechanism of anti-tumor drugs in vitro.Methods:(1)RSM response surface test was used to determine the optimal process conditions for heat-processed hydrolysate Rk1 and Rg5.TLC thin-layer analysis was used to verify the pyrolysis phenomenon and screen the influencing factors,HPLC was used to verify the pyrolysis components,and single-factor experiment was used to complete the three-level parameter setting.(2)the study of heat-processed hydrolysate Rk1 of Hela apoptosis induction effect analysis: after dosing Rk1 CCK 8 method for cell inhibitory effect,detecting the changes of the cell cycle of the dosing Rk1,observe the cell apoptosis after Hoechst33258 dye state,quantitative detection of apoptosis rate before and after dosing Rk1,Western blot analysis after dosing Rk1 effect on the regulation of apoptosis protein levels(3)RNA-seq Rk1 cervical cancer resistant method research preliminary mechanism:Differential gene proteins were identified,GO and KEGG enrichment analysis was performed,RT-q PCR was used to verify the accuracy of sequencing trends,Western-blot was used to verify the protein expression content,and potential new targets for the treatment of cervical cancer were found.Results:(1)TLC thin-layer analysis showed that ginsenoside Rb1 could be pyrolyzed,and ginsenoside pyrolyzation was mainly determined by acid concentration,reaction temperature and reaction time.The results of HPLC verification showed that ginsenoside Rb1 could produce ginsenoside 20(R)-Rg3,20(S)-Rg3,Rk1 and Rg5.The preparation process of ginsenoside Rk1 and Rg5 was optimized by single factor experiment and response surface method.Considering the feasibility of practical process setting,the optimum conditions for the pyrolytic reaction of ginsenoside were selected as 1.6% acid concentration A,46 min time B and 111℃ temperature C.(2)The optimal concentration of Rk1 was 10-30 μm,and the optimal treatment time was 24 h.It is concluded that Rk1 blocks Hela cells in the G0/G1 stage in a dose-dependent manner,and that the dose of 20 sm and 30 m can lead to a significant increase in cell content in stage G0/G1(p<0.01),and a decrease in cell content in stage S and G2/M,showing significant concentration dependence,inhibiting cell division and thus inhibiting cell proliferation.Hoechst33258 staining of Hela cells treated with Rk1 significantly increased the proportion of apoptosis with the increase of Rk1 administration concentration.Quantitative determination of cell apoptosis rate showed that Rk1 had a significant inhibitory effect on the proliferation of Hela cells.With the increase of Rk1 concentration,the percentage of cell apoptosis rate increased in a dose-dependent manner,from 4.28±0.47% to 12.39±0.99%(P <0.001).The inhibitory effect of RK1 was mainly attributed to the induction of apoptosis of Hela cells and the increase of early and late apoptosis rate of Hela cells.Finally,the expression of apoptotic proteins was detected by western blot analysis.Ginsenoside Rk1 could significantly increase the protein expression levels of Caspase-3,LC3 B,Caspase-6 and PARP in Hela cells after treatment for 24 h.The results showed that ginsenoside Rk1 could significantly activate the apoptosis signaling pathway of Caspase 3,PARP and Caspase 6,thus exerting pro-apoptosis effects.Ginsenoside Rk1 can significantly increase the expression level of autophagy marker LC3 B protein,which proves that Rk1 promotes autophagy cell death through autophagy signaling pathway.(3)GO analysis showed that three concentrations of Rk1(10 μM,20μM,30μM)treated Hela cells were mainly upregulated by the following signaling pathways: cytoskeleton,transferase activity,ribonucleic acid binding,cell response to DNA damage.The down-regulated signaling pathways mainly include: negative regulation of apoptosis,ER stress,ER protein folding,protein transport,calcium binding,cytoskeleton,etc.KEGG analysis showed that the expression of most genes related to cell proliferation,cell cycle and cell senescence were down-regulated,and the endoplasmic reticulum protein processing signaling was down-regulated.Through GO enrichment,KEGG enrichment,Western blot experiment and real-time quantitative RT-q PCR verification,we found that YOD1 protein was the most significantly dose-inhibited by Rk1 among all the proteins,and we found a potential new target for the treatment of cervical cancer-YOD1.Conclusion: Ginsenosides can be transformed into secondary ginsenosides by heatprocessed hydrolysate method,and these secondary ginsenosides have higher activity.By investigating the mechanism of Rk1,heat-processed hydrolysate of ginsenosides Rb1,we concluded that Rk1 showed significant anti-tumor effects in in vitro anti-tumor experiments,and Rk1 could induce apoptosis of Hela cells on cancer cells.