Study On The Process Optimization Of Shenshan Granules And Their Anti-tumor And Immune Effects On CT26.WT Colon Cancer-bearing Mic

Obiective: On the basis of the research and development of the prescription,process and quality standard of functional food Shenshan Granules in the early stage and the inhibition of HT-29 colon cancer Cell migration through cell experiments,further optimize its rose extraction method and process parameters,optimize its moisture-proof excipients and dry granulation process parameters,and study the hygroscopic properties of raw materials and finished products,to provide a basis for industrial production;To further explore the immune regulation and anti-tumor effect of Shenshan Granules on CT26.WT colon cancer bearing mice,and provide theoretical basis and experimental support for the functional positioning,clinical application and market promotion of Shenshan Granules.Methods: 1.The differences between flash extraction and immersion extraction were compared by using hyperin and rutin as the components evaluation indexes,and the taste,bitterness and sweetness determined by electronic tongue as the flavor evaluation indexes.Taking the rotating speed of flash extraction,water addition times and soaking time as the factors to be examined,the optimal process parameters of flash extraction were optimized by orthogonal design test,and the effects of different extraction process conditions on its active ingredients and flavor were explored;The optimal extraction process parameters were obtained through statistical analysis.Compared with the best process parameters of soaking extraction,95% confidence interval overlap method was used to compare the differences between flash extraction and soaking extraction;The correlation between flash extraction components and flavor indexes was analyzed.2.Using the experimental method of drug hygroscopicity,select the optimal moisture-proof combination of raw materials and excipients;The optimal process parameters for dry granulation are selected through orthogonal design based on the examination factors of dry granulation,such as the speed of the pressure wheel,the pressure wheel,the feeding speed,and the whole granulation speed;Measure the moisture absorption percentage of Shenshan granules raw materials and finished products in different humidity environment solutions,establish a mathematical model,and calculate their critical relative humidity(CRH),as well as the longest operating time under this relative humidity condition.3.A mouse model of colon cancer was prepared by axillary injection of CT26.WT cells,which were divided into three groups: the blank control group(control),the model group(model),the Shenshan granules group(SSG),the positive drug group(5-fluorouracil,5-FU),6 mice per group,and continuous administration for14 days.The tumor volume,tumor inhibition rate were calculated.IL-2,TNF in serum cell factors-α And IFN-γ The expression levels,the proportion of T lymphocyte subsets in the spleen,the pathological changes in the tumor groups and the expression of Bcl-2,Bax and caspase-3 m RNA and protein in the tumors were determined by ELISA,flow cytometry,he staining,RT-PCR and immunohistochemistry,respectively.Results: 1.The optimal process parameters for flash extraction were as follows: rotation rate2000 r / min,water addition 30 times,time 10 min;Statistically different from the optimal soaking extraction;Comparison of flash extraction and soaking extraction on compositional indexes showed that the flash extraction yields of hyperin and rutin were 1.529 fold,1.393 fold higher than those of soaking extraction,respectively;Comparing on flavor,the bitter soaking extraction was 6.532 times that of the flash extraction;Correlation analysis found that flavor exhibited different degrees of correlation with the contents of some components.2.The prescription ratios of the formulations were 10.0 g of raw materials,6.5 g of maltodextrin,and1.5 g of silica,which significantly(P<0.05)reduced the hygroscopicity of the raw materials;The optimal process parameters for the finished product were the press wheel speed of 8.0 r/min,the press wheel pressure of 6 MPa,the feed rate of 25 r/min,and the whole grain speed of 40 r/min;53.34%,68.26% for raw and finished CRH,respectively;The longest operation time was 2.075 h,4.573 h,respectively,which guaranteed that the water uptake of Shenshan granules was lower than 8%.3.Mouse experiments showed that compared with the model group,the 5-FU and SSG groups had significantly smaller tumor volumes,significantly lower tumor masses,and significantly higher tumor suppressive rates(P < 0.01),and the 5-FU group,serum levels of the cell factors IL-2 and TNF-α And IFN-γ Levels,the ratio of CD4+/CD8+ in the spleen cell suspension and the expression of bcl-2 m RNA and protein in the tumor tissue were significantly decreased(P < 0.01),and the expression of Bax and caspase-3 m RNA and protein was significantly increased(P < 0.01);Serum levels of cell factors IL-2 and TNF in SSG group-αAnd IFN-γ Levels,ratios of CD4+/CD8+ in splenocyte suspensions and Bax and caspase-3m RNA and protein expression in tumor tissues were significantly increased(P < 0.01)and bcl-2m RNA and protein expression was significantly decreased(P < 0.01).Conclusion: To provide a basis and reference for the mode of rosette extraction during industrial production of Shenshan granules and for the maximum time period of humidity conditions and operation of the operating environment between the raw material and the finished product.Animal experiments indicated that Shenshan granules might inhibit tumor growth in CT26.WT colon cancer tumor bearing mice by enhancing the immune antitumor and activating the BCL-2 /Bax/ caspase-3 apoptotic pathway.