| Objective:Optimize the fermentation process and quality standard of ginseng-cordyceps fermentation fungus（ginseng cordyceps fungal substance）,establish the preparation process of compound ginseng cordyceps fungal substance granules,and reveal the anti-tumor activity and mechanism of compound ginseng cordyceps fungal substance granules.In order to lay the foundation for the subsequent research and development of compound ginseng cordyceps fungal substance granules,it provides a new theoretical basis for anti-tumor of traditional Chinese medicine compound.Method:1.Systematic optimization of the process of fermenting ginseng herbs with Cordyceps sinensis using single factor experiments and statistical methods such as response surfaces.The qualitative analysis of the main saponins in the ginseng cordyceps fungal substance was carried out by HPLC-Q-TOF-MS.The MRM model in HPLC-QQQ-MS technology was introduced,and 11 saponins in the ginseng cordyceps fungal substance were quantitatively analyzed.Ten batches of ginseng cordyceps fungal substance were prepared according to the preferred method to determine the range of content determination.2.MTT assay was used to detect the cell viability of ginseng-containing compound and ginseng cordyceps fungal substance-containing compound in A549 cells,and the anti-tumor activity of the two compounds was compared.3.The orthogonal test and other methods were used to study the extraction and separation process,concentration and drying process,and preparation molding process of compound ginseng cordyceps fungal substance granules,prepare the pilot samples,and carry out quality inspection on different batches of samples.4.In the pharmacodynamic study,the killing effect of different concentrations of compound ginseng cordyceps fungal substance granules on A549 cells was detected by MTT method;The migration and invasion of A549 cells by compound ginseng granules were detected by scratch test and Transwell test;The flow cytometry was used to detect cell cycle and apoptosis of A549 cells after drug treatment;The expression of apoptosis-related proteins Caspase3,Bax,Bcl-2 and the related pathway proteins TGF-B,smad2,p-smad2、smad3、p-smad3、E-cadherin were detected by Western Blot.Furthermore,nude mice inoculated with lung cancer cells were used as in vivo models to detect changes in body weight and tumor volume of nude mice after drug treatment;Flow cytometry was used to detect the expression of related surface proteins CD4,CD8a,CD11b,Ly-6G/Ly-6c in blood and spleen cells.5.Research using TMT labeling,high performance liquid chromatography grading and mass spectrometry-based quantitative proteomics to perform quantitative proteomic studies on A549 cell samples before and after treatment with aqueous solution of compound ginseng cordyceps fungal substance granules.Result:1.A stable preparation process of ginseng cordyceps fungal substance was established,and the average yield of ginseng cordyceps fungal substance was about 69.8%.Through qualitative and quantitative analysis of the main saponins of ginseng cordyceps fungal substance,11 saponins were found.The average contents of Rg1,Re,Rb1,Rh1,Ro,Rd,Rf,Rb2,Rg3,F2 and Rg2 can reach 1.50 mg/g,1.32 mg/g,0.64 mg/g,1.12 mg/g,1.79 mg/g,,3.56mg/g,0.37mg/g,0.48mg/g,0.33mg/g,0.51mg/g and 0.70mg/g;Determine the range of content determination of the corresponding component,it is prescribed that the ginsenoside Rd in ginseng cordyceps fungal substance should not be less than 0.3%,Rh1 should not be less than 0.1%,the total amount of Rb1,Re,Rg1 should not be less than 0.3%,and finally developed the quality standard of ginseng cordyceps fungal substance.2.Comparing the survival rate of A549 cells treated with the ginseng-containing compound and the ginseng cordyceps fungal substance-containing compound,the anti-tumor effect of the compound containing ginseng cordyceps fungal substance was significantly better than that of the ginseng-containing compound;following the preparation characteristics of the traditional Chinese medicine preparation,extracted by water decoction,Taking the amount of extraction of total flavonoids as inspected index,the optimal extraction conditions and preparation molding process of compound ginseng cordyceps fungal substance granules were determined.The quality of the three batches of pilot samples was in accordance with the relevant provisions of the Chinese Pharmacopoeia 2015 edition four appendix 0104 granules.3.Compound ginseng cordyceps fungal substance granules significantly inhibited the proliferation of A549 lung cancer cells.When the concentration reached 100μg/m L,the effect was the most significant（P<0.05）.The results of flow cytometry showed that the compound ginseng cordyceps fungal substance granules could inhibit cells enter to G1phase,while decreasing G1/S phase,prolonging G2 phase,preventing cell proliferation,and inducing apoptosis of A549 cells;Tumor-associated protein detection showed that compound ginseng cordyceps fungal substance granules can effectively regulate the expression of apoptosis-related proteins;improve the Caspase3,Bcl-2 and E-cadherin expression,while reducing the expression of TGF-β1,Bax.4.Compound ginseng cordyceps fungal substance granules had significant inhibitory effect on the migration and invasion of A549 cells（P<0.01）.It has a certain therapeutic effect on subcutaneous transplanted tumor of A549 lung cancer,and can effectively change the immune protein cells CD4 and CD8,cells Ly-6G/Ly-6C and CD11b in nude mouse blood and spleen cells.The stimulation of immune proteins and immune cells was dose dependent.5.Quantitative proteomics analysis was performed on A549 cells before and after treatment with compound ginseng cordyceps fungal substance granules.A total of 5641proteins were identified,of which 4569 proteins contained quantitative information.The changed threshold of differential expression is 1.2 times and the statistical test t-test p-value<0.05 is the significance threshold.Compared with the control group,the expression of 192proteins was up-regulated and the expression of 272 proteins was down-regulated.Systematic bioinformatics analysis（protein function annotation）was performed on all identified proteins,and functional classification,functional enrichment,and functional enrichment-based cluster analysis were performed on all differentially expressed proteins.Furthermore,the expression level of the target protein HDAC3 of compound ginseng cordyceps fungal substance granules was verified by immunohistochemistry and reverse transcription PCR.Conclusion:This study optimized the fermentation process of ginseng cordyceps fungal substance,and established the quality standard of ginseng cordyceps fungal substance,which provided a theoretical basis for the quality control of raw materials in the formulation development process.The preparation process of compound ginseng cordyceps fungal substance granules was established.It laid the foundation for subsequent research and expanded production.It confirmed the anti-tumor activity of compound ginseng cordyceps fungal substance granules at the cellular level,indicating that the compound ginseng cordyceps fungal substance granules have a significant role in inhibiting non-small-cell lung cancer,and further explain its mechanism by proteomics,screen out the potential target HDAC3,providing a new direction for the clinical treatment of lung cancer. |
