Effects Of Total Flavonoids From Seabuckthorn Pomace In Inner Mongolia On Biological Behavior Of Gastric Carcinoma Cells

Objective:Gastric cancer is a malignant tumor with a lower incidence than lung cancer in China.At present,various methods for the treatment of gastric cancer have certain limitations.After treatment,they still face problems such as postoperative recurrence and metastasis,low immunity after radiotherapy and chemotherapy,and tumor resistance.Traditional Chinese medicine has a unique advantage in anti-tumor,which can alleviate tumor pain in middle and late stage patients,improve immune function after radiotherapy and chemotherapy,and enhance anti-tumor characteristics of chemotherapy drugs.The purpose of this study is to explore the effects and the possible mechanism of total flavonoids from sea buckthorn pomace in Inner Mongolia on the biological behavior of gastric cancer cell line SGC-7901.Methods:In this study,total flavonoids are extracted from seabuckthorn pomace by ethanol extraction and determined.The effect of different concentrations of total flavonoids from Hippophae rhamnoides L.on the proliferation of gastric cancer cells was detected by the cck8 assay,and the half-maximal inhibitory concentrations is calculated.Using the obtained IC50 values,cell migration,Transwell chamber,flow cytometry and real-time fluorescent quantitative PCR are used to detect the migration ability and cell cycle of gastric cancer cell line SGC-7901,and the expression of Wnt/?-catenin signaling pathway-related molecules in tumor cells before and after drug treatment.Results:(1)The optimal extraction conditions were obtained by orthogonal optimization experiments:the volume fraction of ethanol was 60%,the extraction time was 2.0h,the ratio of material to liquid was 1:20,and extraction was performed twice.The total flavonoids of the seabuckthorn have a high yield and the content can reach to 32.22 mg/g.(2)Cytological results showed that the total flavonoids of Hippophae rhamnoides could inhibit the proliferation of human gastric cancer cell line SGC-7901 in a dose-dependent and time-dependent manner to varying degrees;The IC50 values on SGC-7901 cells at 24h,48h or 72h post treatment were4.578mg/ml,3.676mg/ml and 3.795mg/ml respectively;Scratch test showed that scratch wound healing was inhibited to varying degrees compared with the control group;Migration experiments showed that the total flavonoids of Hippophae rhamnoides were significantly different from those of the control group in a dose-dependent manner after 48 hours of treatment.The results of PI single staining by flow cytometry showed that total flavonoids of Hippophae rhamnoides blocked cell cycle checkpoints at the S phase.(3)After 48 hours of treatment,real-time fluorescence quantitative PCR showed that total flavonoids of Hippophae rhamnoides increased the expression of SRFP and decreased the expression of E-cadherin and GSK3?in gastric cancer cell line SGC-7901.The values of 2~-??CT??CT of E-cadherin,GSK-3?,or SFRP are 0.34±0.07,0.35±0.05,2.39±0.19.That is,when the total flavonoid concentration of Hippophae rhamnoides was 4 mg/ml,the content of SFRP was 2.39±0.19 times that of the control group.Conclusion:(1)Based on the results of single factor and orthogonal experiments,the optimal extraction conditions of total flavonoids in seabuckthorn pomace were determined:the volume fraction of ethanol was 60%,the extraction time was 2.0h,the ratio of material to liquid was 1:20,and extraction was performed twice.Under the condition,the total flavonoids extracted from seabuckthorn pomace can reach to 32.22 mg/g.(2)The scratch test and Tanswell chamber experiments confirmed that the total flavonoids from seabuckthorn pomace can attenuate the migration ability of gastric carcinoma cell line SGC-7901.In addition,the total flavonoids of seabuckthorn pomace can inhibit the proliferation of gastric cancer cells by blocking cell cycle checkpoints at the S phase.(3)The total flavonoids of seabuckthorn pomace can up-regulate the expression of SFRP gene and down-regulate the expression of E-cadherin and gsk3?in gastric cancer cell line SGC-7901.(4)The molecular mechanism of the effect of the total flavonoids from seabuckthorn pomace on gastric cancer cell line SGC-7901 may be related to the Wnt/beta-catenin signaling pathway-related molecules,including the high expression of SFRP and the low expression of gsk3?and E-cadherin.