| Saussurea involucrate is a traditional valuable medicinal plant grown in high altitude areas in China,and it is rich in flavonoids.Esophageal cancer is one of the common malignant tumors,which is difficult to cure.It seriously affects people's quality of life.Some studies have reported that anthocyanins obtained from many plants have highly resistance to esophageal cancer.Therefore,in this study,the callus of Saussurea involucrate,which was red after transfer into the anthocyanin synthesis regulatory gene?PAP1 and LC?,was used as material,and we investigated systematically the antitumor activity of the alcohol extracts from the transgenic and non-transgenic callus of Saussurea involucrate against human esophageal cancer CaEs-17 cells from three aspects of chemical composition,pharmacological activity and molecular biology.The results provides theoretical support for the breeding of Saussurea involucrate of high antitumor activity and provides technical support for the full utilization of anti-tumor efficacy of Saussurea involucrate.The main research results obtained are as follows:?1?Determined the total flavonoid content of two kinds of callus of Saussurea involucrate.The results showed that the total flavonoid content in the callus of transgenic Saussurea involucrate?8257.88±759.20?mg·kg-1 DW was that of non-transgenic callus?3039.70±223.48?mg·kg-1 DW 2.72 times.?2?The species and contents of monophenols in the two kinds of callus of Saussurea involucrate were analyzed.The results showed that the species of monophenols in two kinds of callus of Saussurea involucrate were almost the same,but the contents of monophenols in the transgenic callus of Saussurea involucrate was higher.Cyanidin-galactoside was detected in transgenic callus,which makes them appear red,but was not detected in non-transgenic callus.?3?The inhibitory effect of the alcohol extracts from two kinds of callus of Saussurea involucrate on human esophageal cancer CaEs-17 cells were detected by CCK-8 assay and cell morphology observation.The results showed that the alcohol extracts from the callus of transgenic Saussurea involucrate had a stronger inhibitory effect on proliferation of CaEs-17cells,and its IC50 value(0.2874 mg·mL-1)was 26.4%lower than the IC50 value(0.3903mg·mL-1)of non-transgenic callus extracts.Fluorescence inverted microscope observed that the growth of human esophageal cancer CaEs-17 cells was significantly inhibited,the cell morphology changed significantly,the cell volume decreased,the spacing between cells became larger and separated from each other,the nucleus was highly pyknosis,and the cells died.?4?The apoptosis induction effect of the alcohol extracts from two kinds of callus of Saussurea involucrate on human esophageal cancer CaEs-17 cells were analyzed.Flow cytometry analysis results showed that the alcohol extracts from two kinds of callus of Saussurea involucrate both had significantly apoptosis inducing effect on human esophageal cancer CaEs-17 cells,and both were concentration-dependent.But the apoptosis induction effect of the alcohol extracts from the transgenic callus on CaEs-17 cells was more significant.?5?The expression of related apoptotic genes Bcl-2 and Bax were determined by RT-PCR.The results showed that the alcohol extracts from the two kinds of callus of Saussurea involucrate all could up-regulate the expression of pro-apoptotic gene Bax,showing a rising trend,and down-regulate the expression of Bcl-2 gene,showing a decreasing trend.But the regulation effect of the alcohol extracts from the transgenic callus of Saussurea involucrate was more significant.In summary,compared with non-transgenic callus,the antitumor activity of the alcohol extracts from the callus of transgenic Saussurea involucrate on the esophageal cancer CaEs-17 cells was significantly increased,which was closely related to the significant increase of the content of cyanidin-galactoside and its metabolism-related flavonoid compounds in the transgenic callus. |
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