Extraction Of Hydroxy Safflor Yellow A And The Inhibitory Effects On Liver Cancer Cells

ObjectiveHydroxysafflor yellow A（HSYA）is the most effective water-soluble compound in the pharmacological effect of Carthamus tinctorius L.It is widely used in clinical research and treatment.The extraction of HSYA has been very mature.On the basis of previous studies,flash extraction technology was used to extract HSYA,and the suitable range of each extraction factor was determined by single factor test method.Then the optimum extraction conditions of HSYA by flash extraction technology were determined by orthogonal test,which provided a basis for subsequent experiments.HSYA can inhibit the growth of many kinds of cancer cells.We studied the effects of HSYA on the growth,invasion,migration,cell cycle and apoptosis of human hepatocellular carcinoma SMMC-7721 and HepG2 cells,and the expression of HSYA protein.MethodsHSYA in safflower was extracted by flash extraction technology,and the content of HSYA was determined by high performance liquid chromatography（HPLC）.Taking the extraction content of HSYA as an index,the optimum ratio of material to liquid,extraction time and the suitable range of extraction voltage were investigated by single factor test,and then the optimum extraction process of HSYA flash extraction was determined by orthogonal test.HSYA of 0 mg/mL（blank control group）,0.5 mg/mL（low dose group）,1 mg/mL（medium dose group）,1.5 mg/mL（high dose group）was used to treat SMMC-7721 and HepG2cells respectively.MTT method was used to detect the effect of HSYA on the proliferation of hepatocellular carcinoma cells;Transwell method was used to detect the effect of HSYA on the migration and invasion of hepatocellular carcinoma cells;Flow cytometry was used to detect the effect of HSYA on the migration and invasion of hepatocellular carcinoma cells.To determine the effect of HSYA on cell cycle and apoptosis of hepatocellular carcinoma cells,and to analyze the expression of VEGF,PI3K,AKT and Snail protein in hepatocellular carcinoma cells after HSYA intervention by Western blot.Results（1）The optimum extraction process of HSYA by flash extraction is 1:40g/mL of solid-liquid ratio,3 min of extraction time and 80 V of extraction voltage.（2）MTT results showed that HSYA could inhibit the proliferation of SMMC-7721 and HepG2 cells in a dose-time dependent manner compared with the blank dose group（P<0.05）.（3）Transwell assay showed that with the increase of HSYA concentration,the invasion and migration ability of human hepatocellular carcinoma SMMC-7721 and HepG2 cells decreased gradually.HSYA could inhibit the invasion and migration of human hepatocellular carcinoma SMMC-7721 and HepG2 cells（P<0.05）.（4）The results of flow cytometry showed that with the increase of HSYA concentration,the apoptotic rate increased gradually,the proportion of G₀/G₁phase increased significantly（P<0.05）,and the proportion of S phase decreased significantly（P<0.05）.HSYA could promote the apoptosis of hepatocellular carcinoma cells.（5）Western blot analysis showed that HSYA could also decrease the expression of VEGF,PI3K,AKT and Snail（P<0.05）.ConclusionsFlash extraction of HSYA is a simple process with high extraction rate.It can be used to extract HSYA from safflower.At the same time,HSYA can inhibit the proliferation,migration and invasion of SMMC-7721 and HepG2 cells,block cell cycle,make them arrest in G₀/G₁ phase and induce apoptosis of hepatocellular carcinoma cells.The mechanism may be related to inhibiting the activation of PI3K/AKT/Snail signaling pathway,which provides theoretical basis for the clinical application of HSYA in the treatment of hepatocellular carcinoma.