Effect And Molecule Mechanism Of Total Saponins From Xanthoceras Sorbifolia Bunge Kernel On Apoptosis In HepG2 Hepatoma Cells

Xanthoceras sorbifolia Bunge,belonging to the Sapindales,Sapindaceae,Xanthoceras,is a perennial shrub.The oil content of kernels in Xanthoceras sorbifolia Bunge seeds is above 60%.Besides oil,these kernels also contain a lot of biological substances,such as saponins,sterols,flavonoids,vitamins,and other active ingredients,which have a great value for research and utilization.In recent years,the oil from Xanthoceras sorbifolia Bunge was more researched as one kind of high-quality edible oil or biodiesel marterials,and the edible oil from Xanthoceras sorbifolia Bunge also has been saled as commercialized production.As a by-product of oil production,a large number of kernel cake was produced,and its use was very rare.Most of the kernel cake was made into fertilizer or discarded as garbage,which was a great waste of resources.The studies shows that the saponins in kernel cake from Xanthoceras sorbifolia Bunge has a lot of biological activities,such as anti HIV,anti-hyperglycemic,lowering blood sugar,anti-bacterial effect,which has a wide application prospect.In this study,the kernel cake of Xanthoceras sorbifolia Bunge was selected as the test materials,and the total saponins was extracted from the defatted kernel cake.After purification,its basic composition of total saponins were analyzed with methods of MS and HPLC-ESI-MS.The antitumor activity of total saponins was explored through cell experiments in vitro,and its molecular mechanism were further discussed.The main results are as follows:(1)The total content of saponins from the defatted kernel cake was 93.5%after extracting and purifing.The eight components in total saponins was identified using analytical instruments of Liquid Chromatography/Mass Spectrometry.They are bunkankasaponin B in Xanthoceras sorbifolia,3-0-(3-O-?-L-arabinofuranosyl-2-O-?-D-galactopyranosyl)-?-D-glucuronopyranosyl-21,22-di-O-angeloyl-R 1-barrige nol,16-O-acetyl-21-O-(4-O-angeloyl)-a-L-rhamnopy ranosyl-barringtogenol C,28-O-?-D-glucopyranosyl-16-dexoxybarringtogenol C,acetylastragaloside I,timo-saponin B-?,forsythoside B and a few non-saponin substances of myricetin.(2)The human hepatocellular carcinoma HepG2 cells which in logarithmic growth phase and in good shape was selected as the test object,the effects of purified total saponins on cell proliferation was detected by MTT assay using total saponin of Xanthoceras sorbifolia bunge(TSXS)as the test substance.The results showed that TSXS could significantly inhibit the proliferation of HepG2 cells with a dose-effect relationship.The MTT test showed that the inhibition effect of TSXS on HepG2 cells was best at 4 h treatment time,and the IC50 value was 7.894±0.9916 mg/L.At the same time,it was found that the sensitivity of human hepatocellular carcinoma HepG2 cells for TSXS treatment was higher than that of normal liver cells HL-7702.(3)The changes of cell morphology after TSXS treatment,and AO/EB staining,DAPI staining were observed by inverted fluorescence microscope.The resullts showed that TSXS can caused the apoptosis of HepG2 cells,HepG2 cell became rounding,the cells gap increasing,appearing chromatin condensation and DNA fragmentation.The apoptosis rate and cycle changes of HepG2 cells was analyzed by flow cytometry after TSXS treatment.The test results also showed that TSXS can induced the apoptosis of HepG2 cells,and block the cell cycle at the S phase.So the inhibitory effect of TSXS on the HepG2 cells proliferation was achieved through the path of blocking cell cycle and promoting apoptosis.(4)The changes of mitochondrial membrane potential which was detected with JC-1 fluorescence staining were observed through inverted fluorescence microscope.The results showed that the mitochondrial membrane potential of HepG2 cells was decreased after TSXS treatment,and the Bcl-2/Bax expression decreased.The mitochondrial cytochrome C was released into the cytoplasm,then the caspase-3 protein was activated and DNA repair enzyme PARP fragmented.All of those changs above showed a dose-dependent.Experimentl results showed that the apoptotic effect of TSXS on HepG2 cells was achieved through the pathway of mitochondria-mediated endogenous apoptosis.(5)The changes of apoptosis related protein of HepG2 cells was determined by Western blot method after TSXS treatment.The further test results showed that PI3K/Akt signaling pathway may be involved in the apoptosis of HepG2 cell for TSXS induction.It was analyzed that the ratio of Bcl-2/Bax was regulated through PI3K/Akt pathway to affect the the expression of Bcl-2 protein family,further affected the integrity of the structure and function of mitochondria to induce cell apoptosis.(6)The changes of reactive oxygen species(ROS)in HepG2 cells were determined by flow cytometry.It was found from test that TSXS can stimulate the production of ROS in HepG2 cells,and decreased the expression level of oxidative stress regulated protein HO-1.This result indicated that TSXS could lead to apoptosis by stimulating the cells to produce the oxidative stress.This study systematically explored the effect of TSXS on the apoptosis of HepG2 cells and its molecular mechanism.The results have a great significance to fully exploit the medicinal and edible resources potential of Xanthoceras sorbifolia bunge.This study also provide a theoretical basis for further development of the by-product from defatted kernel cake of Xanthoceras sorbifolia bunge and oil plant resource utilization.