| Pomegranate is a plant which could be used for medicine and food. It has been cultivated for a long history in wide planting range and high yield in China. The pomegranate polyphenols, which have antioxidation, antitumor and regulation of blood lipids and other health and medicinal effects, have abundant content in pomegranate peel, pulp and seed. Among them, the polyphenol content of pomegranate peel is the highest. At present, pomegranate juice, pomegranate wine and other products are popular in the market. But in the processing process of these products, the pomegranate peel which accounting for 20%～30% weight of pomegranate and rich of the polyphenol was largely abandoned, resulting in a waste of resources.Therefore, this study selected pomegranate peel polyphenol as the test materials and then we chose the two main active components of pomegranate, punicalagin and ellagic acid as the experimental materials to explore their antitumor activity and molecular mechanism.This study was in vitro test. We selected in human hepatocellular carcinoma HepG2 cells that in logarithmic phase of growth and in good condition as the test object. The survival rates of cells were determined with MTT assay. After Hoechst 33258 staining, the morphology of cells was observed under the inverted fluorescence microscope. The analysis of cell cycle, apoptosisand the ROS level of HepG2 cells after treatment with the test substances were measured by flow cytometry. Detecting the activity of apoptosis related proteins, Caspase-3 and Caspase-9 were used Caspase Activity Assay kit. In additionn, the expression of P53, Bcl-2/Bax, Cyt-c and PARP proteins were determined by Western-Blot. The main results are as follows:(1) Pomegranate peel polyphenols has obvious antitumor activity and showed a dose-dependent effect. The MTT results showed that pomegranate peel polyphenols has obvious inhibition on the growth of HepG2 human hepatoma cells with the half lethal dose (IC50) of 266.90mg/mL. Hoechst 33258 staining results showed that Pomegranate Peel Polyphenols made the cell number decreased and the chromatin pyknosis and serious cell deformation were occurred. The flow cytometry analysis revealed that the Pomegranate Peel Polyphenols could arrest HepG2 cell cycle in S phase and the number of cells into the early apoptosis significantly increased. It effectively promoted the apoptosis. Therefore, The Pomegranate Peel Polyphenols inhibited the growth of HepG2 cells significantly and the effects were achieved by blocking cells cycle and promoting apoptosis.(2) This research also did in-depth analysis on the apoptosis mechanism of HepG2 cells induced by Pomegranate Peel Polyphenols. The results showed that after treatments with Pomegranate Peel Polyphenols in the HepG2 cells, ROS level significantly increased and the mitochondrial membrane potential decreased. The expressions of apoptosis related protein Cyt-c, Caspase-9 and Caspase-3 increased. At the same time, the Bax/Bcl-2 and the expression of p53 increased significantly. The expression of DNA repair enzyme PARP decreased, and the expression of the shear product increased. Therefore, the mechanism of action of Pomegranate Peel Polypheno-ls induced apoptosis in HepG2 cells was involved in mitochondrial pathway of apopto-sis in cells.(3) The two main active components of Pomegranate Peel Polyphenols also show-ed good anti-tumor activities and the effect was dose-dependent. The growth of HepG2 cells was inhibited by PC and PEA with IC50 values of 83.47μM and 94.70μM. The effect of PC was stronger than PEA. Hoechst 33258 staining showed that both PC and PEA could make the cell deformation, cell nuclear margination and presente-d the obvious apoptosis morphology. Flow cytometry analysis of cell cycle distribution and apoptosis by treatments with PC and PEA showed the PC could make the HepG2 cell cycle arrest in S phase, and was arrested in the G0/G1 phase by the PEA. They also induced the number of cells into the late apoptosis increase significantly to promote the apoptosis of HepG2 cells.(4) The results of further investigated in the cell apoptosis mechanism of PC and PEA in the HepG2 cells showed that after the role of PC and PEA, intracellular ROS level raised and the activities of apoptosis related protein Caspase-9 and Caspase-3 also increased significantly. Thus, as the main components of Pomegranate Peel Polyphenols, the mechanism of PC and PEA induced apoptosis of HepG2 cells were also achieved through mitochondrial apoptosis pathway.This study systematically analyzed the antitumor activities and mechanism of the Pomegranate Peel Polyphenols and its main active ingredients, Punicalagin an-d Pomegranate ellagic acid. This research has positive significance for fully exploring the edible, medicinal and health care value of pomegranate, and also provides the theoretical basis for the depth of processing and utilization and the development of new antitumor drugs of pomegranate resources. |
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