| Hawthorn is a superior medicinal and edible tonic with various effects including invigorating stomach and promoting digestion, promoting blood circulation and removing blood stasis, lowering blood pressure and blood lipid, increasing coronary blood flow, preventing coronary heart disease and angina pectoris, preventing cancer, and so on. Hawthorn has a culture history of3000years, and the hawthorn variety C. pinnatifida var. major, the most commonly cultivated variety in China, is increasingly popular as raw materials for nutritional foods because of the abundant antioxidant flavonoids (It is particularly rich in hyperoside, isoquercitrin and epicatechin).Thus, this study selected the C. pinnatifida var. major as the experimental material, aiming at exploring the anticancer effects and the potential vascular nutrition function of peel polyphenolic extract (HPP) and flesh polyphenolic extract (HFP) from hawthorn fruit. Human MCF-7breast carcinoma cells in logarithmic growth phase were subjected to cell experiments to systematically investigate the anticancer effects and the mechanism underlying. Cell proliferation was determined with colorimetric MTT assay. Cytotoxicity was evaluated by LDH assay after the treatment with HPP or HFP. The cell cycle distribution, apoptosis and intracellular ROS level were analyzed by flow cytometry. Caspase3and caspase9activities were assessed by Caspase Activity Assay kit. In addition, the effect of HPP and HFP on the NO release from NO2in simulated gastric juice was evaluated by chemiluminescence assay. The main findings are as follows.(1) The extraction yields of HPP and HFP from hawthorn fruit, which were extracted with80%aqueous ethanol, were27.3%and11.6%, respectively. The total phenolic and total flavonoids contents in HPP were441.2and357.9μg/mg extracts, while which in HFP were176.8and117.8μg/mg extracts, respectively. In addition, the main polyphenols present in HPP and HFP were identified and quantified by HPLC. Ideain, chlorogenic acid, epicatechin, hyperoside, isoquercitrin and quercitin were identified to exist in HPP with the concentration of199.0,22.1,18.5,15.7,13.4,2.3μg/mg, respectively. While only40.5μg/mg of epicatechin.4.3μg/mg of ideain,2.6μg/mg of chlorogenic acid, and1.5μg/mg of quercitin were detected in HFP. (2) It was confirmed by antitumor experiments in vitro that both HPP and HFP exhibited significant anticancer effects on MCF-7cells in a dose-dependent and time-dependent manner. The MTT and LDH results showed that the growth of MCF-7cells was significantly inhibited by HPP or HFP with the IC50values of88.6μg/mL and175.5μg/mL, respectively. In addition, flow cytometric analysis revealed that both HPP and HFP mediated the cell-cycle arrest at the S-phase, and also evidently led to apoptosis of MCF-7cells. More specifically, when MCF-7cells were treated with200μg/mL of HPP,37.8%of cells were induced into early apoptosis. Thus, we concluded that the inhibitory effects of HPP and HFP on MCF-7cells were achieved through the cell cycle arrest and the induction of apoptosis or necrosis.(3) Further investigations of cell apoptosis mechanism showed that the activity of caspase3and caspase9, and the intracellular ROS level were significantly elevated by HPP and HFP treatment, indicating that the mitochondrial pathway was invloved in the apoptosis induced by HPP and HFP.(4) The potential of HPP or HFP-promoted acidic nitrite reduction was measured by gas-phase chemiluminescence assay. It was found that both HPP and HFP effectively facilitated the production of NO from nitrite.This study for the first time systematically analyzed and compared the composition, the anticancer effects and the potential vascular nutrition function of HPP and HFP. This research has positive significance for fully exploring the health benefits and pharmacological activity of hawthorn, and also provides theoretical and experimental bases for developing novel chemoprevention agents and cardiovascular protective drugs with few side effects. |
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