| The red-flesh apple is a unique species of Malus.It has high ornamental value with red colour of whole fruit.The red-flesh apple has juiceless pulp and slightly sour and astringent taste,but it containing a variety of biologically active ingredients,such as polyphenols,flavonoids and polysaccharides.Studies have shown that that the apple polyphenols played an important role in preventing cardiovascular disease,anti-tumor,anti-oxidation,anti-aging.In order to utilization and development the plant resources of red-flesh apple,the extraction and purification conditions,composition analysis,antioxidation and anticancer activity of polyphenols were discussed,the main results are as follows:(1)Ultrasonic-assisted methodology was used to extract the polyphenols from red-flesh apple.Based on the results of four single factor tests,including extraction time,ultrasonic power,extraction temperature and ratio of material-to-liquid,the effects on the yield of polyphenols were discussed.The optimal process parameters of polyphenol extraction were determined by Response surface methodology and they were as follows:extraction time and temperature was 20 min and 62 ℃,ultrasound power and ratio of material-to-liquid was 360 W and 1:4(g/mL)respectively.The polyphenols yield was achieved to 2.135 mg/g for red-flesh apple under the optimized conditions,and it was only 0.22 percent less than the theoretical prediction(2.320 mg/g).Therefore,the model had extremely good fitting degree.(2)The polyphenols extraction from red-flesh apple was purified through macroporous adsorptive resin method.The NKA-Ⅱ resin was chosed to purify the polyphenols through the comparing of the behavior of static adsorption and desorption test in five macroporous resins for red-flesh apple polyphenol.The results of static adsorption and desorption tests showed that the NKA-II resin could reach the absorption and desorption equilibrium at 6 h and 10 h respectively.The optimal separating process parameters were as follows:the concentration of polyphenols and the sampling rate were 0.8 mg/mL and 4.0 BV/h respectively,and the elution concentration and sampling volume were 75%ethanol and 3.5 BV respectively.In this condition,the purity of red-flesh apple polyphenols was increased from 35%to 84%.(3)The composition of purified polyphenols in red-flesh apple of Xinjiang was analyzed by HPLC.Ten phenolic compounds were identified in purified polyphenols,which were protocatechuic acid,epigallocatechin,quercitrin,catechinic,procyanidin B2,chlorogenic acid,4-hydroxybenzoic acid,hyperoside,rutin and quercetin.Among them,the content of chlorogenic acid was the highest with 123.150 μg/mL,followed by quercitrin and proanthocyanidin B2 with 25.284 μg/mL and 21.958μg/mL.(4)The antioxidant capacity of polyphenols from red-flesh apple were investigated by the free radical scavenging test of polyphenols.The results showed that the polyphenols from red-flesh apple had strong reducing capacity,it also had obvious effects on free radical-scavenging for DPPH·,ABTS+,·OH and O2-· radical.It also had obvious protective effect for lipid peroxidation and oxidation damage induced by Cu2+/H2O2 of BSA protein.(5)The inhibitory actions of polyphenols from red-flesh apple on three tumor cells in vitro were studied by MTT colorimetric.The results showed that the polyphenols(0.1,0.3 and 0.5mg/mL)from red-flesh apple had obvious inhibitory capabilities on Hela(human cervical cancer),HepG2(human hepatocellular cancer)and A549(human nonsmall cell lung cancer),and the order of inhibitory effects was Hela>HepG2>A549.The polyphenols from red-flesh apple displayed the strongest cytotoxic activity against Hela cells rather than other cell lines and the IC50 was about 0.41±0.008 mg/mL.Then Hela cells were treated of polyphenols for different time,and 12 h treatment of polyphenols on Hela cells showed a higher inhibitory rate and ecnomic effects in our subsequent study.(6)The nuclear morphology were analyzed with trypan blue,AO-EB and DAPI staining,mitochondrial membrane potential and the contents of ROS,respectively to investigate whether the decreased viability of Hela cells after polyphenols treatment was mainly due to apoptosis.The results showed the polyphenols from red-flesh apple can cause the apoptosis phenomenon of Hela cells with a dose-dependent manner,such as cell rounding,chromatin condensation and the increase of the cells gap,mitochondrial membrane potential collapse and the increase of ROS.The cell apoptosis was further verified.Consequently,the polyphenols from red-flesh apple can induce the apoptosis of Hela cells to inhibit proliferation and have good anticancer activity. |
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