| ObjectiveThis study was to compare the effects of polyphenols in apple peel and apple flesh on cardiovascular risk factors and the expression of vascular endothelial permeability related signal pathway and expression of genes,and aims to explore the mechanism of coronary artery protection.Method(1)The extraction of the total phenol in apple peel and apple pulp was carried out by ultrasonic assisted extraction method.The extraction parameters were optimized by single factor experiment.The total phenol was purified by macroporous resin.The standard curve of gallic acid was prepared by Folin-Ciocalteus(FC)method.The content of polyphenols in apple pulp and apple peel was calculated.Analysis of Phenolic Compounds in PAP and PAF by High Performance Liquid Chromatography.(2)Adult male ICR mice were randomly divided into 4 groups,eight for each group:Control group:mice were given normal diet.Model group:high fructose and high fat(HFHF)diet containing 20%fructose and 5%.PAP group was fed with HFHF diet and 250 mg/kg PAP each day.PAF group was treated with HFHF diet and 250 mg/kg PAF once a day.All mice were fed for 28 days.The mice were weighed every 7 days after the corresponding treatment.Systolic pressures were measured every 3 days.The blood samples of the mice were collected at the 28th day,serum biochemical indicators including endothelia-1(ET-1),NO,malonaldehyde(MDA),atherosclerosis Index(AI),and homeostasis Model assessment-IR(HOMA-IR)were calculated.(3)Primary culture of vascular endothelial cells from ICR mice were isolated and cultured,the cells were divided into four groups:Control group:the cells were cultured in a routine culture without any treatment;HIF-1α group:HIF-1αexpression vector recombinant plasmid was transfected into mouse vascular endothelial cells.HIF-la + PAP group:HIF-la expression vector recombinant plasmid was transfected into mouse vascular endothelial cells and PAP(10 mg/L)was applied to the cells.HIF-la + PAF group:HIF-la expression vector recombinant plasmid was transfected into mouse vascular endothelial cells,followed by PAF(10 mg/L)on the cells.The expression levels of HIF-1αa,ET-1,ETA,ETB,MLCK,p-MLC,ZO-1 mRNA and protein were detected in each group.Results(1)The results showed that the optimum extraction conditions were as follows:the extraction time was 8.13 min,the ultrasonic power was 503.22 W,the extraction temperature was 62.25 ℃,the ratio of material to liquid was 1:28.34,the average extraction rate was 42.39 ± 1.24 mg/g.The extraction time was 10.22 min,the ultrasonic power was 601.15 W,the extraction temperature was 71.73 ℃ and the ratio of material to liquid was 1:34.11.The optimum extraction conditions were as follows:extraction time was 10.22 min,The average extraction rate of polyphenols in apple peel was 9.22 ± 1.06 mg/g using 5 parallel experiments.(2)Compared with Control group,the body weight of Model group was significantly increased(P<0.05).PAP group and PAF group had lower body weight(P>0.05).In Control group,Mice systolic pressure was kept at a relatively stable level throughout the course of the experiment.The HFHF diet resulted in a significant increase in mouse systolic pressure.Compared with the Model group,the levels of systolic pressure in PAP and PAF groups were reduced in different degrees(P<0.05,P>0.05).Compared with Control group,the level of NO in Model group was significantly decreased(P<0.01),while the levels of ET-1 and MDA in serum were significantly increased(P<0.01).The levels serum NO in PAF and PAP groups were higher than those in Model group(P>0.05,P<0.05),while ET-1 and MDA level in serum were lower(P>0.05,P<0.05).Compared with Control group,the levels of AI in Model group were significantly increased(P<0.01),Compared with Model group,the levels of serum AI in PAF and PAP group were significantly decreased(P<0.05,P<0.01).Compared with Control group,the levels of HOMA-IR in Model group were significantly higher(P<0.01).Compared with Model group,the level of HOMA-IR in PAF and PAP group were significantly lower(P<0.05).(3)Compared with Control group,the expression levels of HIF-1α,ET-1,ETA,ETB,MLCK,p-MLC mRNA and protein were significantly increased in HIF-1α group(P<0.05),while ZO-1 mRNA and the expression level of protein was significantly decreased(P<0.05).Compared with HIF-1α group,HIF-la,ET-1,ETA,ETB,MLCK,p-MLC mRNA and protein were significantly decreased in HIF-lα+ PAP group and HIF-1α+ PAF group(P<0.05).The expression level of ZO-1 mRNA and protein were significantly increased(P<0.05).Compared with HIF-1α + PAF group,HIF-1α,ET-1,ETA,ETB,MLCK,p-MLC mRNA and protein were significantly decreased in HIF-1α + PAP group(P<0.05),while ZO-1 mRNA and protein expression levels were significantly increased(P<0.05).Conclusion(1)The contents of total phenol,flavonoids and taxonomy contained in PAP were significantly higher than those in PAF.(2)Apple polyphenols have the effect of protecting cardiovascular function,can effectively reduce the HFHF diet feeding mice blood pressure,improve endothelial function,relieve hyperlipidemia and insulin resistance,but PAP above the role of stronger than PAF,which may be due to their Containing phenolic substances are caused by differences.(3)PAP and PAF can effectively inhibit the HIF-1/ET-1 signaling pathway caused by increased vascular endothelial cell permeability,and thus the role of cardiovascular protection. |
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