Study On The Mechanism Of Lonicera Caerulea Berry Anthocyanin Extracts Regulating Cholesterol Metabolism

Blue honeysuckle(Lonicera caerulea L.var.edulis)belongs to the Caprifoliaceae or honeysuckle(Lonicera)family,which are deciduous shrubs.The 'Wild' type of L.caerulea berries(hereafter referred to as Wild)is widely known in northeast of our country.The L.caerulea fruit contains many anthocyanins,flavonoids,and phenolic acids with a variety of physiological activity.The structure of anthocyanins of L.caerulea berries extract were analyzed,and the effect of components and antioxidant activity of extracts by High hydrostatic pressure(HHP)technologies were analyzed.Moreover,the physiological activity of cholesterol metabolism of L.caerulea berry extracts was studied in vitro and vivo in order to make clear its biological activity,to better provide theoretical basis for development and utilization of L.caerulea fruit resources.1.Anthocyanins extracted optimization research:Extraction ability of ethanol,methanol,acetone and water solution as extraction solvent was compared that Lonicera caerulea anthocyanins were extracted by solvent extraction method.,according to the result of HPLC-DAD separation,there were significant difference of content monomer component between 3,5,10(Cyanidin-3-hexoside-catechin,Cyanidin-3-glucoside,Peonidin-3-rutinoside)in different extraction agents(p<0.05).On the basis of single factor experiment,the response surface methodology(RSM)was employed to find the key parameters for optimization.Results indicated three factors greatly affecting the extracting amount was material-solvent ratio(X1),time(X2)and ethanol concentration(X3).The result showed that material-solvent ratio on extraction yield reached extremely significant level(P<0.01)and extraction time and ethanol concentration reached significant level(P<0.05).The optimal levels of these factors were material-solvent ratio 1:19,time 67 min and ethanol concentration 69%and the other factors were Volatile revolutions of 160 r/min,temperature 30 ?,pH 2.0.The extracting amount of Lonicera caerulea anthocyanins reached 310.36±5.326 mg/100gFW,under the optimized conditions.Results showed that the models fitted well with the experimental data.LCBE after freeze drying to abtain dark red powder,and anthocyanins recovery rate was 86.2%.2.The effect of HHP treatment on anthocyanins extraction:Wild Lonicera caerulea berries were subjected to five different high hydrostatic pressure(HHP)treatments(which resemble the conditions of active component extraction and commercial sterilization).The findings were compared to the control and thermal groups to determine the effects of HHP processing on anthocyanins,phenolics,vitamin C,and antioxidant capacities of the extracts.Thirteen anthocyanins were obtained by chromatography separation and the structure was identified by HPLC-DAD-ESI/MS.Cyanidin-3-glucoside was the major anthocyanin with a content of 82.68%.As HHP increased,a higher loss of active component was observed.However,their retention rate was higher than that of the heat-treated group.The antioxidant capacities of anthocyanins extracts of wild L.caerulea berry were evaluated by 3 different methods(DPPH assay,oxygen radical absorbance capacity assay,and cellular antioxidant activity assay).The results showed that low HHP for a long period of time(400 MPa/20 min)demonstrated better results than that with high HHP for a short time(600 MPa/10 min),as indicated by the higher contents of anthocyanins and phenols and stronger antioxidant capacities.3.Study of LCBE on cholesterol metabolism in vitro:The identification and quantification of a Lonicera caerulea berry extract rich in anthocyanin(LCBE)was conducted using HPLC-DAD-ESI/MS2.The effect of LCBE on cholesterol absorption and transport,as well as on the expression of genes controlling cholesterol synthesis,efflux,and absorption,was investigated in a Caco-2 cell monolayer model.Our results showed that 40 and 80 ?g/mL LCBE can significantly reduce cholesterol absorption and transport,and inhibit the accumulation of cholesterol in a Caco-2 cell monolayer model.Histological analysis was performed using oil red O staining.mRNA and protein were quantified using real-time PCR and western blot,respectively.Gene and protein expression of synthetic critical of cholesterol(SREBP2)and absorption(NPC1L1 and ACAT2)were significantly inhibited by LCBE(p<0.01).Furthermore,LCBE showed a potential function promoting cholesterol efflux through upregulation of mRNA and/or protein levels of ABCA1 and ABCG5/G8(p<0.01).It was therefore concluded that LCBE is hypocholesterolemic via multiple mechanisms,including modulation of cholesterol biosynthesis,absorption,and efflux-related gene expression.4.Study of LCBE on cholesterol metabolism in vivo:This study aimed to examine the hypolipidemic effect of Lonicera caerulea berry extract on high cholesterol diet(HCD)-induced hypercholesterolemia and serum metabolite changes in rats.Rats were fed an HCD containing LCBE(75 mg/kg,150 mg/kg,and 300 mg/kg body weight intragastrically once daily)or not for 12 weeks.The effects of LCBE on mRNA levels of cholesterol related genes in HCD rats were analyzed using RT-PCR.These include SREBP2,miR-33,miR-122,and their respective target genes ABCA1,FAS,and NPC1,as well as genes related to the metabolism of cholesterol to bile acids,PPARy,LXRa,and CYP7A1.Moreover,NPC1L1,ACAT2,MTP,and ABCG5/8 of intestine were performed.In the present study,LCBE supplementation decreased body and organ weights significantly(p<0.01).In addition,it normalized abnormal plasma lipoprotein levels and decreased liver cholesterol levels in hypercholesterolemic rats(p<0.01).LCBE also improved histopathological features of the hepatic and heart aortas in rats fed an HCD.Compared to the HCD control group,LCBE supplementation significantly decreased levels of TC,TG,LDL-C,increased HDL-C.LCBE treatment promoted greater neutral and acidic sterol excretion(p<0.01),and improved the antioxidant capacity of colon tissue,colon contents,and blood.Moreover,trimethylamine N-oxide(TMAO)levels also decreased in plasma(p<0.01 vs HCD group).Dietary LCBE supplementation promoted the metabolism of cholesterol to bile acids and their efflux by activating PPAR?-LXR?-ABCA1/CYP7A1 pathways and downregulating the mRNA and protein expression of cholesterol biosynthesis gene SREEBP2.Moreover,NPC1L1,ACAT2,and MTP mRNA and protein expression reduced and ABCG5/8 expression increased(p<0.01)after LCBE treatment.These results indicated that the therapeutic effect of LCBE is associated with efflux metabolism and reduced absorption of dietary cholesterol and biosynthesis.