Study On Active Components Of Blueberry Leaves And Healthy Functions In Vitro

To develop blueberry resources,this thesis systematically evaluated the healthy functions and active components of blueberry leaves from different cultivars in the same season and different cultivars in different season,and deeply studied the isolation,purification,structural identification and beneficial health properties of phenolic and polysaccharides.The major research methods and results were as follows:(1)The study was designed to examine total phenolic,total polysaccharides,a-glucosidase inhibition,a-amylase inhibition,pancreatic lipase inhibition,bile salt binding rate and DPPH radical scavenging rate of blueberry leaves of twelve different cultivars in the same season from Zhejiang Province.The results showed that in different cultivars the ’Premier’ leaves had the highest total phenolic content at(12.8510.18)%,the’Star’ leaves contained the highest total polysaccharides level of(8.90±0.12)%.At thesame concentration of extract,the ’Jersey’ and ’Chandler’ leaves had the highest a-glucosidase inhibitory activity,the ’O’neal’ leaves had the best a-amylase inhibitory activity,the ’Misty’leaves had the best pancreatic lipase inhibitory and sodium cholate binding activity,the ’Star’ leaves had the best sodium taurocholate binding activity,and’Premier’ and ’Bluerain’ leaves had the strongest DPPH radical scavenging activity.(2)A comparison of blueberry leaf extracts from different cultivars and seasonal was investigated regarding their proanthocyanidin,flavonoids,total polysaccharides,DPPH radical scavenging,ABTS radical scavenging and OH radical scavenging activity.The results showed that the content of proanthocyanidins,flavonoids and total sugar in blueberry leaves were gradually increased from spring to winter.Among these cultivars,the ’Bluerain’ leaves of winter had the highest total proanthocyanidins content at(14.14±0.03)%,the ’Premier’ leaves of winter had the highest flavonoids content at (1.62±0.02)%,and the ’Misty’ leaves of winter had the highest total polysaccharides content at(15.42±0.06)%.The results of the free radical scavenging assay showed that polyphenol crude extract and crude polysaccharide solution of ’Premier’ leaves in winter had significantly better DPPH radical scavenging ability.Polyphenol crude extract of the’Premier’ leaves in autumn,crude polysaccharide solution of the ’Misty’ leaves in autumn and the ’Bluerain’ leaves in summer had significantly better ABTS radical scavenging ability.Polyphenol crude extract of the ’Bluerain’ leaves in autumn and crude polysaccharide solution of the ’Misty’leaves in autumn had significantly better OH radical scavenging ability(P<0.05).In addition,polysaccharide extract possessed significant antioxidant activity in a concentration-dependant manner,in the tested concentration range.Antioxidant activity was positively correlated with the content of proanthocyanidins and flavonoids,and more significant with flavonoids(The correlation coefficient r was greater than 0.75).(3)The ’Premier’ leaves were selected as materials.The active components were determinated by activity-directed isolation method,what were isolated by organic solvent sequential extraction,silicagel and sephadex LH-20 chromatographic methods and so on.Their structures were elucidated by physiochemical properties and spectral analysis.The results showed that the n-butyl alcohol fraction(LBF)had the highest DPPH radical scavenging,a-glycosidase and a-amylase inhibition activity,and their IC50 values were(11.0810.21)μg/mL,(9.60±.21)μg/mL and(176.64±5.53)μg/mL.The ethyl acetate fraction(LEAF)showed the largest pancreatic lipase inhibition and bile salt binding activity,and their IC50 values were(0.591±0.01)mg/mL,(1.87±0.11)mg/mL(sodium cholate)and(3.2310.03)mg/mL(sodium taurocholate).Seven and four compounds were isolated from LEAF and LBF respectively,and their structures were identified as P-sitosterol(Cl),quercetin-3-O-a-L-arabinofuranoside(C3),quercetin(C4),quercetin-3-O-β-D-glucopyranoside(C6)and 1-O-caffeoylquinic acid(C7).A total of two compounds were isolated from the BF,and their structures were identified as quercetin-3-O-a-L-arabinoside(C8)and quercetin-3-O-β-D-glucuronide(C9).Compounds Cl,C3,C7 and C8 were isolated from blueberry leaves for the first time.The results of the activities of compounds showed that Cl,C4 and C5 had a good a-glucosidase and pancreatic lipase inhibitory activity.Cl,C3,C4,C6,C7 and C8 showed different degrees of pancreatic lipase inhibitory activity,and C4 had more stronger activity than others and positive control-orlistat.Cl,C4,C5,C7 and C8 showed strong DPPH radical scavenging capacity,which C4 was stronger than positive control-VC and C7 was equal to VC.Inhibition effect of C4 on pancreatic lipase activity was researche.The inhibition constant for enzyme-substrate complex(Ki)was 0.32 mmol/L,and that for free enzyme(Ki)was 0.88 mmol/L.The mechanism was defined as a mixed-type reversible.(4)Taking ’Premier’leaves as raw materials,the polysaccharides were determinated by activity-directed isolation method,what were isolated by Sevage,membrane separation,DEAE cellulose column and Sephadex G-100 chromatographic methods and so on.The results showed that the four polysaccharide fractions(BLP1-BLP4)were isolated from blueberry leaf polysaccharides.BLP3 showed the best DPPH radical scavenging and a-glucosidase inhibitory activity.The uniform polysaccharide BLP3-1 was obtained by further purified.Its number-average weight-average molecular weight(Mw),molecular weight(Mn)and Z average molecular weight(Mz)were 54192 Da,4880 Da and 1.19×108 Da,respectively.The characteristic functional groups of BLP3-1 were analyzed by Infrared spectra analysis.Monosaccharide analysis by high-performance liquid chromatography with precolumn derivatization of PMP showed that BLP3-1 was possibily composed of galacturonic acid,galactose,xylose,arabinose and fucose.