玫瑰花中原花青素的分离分析及抗氧化活性研究
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摘要
玫瑰花是我国传统药材之一,《本草纲目拾遗》载:“玫瑰花活血、行血、健脾降火、理气调经、滋补养颜”,其养颜美容的功效被广泛接受。原花青素是具有极强抗氧化能力的多酚类物质,药理作用广泛,目前国外已制成多种药品、保健品、化妆品上市,是一种很有开发前途的天然抗氧化剂。本文主要研究玫瑰花中原花青素的提取及纯化工艺,并建立玫瑰花原花青素中单体和二聚体的HPLC分析方法,最后对玫瑰花原花青素的抗氧化能力进行评价。
本论文首先采用微波法从玫瑰花中提取原花青素,通过单因素实验考察了微波反应时间、反应次数和料液比对提取率的影响,最终确定提取条件为:60%乙醇为溶剂,料液比1:20,微波功率5.00V,微波反应时间20min,反应次数为1次,原花青素的提取率达到1.59%。并将微波法与传统溶剂浸提法进行了对比,结果表明微波法下原花青素的提取率高于溶剂浸提法。
继而采用大孔吸附树脂柱进一步纯化初提液,首先通过静态实验从五种树脂(D101、D1300、NKA,AB-8和NKA-Ⅱ)中筛选出适合纯化原花青素的树脂型号,以饱和吸附量和解吸率为指标综合考虑,结果显示:D101型树脂综合性能最好,其静态饱和吸附量为9.3mg/g湿树脂,解吸率为77.20%。论文进一步通过正交实验考察了纯化工艺参数,包括上样浓度、上样pH、洗脱液浓度、洗脱流速四个因素,以原花青素纯度和回收率为指标,极差分析和方差分析结果表明:上样pH是影响最显著的因素,最优纯化工艺条件为:上样浓度为1.25mg/ml,上样pH为2,洗脱液浓度为70%,洗脱流速为2ml/min。本文还对树脂纯化过程的动力学特征进行了初步探索,绘制了渗漏及洗脱曲线,并用数学方法将渗漏曲线拟合为~(y=1.18(e~(0.31x)-e~(-0.79x))),由此方程积分可得出树脂的动态饱和吸附量即最大上样浓度。
论文进一步建立了玫瑰花原花青素中单体及二聚体的HPLC分析方法,具体色谱条件为:分析柱:Eslipse XDB-C18,4.6mm×150mm,5μm;流动相:A:乙腈,B:1.0%冰醋酸(pH=2.71);洗脱梯度:0-10min:5-10%A;10-100min:10-25%A;流速:1.0ml/min;检测波长:280nm;柱温:30℃;进样量:20μl。在此条件下,四种物质(儿茶素、表儿茶素、表儿茶素没食子酸酯和原花青素B2)的分离度、方法的线性、精密度和加样回收率均符合要求。定量分析结果显示:原花青素B2的含量最高,达2.54%,表儿茶素含量为0.612%,表儿茶素没食子酸酯含量为0.257%。
论文最后将纯化得到的玫瑰花原花青素产品进行抗氧化活性研究,采用DPPH法测定其抗氧化能力,以维生素C为对照,实验测得原花青素对[DPPH·]的半抑制量EC50=0.053mg/ml,维生素C对[DPPH·]的半抑制量EC50=0.066mg/ml,结果表明:原花青素的抗氧化能力强于维生素C。
Flos Rosae Rugosae is a kind of chinese traditional medicine material.It is recorded in 'Compendium of Materia Medica' that it has great effectiveness on keeping young,and this is recepted extensively.Procyanidins are kinds of polyphenol with great antioxidant activity and then extensive pharmacological activity. Procyanidins are natural antioxidants which have prospects of development and hasve been marketed by a lot of countries as medicine,health food and cosmetics.This paper studied on the extraction and isolation of procyanidins from Flos Rosae Rugosae,developed a HPLC analysis method for determination of them and evaluated the antioxidant activity of procyanidins comparing with that of VC.
Procyanidins were extracted from Flos Rosae Rugosae with microwave assisted. The effection of microwave reaction time,times and ratio of solid/solvent on the percentage of procyanidins extraction was investigated by the single-factor experiment.The best extracting conditions were as follows:60%ethanol as the extracting solvent,ratio of solid/solvent 1:20,microwave power 5.00V,microwave reaction time 20min,one times,percentage of procyanidins extraction can achieve to 1.59%.Compared with solvent extraction only,extraction with microwave assisted had better performance.
The extracted crude procyanidins powder was purified by macroporous adsorbent resin.In the beginning,a suitable type of macroporous adsorbent resin had been selected from five types including D101,D1300,NKA,AB-8 and NKA-Ⅱ.Evaluated by static saturated adsorption ability and elution percentage,D101 was thought to be the most suitable type of macroporous adsorbent resin for the purification of procyanidins.Its static saturated adsorption ability was 9.3mg/g wet resin and elution percentage 7.2%.Evaluated by product purity and recovery,the influence of concentration of sample,pH of sample,concentration of eluant and flow rate of elution had been investigated by orthogonal experimental design.Analysis of range and analysis of variance were used to determine the optimal parameters of the purification process,the results were:1.25mg/ml as the concentration of sample,2 as the pH value of sample,70%ethanol aqueous solution as the eluant and 2ml/min as the flow rate of elution.Kinetics about process of purification had also been studied.The curves of dynamic adsorption-elution process had been plotted,and the kinetic equation was ~(Y = 1.18(e~(0.31x) -e~(-0.79x)))
A HPLC method was developed for analysing monomers and dimers of procyanidins from Flos Rosae Rugosae.The column was Eslipse XDB-C18, 4.6mm×150mm,5μm,and thermostated at 30℃.The elution conditions were as follows:flow rate,1 ml/min;solvent A,1.0%AcOH in H_2O;solvent B,CH_3CN; elution starting with 5%B and continuing with a linear gradient from 5 to 10%B in 10min,then 10%to 25%in 100min.The method was validated with respect to resolution,linearity,precision and recovery.Epicatechin,epicatechingallate and procyanidins B2 had been quantitatively analysised,and their contents respectivly were 0.612%,0.257%,2.54%.
The antioxidant activity of procyanidins was evaluated by the amount of antioxidant necessary to decrease by 50%the initial DPPH concentration.The study showed that EC50 for procyanidins was 0.052mg/ml while for VC was 0.066 mg/ml, the conclusion can be drawn that the antioxidant activity of procyanidins is strong than that of VC.
本论文首先采用微波法从玫瑰花中提取原花青素,通过单因素实验考察了微波反应时间、反应次数和料液比对提取率的影响,最终确定提取条件为:60%乙醇为溶剂,料液比1:20,微波功率5.00V,微波反应时间20min,反应次数为1次,原花青素的提取率达到1.59%。并将微波法与传统溶剂浸提法进行了对比,结果表明微波法下原花青素的提取率高于溶剂浸提法。
继而采用大孔吸附树脂柱进一步纯化初提液,首先通过静态实验从五种树脂(D101、D1300、NKA,AB-8和NKA-Ⅱ)中筛选出适合纯化原花青素的树脂型号,以饱和吸附量和解吸率为指标综合考虑,结果显示:D101型树脂综合性能最好,其静态饱和吸附量为9.3mg/g湿树脂,解吸率为77.20%。论文进一步通过正交实验考察了纯化工艺参数,包括上样浓度、上样pH、洗脱液浓度、洗脱流速四个因素,以原花青素纯度和回收率为指标,极差分析和方差分析结果表明:上样pH是影响最显著的因素,最优纯化工艺条件为:上样浓度为1.25mg/ml,上样pH为2,洗脱液浓度为70%,洗脱流速为2ml/min。本文还对树脂纯化过程的动力学特征进行了初步探索,绘制了渗漏及洗脱曲线,并用数学方法将渗漏曲线拟合为~(y=1.18(e~(0.31x)-e~(-0.79x))),由此方程积分可得出树脂的动态饱和吸附量即最大上样浓度。
论文进一步建立了玫瑰花原花青素中单体及二聚体的HPLC分析方法,具体色谱条件为:分析柱:Eslipse XDB-C18,4.6mm×150mm,5μm;流动相:A:乙腈,B:1.0%冰醋酸(pH=2.71);洗脱梯度:0-10min:5-10%A;10-100min:10-25%A;流速:1.0ml/min;检测波长:280nm;柱温:30℃;进样量:20μl。在此条件下,四种物质(儿茶素、表儿茶素、表儿茶素没食子酸酯和原花青素B2)的分离度、方法的线性、精密度和加样回收率均符合要求。定量分析结果显示:原花青素B2的含量最高,达2.54%,表儿茶素含量为0.612%,表儿茶素没食子酸酯含量为0.257%。
论文最后将纯化得到的玫瑰花原花青素产品进行抗氧化活性研究,采用DPPH法测定其抗氧化能力,以维生素C为对照,实验测得原花青素对[DPPH·]的半抑制量EC50=0.053mg/ml,维生素C对[DPPH·]的半抑制量EC50=0.066mg/ml,结果表明:原花青素的抗氧化能力强于维生素C。
Flos Rosae Rugosae is a kind of chinese traditional medicine material.It is recorded in 'Compendium of Materia Medica' that it has great effectiveness on keeping young,and this is recepted extensively.Procyanidins are kinds of polyphenol with great antioxidant activity and then extensive pharmacological activity. Procyanidins are natural antioxidants which have prospects of development and hasve been marketed by a lot of countries as medicine,health food and cosmetics.This paper studied on the extraction and isolation of procyanidins from Flos Rosae Rugosae,developed a HPLC analysis method for determination of them and evaluated the antioxidant activity of procyanidins comparing with that of VC.
Procyanidins were extracted from Flos Rosae Rugosae with microwave assisted. The effection of microwave reaction time,times and ratio of solid/solvent on the percentage of procyanidins extraction was investigated by the single-factor experiment.The best extracting conditions were as follows:60%ethanol as the extracting solvent,ratio of solid/solvent 1:20,microwave power 5.00V,microwave reaction time 20min,one times,percentage of procyanidins extraction can achieve to 1.59%.Compared with solvent extraction only,extraction with microwave assisted had better performance.
The extracted crude procyanidins powder was purified by macroporous adsorbent resin.In the beginning,a suitable type of macroporous adsorbent resin had been selected from five types including D101,D1300,NKA,AB-8 and NKA-Ⅱ.Evaluated by static saturated adsorption ability and elution percentage,D101 was thought to be the most suitable type of macroporous adsorbent resin for the purification of procyanidins.Its static saturated adsorption ability was 9.3mg/g wet resin and elution percentage 7.2%.Evaluated by product purity and recovery,the influence of concentration of sample,pH of sample,concentration of eluant and flow rate of elution had been investigated by orthogonal experimental design.Analysis of range and analysis of variance were used to determine the optimal parameters of the purification process,the results were:1.25mg/ml as the concentration of sample,2 as the pH value of sample,70%ethanol aqueous solution as the eluant and 2ml/min as the flow rate of elution.Kinetics about process of purification had also been studied.The curves of dynamic adsorption-elution process had been plotted,and the kinetic equation was ~(Y = 1.18(e~(0.31x) -e~(-0.79x)))
A HPLC method was developed for analysing monomers and dimers of procyanidins from Flos Rosae Rugosae.The column was Eslipse XDB-C18, 4.6mm×150mm,5μm,and thermostated at 30℃.The elution conditions were as follows:flow rate,1 ml/min;solvent A,1.0%AcOH in H_2O;solvent B,CH_3CN; elution starting with 5%B and continuing with a linear gradient from 5 to 10%B in 10min,then 10%to 25%in 100min.The method was validated with respect to resolution,linearity,precision and recovery.Epicatechin,epicatechingallate and procyanidins B2 had been quantitatively analysised,and their contents respectivly were 0.612%,0.257%,2.54%.
The antioxidant activity of procyanidins was evaluated by the amount of antioxidant necessary to decrease by 50%the initial DPPH concentration.The study showed that EC50 for procyanidins was 0.052mg/ml while for VC was 0.066 mg/ml, the conclusion can be drawn that the antioxidant activity of procyanidins is strong than that of VC.
引文
[1]国家中医药管理局.中华本草(4).上海:上海科学技术出版社.1999,238.
[2]江苏新医学院编.中药大词典(上册).上海:上海科技出版社.1997,1224.
[3]国家药典委员会编.中华人民共和国药典(2000年版一部).北京:化学工业出版社.2000,153.
[4]徐怀德,刘邻渭,李元瑞等.几种干花成分分析及玫瑰花饮料加工技术研究[J].西北农林科技大学学报(自然科学版).2003,31(3):67-69.
[5]王兴国,王亚平,赵青等.CGC/IR/MS方法对玫瑰精油化学成分的分析[J].兰州大学学报(自然科学版).1996,32(1):79-81.
[6]谢琼,张益娜,李敏等.紫外分光光度法测定玫瑰花和玫瑰花渣中的总黄酮含量[J].农产品加工·学刊.2007,4:19-20.
[7]牛淑敏,朱颂华等.中药材玫瑰花抗氧化及作用机制研究[J].南开大学学报(自然科学版).2004,37(2):90-92.
[8]Porter L J,Hrstich L N,Chan B J.The conversion of Procyanidins and rodelphinidins to cyaniding and delphinidin[J].Phytochemistry,1986,25(1):223-225.
[9]Bate Smith,E.Astringent tannins of the leaves of Geranium species[J].Phytochemistry,1981,20:211-216.
[10]Santos Buelga,C Scalbert A.Review:Proanthocyanidins and tannin-like compounds-nature,occurrence,dietary intake and effects on nutrition and health[J].J.Sci.Food Agric.2000,80(7):1094-1117.
[11]石碧,狄莹.植物多酚[M].科学出版社,2000.
[12]Prieur C,Rigaud J,Cheynier V,et al.Oligomeric and polymeric procyanidins from grape seeds[J].Phytochemistry,1994,36(3):781-784.
[13]Jarkko H J,Maarit K,Pirjo M.Isolation and Structure Elucidation of Procyanidin Oligomers from Saskatoon Berries(Amelanchier alnifolia)[J].J.Agric.Food Chem.2007,55,157-164.
[14]Liu L,Xie B J,Cao S Q,et al.A-type procyanidins from Litchi chinensis pericarp with antioxidant activity[J].Food Chemistry.2007(105):1446-1451.
[15]Victor AP de Freitas,Yves Glories,Guy Bourgeois,et al.Characterisation of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry[J].Phytochemistry.1998,49(5):1435.
[16]孙达旺.植物单宁化学[M].北京:中国林业出版社,1992,130-142.
[17]国植,徐莉.原花青素:具有广阔发展前景的植物药[J].国外医药-植物药分册.1996,11(5):196-204.
[18]黄红霞,戚向阳,肖俊松.大孔吸附树脂对苹果原花青素吸附分离的特性[J].食品与发酵工业,2004,30(7):58.
[19]石翠芳,孙智达,谢笔钧.沙枣果肉原花青素的提取、纯化及抗氧化性能的研究[J].农业工程学报.2006,22(3):158.
[20]张泽生,赵春艳,曹力心.大孔吸附树脂分离纯化山楂果中原花青素的研究[J].现代食品科技.2006,22(2):16.
[21]刘睿,段玉清,谢笔钧等.高粱外种皮中原花青素的提取工艺及其组分鉴定[J].农业工程学报.2004,20(1):242.
[22]Wollgast J,Pallaroni L,Agazzi M E,et al.Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection[J].J.Chromatogr.A,2001,926:211-220.
[23]Guyot S,Marnet N,Laraba D,et al.Reversed-phase HPLC following thiolysis for quantitative estimation and characterization of the four main classes of phenolic compounds in different tissue zones of a French cider apple variety(Malus domestica var.Kermerrien)[J].J.Agric.Food Chem.1998,46(5):1698-1705.
[24]Maria J,Ariadna S,Jorge S,et al.A comparison between bark extracts from Pinus pinaster and Pinus radiata:Antioxidant activity and procyanidin composition.Food Chemistry,2007,100,439-444.
[25]Fuleki T,Ricardo da silva,J.M.Catechin and procyanidin composition of seeds from grape cultivars grown in Ontario[J].J.Agric.Food Chem.1997,45,1156-1160.
[26]Ling Z Q,Xie B J,Yang E L.Isolation,Characterization and Determination of Antioxidative Activity of Oligomeric Procyanidins from the Seedpod of Nelumbo nucifera Gaertn[J].Agric.Food Chem,2005,53,2441-2445.
[27]Ulla S,Heikki V,Risto K,et al.Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers[J].Phytochemistry,2002,.60:821-825.
[28]Xu X Y,Xie B J,Pan S Y,et al.A new technology for extraction and purification of proanthocyanidins derived from sea buckthom bark[J].J Sci Food Agric.86:486-492(2006).
[29]戴晶晶,刘文洁,曾凡骏.原花青素低温提取工艺研究.食品工业,2008,1:18-20.
[30]赵文军,吴雪萍,王旭.葡萄籽中低聚原花青素提取条件研究[J].食品科学,2004,25(2):117-120.
[31]王翔飞,周文明,傅建熙.沙棘籽中原花青素的提取工艺.西北农业学报,2006,15(3):204-207.
[32]李凤英,崔蕊静,李春华.采用微波辅助法提取葡萄籽中的原花青素[J].食品与发酵工业.2005,31(1):39-42.
[33]赵春艳,张泽生,刘海俊.微波提取山楂果中原花青素工艺的研究.食品研究与开发,2005,26(6):37-40.
[34]白光辉,张辉,王克亮等.微波场作用下原花青素浸取工艺研究应用化工,2006,35(9):685-687.
[35]姚方耀.葡萄籽中葡萄籽油和原花青素的提取分离工艺研究.天津大学硕士学位论文.2004.
[36]吴朝霞,孟宪军,张红等.超临界CO2萃取葡萄籽中低聚原花青素的初探.沈阳农业大学学报,2007,38(2):241-244.
[37]吕丽爽.葡萄籽低聚原花青素的研制.无锡轻工大学硕士论文.2000.
[38]石翠芳,孙智达,刘畅等.沙枣果肉原花青素的分离纯化.食品与发酵工业,2007,33(4):137-140.
[39]魏孝义,谢海辉,周文华等.原花青素的制备工艺[P].CN1273985A.2000,11.22.
[40]JP5623415.
[41]吴朝霞,孟宪军,金嫘.聚酰胺分离纯化葡萄籽原花青素及部分产物组分构成的初步研究.食品研究与开发,2007,28(2):46-48.
[42]He J R,Celestino S B,Nuno M,et al.Isolation and quantification of oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of column chromatographic techniques[J].J.Agric.Food Chem.2005,53,2441-2445.
[43]吴朝霞,吴朝晖.大孔吸附树脂纯化葡萄籽原花青素的研究,食品与机械,2006,22(4):46-48.
[44]徐晓云,潘思轶,谢笔钧等.树脂法精制沙棘籽原花色素的研究.农业工程学报.2005,21(1):152-154.
[45]Oszmianski J,Sapis J C.Fractionation and identification of some low molecular weight grape seed phenolics[J].J.Agric.Food Chem.1989,37(5):1293-1297.
[46]李春阳,许时婴,王璋.利用区带毛细管电泳分离、分析葡萄籽原花青素.分析检测,2005,4:172-175.
[47]Broadhurst R B,Jones W T.Analysis of condensed tannins using acidified vanillin[J].J.Sci.Food Agric.1978,29,788-794.
[48]李春阳,许时婴,王璋.低浓度香草醛-盐酸法测定葡萄籽、梗中原花青素含量的研究[J].食品工业科技,2004,25(6):128-130.
[49]孙芸,谷文英.硫酸-香草醛法测定葡萄籽原花青素含量[J].生产与科研经验,2003,29(9):43-46.
[50]傅武胜,蔡一新,林丽玉等.铁盐催化比色法测定葡萄籽提取物中的原花青紊[J].食品与发酵工业,2001,27(10):57-61.
[51]马亚军,杨秉勤,郎惠云.钼酸铵分光光度法测定葡萄籽提取物中的原花青素[J].食品科学,2003,24(5):135-137.
[52]马亚军,郎惠云,贾婴琦.硫酸高铈铵分光光度法测定葡萄籽提取物中原花青素[J].分析试验室,2004,25(4):42-44.
[53]马亚军,杨秉勤,郎惠云.高铁盐-铁氰化钾分光光度法测定葡萄籽原花青素的含量.西部粮油科技.2003,(1):60-61.
[54]马亚军,郎惠云.原子吸收法间接测定葡萄籽提取物中的原花青素.分析试验室,2004,23(9):55-57.
[55]韩志萍,郭枫林.电化学方法测定葡萄籽原花青素含量.榆林学院学报,2005,15(3):18-20.
[56]韩志萍,卢翠荚.示波极谱法测定葡萄籽提取物中原花青索.理化检验·化学分册,2005,41(4).56-58.
[57]卢翠英.流动注射-抑制化学发光法测定葡萄籽提取物中原花青素.分析测试学报,2004,23(2):75-77.
[58]Ricardo-da-Silva J M,Cheynier V,Souquet J M.Interaction of Grape Seed Procyanidins with Various Proteins in Relation to Wine Fining.J.Sci.Food and Agric.1991,57(1).
[59]Bachi D,Garg A,Krohn RL.et al.Oxygen free radical scavenging abilities of vitamins C and E,and a grape seed proanthocyanidin extract in vitro.Res.Commun.Mol.Pathol[J].Pharmacol.1997,95.
[60]余莹,粟武,魏东芝.原花青素体外清除自由基活性的研究[J].华东理工大学学报.2002,6(28).
[61]高军涛.葡萄籽中原花青素类物质的提取和分离及其抗氧化性质的研究.中国科学院硕士学位论文,2000.
[62]张竣,吉伟之,齐欣.葡萄籽中多酚类物质的提取及其对油脂的抗氧化作用.食品科学,2001,22(10):43-45.
[63]凌智群,谢笔钧.莲房原花青素抗氧化损伤作用的研究[J].食品科学,2002,23(7):98-100.
[64]Jeun AB,et al.CA,1995,123:107837p.
[65]Bas J M D,FEMANDEZ-LARREA J,BLAY M,et al.Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats[J].J Express Article,2005,10:1096-3095.
[66]Huynh T H,Teel R W.Selective induction of apoptosis in human mammary cancer cells(MCF-7) by pycnogenol[J].An ticancer Res,2000,20(4):2417-2420.
[67]Dongrno A B,Kamanyi,Anchang M S.Anti-inflammatory and analgesic properties of the stem bark extracts of Ery.throphleum suaveolens (Caesatpiniaceae) Guillemin & Perrottet[J].J Ethnopharmacol,2001,77(2-3):137-141.
[68]Dauer A,Metzner P,Schimmer O.Proanthocyanidins from the bark of Hamam elis virginian a exhibit antlmutagenic properties a-gainst nitroaromatic compounds.Planta Med,1998,64(4):324-327.
[69]胡静.玫瑰花总原花青素的分离、纯化及分析.浙江大学硕士学位论文.2006.
[70]陈菁菁,李向荣等.大孔吸附树脂分离纯化桑叶总黄酮及其动力学研究[J].浙江大学学报,2006,35(2):219-223.
[71]向大雄,李焕德等.大孔吸附树脂分离纯化葛根总黄酮的研究[J].中国药学杂志,2003,38(1):35-37.
[72]Gao M,Huang W,Liu C Z.Separation of scutellarin from crude extracts of Erigeron breviscapus(vant.) Hand.Mazz.by macroporous resins[J].J chromatography B,2007,858(1-2):22-26.
[73]Fu B Q,Liu J,Li H,Li L,et al.The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid[J].Journal of Chromatography A,2005,1089:18-24.
[74]Concepcion S M,Jose A L,Fulgencio S C.A Procedure to Measure the Antiradical Efficiency of Polyphenols[J].J Sci Food Agric 1998,76,270-276.
[2]江苏新医学院编.中药大词典(上册).上海:上海科技出版社.1997,1224.
[3]国家药典委员会编.中华人民共和国药典(2000年版一部).北京:化学工业出版社.2000,153.
[4]徐怀德,刘邻渭,李元瑞等.几种干花成分分析及玫瑰花饮料加工技术研究[J].西北农林科技大学学报(自然科学版).2003,31(3):67-69.
[5]王兴国,王亚平,赵青等.CGC/IR/MS方法对玫瑰精油化学成分的分析[J].兰州大学学报(自然科学版).1996,32(1):79-81.
[6]谢琼,张益娜,李敏等.紫外分光光度法测定玫瑰花和玫瑰花渣中的总黄酮含量[J].农产品加工·学刊.2007,4:19-20.
[7]牛淑敏,朱颂华等.中药材玫瑰花抗氧化及作用机制研究[J].南开大学学报(自然科学版).2004,37(2):90-92.
[8]Porter L J,Hrstich L N,Chan B J.The conversion of Procyanidins and rodelphinidins to cyaniding and delphinidin[J].Phytochemistry,1986,25(1):223-225.
[9]Bate Smith,E.Astringent tannins of the leaves of Geranium species[J].Phytochemistry,1981,20:211-216.
[10]Santos Buelga,C Scalbert A.Review:Proanthocyanidins and tannin-like compounds-nature,occurrence,dietary intake and effects on nutrition and health[J].J.Sci.Food Agric.2000,80(7):1094-1117.
[11]石碧,狄莹.植物多酚[M].科学出版社,2000.
[12]Prieur C,Rigaud J,Cheynier V,et al.Oligomeric and polymeric procyanidins from grape seeds[J].Phytochemistry,1994,36(3):781-784.
[13]Jarkko H J,Maarit K,Pirjo M.Isolation and Structure Elucidation of Procyanidin Oligomers from Saskatoon Berries(Amelanchier alnifolia)[J].J.Agric.Food Chem.2007,55,157-164.
[14]Liu L,Xie B J,Cao S Q,et al.A-type procyanidins from Litchi chinensis pericarp with antioxidant activity[J].Food Chemistry.2007(105):1446-1451.
[15]Victor AP de Freitas,Yves Glories,Guy Bourgeois,et al.Characterisation of oligomeric and polymeric procyanidins from grape seeds by liquid secondary ion mass spectrometry[J].Phytochemistry.1998,49(5):1435.
[16]孙达旺.植物单宁化学[M].北京:中国林业出版社,1992,130-142.
[17]国植,徐莉.原花青素:具有广阔发展前景的植物药[J].国外医药-植物药分册.1996,11(5):196-204.
[18]黄红霞,戚向阳,肖俊松.大孔吸附树脂对苹果原花青素吸附分离的特性[J].食品与发酵工业,2004,30(7):58.
[19]石翠芳,孙智达,谢笔钧.沙枣果肉原花青素的提取、纯化及抗氧化性能的研究[J].农业工程学报.2006,22(3):158.
[20]张泽生,赵春艳,曹力心.大孔吸附树脂分离纯化山楂果中原花青素的研究[J].现代食品科技.2006,22(2):16.
[21]刘睿,段玉清,谢笔钧等.高粱外种皮中原花青素的提取工艺及其组分鉴定[J].农业工程学报.2004,20(1):242.
[22]Wollgast J,Pallaroni L,Agazzi M E,et al.Analysis of procyanidins in chocolate by reversed-phase high-performance liquid chromatography with electrospray ionisation mass spectrometric and tandem mass spectrometric detection[J].J.Chromatogr.A,2001,926:211-220.
[23]Guyot S,Marnet N,Laraba D,et al.Reversed-phase HPLC following thiolysis for quantitative estimation and characterization of the four main classes of phenolic compounds in different tissue zones of a French cider apple variety(Malus domestica var.Kermerrien)[J].J.Agric.Food Chem.1998,46(5):1698-1705.
[24]Maria J,Ariadna S,Jorge S,et al.A comparison between bark extracts from Pinus pinaster and Pinus radiata:Antioxidant activity and procyanidin composition.Food Chemistry,2007,100,439-444.
[25]Fuleki T,Ricardo da silva,J.M.Catechin and procyanidin composition of seeds from grape cultivars grown in Ontario[J].J.Agric.Food Chem.1997,45,1156-1160.
[26]Ling Z Q,Xie B J,Yang E L.Isolation,Characterization and Determination of Antioxidative Activity of Oligomeric Procyanidins from the Seedpod of Nelumbo nucifera Gaertn[J].Agric.Food Chem,2005,53,2441-2445.
[27]Ulla S,Heikki V,Risto K,et al.Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers[J].Phytochemistry,2002,.60:821-825.
[28]Xu X Y,Xie B J,Pan S Y,et al.A new technology for extraction and purification of proanthocyanidins derived from sea buckthom bark[J].J Sci Food Agric.86:486-492(2006).
[29]戴晶晶,刘文洁,曾凡骏.原花青素低温提取工艺研究.食品工业,2008,1:18-20.
[30]赵文军,吴雪萍,王旭.葡萄籽中低聚原花青素提取条件研究[J].食品科学,2004,25(2):117-120.
[31]王翔飞,周文明,傅建熙.沙棘籽中原花青素的提取工艺.西北农业学报,2006,15(3):204-207.
[32]李凤英,崔蕊静,李春华.采用微波辅助法提取葡萄籽中的原花青素[J].食品与发酵工业.2005,31(1):39-42.
[33]赵春艳,张泽生,刘海俊.微波提取山楂果中原花青素工艺的研究.食品研究与开发,2005,26(6):37-40.
[34]白光辉,张辉,王克亮等.微波场作用下原花青素浸取工艺研究应用化工,2006,35(9):685-687.
[35]姚方耀.葡萄籽中葡萄籽油和原花青素的提取分离工艺研究.天津大学硕士学位论文.2004.
[36]吴朝霞,孟宪军,张红等.超临界CO2萃取葡萄籽中低聚原花青素的初探.沈阳农业大学学报,2007,38(2):241-244.
[37]吕丽爽.葡萄籽低聚原花青素的研制.无锡轻工大学硕士论文.2000.
[38]石翠芳,孙智达,刘畅等.沙枣果肉原花青素的分离纯化.食品与发酵工业,2007,33(4):137-140.
[39]魏孝义,谢海辉,周文华等.原花青素的制备工艺[P].CN1273985A.2000,11.22.
[40]JP5623415.
[41]吴朝霞,孟宪军,金嫘.聚酰胺分离纯化葡萄籽原花青素及部分产物组分构成的初步研究.食品研究与开发,2007,28(2):46-48.
[42]He J R,Celestino S B,Nuno M,et al.Isolation and quantification of oligomeric pyranoanthocyanin-flavanol pigments from red wines by combination of column chromatographic techniques[J].J.Agric.Food Chem.2005,53,2441-2445.
[43]吴朝霞,吴朝晖.大孔吸附树脂纯化葡萄籽原花青素的研究,食品与机械,2006,22(4):46-48.
[44]徐晓云,潘思轶,谢笔钧等.树脂法精制沙棘籽原花色素的研究.农业工程学报.2005,21(1):152-154.
[45]Oszmianski J,Sapis J C.Fractionation and identification of some low molecular weight grape seed phenolics[J].J.Agric.Food Chem.1989,37(5):1293-1297.
[46]李春阳,许时婴,王璋.利用区带毛细管电泳分离、分析葡萄籽原花青素.分析检测,2005,4:172-175.
[47]Broadhurst R B,Jones W T.Analysis of condensed tannins using acidified vanillin[J].J.Sci.Food Agric.1978,29,788-794.
[48]李春阳,许时婴,王璋.低浓度香草醛-盐酸法测定葡萄籽、梗中原花青素含量的研究[J].食品工业科技,2004,25(6):128-130.
[49]孙芸,谷文英.硫酸-香草醛法测定葡萄籽原花青素含量[J].生产与科研经验,2003,29(9):43-46.
[50]傅武胜,蔡一新,林丽玉等.铁盐催化比色法测定葡萄籽提取物中的原花青紊[J].食品与发酵工业,2001,27(10):57-61.
[51]马亚军,杨秉勤,郎惠云.钼酸铵分光光度法测定葡萄籽提取物中的原花青素[J].食品科学,2003,24(5):135-137.
[52]马亚军,郎惠云,贾婴琦.硫酸高铈铵分光光度法测定葡萄籽提取物中原花青素[J].分析试验室,2004,25(4):42-44.
[53]马亚军,杨秉勤,郎惠云.高铁盐-铁氰化钾分光光度法测定葡萄籽原花青素的含量.西部粮油科技.2003,(1):60-61.
[54]马亚军,郎惠云.原子吸收法间接测定葡萄籽提取物中的原花青素.分析试验室,2004,23(9):55-57.
[55]韩志萍,郭枫林.电化学方法测定葡萄籽原花青素含量.榆林学院学报,2005,15(3):18-20.
[56]韩志萍,卢翠荚.示波极谱法测定葡萄籽提取物中原花青索.理化检验·化学分册,2005,41(4).56-58.
[57]卢翠英.流动注射-抑制化学发光法测定葡萄籽提取物中原花青素.分析测试学报,2004,23(2):75-77.
[58]Ricardo-da-Silva J M,Cheynier V,Souquet J M.Interaction of Grape Seed Procyanidins with Various Proteins in Relation to Wine Fining.J.Sci.Food and Agric.1991,57(1).
[59]Bachi D,Garg A,Krohn RL.et al.Oxygen free radical scavenging abilities of vitamins C and E,and a grape seed proanthocyanidin extract in vitro.Res.Commun.Mol.Pathol[J].Pharmacol.1997,95.
[60]余莹,粟武,魏东芝.原花青素体外清除自由基活性的研究[J].华东理工大学学报.2002,6(28).
[61]高军涛.葡萄籽中原花青素类物质的提取和分离及其抗氧化性质的研究.中国科学院硕士学位论文,2000.
[62]张竣,吉伟之,齐欣.葡萄籽中多酚类物质的提取及其对油脂的抗氧化作用.食品科学,2001,22(10):43-45.
[63]凌智群,谢笔钧.莲房原花青素抗氧化损伤作用的研究[J].食品科学,2002,23(7):98-100.
[64]Jeun AB,et al.CA,1995,123:107837p.
[65]Bas J M D,FEMANDEZ-LARREA J,BLAY M,et al.Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats[J].J Express Article,2005,10:1096-3095.
[66]Huynh T H,Teel R W.Selective induction of apoptosis in human mammary cancer cells(MCF-7) by pycnogenol[J].An ticancer Res,2000,20(4):2417-2420.
[67]Dongrno A B,Kamanyi,Anchang M S.Anti-inflammatory and analgesic properties of the stem bark extracts of Ery.throphleum suaveolens (Caesatpiniaceae) Guillemin & Perrottet[J].J Ethnopharmacol,2001,77(2-3):137-141.
[68]Dauer A,Metzner P,Schimmer O.Proanthocyanidins from the bark of Hamam elis virginian a exhibit antlmutagenic properties a-gainst nitroaromatic compounds.Planta Med,1998,64(4):324-327.
[69]胡静.玫瑰花总原花青素的分离、纯化及分析.浙江大学硕士学位论文.2006.
[70]陈菁菁,李向荣等.大孔吸附树脂分离纯化桑叶总黄酮及其动力学研究[J].浙江大学学报,2006,35(2):219-223.
[71]向大雄,李焕德等.大孔吸附树脂分离纯化葛根总黄酮的研究[J].中国药学杂志,2003,38(1):35-37.
[72]Gao M,Huang W,Liu C Z.Separation of scutellarin from crude extracts of Erigeron breviscapus(vant.) Hand.Mazz.by macroporous resins[J].J chromatography B,2007,858(1-2):22-26.
[73]Fu B Q,Liu J,Li H,Li L,et al.The application of macroporous resins in the separation of licorice flavonoids and glycyrrhizic acid[J].Journal of Chromatography A,2005,1089:18-24.
[74]Concepcion S M,Jose A L,Fulgencio S C.A Procedure to Measure the Antiradical Efficiency of Polyphenols[J].J Sci Food Agric 1998,76,270-276.