摘要
蔷薇红景天(Rhodiola rosea L.)为景天科红景天属植物,是一种具有优良保健功效的珍稀药用植物资源,含有多种生理活性成分,如红景天苷、酪醇、黄酮类等。然而国内外对蔷薇红景天中原花青素的研究报道却极少。为此,本文对蔷薇红景天中原花青素的提取、精制工艺以及稳定性进行了较为系统的研究,为进一步工业化生产和精制原花青素提供了理论基础。
本文建立了香草醛-盐酸法测定蔷薇红景天中原花青素的方法。分别采用两种不同的方法从蔷薇红景天中提取原花青素,通过单因素试验及正交试验设计确定了乙醇回流提取法的最佳工艺条件为:提取溶剂为50% (v/v)乙醇,提取温度80℃,提取时间120 min,料液比1:25;超声波-微波协同萃取法的最佳工艺条件为:提取溶剂为50% (v/v)乙醇,料液比1:25,在200 W的微波功率下提取50 s,然后再在50℃恒温条件下超声波处理20 min,两种方法在最佳提取条件下原花青素得率为分别为5.43%、4.72%。
蔷薇红景天原花青素精制采用了大孔吸附树脂精制法,根据吸附解吸性能,从5种树脂中筛选出AB-8树脂作为精制蔷薇红景天原花青素的树脂,并确定了其吸附和洗脱条件。结果表明,经AB-8树脂固定床进行分离富集后,有效的去除了杂质,原花青素纯度得到了提高,试验达到了预期目的。
通过对原花青素精制物进行IR、HPLC分析,明确了蔷薇红景天中的原花青素是以表棓儿茶素-3-O-棓酸酯(EGCG)为基本结构单元的原翠雀定类原花青素。
蔷薇红景天原花青素的稳定性研究表明,光照可使原花青素含量逐渐下降,较低的温度和较低的pH值条件下原花青素比较稳定;防腐剂苯甲酸钠、山梨酸钾对原花青素的稳定性无太大影响;H_2O_2可导致原花青素的稳定性下降,而Vc对原花青素的稳定性有保护作用;原花青素对K~+、Mg~(2+)、Na~+和Cu~(2+)表现出很好的稳定性,而Fe~(3+)、Sn~(2+)、Al~(3+)可破坏原花青素的稳定性。
Rhodiola rosea is a species which comes from Rhodiola genus of Crassulaceae. As a valuable resource of the medicinal plants, Rhodiola rosea has the superordinary efficiency in health. It contains many kinds of chemical composition with physiological activation, such as salidroside, tyrosol, flavonoids and so on. However, there are only few literatures about proanthocyanidins in Rhodiola rosea at home and abroad. In this paper, the optimum extracting condition and purification method of proanthocyanidins from Rhodiola rosea, and the stability of the purified proanthocyanidins were studied. The study laid theoretical foundation for large-scale industrialization manufacture and purification of proanthocyanidins.
The method of analyzing proanthocyanidins of Rhodiola rosea by spectrophotometric determination of the vanillin-hydrochloric acid method was founded. Two different methods were adopted to extract the proanthocyanidins. Through the single factor experiment and the orthogonal experimental design, the optimum extraction conditions of proanthocyanidins from Rhodiola rosea by ethanol circumfluence were showed as follows: extraction solvent was 50% ethanol (v/v), extraction temperature was 80℃, the solid- solvent ratio was 1:25, extraction time was 120 min. The optimum extraction conditions of proanthocyanidins from Rhodiola rosea by ultrasonic-microwave synergistic extraction were obtained as follows: extraction solvent was 50% ethanol (v/v), the solid-solvent ratio was 1:25, the microwave power was 200 W, the microwave time was 50 s, then ultrasonic wave treatment 20 min under the 50℃constant temperature. Under the optimum condition, the extracting ratio of the two methods were 5.43% and 4.72%, respectively.
Proanthocyanidins of Rhodiola rosea was refined by macroreticular resins. According to the adsorption and desorption capacity, AB-8 was the best in five kinds of selected macroreticular resins. The adsorption and elution conditions were determined. The results showed that much impurity was removed and the purity of the proanthocyanidins was raised by separation and enriched with AB-8 resin fixed bed. The experiment was successful.
The purified proanthocyanidins determined by IR and HPLC, the result showed that proanthocyanidins of Rhodiola rosea was mainly composed of (-)-epigallocatechin-3-O -gallate (EGCG), and it belonged to prodelphinidins.
The stability of proanthocyanidins was studied. The results showed that the amount would decrease under the sunlight, the proanthocyanidins was likely stable at low temperature and low pH. The proanthocyanidins was stable against to antiseptic such as sodium benzoate and potassium sorbate. H_2O_2 had obvious effect on the stability of proanthocyanidins, on the contrary, Vc could protect its stability. Among different metal ions, K~+, Mg~(2+), Na~+ and Cu~(2+) had little influence on the stability of proanthocyanidins, but Fe~(3+), Sn~(2+) and Al~(3+) could destroy its stability.
引文
[1]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.
[2]石碧,狄莹.植物多酚[M].北京:科学出版社,2000,10:137.
[3]王忠合,朱俊晨,陈惠音.葡萄籽原花青素提取物的保健功能与应用[J].食品科技,2006(4):135~139.
[4]步文磊,王茵.原花青素的生物活性及作用机制研究进展[J].国外医学卫生学分册,2007,34 (5):311~315.
[5]罗冠中.原花青素的提取、分离及抗氧化性、稳定性的研究[D].天津:天津科技大学,2006.
[6]陈笳鸿,汪咏梅,吴冬梅,等.毛杨梅树皮提取物抗氧化及清除自由基活性初步研究[J].林产化学与工业,2007,27:1~7.
[7]杨其蒀.天然药物化学[M].北京:中国医药科技出版社,1996,6.
[8]何钊,任其龙.层析法分离葡萄籽中原花青素的研究[J].食品科学,2004,25(8):70~73.
[9]孙达旺.植物单宁化学[M].北京:中国林业出版社,1992:130~142.
[10]韩菊,魏福祥,瞿丽萍,等.葡萄籽中低聚原花色素的性能研究[J].食品科学,2003,24(2):36~38.
[11]Andrew K. Smith,Helen June,Ann C. Noble.Effect of viscosity on the bittemess and astringency of grape seed tannin [M].Food quality and preference,1996,7:161~166.
[12]Kathi J,Kemper M D.Oligomeric proanthocyanidin complexes (OPCs) (Pycnogenols,Pine bark extract,Grape seed extract) [J].Longwood Herber Task Force,1999,10:1~21.
[13]Svedstrom U,Vuorela H,Kostiainen R,et al.Isolation and identification of oligomeric procyanidins from Crataegus leaves and flowers [J].Phytochemistry,2002,60:821~825.
[14]Packer L,Rimbach G,Virgili F.Antioxidant activity and biologic properties of procyanidin-rich extract from pine (Pinus Maritima) bark,Pycnogenol [J].Free Radical Biology and Medicine,1999,27(5-6):704~724.
[15]杜晓.落叶松原花色素的分级及精细化利用研究[D].成都:四川大学,2006.
[16]Shoji T,Masumoto S,Moriichi N,et al.Apple (Malus pumila) procyanidins fractionated according to the degree of polymerization using normal-phase chromatography and characterized by HPLC- ESI/MS and MALDI-TOF/MS[J].Chromatography A,2006,1102:206~213.
[17]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):242~245.
[18]Adamson G E,Lazarus S A,Mitchell A E.HPLC method for the quantification of procyanidins in cocoa and chocolate samples and correlation to total antioxidant capacity [J].Agric Food Chem,1999,47:4184~4188.
[19]Liwei G,Kelm M,Hammerstone J F,et al. Fractionation of polymeric procyanidins from lowbush blueberry and quantification of procyanidins in selected foods with an optimized normal-phaseHPLC-MS fluorescent detection method [J].Agric Food Chem,2002,50:4852~4860.
[20]Karonen M,Loponen J,Ossipov V,et al.Analysis of procyanidins in pine bark with reversed-phase and normal-phase high-performance liquid chromatography-electrospray ionization mass spectrometry [J].Analytica Chimica Acta,2004,522(1):105~112.
[21]孙芸,陶冠军,谷文英.葡萄籽原花青素的反相液相色谱-质谱分析[J].中草药,2003,34(6):493~496.
[22]Mc Murrough I,Baert T.Identification of proanthocyanidins in beer and their direct measurement with dual electrode electrochemical detector [J].Inst Brew,1994,100:409~416.
[23]李敏,向平,杨志伟,等.杨梅不同部位单宁含量与结构研究[J].林产化学与工业,2008,28(3):55~60.
[24]Vivas N,Nonier M F,Pianet I,et al.Proanthocyanidins from Quercus petraea and Q. robur heartwood: quantification and structures [J].Comptes Rendus Chimie,2006(9):120~126.
[25]魏冠红.高聚原花青素的水解工艺研究[D].杭州:浙江大学,2006.
[26]Porter L J,Hrstich J N,Chan B G.The conversion of procyanidins and prodelphinidins to cyaniding and delphinidin [J].Phytochemistry,1986,25(1):223~230.
[27]Ryoyasu Saijo.Isolation and chemical structures of two new catechins from fresh tea leaf [J].Agric Biol Chem,1982,46(7):1969~1970.
[28]Kennedy J A,Jones G P.Analysis of proanthocyanidins cleavage products following acid-catalysis in the presence of excess phloroglucinol [J].Agric Food Chem,2001,49(4):1740~1746.
[29]Meagher L P,Lane G,Sivakumaran S,et al.Characterization of condensed tannins from Lotus species by thiolytic degradation and electrospray mass spectrometry [J].Animal Feed Science and Technology,2004,117:151~163.
[30]冯建光.葡萄籽提取物中有效成分不同测定方法的比较[J].中国食品添加剂,2003,5:100~102.
[31]鲍俊竹,陈月坤,徐桂花.测定葡萄籽提取物中原花青素含量的方法[J].农业科学研究,2005,26(1):43~45.
[32]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.
[33]姚开,何强,吕远平,等.葡萄籽提取物中原花青素含量的测定[J].食品与发酵工业,2002,28(3):17~19.
[34]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素[J].食品与发酵工业,2001,27(10):57~61.
[35]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[J].分析试验室,2004,23(9):55~57.
[36]韩志萍,郭枫林.电化学方法测定葡萄籽原花青素含量[J].榆林学院学报,2005,15(3):18~20.
[37]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[J].食品工业科技,2005,26(4):172~174.
[38]吴琼英,周卫靖.苹果中原花青素的提取及其抗氧化活性研究[J].食品科技,2006(12):42~45.
[39]孟宪军,黄朝晖,陆平,等.高粱原花青素提取工艺研究[J].食品科技,2008(4):123~126.
[40]张代佳,刘传斌,修志龙.微波技术在植物胞内有效成分提取中的应用[J].中草药,2000,31(9):5~6.
[41]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.
[42]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[J].食品研究与开发,2005,26(6):37~40.
[43]Athanasios C K,Nikoloas G S,Dimitra J D.Comparison of distillation and ultrasound-assisted extraction methods for the isolation of sensitive aroma compounds from garlic (Allium sativum) [J]. Ultrasonics Sonochemistry,2006,13:54~60.
[44]刘春辉,周亚平,祝军,等.苹果果肉中原花青素超声波的辅助浸提[J].食品与生物技术学报,2008,27(1):80~83.
[45]王军,王敏,于智峰,等.基于响应曲面法的苦荞麸皮总黄酮提取工艺优化[J].农业机械学报,2007,38(7):205~208.
[46]钟振声,冯焱,孙立杰.超声波法从葡萄籽中提取原花青素[J].精细化工,2005,22(1):41~43.
[47]徐怀德,闫宁环,陈伟,等.黑莓原花青素超声波辅助提取优化及抗氧化性研究[J].农业工程学报,2008,24(2):264~269.
[48]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[D].天津:天津大学,2003.
[49]成智涛,王仁才,熊兴耀,等.超临界CO2萃取刺葡萄籽原花青素的工艺研究[J].现代生物医学进展,2007,7(3):363~366.
[50]张长贵.蚕豆壳中原花色素提取、纯化及抗氧化性质研究[D].重庆:西南大学,2007.
[51]Ariga Toshiaki.Extraction of proanthocyanidins [P]. JP 5623415.
[52]罗艳玲,欧仕益.大孔树脂在食品活性成分分离中的应用[J].食品与机械,2005,21(5):81~83.
[53]汪洪武,刘艳清.大孔吸附树脂的应用研究进展[J].中药材,2005,28(4):353~356.
[54]孙芸,谷文英.大孔吸附树脂对葡萄籽原花青素的吸附研究[J].离子交换与吸附,2003,19(6):561~566.
[55]樊丽华.葡萄籽中原花青素提取方法的进一步研究[J].新疆大学学报(自然科学版),2004,21(l): 102~104.
[56]李瑞丽,马润宇.层析法分离提取葡萄籽原花青素进一步研究[J].化工时刊,2006,20(6):3~6.
[57]杨迎花,李晓丽.大孔吸附树脂对葡萄籽原花青素的吸附特性[J].精细化工,2007,24(9):899 ~902.
[58]孙芸,徐宝才,谷文英,等.AB-8树脂对葡萄籽原花青素按聚合度分离特性的研究[J].食品科技,2007(6):60~64.
[59]李莹,赵谋明,于立梅,等.采用大孔吸附树脂纯化马尾松树皮原花青素及其抗氧化性研究[J].食品与发酵工业,2006,32(12):145~149.
[60]王文雅,李军,岳海燕,等.3种大孔吸附树脂对葡萄酒下脚料中原花青素分离性能的研究[J].食品科技,2008(6):146~149.
[61]朱凤妹,杜彬,李军,等.大孔吸附树脂分离葡萄酒下脚料中原花青素的研究[J].食品工业,2008 (1):6~9.
[62]Toshiaki A,Ikunori K,Danji F.Antioxidative properties of procyanidins B-1 and B-3 from azuki beans in aqueous systems [J].Agri Biol Chem,1988,52(11):2717~2722.
[63]Bagchi D,Bagchi M,Sthos S J,et al.Free radicals and grape seed proanthocyanidin extract: importance in human health and disease prevention [J].Toxicology,2000,148:187~197.
[64]Del-Bas J M,Fernandez-Larrea J,Blay M,et al.Grape seed procyanidins improve atherosclerotic risk index and induce liver CYP7A1 and SHP expression in healthy rats [J].FASEB J,2005,19(3):479~481.
[65]Debasis Bagchi,Chandan K Sen,Sidhartha D Ray,et al.Molecular mechanisms of cardioprotection by a novel grape seed proanthocyanidin extract [J].Mutation Research,2003,523~524.
[66]Huynh T H,Tee R W.Selective induction of apoptaosis in human nummary cancer cell [MCF7] by pycnogenol [J].Anticancer Res,2000,20(4):2417~2420.
[67]段玉清,张海晖,邓乾春,等.莲房原花青素对免疫功能的影响[J].食品研究与开发,2007,28(12):1~5.
[68]Sharma S D,Katiyar S K.Dietary grape-seed proanthocyanidins inhibition of ultraviolet B-induced immune suppression is associated with induction of IL-12[J].Carcinogenesis,2006,2(1):95~102.
[69]周素娟.葡萄籽提取物原花青素的研究概况及其在我国保健食品中的应用[J].中国食品卫生杂志,2007,19(3):284~286.
[70]包文芳,吴维春,张薇.国产玫瑰红景天水溶性成分得分离与鉴定[J].中国药物化学杂志,2000,10(3):209~210.
[71]Thlonen A,Hohtola A,Jalonen J.Comparison of electrospray ionization and atmospheric pressure chemical ionization techniques in the analysis of the main constituents from Rhodiola rosea extracts by liquid chromatography/mass spectrometry [J].Mass spectrum,2003,38(8):845~853.
[72]Linh P T,Kim Y H,Hong S P,et al.Quantitative determination of salidroside and tyrosol from the underground part of Rhodiola rosea by high performance liquid chromatography[J].Arch Pharm Res,2000,23:349~352.
[73]Li Zhang,Huixin Yu,Yang Sun,et al.Protective effects of salidroside on hydrogen peroxide- induced apoptosis in SH-SY5Y human neuroblastoma cells [J].European Journal of Pharmacology,2007,564(1-3):18~25.
[74]Sen Yu,Li Liu,Tao Wen,et al.Development and validation of a liquid chromatographic/electros- pray ionization mass spectrometric method for the determination of salidroside in rat plasma: App- lication to the pharmacokinetics study [J].Journal of Chromatography B,2008,861:10~15.
[75]Gad G. Yousef,Mary H. Grace,Diana M. Cheng,et al.Comparative phytochemical characterize- ation of three Rhodiola species [J].Phytochemistry,2006,67:2380~2391.
[76]李春阳,许时婴,王璋.香草醛-盐酸法测定葡萄籽、梗中原花青素含量的研究[J].食品科学,2004,25(2):157~160.
[77]樊金玲,陶冠军,朱文学.香草醛/硫酸比色法测定两种原花色素含量的比较[J].食品科学,2007,28(9):467~472.
[78]赵小亮,白红进,吴翠云,等.超声-微波协同萃取法提取杜梨果实多糖[J].时珍国医国药,2007,18(9):2151~2152.
[79]吴素仪,丘泰球,范晓丹.超声波在中草药有效成分提取应用中的研究进展[J].江苏中医药,2008,40(7):93~94.
[80]罗锋,汪河滨,杨玲,等.超声-微波协同萃取法提取甘草黄酮的研究[J].食品研究与开发,2006,27(8):127~128.
[81]唐仕荣.微波-超声波协同萃取银杏黄酮的工艺研究[J].云南化工,2007,34(3):13~16.
[82]白红进,汪河滨,褚志强,等.不同方法提取黑果枸杞多糖的研究[J].食品工业科技,2007,28(3):145~146.
[83]赵玉卉,赵小亮.杜梨叶片多糖的超声-微波协同萃取法提取[J].食品科技,2008(8):131~133.
[84]马利华,秦卫东,戴晓娟,等.超声波-微波协同提取牛蒡中类胡萝卜素[J].食品研究与开发,2007,28(1):81~85.
[85]赵文恩,韩桂花,焦凤云.葡萄籽原花素提取工艺研究[J].食品科学,2002,23(8):108~110.
[86]于立梅,赵谋明,崔春,等.山竹壳中原花青素提取、分级及抗氧化活性的研究[J].食品与机械,2007,23(4):60~64.
[87]杜学禹,章正格,王飞.加勒比松树皮中原花青素的提取工艺研究[J].生物质化学工程,2007, 41(6):6~10.
[88]李春阳,许时婴,王璋.从葡萄籽中提取分离原花青素的研究[J].中外葡萄与葡萄酒,2004 (4):20~23.
[89]丁丽,王敏,杜连祥.荔枝核中黄烷-3-醇的鉴定及原花青素的提取[J].食品研究与开发,2006,27(7):67~69.
[90]刘睿,谢笔钧,潘思轶.高粱种子外种皮中原花青素提取、纯化及其抗氧化活性的研究[J].中国粮油学报,2003,18(4):43~47.
[91]付为琳,杨立刚,孙桂菊.微波萃取在菊花黄酮提取工艺中的应用研究[J].食品研究与开发,2008,29(1):1~3.
[92]周忻.大孔吸附树脂分离纯化中草药有效成分的影响因素[J].中国药物应用与监测,2006(2):50~53.
[93]吕洁丽,杨中汉,袁珂.新型凝胶树脂及大孔吸附树脂在中草药成分分离纯化中的应用[J].中药材,2005,28(3):239~242.
[94]Guyot S,Marnet N,Drilleau J F.Thiolysis-HPLC characterization of apple procyanidins covering a large range of polymerization states [J].Agric Food Chem,2001,49:14~20.
[95]樊金玲,丁霄霖,徐德平.沙棘籽中的原花色素化合物[J].中草药,2006,37(4):514~517.
[96]李楠.大孔树脂吸附分离金莲花黄酮类化合物的研究[J].食品研究与开发,2008,29(2):65~67.
[97]赵平,宋学娟,张月萍.大孔树脂对葡萄籽原花青素的吸附研究[J].中国医药杂志,2007,32(20):2196~2201.
[98]张茜,贾冬英,姚开,等.大孔吸附树脂纯化石榴皮多酚[J].精细化工,2007,24(4):345~348.
[99]王正烈,周亚平,李松林,等.物理化学(下册)(第四版)[M].北京:高等教育出版社,2003,167~168.
[100]Fool L Y.Proanthocyanidins: Gross chemical structure by infrared spectra [J].Phytochemistry,1981,20(6):1397~1402.
[101]李春阳,许时婴,王璋.葡萄籽原花青素结构单元的红外光谱分析[J].食品与生物技术学报,2005,24(4):47~51.
[102]李斌,孟宪军,智红涛,等.葡萄籽中原花青素的稳定性研究[J].食品研究与开发,2007,28 (10):71~73.
[103]戚向阳,陈维军,王小红.苹果原花青素的稳定性及其保健饮品的研究[J].食品科技,2003(1):88~90.
[104]郑燕升,莫倩,廖政达.野生毛葡萄籽原花青素的稳定性研究[J].安徽农业科学,2008,36(13):5259~5260.