沙棘叶中黄酮类化合物的高效提取及分离纯化研究硕士学位.doc

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1、【精品文档】如有侵权，请联系网站删除，仅供学习与交流沙棘叶中黄酮类化合物的高效提取及分离纯化研究硕士学位.精品文档.学 位 论 文沙棘叶中黄酮类化合物的高效提取及分离纯化研究Classified Index: U.D.C:Dissertation for the Master Degree in EngineeringSTUDY ON THE PROCESS OF HIGH-EFFECTIVE EXTRACTION AND SEPARATION OF FLAVONOIDS FROM SEA-BUCKTHORN LEAVES 学位论文原创性声明本人郑重声明：所呈交的论文是本人在导师的指导下独立进

2、行研究所取得的研究成果。除了文中特别加以标注引用的内容外，本论文不包含任何其他个人或集体已经发表或撰写的成果作品。对本文的研究做出重要贡献的个人和集体，均已在文中以明确方式标明。本人完全意识到本声明的法律后果由本人承担。作者签名： 日期： 年 月 日学位论文版权使用授权书本学位论文作者完全了解学校有关保留、使用学位论文的规定，同意学校保留并向国家有关部门或机构送交论文的复印件和电子版，允许论文被查阅和借阅。本人授权 大学可以将本学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或扫描等复制手段保存和汇编本学位论文。涉密论文按学校规定处理。作者签名：日期： 年 月 日导师签名：

3、日期： 年 月 日目 录中文摘要I英文摘要III1 引言41.1 沙棘概况41.1.1 沙棘的生物特征41.1.2 沙棘的分布41.2 沙棘的药理作用41.2.1对心血管系统疾病的作用41.2.2对免疫系统的作用41.2.3对消化系统的作用41.2.4对抗氧化、抗衰老的作用41.2.5耐缺氧、耐疲劳的作用41.2.6促进儿童生长发育41.2.7抗肿瘤、抗癌作用41.2.8对呼吸系统的作用41.2.9抗炎症作用41.3沙棘的应用41.3.1应用于食品41.3.2应用于医药41.3.3应用于化妆品41.3.4应用于环境保护41.4沙棘的化学成分41.4.1黄酮类化合物41.4.2多糖41.4.3维

4、生素类41.4.4微量元素41.4.5蛋白质和氨基酸41.4.6酚类和有机酸41.4.7油和脂肪酸41.4.8三萜、甾体类化合物41.5沙棘的研究概况41.6黄酮类化合物的研究41.6.1黄酮类化合物的提取分离41.6.2黄酮类化合物的分析检验方法41.7本课题研究的目的及意义41.8研究的主要内容42 材料与方法42.1 试验材料42.1.1 材料与试剂42.1.2 主要仪器设备42.2 试验方法42.2.1沙棘叶中黄酮类化合物的提取工艺42.2.2超声波水解法提取沙棘叶中黄酮类化合物42.2.3酶解法提取沙棘叶中黄酮类化合物42.2.4超声波水解法与酶解法的比较42.2.5F8大孔吸附树脂

5、分离纯化黄酮类化合物的试验方法42.2.6聚酰胺分离纯化黄酮类化合物的试验方法42.2.7高效液相色谱对收集的样品分析42.2.8F8大孔吸附树脂法与聚酰胺法的比较43 结果与分析43.1超声波水解法提取沙棘叶黄酮类化合物的单因素试验结果43.1.1提取溶剂的选择43.1.2乙醇浓度对沙棘叶黄酮得率的影响43.1.3盐酸浓度对沙棘叶黄酮得率的影响43.1.4水解时间对沙棘叶黄酮得率的影响43.1.5水解温度对沙棘叶黄酮得率的影响43.1.6料液比对沙棘叶黄酮得率的影响43.2超声波水解法提取沙棘叶黄酮类化合物的响应面分析结果43.2.1回归方程的建立与分析43.2.2乙醇浓度(A)和水解时间(

6、B)对沙棘叶黄酮类化合物得率(Y)的影响43.2.3乙醇浓度(A)和盐酸浓度(C)对沙棘叶黄酮类化合物得率(Y)的影响43.2.4水解时间(B)和盐酸浓度(C)对沙棘叶黄酮类化合物得率(Y)的影响43.2.5最优条件下样品的HPLC图谱43.3酶解法提取沙棘叶黄酮类化合物的单因素试验43.3.1酶浓度对沙棘叶黄酮得率的影响43.3.2酶催化的pH值对沙棘叶黄酮得率的影响43.3.3酶解温度对沙棘叶黄酮得率的影响43.3.4酶解时间对沙棘叶黄酮得率的影响43.3.5料液比对沙棘叶黄酮得率的影响43.4酶解法提取沙棘叶黄酮类化合物的响应面分析结果43.4.1回归方程的建立与分析43.4.2酶浓度(

7、A)和酶催化的pH(B)对沙棘叶黄酮类化合物得率(Y)的影响43.4.3酶浓度(A)和酶解温度(C)对沙棘叶黄酮类化合物得率(Y)的影响43.4.4酶催化的pH(B)和酶解温度(C)对沙棘叶黄酮类化合物得率(Y)的影响43.4.5最优条件下样品的HPLC图谱43.5超声波水解法与酶水解法的比较43.6 F8大孔吸附树脂的静态吸附试验43.6.1F8大孔吸附树脂静态吸附动力学曲线43.6.2F8大孔吸附树脂吸附等温线的测定43.7 F8大孔吸附树脂动态吸附和解吸附的研究43.7.1上样流速对吸附黄酮类化合物的影响43.7.2上样浓度对吸附黄酮类化合物的影响43.7.3洗脱剂的选择43.7.4洗脱

8、剂浓度对洗脱效果的影响43.7.5洗脱流速对洗脱效果的影响43.7.6洗脱剂用量对洗脱效果的影响43.8高效液相色谱对样品分析43.9聚酰胺的静态吸附和解吸附试验43.9.1聚酰胺目数的筛选43.9.2聚酰胺静态吸附动力学曲线43.10聚酰胺的动态吸附试验43.10.1上样流速对吸附黄酮类化合物的影响43.10.2上样浓度对吸附黄酮类化合物的影响43.10.3洗脱剂浓度对洗脱效果的影响43.10.4洗脱流速对洗脱效果的影响43.10.5洗脱剂用量对洗脱效果的影响43.11高效液相色谱对样品分析43.12F8大孔吸附树脂法与聚酰胺法的比较44 讨论44.1超声波水解法条件的确定44.2酶水解法条

9、件的确定44.3吸附树脂法条件的确定44.4超声波水解法的工作原理44.5酶解法的基本原理44.6F8大孔吸附树脂分离黄酮类化合物的基本原理44.7聚酰胺分离黄酮类化合物的基本原理45结论4致 谢4参考文献4攻读硕士期间发表的学术论文4CONTENTSChinese AbstractIEnglish AbstractIII1 Introduction11.1 Overview of sea-buckthorn11.1.1 Biotic characteristic of sea-buckthorn11.1.2 Distribution of sea-buckthorn11.2 Pharmaco

10、logical action of sea-buckthorn21.2.1The effect of disease of cardiovascular system21.2.2The effect of immune system21.2.3The effect of alimentary system31.2.4The effect of anti-oxidation and anti-aging31.2.5The effect of hypoxia-fast and defatigation-fast31.2.6Encouraging growth and development for

11、 child31.2.7The effect of anti-tumor and anti-tumous31.2.8The effect of respiratory apparatus41.2.9The effect of anti-inflammatory41.3Application of sea-buckthorn41.3.1Application for foodstuff41.3.2Application for medicine51.3.3Application for cosmetic51.3.4Application for environmental protection5

12、1.4Chemical composition of sea-buckthorn61.4.1Flavanoid61.4.2Polyose71.4.3Vitamins81.4.4Microelement81.4.5Protein and amino acids81.4.6Phenols and organic acid81.4.7Oil and fatty acid81.4.8Chemical compound of triterpene and steroid91.5Investigative overview of sea-buckthorn91.6Investigation of flav

13、anoid91.6.1Abstraction and segregation of sea-buckthorn91.6.2The method of analyses for sea-buckthorn131.7Target and significanc of research131.8Content of Research142 Material and method152.1 Test material152.1.1 Material and agent152.1.2 Main instruments152.2 Test methods162.2.1Extractive technolo

14、gy of flavanoid from sea-buckthorn leaves162.2.2Extraction of flavanoid from sea-buckthorn leaves by ultrasonic wave hydrolization method 182.2.3Extraction of flavanoid from sea-buckthorn leaves by enzymolysis method202.2.4Comparison between ultrasonic waves hydrolysis and enzymolysis212.2.5The test

15、 methods of segregating flavanoid by F8 macroporous adsorptive resins212.2.6The test methods of segregating flavanoid by polyamide232.2.7The analysis for exemplar by HPLC252.2.8Comparison between F8 macroporous adsorptive resins and polyamide253 Result and analysis273.1The result of single-factor te

16、st of flavanoid from sea-buckthorn leaves by ultrasonic waves hydrolysis273.1.1The selection of extraction solvent273.1.2The effect of concentration of alcohol on the total yield of flavonoid273.1.3The effect of density of hydrochloric acid on the total yield of flavonoid 283.1.4The effect of hydrol

17、ytic time on the total yield of flavonoid283.1.5 The effect of hydrolytic temperature on the total yield of flavonoid293.1.6The effect of ratio of material and fluid on the total yield of flavonoid303.2The experiment result of response surface of flavanoid from sea-buckthorn leaves by ultrasonic wav

18、es hydrolysis303.2.1Establishment and analysis of regression equation303.2.2 The effect of concentration of alcohol(A) and hydrolytic time(B) on the total yield(Y) of flavonoid323.2.3The effect of concentration of alcohol(A) and density of hydrochloric acid(C) on the total yield(Y) of flavonoid323.2

19、.4The effect of hydrolytic time(B) and density of hydrochloric acid(C) on the total yield(Y) of flavonoid333.2.5HPLC atlas of exemplar at optima condition343.3The result of single-factor test of flavanoid from sea-buckthorn leaves by enzymolysis method 343.3.1The effect of ratio of enzyme concentrat

20、ion on the total yield of flavonoid343.3.2The effect of ratio of pH on the total yield of flavonoid353.3.3The effect of ratio of temperature on the total yield of flavonoid363.3.4The effect of ratio of time on the total yield of flavonoid363.3.5The effect of ratio of material and fluid on the total

21、yield of flavonoid373.4The experiment result of response surface of flavanoid from sea-buckthorn leaves by enzymolysis method 373.4.1Establishment and analysis of regression equation373.4.2The effect of enzyme concentration(A) and pH(B) on the total yield(Y) of flavonoid393.4.3The effect of enzyme c

22、oncentration(A) and temperature(C) on the total yield(Y) of flavonoid393.4.4The effect of pH(B) and temperature(C) on the total yield(Y) of flavonoid403.4.5HPLC atlas of exemplar at optima condition413.5Comparison between ultrasonic waves hydrolysis and enzymolysis413.6 The test of static adsorb of

23、F8 macroporous adsorptive resins423.6.1The static adsorption kinetics curve of F8 macroporous adsorptive resins423.6.2Adsorption isotherm for F8 macroporous adsorptive resins433.7 Studied on dynamic adsorb and desorb443.7.1The effect of sample injection flow rate on adsorption rate443.7.2The effect

24、of sample injection concentration on adsorption rate443.7.3The selection of eluant453.7.4The effect of eluant concentration on desorption rate and Fneness of product453.7.5The effect of elution flow rate on desorption rate and Fneness of product463.7.6The effect of eluant dosage on desorption rate47

25、3.8The analysis of exemplar by HPLC483.9The test of static adsorb and desorb of polyamide483.9.1Screening of mesh of polyamide483.9.2The static adsorption kinetics curve of polyamide483.10The test of dynamic adsorb493.10.1The effect of sample injection flow rate on adsorption rate493.10.2The effect

26、of sample injection concentration on adsorption rate503.10.3The effect of eluant concentration on desorption rate and Fneness of product503.10.4The effect of elution flow rate on desorption rate and Fneness of product513.10.5The effect of eluant dosage on desorption rate513.11The analysis of exempla

27、r by HPLC523.12Comparison between F8 macroporous adsorptive resins and polyamide534 Discussion554.1Determination of condition by ultrasonic wave hydrolysis method 554.2Determination of condition by enzymolysis554.3Determination of condition by polymeric adsorbent554.4Operating principle of ultrasoni

28、c wave hydrolysis method 564.5Rudiment of enzymolysis method 564.6Rudiment of F8 macroporous adsorptive resins for segregating flavanoid564.7Rudiment of polyamide for segregating flavanoid575 Conclusion59Acknowledgements61Reference63Published Paper during the Studying Course69独创声明本人声明所呈交的学位论文是本人在导师指

29、导下进行的研究工作及取得的研究成果。据我所知，除了文中特别加以标注和致谢的地方外，论文中不包含其他人已经发表或撰写过的研究成果，也不包含未获得(注：如没有其他需要特别声明的，本栏可空)或其他教育机构的学位或证书使用过的材料。与我一同工作的同志对本研究所做的任何贡献均已在论文中作了明确的说明并表示谢意。 学位论文作者签名： 日期： 年 月 日 学位论文版权使用授权书本学位论文作者完全了解学校有关保留、使用学位论文的规定，学校有权保留并向国家有关部门或机构送交论文的复印件和磁盘，允许论文被查阅和借阅。本人授权学校可以将学位论文的全部或部分内容编入有关数据库进行检索，可以采用影印、缩印或扫描等复制手

30、段保存、汇编学位论文。(保密的学位论文在解密后适用本授权书)学位论文作者签名： 日期： 年 月 日导师签名： 日期： 年 月 日摘 要本文以沙棘叶中黄酮类化合物为研究对象，利用超声波水解法和酶解法提取沙棘叶中黄酮类化合物，并对超声波水解法与酶解法提取的黄酮类化合物得率进行比较，确定沙棘叶中黄酮类化合物提取的最佳工艺条件。同时，利用F8大孔吸附树脂、聚酰胺分离纯化出黄酮类化合物单体。在超声波水解法提取沙棘叶中黄酮类化合物的最佳工艺条件建立过程中，选择了乙醇浓度、盐酸浓度、水解时间、水解温度和料液比等因素进行单因素试验，并在单因素的基础上，采用响应面的分析方法，确定了沙棘叶中黄酮类化合物提取的最佳

31、工艺条件：乙醇浓度70.86%，盐酸浓度3.15 mol/L，水解时间29.66 min，温度60,料液比1:20，该条件下，沙棘叶中黄酮类化合物得率为1.10%；酶解法提取黄酮类化合物的最佳工艺条件为：酶浓度80.69 U/mL，酶催化的pH值4.02，酶解温度50.06，酶解时间8 h，料液比1:25，此条件下，沙棘叶中黄酮类化合物的得率为0.82%。因此，超声波水解法提取沙棘叶中黄酮类化合物是酶解法提取工艺得率的1.34倍，不仅得率高于酶解法，且提取时间短、耗能低，可降低生产成本，提高经济效益。本文还建立了F8大孔吸附树脂和聚酰胺分离纯化沙棘叶中黄酮类化合物，通过对上样浓度、上样流速、洗

32、脱剂浓度、洗脱剂流速和洗脱剂用量等工艺参数的分析，得到F8大孔吸附树脂的分离纯化效果好于聚酰胺，F8大孔吸附树脂对沙棘叶中黄酮类化合物的最佳分离纯化条件：上样浓度0.3 mg/mL，上样流速0.5 mL/min，洗脱剂浓度100%，洗脱剂流速0.6 mL/min，洗脱剂用量2个柱体积，此条件下，F8大孔吸附树脂对沙棘叶中黄酮类化合物的解吸率为87.02%；聚酰胺的最优纯化条件：上样流速0.4 mL/min，上样浓度0.4 mg/mL，洗脱剂浓度60%，洗脱剂流速0. 6 mL/min，洗脱剂用量1.5个柱体积，此时解吸率为82.62%。因此，F8大孔吸附树脂法分离纯化黄酮类化合物较好于聚酰胺法

33、，解吸率是聚酰胺法的1.04倍，为应用于工业化生产提供了有益的参考。关键词：沙棘叶；黄酮类化合物；高效液相色谱；F8大孔吸附树脂；聚酰胺Study on the Process of High-Effective and Separation of Flavonoids from Sea-buckthorn leavesAbstractFlavones in Sea-buckthorn leaves was studied in this paper. Total flavones was extracted with ultrasonic wave hydrolysis method and

34、 enzymic hydrolysis method, It compared the yeild of flavones which were extracted with ultrasonic wave hydrolysis method and enzymic hydrolysis method, and demonstrated the best condition of extracting flavones in Sea-buckthorn leaves. The monomer compounds were gained by the F8 macroporous adsorpt

35、ive resins and polyamide. The experiment chose the ultrasonic wave hydrolysis to withdraw ethyl alcohol density,the acid hydroc alcohol density,withdraws factor and so on hydrolysis time hydrolysis temperature and material ratio, on basis of single-factor test, response surface methodology was appli

36、ed. Content of flavonoid in sea-buckthorn leaves was determined by liquid chromatography. The optimal extraction conditions were obtained: concentration of alcohol 70.86%, concentrtion of hydrochloric acid 3.15 mol/L, hydrolytic time 29.66 min, hydrolytic temperature 60, material ratio 1:20, the sea

37、-buckthorn withdraws the total flavane under this condition rate is 1.10%. It is super-1.34 double to the enzymic hydrolysis method. The optimal extraction conditions of enzymic hydrolysis method were obtained: enzyme concentration 80.69%, enzymatic pH 4.02, enzymolysis temperature 50.06, enzymolysi

38、s time 8h, material ratio 1:25, the sea-buckthorn withdraws the total flavane under this condition rate is 0.82%. Therefore, the extraction of flavonoids with ultrasonic wave hydrolysis method not only yield exceeded enzymic hydrolysis method, but the time of extraction was short and the waste energ

39、y was low, thus, this may cut down the production price, and elevate the economic returns.The text also established the method to segregate and cleansing flavonoids by F8 macroporous adsorptive resins and polyamide. Through analyzed the technology parameter such as sample injection concentration、sam

40、ple injection flow rate、elution concentration、elution flow rate 、eluant dosage and so on, gained the result that was the effect of segregation and cleansing flavonoids from sea-buckthorn F8 macroporous adsorptive resins surpass polyamide, the optimal condition of segregation and cleansing by F8 macr

41、oporous adsorptive resins:sample injection concentration 0.3 mg/mL, sample injection flow rate 0.5 mL/min, elution concentration 100%, elution flow rate 0.6 mL/min, eluant dosage double column volume.The adsorption rate of flavonoids from sea-buckthorn leaves by F8 macroporous adsorptive resins was

42、87.02%, it is super-1.04 double to the polyamide method, the optimal condition of polyamide were: sample injection concentration 0.4 mg/mL, sample injection flow rate 0.4 mL/min, elution concentration 60%, elution flow rate 0.6 mL/min, eluant dosage 1.5 double column volume.Key Words: sea-buckthorn

43、leaves; flavonoids; high performance liquid chromatogram; F8 macroporous adsorptive resins; polyamideMaster Candidate: Shen ShuangshuangMajor: Agricultural Products Processing and Storing EngineeringSupervisor: Prof. Han Jianchun1 引言沙棘是一种含有多种维生素、多种微量元素、多种氨基酸和其它生物活性物质的药用食物。也是地球上生存超过两亿年的植物；沙漠和高寒山区的恶劣环

44、境中能够生存的植物； “地球癌症” 砒砂岩地区唯一能生长的植物；西部大开发生态环保价值最高的植物；完全在无污染环境中生长的绿色植物；世界植物群体中公认的 Vc之王；一个被中国中医药典和世界药典广泛入药的植物；被国家卫生部确认为药食同源的植物。 1977年我国卫生部首次将沙棘正式列入中国药典(张鞍灵等，2001)。之后，国家医药局和卫生部联合公布沙棘为药食同源植物。 沙棘的根、叶、花、果、籽均可入药，特别是果实含有人体不能合成的、但又为人身心健康不可少的多种维生素，享有“世界植物之奇”、维生素宝库之称。1.1 沙棘概况1.1.1 沙棘的生物特征沙棘(Sea-Buckthorn)，又名酸柳、酸柳果

45、、其察日嘎茶(蒙语)、吉汉(维吾尔名)、沙枣等(刘锡建，2004；侯冬岩等，2002)，是一种耐寒的、有刺的落叶灌木。沙棘高24 m，最高可达10m。沙棘刺较多，粗壮，顶生或侧生；嫩枝褐绿色，密被银白色而带褐色鳞片，或有时具白色星状毛，老支灰黑色，粗糙；牙大，金黄色或锈色。单叶通常近对生；叶柄极短；叶片纸质，狭披针形或长圆状披针形，长38 cm，宽约1 cm(刘高波，2007)，两端钝形或基部近圆形，上面绿色，初被白色盾形毛或星状毛，下面银白色或淡白色，被鳞片。果实圆球形，直径46 mm，橙黄色或橘红色；果梗长12.5 mm。种子小，黑色或紫黑色，有光泽。花期45月，果期910月(Vahid

46、Bilaloglu Guliyev et al，2004)。1.1.2 沙棘的分布沙棘的地理分布很广，在东经2123北纬2769之间(刘洪章等，1995)，跨欧亚两洲温带地区，分为六个种和十二个亚种。我国是沙棘属植物分布区面积最大，种类最多的国家。 在中国，南起云南西北部和喜马拉雅南坡的亚东，北至新疆阿尔泰山地，东抵内蒙古木盟库伦以东地区，西到帕米尔高原地广大地区。包括有山西、陕西、内蒙古、河北、甘肃、宁夏、辽宁、青海、四川、云南、贵州、新疆、西藏等19个省和自治区都有分布，总面积高达1800万亩。中国沙棘：分布于华北、西北及四川等地；云南沙棘：分布于四川、云南、西藏等地(徐铭渔等，1994)；中国天然生长的沙棘亚种，面积最大，占我国沙棘资源面积的80%以上，主要分布在黄河中游地区。目前，我国在水土流失地区大面积种植的即是这种沙棘；中亚沙棘：主要分布在新疆的天山以南；西藏沙棘：主要分布在青藏高原；蒙古沙棘，主要分布在新疆的天山以北。俄罗斯主要以这种沙棘为育种材料，培育出了大果沙棘两种；柳叶沙棘：主要分布在西藏东南部。开发研究较少；云南沙棘：主要分布在云贵