摘 要
本研究通过单因素与正交试验相结合的方法，旨在筛选出杜仲叶绿原酸的最适浸出溶剂和最适提取工艺并采用高效液相色谱法建立杜仲叶药材指纹图谱。
试验得到如下结果（1）标准曲线绘制结果：紫外分光光度法：y=22.6442x-0.2039,r=0.9999（n=6）。其中 y 为绿原酸浓度（μg/mL），x 为吸光度值，说明绿原酸浓度在5~40μg/mL 区间内线性关系良好；高效液相色谱法：y=0.001x+0.0022 ,r=0.999（n=6）其中 y 为绿原酸质量（μg），x 为绿原酸峰面积，试验证明绿原酸质量在 0.075~0.6μg 区间内呈良好线性关系。（2）浸出溶剂筛选结果：试验结果表明甲醇、乙醇、丙酮最佳体积分数分别为 20%、30%、40%,对应绿原酸得率为 0.8471%,0.9056%,0.9430%,水做浸出溶剂，绿原酸得率为 0.7032%.通过指纹图谱分析可知 20%甲醇、30%乙醇、40%丙酮溶剂的浸提物色谱图峰形一致，且绿原酸峰面积占总峰面积百分比依次为 81.583%,81.168%,80.424%,百分比差异很小。（3）30%乙醇溶液做提取溶剂时，各工艺绿原酸最佳提取条件分别为：①超声提取法：溶剂 pH 值为 4,液料比为 14,超声频率 40KHz,超声时间 80min 此时绿原酸得率最高为 0.9032%.正交试验结果表明，各因素对绿原酸得率影响，递减顺序为：超声功率>超声时间>料液比，即超声功率对绿原酸得率影响最大。②微波提取法：溶剂 pH 值为 4,液料比为 18,微波火力中火，微波辐射时间 60s,此时绿原酸得率最高为 0.9313%.各因素对绿原酸得率影响从大到小顺序为：微波火力>辐射时间>液料比，即微波火力对绿原酸得率影响最大。③酶解提取法：液料比为 14,酶解反应时间为 1.5h,溶剂 pH 值为 4.5,酶用量 0.35%,酶解温度 40℃，此时绿原酸得率最高为 0.9508%.试验各因素对绿原酸得率影响排序为：酶解 pH>酶解温度>酶用量，即酶解 pH 值对绿原酸得率影响最大。④热浸提取法：提取液 pH 值为 4,热浸温度 70℃，热浸时间 0.5h,液料体积质量比 18,此时绿原酸得率最高为 0.9654%.各因素对绿原酸得率影响递减顺序为：液料比>热浸温度>热浸时间，即液料比对绿原酸得率影响最大。（4）绿原酸提取数据及色谱图分析可知，绿原酸最佳提取方法为热浸提取法。（5）用筛选出的绿原酸最适提取工艺所制备的 10 批杜仲叶提取液，指纹图谱相似度均达 0.9 以上。
本研究最终得出，杜仲叶绿原酸最佳提取溶剂为体积分数 30%的乙醇溶液。热浸提取为杜仲叶绿原酸提取的最适工艺。建立了最佳提取工艺条件下，重庆杜仲叶药材指纹图谱。
关 键 词：杜仲叶；绿原酸；提取；指纹图谱。
Abstract:In this study, through the combination of single factor and orthogonal experiments to selectthe best extraction solvent, the best method of extracting chlorogenic acid and to establish thefingerprint of Eucommia ulmoides Oliv leaves by HPLC.
Test results were as follows. （1）The standard curve of Chlorogenic acid obtained byultraviolet spectrophotometric method was obtained as follows: y=22.6442x-0.2039 r = 0.9999（n= 6）。y was chlorogenic acid concentrations（μg/mL），x was the absorbance value.The chlorogenicacid concentration within 5~40μg/mL had good linear relationship; Standard curve of HPLC: y=0.001x+0.0022, r = 0.999（n=6）y was chlorogenic acid mass （μg）， x was the peak area ofchlorogenic acid.It showed that chlorogenic acid quality within the range of 0.075~0.6μg hadgood linear relationship.（2）When the methanol, ethanol, acetone as chlorogenic acid extractionsolvent, their best volume fraction were 20%,30% and 40%, and the corresponding yield ofchlorogenic acid were 0.8471%, 0.9056%, 0.9430%.Water as extraction solvent,chlorogenic acidyield was 0.7032%. Through comparing with the fingerprint peak shape of 20% methanol, 30%ethanol and 20%acetone extraction solvent could be concluded that their peak shapes wereconsistent, and chlorogenic acid peak were respectively took 81.583,81.168,80.424 percents, thedifferences among these percentages were very small. （3）When 30% ethanol （volume fraction）as chlorogenic acid extraction solvent, the best conditions of eatch extraction method just asfollows: ①Ultrasonic method,the solvent pH was 4,the liquid to solid ratio was14,the ultrasonicfrequency was40KHz,the ultrasonic time was80min,the extraction rate of chlorogenic acidreached as high as 0.9032%.Orthogonal experiment results showed that various factors on theimpact of chlorogenic acid yield the sequence was: ultrasonic power>ultrasonic time>solid toliquid ratio, namely the ultrasonic power influenced on the yield of chlorogenic acid was thelargest;②The conditions of microwave method,the solvent pH was 4,the liquid to solid ratio was18, the microwave fire was medium heat, the microwave time was 60s,the extraction rate ofchlorogenic acid reached as high as 0.9313%.Orthogonal experiment results showed that variousfactors influenced chlorogenic acid yield, the descending order as follows: microwavefire>irradiation time>liquid ratio, namely the microwave fire influenced on the yield ofchlorogenic acid was the largest;③The conditions of enzymatic method,the liquid to solid ratiowas 14, reaction time was 1.5 h, the solvent pH was 4.5, enzyme dosage was0.35%,enzymolysistemperature was 40℃，the chlorogenic acid yield as high as 0.9508%.Orthogonal experimentresults showed that various factors influenced on the yield of chlorogenic acid from big to smallorder: pH>reaction temperature>enzyme dosage, pH had the largest influence on the yield of chlorogenic acid;④The conditions of heat immersion method,the solvent pH was 4, heatimmersion temperature was 70 ℃， reaction time was 0.5h,the liquid to solid ratio was 18, thechlorogenic acid yield as high as 0.9654%.Orthogonal experiment results showed that variousfactors influenced on the yield of chlorogenic acid from big to small order: liquid to solidratio>heat immersion temperature>reaction time, liquid to solid ratio had the largest influence onthe yield of chlorogenic acid.（4）Thought analyzed the chromatogram could be concluded that thebest extraction method of chlorogenic acid was heat immersion method.（5）With the bestextraction process to make the HPLC fingerprint of 10 batches of eucommia ulmoides olivleaves, fingerprint similarity were above 0.9.
The study ultimately concluded that the Volume fraction of 30%ethanol solution was thebest solvent for extracting the chlorogenic acid in eucommia ulmoides oliv leaves. Heatingimmersion method was the optimal process for extracting chlorogenic acid in eucommiaulmoides oliv leaves.Tests established HPLC fingerprint of eucommia ulmoides oliv leavesunder the best extraction techonolygy.
Key words: Eucommia ulmoides oliv leaves; Chlorogenic acid; Extraction; Fingerprint.
目 录
第一章 文献综述
1.1 杜仲简介
1.1.1 杜仲历史、地理分布、形态及性状鉴别
1.1.2 杜仲化学成分研究
1.1.3 杜仲产品及功效
1.1.4 杜仲叶指纹图谱研究
1.2 绿原酸简介
1.2.1 绿原酸植物分布、合成及代谢
1.2.2 绿原酸的结构性质、药理作用及应用领域
1.2.3 绿原酸的提取、分离纯化及定量方法研究
1.3 试验的目的、意义
1.3.1 试验的目的
1.3.2 试验的意义
第二章 试验研究
2.1 绿原酸鉴定及检测方法建立
2.1.1 试验材料
2.1.2 试验方法
2.1.3 结果与分析
2.1.4 讨论
2.1.5 小结
2.2 杜仲叶绿原酸浸出溶剂筛选
2.2.1 试验材料
2.2.2 试验方法
2.2.3 结果与分析
2.2.4 讨论
2.2.5 小结
2.3 绿原酸提取工艺研究
2.3.1 试验材料
2.3.2 试验方法
2.3.3 结果与分析
2.3.4 讨论
2.3.5 小结
2.4 重庆杜仲叶指纹图谱建立
2.4.1 试验材料
2.4.2 试验方法
2.4.3 结果与分析
2.3.4 讨论
2.3.5 小结
第三章 结论
附录
参考文献
致谢
