摘 要
本研究通過單因素與正交試驗相結合的方法，旨在篩選出杜仲葉綠原酸的最適浸出溶劑和最適提取工藝並採用高效液相色譜法建立杜仲葉藥材指紋圖譜。
試驗得到如下結果（1）標準曲線繪製結果：紫外分光光度法：y=22.6442x-0.2039,r=0.9999（n=6）。其中 y 為綠原酸濃度（μg/mL），x 為吸光度值，說明綠原酸濃度在5~40μg/mL 區間內線性關係良好；高效液相色譜法：y=0.001x+0.0022 ,r=0.999（n=6）其中 y 為綠原酸質量（μg），x 為綠原酸峰面積，試驗證明綠原酸質量在 0.075~0.6μg 區間內呈良好線性關係。（2）浸出溶劑篩選結果：試驗結果表明甲醇、乙醇、丙酮最佳體積分數分別為 20%、30%、40%,對應綠原酸得率為 0.8471%,0.9056%,0.9430%,水做浸出溶劑，綠原酸得率為 0.7032%.通過指紋圖譜分析可知 20%甲醇、30%乙醇、40%丙酮溶劑的浸提物色譜圖峰形一致，且綠原酸峰面積占總峰面積百分比依次為 81.583%,81.168%,80.424%,百分比差異很小。（3）30%乙醇溶液做提取溶劑時，各工藝綠原酸最佳提取條件分別為：①超聲提取法：溶劑 pH 值為 4,液料比為 14,超聲頻率 40KHz,超聲時間 80min 此時綠原酸得率最高為 0.9032%.正交試驗結果表明，各因素對綠原酸得率影響，遞減順序為：超聲功率>超聲時間>料液比，即超聲功率對綠原酸得率影響最大。②微波提取法：溶劑 pH 值為 4,液料比為 18,微波火力中火，微波輻射時間 60s,此時綠原酸得率最高為 0.9313%.各因素對綠原酸得率影響從大到小順序為：微波火力>輻射時間>液料比，即微波火力對綠原酸得率影響最大。③酶解提取法：液料比為 14,酶解反應時間為 1.5h,溶劑 pH 值為 4.5,酶用量 0.35%,酶解溫度 40℃，此時綠原酸得率最高為 0.9508%.試驗各因素對綠原酸得率影響排序為：酶解 pH>酶解溫度>酶用量，即酶解 pH 值對綠原酸得率影響最大。④熱浸提取法：提取液 pH 值為 4,熱浸溫度 70℃，熱浸時間 0.5h,液料體積質量比 18,此時綠原酸得率最高為 0.9654%.各因素對綠原酸得率影響遞減順序為：液料比>熱浸溫度>熱浸時間，即液料比對綠原酸得率影響最大。（4）綠原酸提取數據及色譜圖分析可知，綠原酸最佳提取方法為熱浸提取法。（5）用篩選出的綠原酸最適提取工藝所製備的 10 批杜仲葉提取液，指紋圖譜相似度均達 0.9 以上。
本研究最終得出，杜仲葉綠原酸最佳提取溶劑為體積分數 30%的乙醇溶液。熱浸提取為杜仲葉綠原酸提取的最適工藝。建立了最佳提取工藝條件下，重慶杜仲葉藥材指紋圖譜。
關 鍵 詞：杜仲葉；綠原酸；提取；指紋圖譜。
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 錄
第一章 文獻綜述
1.1 杜仲簡介
1.1.1 杜仲歷史、地理分布、形態及性狀鑑別
1.1.2 杜仲化學成分研究
1.1.3 杜仲產品及功效
1.1.4 杜仲葉指紋圖譜研究
1.2 綠原酸簡介
1.2.1 綠原酸植物分布、合成及代謝
1.2.2 綠原酸的結構性質、藥理作用及應用領域
1.2.3 綠原酸的提取、分離純化及定量方法研究
1.3 試驗的目的、意義
1.3.1 試驗的目的
1.3.2 試驗的意義
第二章 試驗研究
2.1 綠原酸鑑定及檢測方法建立
2.1.1 試驗材料
2.1.2 試驗方法
2.1.3 結果與分析
2.1.4 討論
2.1.5 小結
2.2 杜仲葉綠原酸浸出溶劑篩選
2.2.1 試驗材料
2.2.2 試驗方法
2.2.3 結果與分析
2.2.4 討論
2.2.5 小結
2.3 綠原酸提取工藝研究
2.3.1 試驗材料
2.3.2 試驗方法
2.3.3 結果與分析
2.3.4 討論
2.3.5 小結
2.4 重慶杜仲葉指紋圖譜建立
2.4.1 試驗材料
2.4.2 試驗方法
2.4.3 結果與分析
2.3.4 討論
2.3.5 小結
第三章 結論
附錄
參考文獻
致謝
本研究通過單因素與正交試驗相結合的方法，旨在篩選出杜仲葉綠原酸的最適浸出溶劑和最適提取工藝並採用高效液相色譜法建立杜仲葉藥材指紋圖譜。
試驗得到如下結果（1）標準曲線繪製結果：紫外分光光度法：y=22.6442x-0.2039,r=0.9999（n=6）。其中 y 為綠原酸濃度（μg/mL），x 為吸光度值，說明綠原酸濃度在5~40μg/mL 區間內線性關係良好；高效液相色譜法：y=0.001x+0.0022 ,r=0.999（n=6）其中 y 為綠原酸質量（μg），x 為綠原酸峰面積，試驗證明綠原酸質量在 0.075~0.6μg 區間內呈良好線性關係。（2）浸出溶劑篩選結果：試驗結果表明甲醇、乙醇、丙酮最佳體積分數分別為 20%、30%、40%,對應綠原酸得率為 0.8471%,0.9056%,0.9430%,水做浸出溶劑，綠原酸得率為 0.7032%.通過指紋圖譜分析可知 20%甲醇、30%乙醇、40%丙酮溶劑的浸提物色譜圖峰形一致，且綠原酸峰面積占總峰面積百分比依次為 81.583%,81.168%,80.424%,百分比差異很小。（3）30%乙醇溶液做提取溶劑時，各工藝綠原酸最佳提取條件分別為：①超聲提取法：溶劑 pH 值為 4,液料比為 14,超聲頻率 40KHz,超聲時間 80min 此時綠原酸得率最高為 0.9032%.正交試驗結果表明，各因素對綠原酸得率影響，遞減順序為：超聲功率>超聲時間>料液比，即超聲功率對綠原酸得率影響最大。②微波提取法：溶劑 pH 值為 4,液料比為 18,微波火力中火，微波輻射時間 60s,此時綠原酸得率最高為 0.9313%.各因素對綠原酸得率影響從大到小順序為：微波火力>輻射時間>液料比，即微波火力對綠原酸得率影響最大。③酶解提取法：液料比為 14,酶解反應時間為 1.5h,溶劑 pH 值為 4.5,酶用量 0.35%,酶解溫度 40℃，此時綠原酸得率最高為 0.9508%.試驗各因素對綠原酸得率影響排序為：酶解 pH>酶解溫度>酶用量，即酶解 pH 值對綠原酸得率影響最大。④熱浸提取法：提取液 pH 值為 4,熱浸溫度 70℃，熱浸時間 0.5h,液料體積質量比 18,此時綠原酸得率最高為 0.9654%.各因素對綠原酸得率影響遞減順序為：液料比>熱浸溫度>熱浸時間，即液料比對綠原酸得率影響最大。（4）綠原酸提取數據及色譜圖分析可知，綠原酸最佳提取方法為熱浸提取法。（5）用篩選出的綠原酸最適提取工藝所製備的 10 批杜仲葉提取液，指紋圖譜相似度均達 0.9 以上。
本研究最終得出，杜仲葉綠原酸最佳提取溶劑為體積分數 30%的乙醇溶液。熱浸提取為杜仲葉綠原酸提取的最適工藝。建立了最佳提取工藝條件下，重慶杜仲葉藥材指紋圖譜。
關 鍵 詞：杜仲葉；綠原酸；提取；指紋圖譜。
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 錄
第一章 文獻綜述
1.1 杜仲簡介
1.1.1 杜仲歷史、地理分布、形態及性狀鑑別
1.1.2 杜仲化學成分研究
1.1.3 杜仲產品及功效
1.1.4 杜仲葉指紋圖譜研究
1.2 綠原酸簡介
1.2.1 綠原酸植物分布、合成及代謝
1.2.2 綠原酸的結構性質、藥理作用及應用領域
1.2.3 綠原酸的提取、分離純化及定量方法研究
1.3 試驗的目的、意義
1.3.1 試驗的目的
1.3.2 試驗的意義
第二章 試驗研究
2.1 綠原酸鑑定及檢測方法建立
2.1.1 試驗材料
2.1.2 試驗方法
2.1.3 結果與分析
2.1.4 討論
2.1.5 小結
2.2 杜仲葉綠原酸浸出溶劑篩選
2.2.1 試驗材料
2.2.2 試驗方法
2.2.3 結果與分析
2.2.4 討論
2.2.5 小結
2.3 綠原酸提取工藝研究
2.3.1 試驗材料
2.3.2 試驗方法
2.3.3 結果與分析
2.3.4 討論
2.3.5 小結
2.4 重慶杜仲葉指紋圖譜建立
2.4.1 試驗材料
2.4.2 試驗方法
2.4.3 結果與分析
2.3.4 討論
2.3.5 小結
第三章 結論
附錄
參考文獻
致謝
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