Development of simultaneous methods for diosgenin and phytosterols analysis in yam

山藥為常見之保健食品或藥用材料，其含有同屬固醇類之薯蕷皂配基(diosgenin)與植物固醇(phytosterols)兩類重要活性物質。一般分析此兩類固醇類化合物之樣品製備方法包括了酸鹼水解和萃取等步驟，再分別以氣相層析法或液相層析法定量。本論文以氣相層析與高效能液態相層兩種分析方法同步分析薯蕷皂配基與植物固醇兩類化合物。氣相層析使用DB-5(5%phenyldimethylpolysiloxane)的毛細管柱，可將薯蕷皂配基之波峰調整在植物固醇之間。高效能液相層析使用C18之管柱，以氰甲烷與甲醇混合溶液為流洗液，以梯度的方式先將極性較高之薯蕷皂配基於氰甲烷比甲醇為95：5(v/v)時流洗出來，再於65：35(v/v)時流洗出植物固醇，主要之植物固醇為β-sitosterol、stigmasterol和campesterol。本研究並比較使用紫外光波長205 nm（UV205）與蒸發式光散射（ELSD）兩個液相層析偵測器應用於分析固醇類化合物之差異：以UV205具有較佳的偵測靈敏度，其偵測極限（0.03~0.09μg）為ELSD的十分之一；但ELSD具有可容忍雜質干擾及不受溶劑梯度影響之優點。另外，本研究亦探討於酸鹼水解之前之溶劑預萃取步驟之必要性，結果顯示，包含粗纖維之山藥水解物具有吸附此兩類化合物之作用，因此溶劑預萃取步驟是正確分析山藥薯蕷皂配基與植物固醇含量所必須的前處理。溶劑預萃取方法使用索氏萃取器分別以正己烷-甲醇系列萃取與正己烷-異丙醇(1:2, v/v)混合溶液直接萃取，可獲得與常使用之氯仿-甲醇(2:1, v/v)混合溶液萃取方式有相似的結果。利用所建立之方法分析台灣常見的三種山藥：屬長薯之基隆山藥（D. pseudojaponica），屬塊薯之台農二號（D. alata L.）與屬紅色肉之名間長紅（D. alata purpurea）；發現三種樣品均含有植物固醇，含量範圍在9.9~30.1 mg/100g，只有在基隆山藥含有薯蕷皂配基，含量為15.2 mg/100g，且其葉、莖蔓及塊莖削皮均含有植物固醇與薯蕷皂配基，為具有開發潛力之廢棄物。Dioscorea (or Chinese yam) is a common raw material for drug and health food manufacture. Similar but separated analytical assays were generally conducted to determine contents of two groups of biological active compounds, phytosterols and diosgenin. One gas-liquid and one high performance of liquid chromatographic methods were established to simultaneously determine these two groups of compounds both belonging to steroids. The methods included acidic and alkali hydrolysis, solvent extraction and chromatographic analysis. A DB-5 (poly(phenylmethyl) siloxane, 5% phenyl) capillary column was selected for gas-liquid chromatography. The retention time of diosgenin peak was among those of phytosterols. A C18 column was selected for liquid chromatography and gradient of acetonitrile-methanol mixed solvent was used as eluant. The diosgenin with high polarity was eluted out at higher acetonitrile ratio 95:5 (v/v) and the phytosterols were eluted out at lower ratio 65:35 (v/v). The major phytosterols found in yam were beta-sitosterol, stigmasterol and campesterol. Two detection systems, ultraviolet absorption at 205nm (UV205) and evaporative light scattering detection (ELSD), were used and compared for detection sensitivity and stability. The UV205 had higher sensitivity with detection limits in the range of 0.03~0.09 ug which was ten-times higher than ELSD, but ELSD tolerant higher level of noise and did not have baseline interference caused by solvent gradient. Lipid extraction prior acid hydrolysis was found to be necessary to avoid absorption effects of fibers and acid resistant residues. Trails for extracting efficiency were conducted on Soxhlet extracting apparatus and on soak-and-shake extraction. The results indicated that sequential extraction using hexane and methanol as solvent on Soxhlet apparatus yielded the highest determining values. Hexan-isopropanol (1:2, v/v) mixture also served as an efficient solvent as well as mixture of chloroform-methanol (2:1, v/v). Phytosterol contents were determination on three representative Taiwanese yam cultivars, D. pseudojaponica, D. alata L., Tainun 2, and D. alata L. var. purpurea). Phytosterols were found in all samples and the total phytosterol contents were in the range of 9.9~30.1mg/100g. Diosgenin was only found in D. pseudojaponica samples included tubers, leaves, vines and the content of edible tuber portion was 15.2 mg/100g.中文摘要………………………………………………………………Ⅰ 英文摘要………………………………………………………………Ⅲ 目錄……………………………………………………………………Ⅳ 表次……………………………………………………………………Ⅵ 圖次……………………………………………………………………Ⅶ 壹、前言………………………………………………………………1 貳、文獻回顧…………………………………………………………2 一、山藥………………………………………………………………2 (一)簡介……………………………………………………………2 (二)化學組成與營養成份…………………………………………3 (三)生理活性物質…………………………………………………5 二、植物固醇…………………………………………………………7 (一)簡介………………………………………………………… 7 1.來源與天然分佈…………………………………………7 2.種類與存在形式…………………………………………9 3.吸收與代謝………………………………………………11 (二)生理功能……………………………………………………12 1.對膽固醇的影響…………………………………………12 2.抗腫瘤活性………………………………………………12 3.抗氧化活性………………………………………………13 4.其他生理功效……………………………………………13 (三)安全性………………………………………………………14 (四)植物固醇的定量分析………………………………………15 1.植物固醇總含量之分析…………………………………15 2.植物固醇的萃取與分離…………………………………18 3.完整植物固醇的分析……………………………………19 4.不同形態植物固醇單一分子化合物之分析……………23 三、皂素與皂素配基…………………………………………………25 (一)簡介…………………………………………………………25 (二)生理活性……………………………………………………28 (三)安全性………………………………………………………30 參、材料與方法………………………………………………………31 一、實驗材料…………………………………………………………31 二、實驗方法…………………………………………………………33 (一)直接水解、皂化測定總量之方法…………………………33 (二)薯蕷皂配基與植物固醇之萃取……………………………34 1.索氏萃取…………………………………………………34 2.單一混合溶劑之萃取……………………………………34 (三)分析樣品的製備……………………………………………35 1.氣相層析分析之樣品……………………………………35 2.液相層析分析之樣品……………………………………35 (四)氣相層析法分析薯蕷皂配基與植物固醇…………………36 1.氣相層析之分析條件……………………………………36 2.植物固醇與薯蕷皂配基的定量…………………………37 3.蕷皂配基與植物固醇回收率之確校…………………38 (五)液相層析法分析薯蕷皂配基與植物固醇…………………38 1.高效能液相層析分析條件………………………………38 2.薯蕷皂配基與植物固醇之定量(檢量線與偵測極限)…38 (六)統計分析……………………………………………………39 肆、結果與討論………………………………………………………44 一、氣相層析法與液相層析法分析薯蕷皂配基與植物固醇方法之建立 (一)氣相層析法…………………………………………………44 (二)液相層析法…………………………………………………46 二、評估溶劑預萃取步驟之必要性…………………………………52 三、最適萃取溶劑之探討……………………………………………55 四、台灣山藥薯蕷皂配基與植物固醇含量之分析…………………58 (一)不同品種山藥之含量………………………………………58 (二)基隆山藥各部分之含量……………………………………60 五、氣相層析法與液相層析法之比較………………………………65 伍、結論………………………………………………………………69 陸、參考文獻…………………………………………………………7