利用超高效能液相層析串聯高解析質譜法建立薯蕷與葫蘆巴中的固醇類皂苷分析平臺

薯蕷(Dioscorea spp.)與葫蘆巴(Trigonella foenum-graecum.)是中國傳統食藥材料，其生理活性差異來自於其中主要活性物質——固醇類皂苷(steroidal saponins)的多元結構，因此需要鑑別其組成的分析平臺。本研究使用超高效能液相層析串聯高解析質譜法(UHPLC-HRMS)建立鑑別47種固醇類皂苷之方法。高解析質譜分析平台藉由化合物層析性質、精確分子量、同位素分佈與分子斷裂模式可以鑑別出35種furostanol型皂苷與12種spirostanol型皂苷，其中包含10對同分異構物，有39種採用定量或半定量分析。食用薯蕷中皂苷含量遠低於藥用薯蕷與葫蘆巴，所有樣品中普遍存在的皂苷為protodioscin。葫蘆巴和薯蕷中只有43.6%的皂苷是相同的。葫蘆巴豆芽(HLB_Bu)具有29種固醇類皂苷，主要成分為furostanol型皂苷，其含量佔了總皂苷含量的95%以上。diosgenin、trillin、deltonin、protodeltonin僅存在於特定樣品中。zingiberenoside B為盾葉薯蕷中獨有的成分。三種萆薢中，只有粉萆薢中含有trigoneoside XIIIb與zingiberensis New Saponin。trigoneoside XVIIa、trigoneoside XVIIb、FU5、trigoneoside IIa與trigoneoside IIb是區分葫蘆巴豆與葫蘆巴豆芽的指標化合物。薯蕷中皂苷含量最高的是皮，其次是肉，最少的是葉子和梗。基隆山藥皮中的皂苷含量為梗中的9.39倍。由於雙葡萄醣皂苷(diosgenin diglucoside)、單醣皂苷和皂苷元等spirostanol型皂苷在天然植物中的含量低，因此需要使用三氟乙酸(trifluoroacetic acid, TFA)水解上述萃取物來增加。薯蕷中固醇皂苷不會受光照與熱加工的影響，但容易受到微生物群落(microbial community)與酵素的作用而產生結構的變化。本研究利用UHPLC-HRMS所建立的分析平台，可以有效篩選鑑別薯蕷與葫蘆巴中的固醇類皂苷，通過定量與半定量的方式靈敏地檢測出不同品種、部位以及加工方式樣品中的皂苷差異與含量，可協助薯蕷育種篩選與製程品質管控需求之應用。

關鍵字

薯蕷；葫蘆巴；固醇類皂苷；超高效能液相層析串聯高解析質譜法

並列摘要

Dioscorea (Dioscorea spp.) and Fenugreek (Trigonella foenum-graecum.) are traditional Chinese food and medicine materials. The difference in their physiological activities comes from the multiple structure of the main active substance—steroidal saponins, so it is very necessary to establish an analysis platform to identify their composition. In this study, ultra-high performance liquid chromatography tandem high-resolution mass spectrometry (UHPLC-HRMS) was used to establish a method to identify 47 sterol saponins. The high-resolution mass spectrometry platform can identify 35 furostanol-type saponins and 12 spirostanol-type saponins based on compound chromatographic properties, precise molecular weight, isotope distribution and molecular fragmentation patterns, including 10 pairs of isomers, 39 of which are used quantitative or semi-quantitative analysis. The contents of saponins in edible dioscorea is much lower than medicinal dioscorea and fenugreek. The most common saponin in samples is protodioscin. Only 43.6% of saponins in fenugreek and yam are the same. Fenugreek bud (HLB_Bu) have 29 sterol saponins, in which the main component is furostanol-type saponins, and its contents accounts for more than 95% of the total saponins. Diosgenin, trillin, deltonin, and protodeltonin are only present in certain samples. Zingiberenoside B is a unique ingredient in D.zingiberensis. Among the three types of Bixie, only the D. collettii var. hypoglauca contains trigoneoside XIIIb and zingiberensis New Saponin. Trigoneoside XVIIa, trigoneoside XVIIb, FU5, trigoneoside IIa, and trigoneoside IIb are index compounds that distinguish fenugreek bean from fenugreek bud. The highest contents of saponins in yam is the skin, followed by the flesh, and the least is the leaves and stems. The contents of saponins in the skin of D.pseudojaponia Yamam is 9.39 times higher than the stem. Due to the low contents of spirostanol-type saponins, such as diosgenin diglucoside, monosaccharide saponins and diosgenin in natural plants, it is necessary to hydrolyze the above-mentioned extracts with trifluoroacetic acid (TFA) to increase it. The steroidal saponins in yam are not affected by light and thermal processing, but changes because of the microbial community and enzymes. In this study, the analysis platform established by UHPLC-HRMS can effectively select and identify the steroidal saponins in yam and fenugreek. Platform can sensitively detect the differences in saponins in samples of different varieties, parts and processing methods through quantitative and semi-quantitative analysis, which can assist the application of yams selection and process quality control requirements.