以液相層析串聯高解析質譜法建立靈芝三萜類輪廓分析

Abstract

靈芝三萜類(Ganoderma triterpenoids)為靈芝中重要生理活性成分，本研究目的為使用液相層析串聯高解析軌道式質譜儀(liquid chromatography-high resolution (Orbitrap) mass spectrometry, LC-MS/MS)，建立可鑑別其多元結構特徵化合物組成分析平台，成功定性出共53種靈芝三萜，其中結構分為含30個碳原子的主要型(M type)、二烯型(D type)、含27個碳原子的赤芝酸型(L type)和含24個碳原子的短鏈型(S type)。在電噴灑游離中，通過切換負電和正電模式運行，並經過高能碰撞解離下，觀察到靈芝三萜規律的特徵斷片模式，在不同品種、部位、形態、生長期的樣品中，建立了分析靈芝三萜輪廓的平台。定性策略藉由比對文獻的UV吸收、質量準確度、化合物滯留因子、同位素波峰離子質量與相對比例、特徵斷片離子質量與比例，最後比對標準品以辨識之，並利用內標校正進行定量分析了49種靈芝三萜。研究結果顯示，赤芝以L型三萜類含量最高，而松杉靈芝為M型，樣化程度M>L>D>S，故松杉靈芝較赤芝氧化程度可能更高；鹿角赤芝生長過程化合物持續氧化，氧化程度較低的為差異表達的代謝產物，在第3個月生長期到達顛峰，而第5個月含量會變低，表示鹿角赤芝成熟以後氧化程度將會上升；在正電游離的M3-4化合物在孢子樣品中最顯著的高，因為在正電模式下，氧化程度較低，故孢子萌發成為靈芝，氧化程度會上升；鹿角赤芝的頂端含有差異表達的代謝產物，故可能含有其他部位也找不到的稀有靈芝三萜；另一方面，紫芝菌柄中M1-30, M3-4與 D2-4含量最高且為差異表達的代謝產物，故紫芝菌柄能夠提供的靈芝三萜是不可或缺的部位。本研究建立分析平台可以協助了解不同三萜類在不同靈芝樣品中的含量與分布，也是靈芝產業監控與管理產品中三萜含量的利器，適用於品質管制用途。

The beneficial health effects of Ganoderma spp might be attributed to the bioactive compounds such as triterpenoids. This study is to develop a triterpenoids profile for quality assessment by ultra-high liquid chromatography-high resolution MS (UPLC-HRMS). There are 53 Ganoderma triterpenoids in the QC sample for qualitative. What’s more, there are 53 compounds for qualitative, and 50 compounds for quantitative. Amound them, the structure is divided into the main type, containing 30 carbon atoms (M type), the diene type (D type), containing the 27 carbon atoms lucidenic acid type (L type) and containing 24 carbon atoms short chain type (S type). Operated by switching between negative and positive mode, all the compounds can analyze at once. Furthermore, while higher-energy collisional dissociation occurs, it will generate fragment ions. This study developed Ganoderma triterpenoids profile in different species, parts, forms and growth stage for qualitative analysis along with UV, mass accuracy, isotope pattern, retention factor, and authentic compounds with internal standards. This study quantitative 49 triterpenoids. The result indicates that G. lucidum are composed of L type triterpenoids, on the contrary, G.tsugae are composed of M type triterpenoids. If the oxidized degree M>L>D>S, then G.tsugae may stand out for its oxidized degree. During the growth stage, the oxidized degree has improved. For example, positive ionization compounds are dominant compounds in spore samples. Furthermore, during the growth process of antler-shaped G. lucidum, the compounds continue to oxidize. The compounds reached the peak during the third month of growth, and the content would decrease in the fifth month, indicating that the degree of oxidation would be increase after it became mature. M3-4 compound which ionized in positive mode is the significantly highest in the spore samples. The top of antler-shaped G. lucidum contained differentially expressed metabolites that cannot be found in other parts. On the other hand, G. formasanum stipe has the highest content of M1-30, M3-4 and D2-4. They are differentially expressed metabolites, so the stipe is an indispensable part. The established analytical platform can be an effective tool for monitoring the distribution and content of triterpenoids in different samples and also a tool for quality control.