采用超高效液相色谱-四极杆静电场轨道阱高分辨质谱(UPLC-Orbitrap-HRMS)联用技术对灵芝醇提物的化学成分进行系统分析，总结其代表性化学成分的裂解规律，并对灵芝潜在作用于法尼醇X受体(farnesoid X receptor，FXR)靶点的抗肝纤维化活性物质进行研究，阐明其药效物质基础。初步鉴定出灵芝醇提物95种化学成分，包括灵芝酸类24种、灵芝烯酸类9种、赤芝酸类13种、丹芝酸类3种、灵芝内酯类1种、其他三萜类成分40种、脂肪酸类成分4种、其他类1种，并分析了其裂解规律。如灵芝酸、灵芝烯酸类成分的结构特征以C30为骨架，含有游离的-COOH及-OH，易失去H2O、CO2分子形成碎片离子，D环多为五元环，易发生断裂;赤芝酸类成分属于C27骨架的羊毛甾烷型，与灵芝酸类成分相比其侧链结构变短，由8个碳减少为5个，同灵芝酸类含有常见的游离-COOH、-OH，容易失去H2O、CO2分子。通过选取已报道的6种FXR受体激动剂组成训练集，建立基于FXR配体的药效团模型，以鉴定出的95种灵芝化学成分与药效团进行匹配，通过测试集分子对模型进行验证，选出最优药效团模型02(敏感性=0。750 00，特异性=0。555 56，ROC=0。750)用于对灵芝化合物库的虚拟筛选，经匹配筛选得到31种潜在的可用于激活FXR的灵芝活性成分，随后ADMET预测结果表明灵芝酸H和赤芝酸J与血浆蛋白的结合率低于90％，且无肝毒性，可作为FXR激活剂开发单用或联用治疗肝纤维化的临床药物。
Chemical composition analysis of Ganoderma lucidum based on UPLC-Orbitrap-HRMS and virtual screening of FXR activators for treatment of liver fibrosis
The chemical composition of Ganoderma lucidum ethanol extracts was systematically analyzed and identified by ultra-high performance liquid chromatography-quadrupole electrostatic field orbitrap high-resolution mass spectrometry ( UPLC-Orbitrap-HRMS) .The fragmentation pattern of the representative chemical compounds was summarized,and the potential anti-liver fibrosis active com-pounds of G. lucidum acting on the farnesoid X receptor (FXR) target were studied to elucidate its pharmacodynamic substance basis.Preliminarily,95 chemical constituents of G. lucidum ethanol extracts were identified,including 24 ganoderic acids,9 ganoderenic acids,13 lucidenic acids,3 ganolucidic acids,1 ganoderma lactone,40 other triterpenoids,4 fatty acids,and 1 other constituent. In addition,the fragmentation patterns of the representative compounds were also analyzed. The structural characteristics of ganoderic acids and ganoderenic acids were the C30 skeleton,containing free-COOH and-OH groups,which could easily lose H2O and CO2 to form fragment ions. The D-ring was mostly a five-membered ring,which was prone to breakage. Lucidenic acids were the lanos-terolane-type of the C27 skeleton,and the side-chain structure became shorter and contained the same free-COOH and-OH compared with ganoderic acids,which had been reduced from 8 to 5 cartons and prone to lose H2O and CO2. Then,six reported FXR receptor agonists were selected to form a training set for establishing a pharmacophore model based on FXR ligands. The 95 identified chemical constituents of G. lucidum were matched with the pharmacophore,and the optimal pharmacophore model 02 (sensitivity=0. 750 00,specificity=0. 555 56,ROC=0. 750) was selected for the virtual screening of the G. lucidum compound library through the validation of the test set. Finally,31 potential G. lucidum active constituents were screened and chosen to activate the FXRs. The ADMET re-sults showed that ganoderic acid H and lucidenic acid J had less than 90％ plasma protein binding rate and no hepatotoxicity,which could be used as FXR activators for developing clinical drugs for the treatment of liver fibrosis,either alone or in combination.
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