灵芝孢子中三萜类成分UPLC-Q-TOF-MS/MS定性分析和HPLC含量测定研究  

UPLC-Q-TOF-MS/MS qualitative analysis and HPLC determination of triterpenoids in Ganoderma lucidum spores

作　　者：乔灵芝 卢晨娜 刘晓谦[2] 陈两绵[2] 冯伟红[2] 梁曜华[2] 李春[2] 王智民[2] QIAO Ling-zhi;LU Chen-na;LIU Xiao-qian;CHEN Liang-mian;FENG Wei-hong;LIANG Yao-hua;LI Chun;WANG Zhi-min(Tianjin University of Traditional Chinese Medicine,Tianjin 301617,China;National Engineering Laboratory for Quality Control Technology of Chinese Herbal Medicines,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]天津中医药大学,天津301617 [2]中国中医科学院中药研究所、中药质量控制技术国家工程实验室,北京100700

出　　处：《中国中药杂志》2024年第20期5487-5496,共10页China Journal of Chinese Materia Medica

基　　金：国家重点研发计划项目(2023YFC3504000);中国中医科学院科技创新工程项目(CI2023E001TS03,CI2021A04402,CI2021A04412)。

摘　　要：灵芝孢子为多孔菌科真菌赤芝Ganoderma lucidum或紫芝G.sinense的干燥成熟孢子,在提高免疫力、抗肿瘤、抗氧化等多方面有较强的生理活性,三萜是其活性成分之一,但目前没有可靠的含量测定方法。因此,该研究采用UPLC-Q-TOF-MS/MS定性分析灵芝孢子中的三萜类成分,并建立了同步测定灵芝孢子中5种三萜类成分含量的HPLC方法。在负离子模式下,通过与对照品保留时间、紫外光谱、裂解碎片比对,从灵芝孢子中确认了9种三萜类成分,分别是灵芝酸I、灵芝烯酸C、灵芝酸C2、灵芝酸C6、灵芝酸G、灵芝烯酸B、灵芝酸B、灵芝酸A、灵芝酸H;通过与文献比较,从灵芝孢子中推测了3种三萜成分,分别为赤芝酸E、(Z)-6-[(3S,5R,7S,10S,12S,13R,14R,17R)-12-乙酰氧基-3,7-二羟基-4,4,10,13,14-五甲基-11,15-二氧代-2,3,4,5,6,7,10,11,12,13,14,15,16,17-十四氢-1H-环戊烯并[α]菲-17-基]-2-甲基-4-氧代庚-5-烯酸和ganoderin A。以Ultimate HPLC XS-C_(18)色谱柱(4.6 mm×250 mm, 5μm)为固定相,乙腈-0.072%磷酸水为流动相,梯度洗脱,流速1.2 mL·min^(-1),检测波长257 nm,柱温40℃,该条件下5种三萜类成分可实现同步定量测定,方法学考察结果符合含量测定要求。灵芝烯酸C、灵芝酸C2、灵芝酸C6、灵芝烯酸B和灵芝酸H这5种成分分别在0.93~16.08、3.44~23.88、4.28~59.40、1.40~16.16、3.53~61.20μg·mL^(-1)呈良好的线性关系(R~2>0.999),平均回收率在79.53%~93.60%,RSD在4.7%~7.6%。15批不同产地的灵芝孢子原粉和4批不同产地破壁灵芝孢子中5种三萜类成分的含量差别较大,15批不同产地原粉的5种成分总量在0.44~16.25μg·g^(-1);4批不同产地的破壁灵芝孢子三萜总量在0.52~3.57μg·g^(-1)。该文建立的新方法操作简便、结果可靠,可以用于灵芝孢子中三萜类成分的准确测定,为灵芝孢子的质量评价提供了方法依据。Ganoderma spores are the dry and mature spores of G.lucidum or G.sinense of Polyporaceae,with strong immunity-improving,anti-tumor,and anti-oxidant activities.Triterpenoids are one of the main active components in Ganoderma spores,while the method for determining the content of triterpenoids remains to be developed.In this paper,UPLC-Q-TOF-MS/MS was employed to qualitatively analyze the triterpenoids in G.lucidum spores,and an HPLC method was established for simultaneous determination of five triterpenoids in the spores.The comparison on the retention time,ultraviolet spectrum,and fragmentation with the reference substance in the negative ion mode revealed nine triterpenoids in G.lucidum spores.These nine tri terpenoids were ganoderic acid I,ganoderenic acid C,ganoderic acid C2,ganoderic acid C6,ganoderic acid G,ganoderenic acid B,ganoderic acid B,ganoderic acid A,and ganoderic acid H.Three tri terpenoids from the spores were inferred by comparison with the available articles,including lucidenic acid E,(Z)-6-[(3S,5R,7S,10S,12S,13R,14R,17R)-12-acetoxy-3,7-dihydroxy-4,4,10,13,14-pentamethyl-11,15-dioxo-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetrahydro-1H-cyclopenteno[α]phenanthrene-17-yl]-2-methyl-4-oxoheptyl-5-enoic acid,and ganoderin A.With Ultimate HPLC XS-C18 column(4.6 mm×250 mm,5μm)as the stationary phase and acetonitrile-0.072%phosphoric acid solution as the mobile phase,gradient elution was carried out at a flow rate of 1.2 mL·min^(-1),detection wavelength of 257 nm,and column temperature of 40℃.These conditions enabled the simultaneously quantitative analysis of the five triterpenoids,and the results of methodological investigation showed that the method met the requirements of content determination.Ganoderenic acid C,ganoderic acid C2,ganoderic acid C6,ganoderenic acid B,and ganoderic acid H showed a good linear relationship(R2>0.999)within the concentration ranges of 0.93-16.08,3.44-23.88,4.28-59.40,1.40-16.16,and 3.53-61.20μg·mL^(-1),respectively,with the average recovery rate of 79.53%-93.60%and R

关 键 词：灵芝孢子 三萜 定性分析 含量测定 

分 类 号：R284.1[医药卫生—中药学]