乌头碱的代谢产物去氧乌头碱的生物转化及其电喷雾串联质谱

采用人肠内细菌和乌头碱体外温孵的方法, 探讨去氧乌头碱在人肠内的生物转化. 利用离子阱和傅里叶变换离子回旋共振质谱直接分析去氧乌头碱的转化产物. 乌头类生物碱及其代谢产物在正离子电喷雾质谱条件下形成质子化分子([M+H]+), 通过多级串联质谱进行结构表征. 去氧乌头碱可被人肠内细菌转化, 通过脱酰基、脱甲基脱羟基以及酯化反应产生新型的单酯型、双酯型和脂类生物碱等10余种代谢产物. 双酯型的去氧乌头碱的毒性较高, 当它被肠内细菌转化为单酯型和脂类生物碱时会使其毒性降低.     

16-O-去甲基乌头碱的生物转化及电喷雾质谱研究

采用乌头碱和人肠内细菌体外温孵的方法, 探讨乌头碱在肠内的生物转化规律. 乌头类生物碱在ESI正离子模式条件下形成质子化分子[M＋H]^＋, 利用离子阱电喷雾串联质谱和傅立叶离子回旋共振电喷雾串联质谱方法可以直接分析乌头碱的转化产物. 本文首次报道了乌头碱在人肠内菌群环境中产生16-O-去甲基乌头碱, 16-O-去甲基乌头碱可进一步被肠内细菌转化, 通过脱乙酰基、脱苯甲酰基、脱甲基、脱羟基以及酯化反应, 产生新型的单酯型、双酯型和20余种脂类生物碱等转化产物.     

16-O-去甲基去氧乌头碱在肠内细菌中的生物转化研究

采用人肠内细菌和乌头碱体外温孵的方法,探讨乌头碱的代谢产物16-O-去甲基去氧乌头碱在人肠内的生物转化。利用离子阱电喷雾串联质谱(ESIMS/MSn)方法直接分析16-O-去甲基去氧乌头碱的代谢产物。乌头类生物碱在ESI正离子模式条件下形成质子化分子[M H] 。16-O-去甲基去氧乌头碱可被人肠内细菌转化,通过脱乙酰基、脱苯甲酰基、脱甲基、脱羟基以及酯化反应产生新型的单酯型、双酯型和脂类生物碱等10余种代谢产物。双酯型生物碱具有较高的毒性,相对应的单酯型和脂类生物碱毒性较低。16-O-去甲基去氧乌头碱被肠内细菌转化为单酯型和脂类生物碱会使其毒性降低。     

复方中药四逆汤中乌头碱类二萜生物碱的电喷雾串联质谱研究

利用电喷雾串联质谱方法不经柱分离而直接分析复方中药四逆汤中二萜生物碱的组成,乌头碱类二萜生物碱在质谱条件下形成质子化分子,一种生物碱对应一个分子离子峰.检测到了苯甲酰单酯型、双酯型和脂类生物碱等18种二萜生物碱,其中苯甲酰中乌头原碱等单酯型生物碱是四逆汤中的主要生物碱成分.     

利用液质联用技术分析干姜对乌头类生物碱在大鼠肠内吸收的影响

为从吸收的角度考察干姜对乌头类双酯型生物碱的解毒机理, 采用外翻肠囊法展开实验. 利用超高液相与三重四极杆质谱联用技术定量检测双酯型生物碱成分, 采用标准曲线法计算乌头碱、中乌头碱、次乌头碱在肠囊内吸收的绝对含量, 采用质谱峰面积直接分析其它双酯型生物碱的相对变化, 结果加入干姜提取液后, 乌头碱、中乌头碱、次乌头碱的单位肠管面积累计吸收量均降低, 10-羟基中乌头碱的的累积峰面积降低; 加入维拉帕米后, 双酯型生物碱的单位肠管面积累计吸收量及累积峰面积均增加; 向含有地高辛的肠营养液中加入干姜提取液后, 地高辛在各实验时间点的单位肠管面积累计吸收量均降低, 根据以上结果推测干姜抑制乌头类双酯型生物碱在大鼠肠囊内吸收的可能机制是通过诱导肠内P-葡糖蛋白, 从而抑制作为P-葡糖蛋白底物的双酯型生物碱的吸收, 最终起到减毒作用.     

"甘草附子汤"和"术附汤"肠内生物转化的电喷雾质谱研究

以电喷雾质谱法作为研究方法,以内标化合物为切入点,对复方中双酯型、单酯型及脂型生物碱的生物转化进行了深入研究,建立了电喷雾质谱对代谢前后生物碱成分的半定量分析方法.分析了中药复方甘草附子汤和术附汤经大鼠肠内菌群代谢主要生物碱的含量变化,研究结果表明,配伍中药甘草和白术可以有效地降低共煎液中双酯型生物碱含量,在代谢过程中...     

乌头类双酯型生物碱组分转化为单酯水解型及脂型生物碱组分的研究

建立了乌头类双酯型生物碱组分专一的转化为单酯水解型生物碱组分和脂型生物碱组分的方法,其转化率在90%以上,应用电喷雾多级串联质谱方法监测反应进程并对反应产物进行鉴定,同时可以考察反应中生成的副产物.     

草乌中二萜类生物碱的电喷雾串联质谱研究

利用电喷雾质谱技术对传统中药草乌中的二萜类生物碱进行直接分析鉴定.通过实验数据并对比文献发现,草乌中含有单酯型、双酯型、三酯型和脂类等共4种类型生物碱,其中三酯型和脂类生物碱在草乌中为首次发现.由于这些生物碱的结构相似,在电喷雾串联质谱中碎裂方式相同,因此根据电喷雾串联质谱结果确定了这些生物碱的结构.     

电喷雾串联质谱分析附子炮制中的化学成分变化

利用电喷雾质谱方法(ESI-MS)分析了附子加辅料(甘草)炮制前后水煎液中二萜类生物碱在种类和含量方面的变化,通过加入内标化合物,建立了电喷雾质谱的半定量分析方法。此方法具有快速、准确、灵敏的特点,能够更加全面地反映中药配伍炮制过程中多种化学成分的含量变化,并能根据电喷雾串联质谱的分析结果鉴定配伍后产生的新的化学成分,在共煎液中的次乌头碱、中乌头碱和乌头碱的相对含量分别是单煎液中的5.67%、4.05%和4.88%。通过研究附子与甘草的单煎液、共煎液以及药渣中化学成分的变化,揭示了甘草作为辅料,在炮制过程中对附子减毒作用机理。     

雪上一支蒿中乌头碱类生物碱的电喷雾串联质谱分析

利用电喷雾串联质谱(ESI-MS／MS)对雪上一支篙的乙醇提取液进行了直接分析，方法简便，直观，用样量少。ESL-MS可以给出分子量信息，MS／MS方法则可以从复杂体系中获得结构信息。在雪上一支蒿中发现乌头碱、去氧乌头碱及它们的水解产物和脂类生物碱等共19种二菇生物碱，其中脂类生物碱为首次在该植物中发现。     

Effect of pH on the Metabolism of Aconitine under Rat Intestinal Bacteria and Analysis of Metabolites Using HPLC/MS-MSn Technique

A semi‐quantitative method of mass spectrometry (MS) has been described for the analysis of metabolites of aconitine by rat intestinal bacteria at different pH. At pH 7.0, the rat intestinal bacteria exhibit optimal activity for the metabolism of aconitine. A high‐performance liquid chromatography‐electrospray ionization multiple‐stage mass spectrometry (HPLC/ESI‐MSⁿ) method has been applied to investigate the characteristic product ions of metabolites. Then, the logical fragmentation pathways of metabolites have been proposed. By comparing the retention time (t_R) of HPLC and the ESI‐MSⁿ data with the data of standard compounds and reports from literature, ten metabolites have been identified and a distinctive metabolite (15‐deoxyaconitine) has been deduced first time. The experimental results demonstrate that HPLC/ESI‐MSⁿ is a specific and useful method for the identification of metabolites of aconitine. Also, in the present paper, the HPLC‐MS method was introduced to determine the synthetical metabolite prior to the study of the toxicity by the method of Bliss.     

乌头碱的LC-MS定量分析

乌头生物碱各成分毒性差异很大,其中乌头碱的毒性为其它成分的100-2000倍,是引起中毒和死亡的主要原因。乌头生物碱种类多,在煎煮或泡制过程中易水解产生不同水解产物,进入体内后代谢情况又不明,因此采用液相色谱方法对体内检材乌头碱成分仅靠保留时间确定依据不足,定量工作更是无法开展。但在现实生活中炮制后的乌头植物可入药,且炮制过的乌头植物也可检出少量原碱。遇到体内检材中检验出乌头生物碱成分时,办案单位往往希望有一个量的甄别。经查阅资料,未见体内检材(如血、肝、尿等)中乌头碱含量的报道。我们应用LC-MS,采用646.4单离子扫描方式对实际案例血中乌头碱含量进行了测定,为今后的进一步研究和同行提供数据积累。     

New understanding of aconitine hydrolysis pathway: Isolation,identification and toxicity evaluation based on intermediate products

Aconitine hydrolysis is deemed to be the guarantee for the safe application of Aconitum phytomedicine. Studies have suggested that hydrolysates of aconitine not only include benzoylaconitine and aconine, but other hydrolysates. Moreover, these hydrolysates maybe have a mutual transformation relationship, which has not been confirmed. Herein, hydrolysates of aconitine and their mutual transformation relationship were studied by the theoretical quantum chemistry, UPLC-Q-TOF-MS, the separation and identification of target products, etc. Then the toxicity of its hydrolysates was evaluated. The results demonstrate that the probability is the same for aconitine hydrolysis to pyroaconitine and benzoylaconitine, but they are difficult to convert to each other. Aconitine hydrolysis has three independent hydrolysis pathways, 1) to indaconitine, 2) to benzoylaconitine, and aconine, 3) to pyroaconitine and to 16-epi-pyroaconine. The result of embryotoxicity evaluation on zebrafish was aconitine > indaconitine > benzoylaconitine > α-pyroaconitine > β- pyroaconitine > aconine > 16-epi-pyroaconine. In conclusion, aconitine have three independent hydrolysis pathways and the hydrolysates of different pathways cannot be transformed into each other. Pyroaconitine is a hydrolysate of aconitine except for benzoylaconitine, and its toxicity is lower than benzoylaconitine. More importantly, it clarifies the long-standing debate and provides scientific evidence for the processing and detoxification of Aconitum phytomedicine.     

Simultaneous quantification and semi‐quantification of amentoflavone and its metabolites in human intestinal bacteria by liquid chromatography Orbitrap high‐resolution mass spectrometry

A quick, easy, effective method followed by ultra‐high‐pressure liquid chromatography coupled with linear ion trap–Orbitrap tandem mass spectrometry (UHPLC‐LTQ‐Orbitrap MS) was developed for the simultaneous identification and quantification of the metabolites produced by amentoflavone (AMF) in human intestinal bacteria from human feces. The method validated for quantification of AMF concerning precision, accuracy, recovery, matrix effect, stability and limits showed acceptable results. Compared with blank human intestinal bacteria chromatography, three metabolites were identified based on high‐accuracy protonated precursors and multi‐stage mass spectrometry (MSⁿ ) using the proposed strategy. At the same time, a new method was developed for semi‐quantification of three metabolites. We describe the trend over 24 h of concentration–time curves for AMF and its metabolites. Moreover, the main metabolic pathway of AMF was clarified in human intestinal bacteria. The method was validated and successfully applied to the detection and quantification of AMF and its metabolites.     

Rapid separation and determination of aconitine alkaloids in traditional Chinese herbs by capillary electrophoresis using 1-butyl-3-methylimidazoium-based ionic liquid as running electrolyte

A novel and very simple capillary electrophoretic method for analyzing aconitine components in Aconitum plants was developed using 1-butyl-3-methylimidazoium tetrafluoroborate (1B-3MI-TFB)-based ionic liquid as running electrolyte solution for the first time. The optimum conditions were 35 mM 1B-3MI-TFB solution (pH 8.50) and 15 kV applied voltage. The detection was performed at 254 nm. Aconitine, meaconitine and hypaconitine in Aconitum plants were separated and identified within 5 min. The recoveries were 91.0-103.0% for hypaconitine, 92.8-96.2% for aconitine and 96.0-106.6% for mesaconitine, respectively. Compared with other methods, the analytical time was decreased 4-8-fold and the effect of Joule heating was weaker because the current was smaller.     

Milde alkalische Hydrolyse von Aconitin

Mild Alkaline Hydrolysis of Aconitine Hydrolysis of aconitine ( 1 ) with 0.04N K₂CO₃ in 90% MeOH at room temperature yields, besides the alkamine aconine 3 considerable amounts of 8-O-methylaconine ( 6 ) and smaller quantities of desbenzoyl-pyroaconitine ( 4 ) and 16-epi-desbenzoyl-pyroaconitine ( 5 ). Better yields of 4 and 5 are obtained when heating a solution of aconitine in 0.04N K₂CO₃ in 90% EtOH.     

Metabolism of gentiopicroside (gentiopicrin) by human intestinal bacteria

As a part of our studies on the metabolism of crude drug components by intestinal bacteria, gentiopicroside (a secoiridoid glucoside isolated from Gentiana lutea), was anaerobically incubated with various defined strains of human intestinal bacteria. Many species had ability to transform it to a series of metabolites. Among them, Veillonella parvula ss parvula produced five metabolites, which were identified as erythrocentaurin, gentiopicral, 5-hydroxymethylisochroman-1-one,5-hydroxymethylisochromen-1- one and trans-5,6-dihydro-5-hydroxymethyl-6-methyl-1H,3H-pyrano[3,4-c]pyra n-1-one.