大川芎方部分药效物质的检测及标准品的制备研究

【作者】 王明霞；

【导师】 肖红斌；

【作者基本信息】 北京中医药大学 ， 中药化学， 2019， 硕士

【摘要】 大川芎方是川芎:天麻按4:1比例配伍组成的,其为临床常用来治疗偏头痛的经典中药复方。天麻中分离鉴定的化学成分主要有酚类、有机酸类、多糖类及甾体类,其文献报道有效成分为天麻素、对羟基苯甲醇、巴利森苷类等成分;川芎主要含有苯酞类、酚酸类和生物碱类等成分。基于课题组前期发现,天麻中的羟苄醇酯成分进入体内后99%代谢转化为天麻素,巴利森苷类成分在体内为天麻素的前药,但是天麻素很难透过血脑屏障,进入体内的起效物质一直引起质疑;川芎进入体内的主要为苯酞类成分且烷基苯酞类化合物在体内易转化为羟基苯酞类化合物,因此推测进入血浆的这些苯酞类成分可能是药效成分。为进一步明确天麻药材的质量以及天麻素和川芎在体内的主要药效成分。本论文对其大麻药材的主要活性成分进行含量分析、天麻素体内代谢产物进行鉴定以及采用电化学模拟系统和分离制备技术对其天麻素的代谢产物和川芎苯酞类的成分进行了制备。主要研究内容如下:(1)为进一步明确天麻药材的质量,本研究建立了高灵敏度的荧光检测方法对其28批不同产地天麻中天麻素、对羟基苯甲醇、巴利森苷A、巴利森苷B、巴利森苷C 5个成分进行了同时定量分析。其结果表明:天麻素和对羟基苯甲醇定量下限为3 ng·mL-1,巴利森苷A、巴利森苷B、巴利森苷C的定量下限为10 ng·mL-1,精密度良好(RSD<5.0%),重复性良好(RSD<5.0%),平均回收率(n=5)分别为 97.4%、96.9%、99.1%、98.5%、96.3%。28批样品中上述5个成分的含量分别为0.48～20.01、0.08～2.54、0.49～15.44、1.65～8.20、0.73～2.12 mg·g-1。(2)为进一步阐明天麻素在体内的代谢产物以及代谢路径,本研究采用超高效液相色谱-飞行时间质谱(UPLC-Q-TOF-MS/MS)联用技术全面鉴定了天麻素大鼠入血、肝组织以及脑组织的代谢产物。结果表明:通过代谢产物的的精确分子质量、质谱裂解规律以及特征碎片离子,根据结合诊断离子策略、中性丢失策略以及Agilent Metabolite ID软件,鉴定出天麻素在体内的12个代谢产物,其发生的主要代谢反应有氧化、葡萄糖醛酸化、硫酸酯化等。(3)本研究建立了电化学模拟系统模拟天麻素体内的I相氧化代谢产物。结果表明:在最佳电压条件下,通过电化学和质谱联用(EC-MS)在线模拟和电化学制备型电极富集得到天麻素的I相氧化产物,通过分离纯化得到77 mg天麻素的I相氧化产物,最终确定结构为4-O-β-D-吡喃葡萄糖基苯甲醛。(4)本研究采用多种常压色谱柱对川芎苯酞类的成分进行了分离制备。结果表明:从50%乙醇部位分离得到了 9个化合物,分别为阿魏酸、洋川芎内酯I、洋川芎内酯A、洋川芎内酯N、欧当归内酯A。由于化合物6和化合物7在高效液相上无法达到基线分离,制备型液相无法实现其分离,因此,本实验建立了 SFC方法分别对化合物6和化合物7进行分离,化合物和化合物7的各2个单体成分达到了基线分离,后期将利用SFC对其4个单体成分进行富集和分离,并对其结构进行确认。结论:本研究建立的高灵敏度的荧光检测方法,杂质干扰少,分离度好,灵敏度高,线性范围宽,适合天麻成分分析及药材质量控制,为进一步完善天麻药材的质量标准评价方法提供参考依据。采用超高效液相色谱-飞行时间质谱(UPLC-Q-TOF-MS/MS)联用技术全面筛选出天麻素灌胃大鼠在血液、肝组织以及脑组织的12个代谢产物,其发生的主要代谢反应有氧化、葡萄糖醛酸化、硫酸酯化等,为进一步研究天麻素在体内的起效物质奠定了基础。建立的电化学模拟系统和质谱联用技术在线模拟天麻素体内的2个I相氧化代谢产物,并可通过制备型电极进行氧化代谢产物的放大富集,建立的此方法,干扰物少,快速,不需要很多的动物等优点,为中药代谢产物的制备开拓了新思路以及方法。利用多种常压色谱柱从川芎中分离得到9个化合物,为进一步明确川芎药效成分奠定了基础。更多还原

【Abstract】 Dachuanxiong prescription is composed of chuanxiong Rhizoma:gastrodiae Rhizoma in a ratio of 4:1.It is a traditional Chinese medicine compound commonly used to treat migraine.The chemical components isolated and identified in gastrodiae Rhizoma are mainly phenols,organic acids,polysaccharides and steroids.In addition to gastrodin,Parishin components in gastrodia elata were also reported to have good neuroprotective effects.chuanxiong Rhizoma mainly contains phthalide,phenolic acid and alkaloid etc.Based on the previous findings of the research group,it was found that the hydroxybenzyl alcohol ester in gastrodiae Rhizoma was metabolized into gastrodin by 99%after entering the body,and Parishin was the prodrug of gastrodin in the body.However,gastrodin was difficult to pass the blood-brain barrier,and the active substance in the body has always been questioned.chuanxiong Rhizoma is mainly composed of phthalide in vivo and is unstable and easily oxidized in vivo,so it is speculated that these phthalides entering the plasma may be pharmacodynamic components.Therefore,it is important to further clarify the quality of gastrodiae Rhizoma and the active ingredients of gastrodin and chuanxiong Rhizoma.In this paper,the quality of gastrodiae Rhizoma,the identification of the metabolites in gastrodin,the electrochemical and mass spectrometry techniques and the separation and preparation techniques were used to prepare the metabolites of gastrodin and the components of ligustrum chuanxiong Hort phthalide.The main research contents are as follows:(1)In this study,A high-sensitivity fluorescence detection method was established for simultaneous quantitative analysis of gastrodin,p-hydroxybenzenyl alcohol,Parishin A,Parishin B and Parishin C in 28 batches of gastrodiae Rhizoma from different origins.The results showed that the quantitative lower limit of gastrodin and p-hydroxybenzyl alcohol was 3 ng·ml-1,and the quantitative lower limit of Parishin A,Parishin B and Parishin C was 10 ng·ml-1,with good precision(RSD<5.0%)and good repeatability(RSD<5.0%),and the average recovery(n=5)was 97.4%,96.9%,99.1%,98.5%and 96.3%,respectively.The contents of the above five components in 28 batches of samples were 0.48～20.01,0.08～2.54,0.49～15.44,1.65～8.20,0.73～2.12 mg·g-1,respectively.(2)In this study,UPLC-Q-TOF-MS was used to comprehensively identify the metabolites of gastrodin in blood,liver and brain tissues of rats.Results show that through the metabolites of precise molecular mass,mass spectrometry cracking rule and characteristic fragment ions,according to the diagnostic ion binding,neutral loss and Agilent Metabolite ID software,select 12 gastrodin metabolites in the body,its the main metabolic reactions such as oxidation,glucose aldehyde acid,sulfuric acid ester.(3)This study established the electrochemical and mass spectrometry technology preparation of gastrodin phase I metabolites.The results showed that,under the optimal electrochemical conditions,the phase I oxidation products of gastrodin were obtained by on-line electrochemical and mass spectrometry(EC-MS)simulation and electrochemical preparation electrode enrichment,and then 77 mg phase I oxidation products of gastrodin were obtained by preparative high performance liquid chromatography separation and purification.(4)In this study,a variety of atmospheric pressure chromatographic columns were used to separate the components of the chuanxiong Rhizoma.The results showed that 9 chemical components were obtained from 50%ethanol:ferulic acid,senkyunolide I,senkyunolide A,senkyunolide N,levistilide A.compounds 6 and 7 could not achieve the baseline separation in the high performance liquid phase,the separation in the preparative liquid phase could not be realized,and the monomer structure could not be confirmed.In this experiment,SFC method was established to separate compounds 6 and 7 respectively.Two monomers of compounds 6 and 7 reached the baseline separation.In the later stage,SFC was used to enrich and separate four monomers and confirm their structures.Conclusion:The fluorescence detection method with high sensitivity established in this study has the advantages of less impurity interference,good separation degree,high sensitivity and wide linear range,which is suitable for gastrodia elata composition analysis and quality control of medicinal materials,and provides reference for further improving the quality standard evaluation method of gastrodiae Rhizoma.Twelve metabolites of gastrodin in blood,liver and brain tissues were screened by UPLC-Q-TOF-MS/MS,and the main metabolic reactions were oxidation,gluconaldehyde acidification and sulfuric acid esterification.The established electrochemical simulation system can be combined with mass spectrometry to detect the generation of metabolites online,which has the advantages of less interferences,fast speed and no need for many animals,etc.thus opening up new ideas and methods for the preparation of metabolites of traditional Chinese medicine.The separation of 9 chemical components from chuanxiong Rhizoma by various normal pressure chromatographic columns has laid a foundation for the search of active components of chuanxiong Rhizoma.更多还原