辅料因素对蒙药诃子制草乌化学成分的影响及炮制减毒原理研究

发布时间：2018-03-07 08:11

本文选题：草乌　切入点：诃子　出处：《北京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：草乌在传统中、蒙和藏医药中均有悠久的使用历史,三者都认识到草乌的毒性,为保证临床用药的安全、有效,都强调内服要炮制后使用,但炮制方法有所不同。中药炮制草乌主要采用蒸、煮法,而蒙、藏医炮制草乌常采用加辅料制法,炮制辅料常见诃子汤、甘草汤、酸奶、童便等,其中以诃子汤制最为常见,认为诃子可解草乌毒。对蒙医药诃子制草乌的化学成分及炮制减毒原理进行深入研究分析,可为临床安全、有效的使用草乌、扩大草乌的应用范围提供新的思路。目的从生物碱类成分及特征图谱等角度,研究诃子作为炮制辅料对诃子制草乌化学成分的影响;结合诃子制草乌在人工胃肠液溶出规律、煎煮过程中生物碱动态变化、急毒、细胞毒等研究,为诃子制草乌工艺优化及炮制减毒机理阐释提供依据。方法1.辅料因素对诃子制草乌生物碱含量的影响HPLC检测不同比例的诃子制草乌中6种生物碱含量,并对不同浓度诃子汤pH值、总鞣质含量、以及诃子汤新鲜程度等相关因素进行考察,探讨辅料因素对诃子制草乌毒效成分含量的影响。2.特征图谱研究建立包含辅料信息的诃子制草乌的HPLC特征指纹图谱检测方法,并同时定量检测诃子和草乌中的指标性成分,研究诃子制草乌炮制前后化学成分变化,为诃子制草乌的定性鉴别及质量控制提供方法依据。3.人工胃、肠液中溶出率研究用人工胃、肠液分别孵育生草乌和诃子制草乌粉末,通过检测含药人工胃、肠液不同时间点样品中生物碱浓度,分析诃子炮制对草乌中生物碱动态溶出规律的影响。4.煎煮过程中生物碱动态变化规律以生草乌和诃子制草乌为研究对象,检测二者在煎煮过程中酯型生物碱成分的含量变化,分析草乌经诃子汤炮制后酯型生物碱在煎煮液中动态溶出规律,为临床应用提供参考。5.急毒、细胞毒(或体内外毒性)影响首先通过急性毒性试验测定生草乌和诃子制草乌对小鼠的LD50值,并结合生草乌和诃子制草乌样品生物碱含量检测结果,分析诃子制草乌的减毒作用与其所含生物碱成分之间的量——效关系。利用HCA心肌毒性评价方法,分别评价乌头碱、诃子鞣酸及乌头碱诃子鞣酸联合给药的心肌细胞毒性及其机制,探讨诃子中活性成分诃子鞣酸对乌头碱心肌毒性的影响,分析诃子制草乌可能存在的减毒机制。结果1.辅料因素对诃子制草乌生物碱含量的影响随炮制辅料诃子汤浓度增加,诃子汤所含总鞣质含量不断升高,pH值先减小后基本维持稳定;炮制品诃子制草乌中6种生物碱总量先逐渐升高后变化不规律。其中草乌、诃子用量比例为2:1时,诃子制草乌中生物碱含量达到最低,该比例与1986年《内蒙古蒙药材标准》中对诃子、草乌炮制用量的比例一致。2.特征图谱研究建立了诃子制草乌的HPLC特征指纹图谱,共确定了 26个共有峰,其中17个来自生草乌,9个来自炮制辅料诃子。对照品指认了新增加的6号峰和16号峰分别为诃子所含有的没食子酸和诃子鞣酸;建立的HPLC特征图谱分析方法还可用于定量检测诃子制草乌中没食子酸、诃子鞣酸、苯甲酰新乌头原碱和新乌头碱的含量。3.人工胃、肠液中溶出率研究在人工胃、肠液中孵育30min时,酯型生物碱溶出率已经达到60%以上;无论在人工胃液还是人工肠液中,诃子制草乌中单、双酯型生物碱溶出率均低于生草乌。4.煎煮过程中生物碱动态变化规律生草乌在水煎煮过程中,双酯型生物碱迅速水解,在煎煮液沸腾3min双酯型生物碱含量即开始快速下降,150min后水煎液中已检测不到双酯型生物碱含量。诃子制草乌水煎液中双酯型生物碱含量在30min达到最高,之后不断降低,但降低幅度较慢,煎煮6小时后水煎液中依然保持较高浓度的双酯型生物碱。5.急毒、细胞毒(或体内外毒性)影响草乌经诃子炮制前后,急性毒性LD50值表示分别为224.3 mg.kg-1和320.5mg.kg-1,诃子制草乌粉末毒性明显小于生草乌,降低幅度约为30%,远大于生物碱含量降低幅度4%。基于高内涵分析技术(HCA)的乌头碱、诃子鞣酸心肌毒性评价结果为,乌头碱浓度20 μ mol/L时会造成心肌细胞线粒体膜电位增高和胞内钙离子超载,当乌头碱和诃子鞣酸联合给药时,各剂量组心肌细胞线粒体膜电位和心肌细胞内钙离子超载现象均消失,和正常组无显著性差异。结论草乌经诃子汤炮制后,其生物碱类成分含量发生了变化,并引入了炮制辅料诃子中的化学成分,其成分组成发生了量和质的变化。诃子制草乌确有减毒的作用,但其炮制减毒的原理与传统中医药蒸、煮法减毒原理不同,并不是通过直接降低草乌中双酯型生物碱含量来达到炮制减毒的作用。诃子制草乌由于引入了炮制辅料诃子中的活性成分,如鞣质类成分等,影响草乌中生物碱在胃、肠液中的溶出规律,起到一定的缓释(减毒)作用;在煎煮过程中,减缓双酯型生物碱的水解,起到保留药效成分(存效)的作用。另一方面,诃子具有很强的药理活性,如强心作用,草乌经诃子汤炮制后急性毒性降低,或许是诃子中的活性成分如诃子鞣酸等,能够减轻草乌中生物碱导致的心肌毒性,从而起到减毒作用。
[Abstract]:Aconitum in the tradition, has a long history of using Mongolian and Tibetan medicine, the three researchers have been aware of the toxicity of Aconitum, in order to ensure the safety of clinical medication, effective, emphasize to use oral after processing, but the processing method is different. Traditional Chinese medicine Aconitum mainly by steaming, boiling, and Mongolia, the Tibetan medicine processing Caowu by adding accessories preparation, processing materials of common myrobalan licorice soup, soup, yogurt, etc. the soup of Terminalia Tongbian, were the most common, that can be toxic. Solutions of Aconitum myrobalan chemical composition and processing of Mongolian medicine Terminalia kusnezoffii attenuated the principle of in-depth research and analysis, for the clinical safe, effective use Aconitum, provide a new way to expand the scope of application of Aconitum alkaloids. From the angle of influence and characteristics, research as processing materials of myrobalan Terminalia kusnezoffii chemical composition; combination of Terminalia kusnezoffii dissolved in artificial gastrointestinal fluid Law of changes in the decocting process of dynamic alkaloids acute toxicity study, cytotoxic, provide the basis for process optimization and processing of Terminalia kusnezoffii attenuated mechanism explanation. Methods 1. accessories influence factors of prepared aconite alkaloids of myrobalan Terminalia kusnezoffii HPLC detection in different ratio of 6 kinds of alkaloid content, and different concentrations of Terminalia Decoction the pH value, the total tannin content, investigated related factors and fresh degree of Kezi decoction, excipients on Terminalia kusnezoffii toxic ingredients were studied to establish HPLC fingerprint detection methods include materials information system of grass Ukraine of Terminalia.2. characteristic spectrum, and the index components and quantitative detection of myrobalan and Caowu and the changes of chemical components before and after the Terminalia kusnezoffii processing methods based on the.3. artificial stomach for qualitative identification and quality control of Fructus kusnezoffii, the dissolution rate of juice Artificial gastric juice, incubated Caowu and Myrobalam prepared aconite powder, through the detection of drug containing artificial gastric juice, the concentration of alkaloids in samples at different time points, analysis on the dynamic processing of Terminalia Aconitum alkaloids in the dissolution process of dynamic changes of alkaloids in the decoction of.4. and Radix Aconiti kusnezoffii chebulinic as the research object, content changes the two methods in the boiling process of ester type alkaloids, Aconite Decoction after processed by chebulinic analysis of ester alkaloids in decoction solution in dynamic dissolution law, provide the reference for the clinical application of.5. acute toxicity, cytotoxicity (or in vivo) first effect by measuring LD50 Caowu and Myrobalam kusnezoffii on mice value the acute toxicity test, and combined with the detection results of Radix Aconiti kusnezoffii and myrobalan samples prepared aconite alkaloid content analysis, reducing the toxicity of Terminalia and prepared aconite alkaloid components between the quantity of Effect evaluation method of HCA. Using the myocardial toxicity were evaluated, aconitine, aconitine and tannin myrobalan Terminalia tannic acid combine to give myocardiac cytotoxic drugs and its mechanism, effect of active constituents in Fructus tannic acid on myocardial toxicity of aconitine, Terminalia kusnezoffii can analysis the toxic mechanism exists. Results of the 1. reduction effect of excipients the prepared aconite alkaloid content of Terminalia increased with the concentration of processing materials of Terminalia decoction, Kezi decoction containing the total tannin content increased, pH value decreased first and then remained stable; 6 of total alkaloids in processed products of Fructus kusnezoffii first increased gradually after the change. The proportion of Terminalia Aconitum, 2:1, alkaloid content in chebulinic prepared aconite reached the lowest level, with the ratio in 1986 of < Inner Mongolia Mongolian medicine standard of > myrobalan, of uniform.2. characteristics of the proportion of a processed Aconitum He Zi grass HPLC fingerprint of Ukraine, a total of 26 common peaks were confirmed, including 17 from kusnezoffii, 9 from Terminalia. The control of processing materials identified the new peak 6 and peak 16 were chebulinic contains gallic acid and tannic acid chebulinic; HPLC fingerprint analysis method can be established for the quantitative detection of gallic acid, Terminalia kusnezoffii in Terminalia tannic acid, benzoylmesaconine and mesaconitine in artificial gastric.3., the dissolution rate of artificial gastric juice in intestinal fluid, incubated in 30min, the dissolution rate has reached more than 60% ester type alkaloids; both in artificial gastric juice or artificial intestinal juice in the single Terminalia kusnezoffii, were lower than the rate of variation of alkaloids of Radix Aconiti kusnezoffii.4. dynamic in the decoction of Radix Aconiti kusnezoffii in water decoction process of dissolution of diester alkaloids, diester alkaloids rapid hydrolysis, boiling 3min diester alkaloids containing in Decoction The amount that is declining rapidly, after 150min decoction has not detected the content of diester alkaloids. The content of diester chebulinic prepared aconite Decoction of alkaloids reached the highest at 30min, then decreased, but the decrease is slow, boiling 6 hours after the decoction remains of diester alkaloids.5. high the concentration of acute toxicity, cytotoxicity (or in vivo) by chebulinic effects of Radix Aconiti kusnezoffii before and after processing, the acute toxicity of LD50 value were 224.3 mg.kg-1 and 320.5mg.kg-1, Terminalia kusnezoffii powder significantly less than the toxicity of Radix Aconiti kusnezoffii, reduced by about 30%, larger than the alkaloid content decreased 4%. analysis technology based on high content (HCA). Aconitine, for evaluation of myocardial toxicity of aconitine chebulinic tannic acid, concentration of 20 mol/L induced calcium increase of mitochondrial membrane potential and intracellular overload, when aconitine and Myrobalam tannic acid combination when administered by The phenomenon of calcium overload in myocardial cell membrane potential and mitochondrial dose disappeared, there was no significant difference between the normal group and the conclusion. Aconitum Kezi decoction after processing, changed its alkaloid content, and introduces the chemical composition of processing materials in the composition of Myrobalam, changes in quantity and quality there are attenuated. Terminalia kusnezoffii effect, but its processing attenuated principle with traditional Chinese medicine steaming, boiling attenuated principle is different, and not by directly reduce the content of diester type Aconitum alkaloids to achieve the processing effect of attenuated. He sub kusnezoffii due to the introduction of the active components of processing materials from Terminalia such as, tannins, alkaloids in Aconitum of stomach, intestinal digestion law, plays a certain role; sustained release (attenuated) in the boiling process, slow hydrolysis of diester alkaloids, to retain active components ( Save effect) effect. On the other hand, myrobalan has strong pharmacological activity, such as cardiac function after acute toxicity of Aconitum Kezi decoction after processing is reduced, may be the active ingredients such as tannic acid in Fructus Chebulae Fructus Chebulae, can relieve the myocardial toxicity of lead Aconitum alkaloids in, so as to reduce the toxicity of.

【学位授予单位】：北京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R29

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