本文選題：草烏　切入點(diǎn)：訶子　出處：《北京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：草烏在傳統(tǒng)中、蒙和藏醫(yī)藥中均有悠久的使用歷史,三者都認(rèn)識(shí)到草烏的毒性,為保證臨床用藥的安全、有效,都強(qiáng)調(diào)內(nèi)服要炮制后使用,但炮制方法有所不同。中藥炮制草烏主要采用蒸、煮法,而蒙、藏醫(yī)炮制草烏常采用加輔料制法,炮制輔料常見訶子湯、甘草湯、酸奶、童便等,其中以訶子湯制最為常見,認(rèn)為訶子可解草烏毒。對(duì)蒙醫(yī)藥訶子制草烏的化學(xué)成分及炮制減毒原理進(jìn)行深入研究分析,可為臨床安全、有效的使用草烏、擴(kuò)大草烏的應(yīng)用范圍提供新的思路。目的從生物堿類成分及特征圖譜等角度,研究訶子作為炮制輔料對(duì)訶子制草烏化學(xué)成分的影響;結(jié)合訶子制草烏在人工胃腸液溶出規(guī)律、煎煮過程中生物堿動(dòng)態(tài)變化、急毒、細(xì)胞毒等研究,為訶子制草烏工藝優(yōu)化及炮制減毒機(jī)理闡釋提供依據(jù)。方法1.輔料因素對(duì)訶子制草烏生物堿含量的影響HPLC檢測(cè)不同比例的訶子制草烏中6種生物堿含量,并對(duì)不同濃度訶子湯pH值、總鞣質(zhì)含量、以及訶子湯新鮮程度等相關(guān)因素進(jìn)行考察,探討輔料因素對(duì)訶子制草烏毒效成分含量的影響。2.特征圖譜研究建立包含輔料信息的訶子制草烏的HPLC特征指紋圖譜檢測(cè)方法,并同時(shí)定量檢測(cè)訶子和草烏中的指標(biāo)性成分,研究訶子制草烏炮制前后化學(xué)成分變化,為訶子制草烏的定性鑒別及質(zhì)量控制提供方法依據(jù)。3.人工胃、腸液中溶出率研究用人工胃、腸液分別孵育生草烏和訶子制草烏粉末,通過檢測(cè)含藥人工胃、腸液不同時(shí)間點(diǎn)樣品中生物堿濃度,分析訶子炮制對(duì)草烏中生物堿動(dòng)態(tài)溶出規(guī)律的影響。4.煎煮過程中生物堿動(dòng)態(tài)變化規(guī)律以生草烏和訶子制草烏為研究對(duì)象,檢測(cè)二者在煎煮過程中酯型生物堿成分的含量變化,分析草烏經(jīng)訶子湯炮制后酯型生物堿在煎煮液中動(dòng)態(tài)溶出規(guī)律,為臨床應(yīng)用提供參考。5.急毒、細(xì)胞毒(或體內(nèi)外毒性)影響首先通過急性毒性試驗(yàn)測(cè)定生草烏和訶子制草烏對(duì)小鼠的LD50值,并結(jié)合生草烏和訶子制草烏樣品生物堿含量檢測(cè)結(jié)果,分析訶子制草烏的減毒作用與其所含生物堿成分之間的量——效關(guān)系。利用HCA心肌毒性評(píng)價(jià)方法,分別評(píng)價(jià)烏頭堿、訶子鞣酸及烏頭堿訶子鞣酸聯(lián)合給藥的心肌細(xì)胞毒性及其機(jī)制,探討訶子中活性成分訶子鞣酸對(duì)烏頭堿心肌毒性的影響,分析訶子制草烏可能存在的減毒機(jī)制。結(jié)果1.輔料因素對(duì)訶子制草烏生物堿含量的影響隨炮制輔料訶子湯濃度增加,訶子湯所含總鞣質(zhì)含量不斷升高,pH值先減小后基本維持穩(wěn)定;炮制品訶子制草烏中6種生物堿總量先逐漸升高后變化不規(guī)律。其中草烏、訶子用量比例為2:1時(shí),訶子制草烏中生物堿含量達(dá)到最低,該比例與1986年《內(nèi)蒙古蒙藥材標(biāo)準(zhǔn)》中對(duì)訶子、草烏炮制用量的比例一致。2.特征圖譜研究建立了訶子制草烏的HPLC特征指紋圖譜,共確定了 26個(gè)共有峰,其中17個(gè)來自生草烏,9個(gè)來自炮制輔料訶子。對(duì)照品指認(rèn)了新增加的6號(hào)峰和16號(hào)峰分別為訶子所含有的沒食子酸和訶子鞣酸;建立的HPLC特征圖譜分析方法還可用于定量檢測(cè)訶子制草烏中沒食子酸、訶子鞣酸、苯甲酰新烏頭原堿和新烏頭堿的含量。3.人工胃、腸液中溶出率研究在人工胃、腸液中孵育30min時(shí),酯型生物堿溶出率已經(jīng)達(dá)到60%以上;無論在人工胃液還是人工腸液中,訶子制草烏中單、雙酯型生物堿溶出率均低于生草烏。4.煎煮過程中生物堿動(dòng)態(tài)變化規(guī)律生草烏在水煎煮過程中,雙酯型生物堿迅速水解,在煎煮液沸騰3min雙酯型生物堿含量即開始快速下降,150min后水煎液中已檢測(cè)不到雙酯型生物堿含量。訶子制草烏水煎液中雙酯型生物堿含量在30min達(dá)到最高,之后不斷降低,但降低幅度較慢,煎煮6小時(shí)后水煎液中依然保持較高濃度的雙酯型生物堿。5.急毒、細(xì)胞毒(或體內(nèi)外毒性)影響草烏經(jīng)訶子炮制前后,急性毒性LD50值表示分別為224.3 mg.kg-1和320.5mg.kg-1,訶子制草烏粉末毒性明顯小于生草烏,降低幅度約為30%,遠(yuǎn)大于生物堿含量降低幅度4%。基于高內(nèi)涵分析技術(shù)(HCA)的烏頭堿、訶子鞣酸心肌毒性評(píng)價(jià)結(jié)果為,烏頭堿濃度20 μ mol/L時(shí)會(huì)造成心肌細(xì)胞線粒體膜電位增高和胞內(nèi)鈣離子超載,當(dāng)烏頭堿和訶子鞣酸聯(lián)合給藥時(shí),各劑量組心肌細(xì)胞線粒體膜電位和心肌細(xì)胞內(nèi)鈣離子超載現(xiàn)象均消失,和正常組無顯著性差異。結(jié)論草烏經(jīng)訶子湯炮制后,其生物堿類成分含量發(fā)生了變化,并引入了炮制輔料訶子中的化學(xué)成分,其成分組成發(fā)生了量和質(zhì)的變化。訶子制草烏確有減毒的作用,但其炮制減毒的原理與傳統(tǒng)中醫(yī)藥蒸、煮法減毒原理不同,并不是通過直接降低草烏中雙酯型生物堿含量來達(dá)到炮制減毒的作用。訶子制草烏由于引入了炮制輔料訶子中的活性成分,如鞣質(zhì)類成分等,影響草烏中生物堿在胃、腸液中的溶出規(guī)律,起到一定的緩釋(減毒)作用;在煎煮過程中,減緩雙酯型生物堿的水解,起到保留藥效成分(存效)的作用。另一方面,訶子具有很強(qiáng)的藥理活性,如強(qiáng)心作用,草烏經(jīng)訶子湯炮制后急性毒性降低,或許是訶子中的活性成分如訶子鞣酸等,能夠減輕草烏中生物堿導(dǎo)致的心肌毒性,從而起到減毒作用。
[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.

【學(xué)位授予單位】：北京中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R29

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本文編號(hào)：1578605