曲克芦丁在大鼠体内和体外的代谢研究

发布时间：2018-02-11 20:55

本文关键词： 曲克芦丁代谢产物曲克芦丁苷元肠道菌群高效液相色谱　出处：《安徽医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：曲克芦丁是天然存在的芦丁经羟乙基化制成的半合成的黄酮类化合物,是心脑血管药维脑路通中最重要的成分,又因具有维生素P4样的作用而被称作维生素P4,化学名为3’,4’,7-三羟乙基芦丁。具有抑制红细胞和血小板的凝集作用;预防血栓的形成;同时能够保护血管内皮细胞,降低血管壁的通透性和消除水肿;抗炎;抗化学性肝损伤等药理作用。临床上常用来治疗脑梗塞及中风后遗症、慢性静脉功能不全、静脉曲张、痔疮、长距离飞行微血管等疾病。尽管曲克芦丁已经在临床上使用多年,但是有关其不良反应的报导却逐渐增多,并且目前有关曲克芦丁在体内的代谢报道较少,仅有一些关于曲克芦丁在人和大鼠体内的药代动力学研究,对于曲克芦丁在体内的代谢情况没有完全的认识。为了研究曲克芦丁在大鼠体内和体外的代谢转化情况,本文对曲克芦丁在大鼠体内和体外代谢的情况进行了较为深入的研究。主要研究内容概括如下:1.曲克芦丁的代谢产物研究Sprague-Dawley大鼠腹腔注射曲克芦丁后,收集24h的尿液和粪便样品,样品处理后用HPLC检测,结果表明:给药尿液与空白尿液没有明显差别,但在粪便样品中检测到一明显的代谢产物峰。采用肠道菌群体外培养法培养曲克芦丁,将得到的肠道菌群培养样品样品处理后用HPLC分析,也检测到一明显的代谢产物峰,且其保留时间与粪便中检测到的保留时间一致。然后用肠道菌群体外大量培养曲克芦丁,将得到的肠道菌群培养液进行萃取后分离纯化,得到一纯代谢产物,并将该物质进行核磁共振、质谱分析,确定其结构和分子式,为曲克芦丁脱去芸香糖得到的苷元,即3′,4′,7-三(-o-羟乙基)槲皮素,分子式为c12h22o10。2.生物样品中曲克芦丁和曲克芦丁苷元定量分析方法的建立本文采用液液萃取的方法提取大鼠肠道菌群样品、尿液、粪便和胆汁中的曲克芦丁和曲克芦丁苷元,建立了曲克芦丁和曲克芦丁苷元的hplc分析方法。色谱柱为shim-packodsc18柱(250mm×4.6mm,5μm);流动相为甲醇-1.7%冰醋酸并采取线性梯度洗脱的方式(0~15min,41:59~41:59;15~30min,41:59~65:35;30~40min;65:35~41:59);流速为1.0ml·min-1;检测波长为350nm;柱温为40℃;内标是芦丁。结果表明,内源性杂质对曲克芦丁和曲克芦丁苷元的测定无干扰。采用本文建立的生物样品预处理方法和hplc法分离测定生物样品中的曲克芦丁和曲克芦丁苷元的含量,生物样品的预处理方法具有很高的回收率,也具有良好的分离选择性。本方法中的线性范围、灵敏度、特异性、精密度和准确度均符合体内药物分析的要求,并且该方法法已经成功地运用于肠道菌群、尿液、粪便和胆汁中曲克芦丁和曲克芦丁苷元的含量检测。3.曲克芦丁和曲克芦丁苷元在大鼠体内和体外的代谢与排泄研究采用体外培养大鼠肠道细菌和腹腔注射给药的的方式代谢曲克芦丁,收集肠道菌群孵育液、尿液、粪便和胆汁样品,并采用hplc法对曲克芦丁及其代谢物曲克芦丁苷元的代谢情况进行研究。结果表明:曲克芦丁在大鼠肠道细菌中的代谢速率较快,曲克芦丁在前6h减少了77、13%,前12h减少了99、14%,24h代谢完全。代谢产物-曲克芦丁苷元自孵育1h时即产生,随后逐渐增多,6h时浓度最大,随后缓慢减少。曲克芦丁在1h时尚有较高浓度,随后浓度迅速下降,12 h时浓度基本降至谷底,说明曲克芦丁在肠道菌群的作用下转化迅速,糖苷键迅速被裂解,绝大部分转化为曲克芦丁苷元。SD大鼠腹腔给药曲克芦丁后,尿液中的排泄结果显示,0~24h的排泄量占总给药量的16.17%。粪便中的排泄结果显示,0~24h的排泄量占总给药量的0.54%。0~24h内从胆汁中排泄的曲克芦丁占总给药量的58.94%。0~24h内粪便中曲克芦丁苷元的产生量占总给药量的22.69%。
[Abstract]:Troxerutin is a semi synthetic flavonoid rutin natural existence made by hydroxyethylation, is the most important cardiovascular drugs Venoruton in composition, and has the role of vitamin P4 is known as vitamin P4, chemical name: 3 ', 4', 7- three hydroxyethyl rutin. With agglutination inhibition of red blood cells and platelets; to prevent thrombosis; at the same time to protect the vascular endothelial cells, decrease the permeability of the blood vessel wall and eliminate edema; anti inflammation; anti chemical liver injury and other pharmacological effects. Clinically to treat cerebral infarction and stroke sequelae, chronic venous insufficiency, varicose veins, hemorrhoids, long the flight distance of microvascular disease. Although troxerutin has been used in clinic for many years, but the adverse reaction reports has gradually increased, and the troxerutin in vivo metabolism reported less, only some of the customs In troxerutin pharmacokinetic studies in humans and rats, for troxerutin without complete understanding in the metabolic situation. In order to study the effect of troxerutin in rats in vivo and in vitro metabolic transformation, this thesis deeply researches on the troxerutin in vivo and in vitro metabolism in rats the main contents are summarized as follows: 1. troxerutin metabolites of Sprague-Dawley rats by intraperitoneal injection of troxerutin, collecting 24h urine and feces samples, samples treated with HPLC detection, the results show that the drug had no significant difference with the blank urine urine, but detected obvious metabolites the peak in fecal samples. The intestinal flora in vitro cultured troxerutin, the intestinal flora analysis of HPLC with training samples after treatment, also detected a significant metabolite peaks and their retention The same retention time and time detected in the faeces. Then the intestinal flora in vitro troxerutin, the intestinal bacteria culture fluid were obtained after extraction separation and purification, a pure metabolite, and the substance of NMR, mass spectrometry analysis, determine its structure and molecular formula, remove glycoside rutinose obtained for troxerutin, 3 ', 4', three 7- (-o- hydroxyethyl quercetin), molecular formula c12h22o10.2. in biological samples and troxerutin troxerutin aglycone quantitative analysis method is established in this paper by using liquid-liquid extraction and extraction of rat intestinal bacteria samples, urine and feces. In the bile and troxerutin troxerutin aglycone, established Troxerutin and troxerutin aglycone HPLC analysis method. The shim-packodsc18 column (250mm * 4.6mm, 5 m); the mobile phase was methanol -1.7% acetic acid and linear gradient elution The way (0~15min, 41:59~41:59; 15~30min, 41:59~65:35; 30~40min; 65:35~41:59); the flow rate was 1.0ml - min-1; the detection wavelength was 350nm; the column temperature is 40 DEG C; the internal standard is rutin. The results showed that endogenous impurities in troxerutin Troxerutin and determination of rutin glycoside without interference. Using the biological samples the pretreatment method and HPLC method for the determination of biological samples in troxerutin Troxerutin and aglycones in isolation, pretreatment methods of biological samples with high recovery rate, but also has good selectivity. The linear range of this method, the sensitivity, specificity, precision and accuracy are character analysis of medicine in the body, and this method has been successfully applied to the intestinal flora, urine, feces and bile for measuring the content of.3. and troxerutin in troxerutin aglycone and troxerutin troxerutin aglycone in rats in vivo and in vitro generation Xie and excretion study using cultured rat intestinal bacteria and intraperitoneal injection of the metabolism of troxerutin in vitro intestinal flora, collecting incubation fluid, urine, feces and bile samples, and uses the HPLC method to troxerutin Troxerutin and its metabolite glycoside metabolism were studied. The results showed that: the metabolic rate of troxerutin on intestinal bacteria in rats rapidly, troxerutin reduced 77,13% in the first 6h, 12h decreased 99,14% and 24h metabolism completely. Metabolite of troxerutin aglycone from incubated 1H is generated, then gradually increased, when the concentration of 6h, and then decreased slowly. Troxerutin with high concentration of 1H in fashion, then the concentration decreased rapidly, 12 h concentration dropped to the bottom, indicating the rapid transformation of troxerutin in intestinal flora under the action of the glycosidic bond cleavage was quickly, most converted to troxerutin aglycone.SD rats intraperitoneal The medicine of troxerutin, urine excretion showed that excretion of 0~24h total dose of 16.17%. in fecal excretion showed that excretion of 0~24h total dose of 0.54%.0~24h in the bile of Troxerutin and total dosage of 58.94%.0~24h in feces of troxerutin aglycone which accounted for the total dosage of 22.69%.

【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R965

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