本文關(guān)鍵詞： 曲克蘆丁 代謝產(chǎn)物 曲克蘆丁苷元 腸道菌群 高效液相色譜　出處：《安徽醫(yī)科大學(xué)》2014年碩士論文　論文類(lèi)型：學(xué)位論文

【摘要】：曲克蘆丁是天然存在的蘆丁經(jīng)羥乙基化制成的半合成的黃酮類(lèi)化合物,是心腦血管藥維腦路通中最重要的成分,又因具有維生素P4樣的作用而被稱(chēng)作維生素P4,化學(xué)名為3’,4’,7-三羥乙基蘆丁。具有抑制紅細(xì)胞和血小板的凝集作用;預(yù)防血栓的形成;同時(shí)能夠保護(hù)血管內(nèi)皮細(xì)胞,降低血管壁的通透性和消除水腫;抗炎;抗化學(xué)性肝損傷等藥理作用。臨床上常用來(lái)治療腦梗塞及中風(fēng)后遺癥、慢性靜脈功能不全、靜脈曲張、痔瘡、長(zhǎng)距離飛行微血管等疾病。盡管曲克蘆丁已經(jīng)在臨床上使用多年,但是有關(guān)其不良反應(yīng)的報(bào)導(dǎo)卻逐漸增多,并且目前有關(guān)曲克蘆丁在體內(nèi)的代謝報(bào)道較少,僅有一些關(guān)于曲克蘆丁在人和大鼠體內(nèi)的藥代動(dòng)力學(xué)研究,對(duì)于曲克蘆丁在體內(nèi)的代謝情況沒(méi)有完全的認(rèn)識(shí)。為了研究曲克蘆丁在大鼠體內(nèi)和體外的代謝轉(zhuǎn)化情況,本文對(duì)曲克蘆丁在大鼠體內(nèi)和體外代謝的情況進(jìn)行了較為深入的研究。主要研究?jī)?nèi)容概括如下:1.曲克蘆丁的代謝產(chǎn)物研究Sprague-Dawley大鼠腹腔注射曲克蘆丁后,收集24h的尿液和糞便樣品,樣品處理后用HPLC檢測(cè),結(jié)果表明:給藥尿液與空白尿液沒(méi)有明顯差別,但在糞便樣品中檢測(cè)到一明顯的代謝產(chǎn)物峰。采用腸道菌群體外培養(yǎng)法培養(yǎng)曲克蘆丁,將得到的腸道菌群培養(yǎng)樣品樣品處理后用HPLC分析,也檢測(cè)到一明顯的代謝產(chǎn)物峰,且其保留時(shí)間與糞便中檢測(cè)到的保留時(shí)間一致。然后用腸道菌群體外大量培養(yǎng)曲克蘆丁,將得到的腸道菌群培養(yǎng)液進(jìn)行萃取后分離純化,得到一純代謝產(chǎn)物,并將該物質(zhì)進(jìn)行核磁共振、質(zhì)譜分析,確定其結(jié)構(gòu)和分子式,為曲克蘆丁脫去蕓香糖得到的苷元,即3′,4′,7-三(-o-羥乙基)槲皮素,分子式為c12h22o10。2.生物樣品中曲克蘆丁和曲克蘆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腸道菌群樣品、尿液、糞便和膽汁中的曲克蘆丁和曲克蘆丁苷元,建立了曲克蘆丁和曲克蘆丁苷元的hplc分析方法。色譜柱為shim-packodsc18柱(250mm×4.6mm,5μm);流動(dòng)相為甲醇-1.7%冰醋酸并采取線性梯度洗脫的方式(0~15min,41:59~41:59;15~30min,41:59~65:35;30~40min;65:35~41:59);流速為1.0ml·min-1;檢測(cè)波長(zhǎng)為350nm;柱溫為40℃;內(nèi)標(biāo)是蘆丁。結(jié)果表明,內(nèi)源性雜質(zhì)對(duì)曲克蘆丁和曲克蘆丁苷元的測(cè)定無(wú)干擾。采用本文建立的生物樣品預(yù)處理方法和hplc法分離測(cè)定生物樣品中的曲克蘆丁和曲克蘆丁苷元的含量,生物樣品的預(yù)處理方法具有很高的回收率,也具有良好的分離選擇性。本方法中的線性范圍、靈敏度、特異性、精密度和準(zhǔn)確度均符合體內(nèi)藥物分析的要求,并且該方法法已經(jīng)成功地運(yùn)用于腸道菌群、尿液、糞便和膽汁中曲克蘆丁和曲克蘆丁苷元的含量檢測(cè)。3.曲克蘆丁和曲克蘆丁苷元在大鼠體內(nèi)和體外的代謝與排泄研究采用體外培養(yǎng)大鼠腸道細(xì)菌和腹腔注射給藥的的方式代謝曲克蘆丁,收集腸道菌群孵育液、尿液、糞便和膽汁樣品,并采用hplc法對(duì)曲克蘆丁及其代謝物曲克蘆丁苷元的代謝情況進(jìn)行研究。結(jié)果表明:曲克蘆丁在大鼠腸道細(xì)菌中的代謝速率較快,曲克蘆丁在前6h減少了77、13%,前12h減少了99、14%,24h代謝完全。代謝產(chǎn)物-曲克蘆丁苷元自孵育1h時(shí)即產(chǎn)生,隨后逐漸增多,6h時(shí)濃度最大,隨后緩慢減少。曲克蘆丁在1h時(shí)尚有較高濃度,隨后濃度迅速下降,12 h時(shí)濃度基本降至谷底,說(shuō)明曲克蘆丁在腸道菌群的作用下轉(zhuǎn)化迅速,糖苷鍵迅速被裂解,絕大部分轉(zhuǎn)化為曲克蘆丁苷元。SD大鼠腹腔給藥曲克蘆丁后,尿液中的排泄結(jié)果顯示,0~24h的排泄量占總給藥量的16.17%。糞便中的排泄結(jié)果顯示,0~24h的排泄量占總給藥量的0.54%。0~24h內(nèi)從膽汁中排泄的曲克蘆丁占總給藥量的58.94%。0~24h內(nèi)糞便中曲克蘆丁苷元的產(chǎn)生量占總給藥量的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%.

【學(xué)位授予單位】：安徽醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類(lèi)號(hào)】：R965

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