【作者】 尹登科；

【导师】 丁虹； 石米扬；

【作者基本信息】 武汉大学 ， 微生物与生化药学， 2004， 硕士

【摘要】 已经报道阿魏酸为当归注射液的主要活性成分,无论在体外还是体内均能抑制ADP诱导的血小板聚集和释放,本文通过利用不同的pH条件,用醋酸乙酯萃取得到两个萃取组分,通过高效液相试验鉴定其中一个组分不含有阿魏酸,体外血小板凝集抑制试验表明去除阿魏酸的组分仍然具有较强的血小板抑制活性。通过气相色谱/质谱技术对当归注射液中醋酸乙酯萃取组分进行成分分析,色谱条件:色谱柱:All tech AT ^TM-5 （30m×0.2mm×0.2μm）,载气为高纯氦气,流速1mL·min^-1,进样口温度220℃,柱温70℃,保持1min,以10℃·min^-1的速率升温到200℃,保持1min,再以20℃·min^-1升温到270℃,保持5min;质谱检测总离子流。气相色谱保留时间为8.06min的峰对应的质谱图经数据库分析可能是5-羟甲基呋喃甲醛（5-Hydroxymethyl-furfural,HMF）。用对照品进行了气相色谱/质谱和高效液相验证,高效液相色谱条件:采用Agilent Zorbax C₁₈色谱柱（150mm×2.1mm×5μm）,流动相:甲醇（B）,1%冰醋酸（A）,10%/B90%A——90%A10%B（25min）线形洗脱,流速:1mL·min^-1,检测波长:320nm,柱温:30℃。结果同样证实当归注射液中含有HMF。并通过对照品建立了当归注射中HMF的高效液相含量测定方法。 谷胱甘肽硫转移酶是体内重要的一种具有解毒作用的酶,在药物代谢过程中起着重要的作用。本文通过转有质粒p^GEX-5X-1的大肠杆菌BL21（DE3）,通过乳糖诱导表达日本血吸虫谷胱甘肽硫转移酶,超声波破碎粗提谷胱甘肽硫转移酶,将谷胱甘肽硫转移酶固定在戊二醛交联的壳聚糖上,对游离酶和固定酶的最适pH、温度,游离酶和固定化酶对底物1-氯-2,4-二硝基苯（CDNB）和谷胱甘肽（GSH）的亲和力,温度的稳定性进行了研究。结果固定化酶活回收率可达41.6%,最适pH6.5～7.0,最适温度37℃,对底物（CDNB和GSH）的亲和力略有下降,对温度稳定性大大提高。在体外固定化酶有很好的催化解毒作用。更多还原

【Abstract】 It has been reported that ferulic acid is an important active compound in Angelica Injection. Ferulic acid can restrain agglutination of blood platelet induced by ADP whether in vitro or vivo. We use ethyl acetate extract Angelica injection at different pH and get two components. Restrain agglutination of blood platelet in vitro and high performance liquid chromatograph indicate that the component without ferulic acid also can restrain agglutination of blood platelet. Ethyl acetate extract of Angelica injection was analyzed with GC/MS, the gas chromatographic conditions was advanced as follows: All tech AT^TM-5 （30 m×0.2mm×0.2μm） column, high purity helium was used as carries gas with flow rate at lmL.min^-1;Injector tempertature:220 ℃; Column temperature:70℃ keeping for 1 min, from 70℃ to 200℃（10℃.min^-1）, keeping for 1 min, then from 200℃ to 270℃（ 20℃.min^-1） keeping for 5 min; MS detected with total ion monitoring. The MS of retention time of 8.06min on GC is matched with referenced standard HMF. It was demonstrated that HMF existed in Angelica Injection through GC/MS and HPLC methods with reference standard HMF. The condition of HPLC as follows: Agilent Zorbax C₁8 column（150mm×2.1mm×5μm）,the mobile phase: methanol（B）, l%acetic acid（A）,10%B90%A-90%A10%B （25min） ,flow rate:1mL.min^-1, detective wave length:320nm, column temperature: 30℃ and the HPLC method for quantitation of HMF was established.Glutathione S transferase is one of the most important enzymes for detoxifcation and play important role in drug metabolization. We use E. coli BL21（DE3） to express GST of Schistosoma japonicum, and the crude extract of GST was immobilized on Chitosan and cross-linked with gluaraldehyde. Optimal pH, temperature, affinity to substrate l-chloro-2,4-dinnitrobenzen（CDNB） and glutathione（GSH） and thermal stability of immobilized GST were investigated and compared with the free enzyme. The activity recovery of immobilized GST reached 41.6%, optimal pH 6.5⁷.0, optimal temperature 37℃ and the thermal stability of immobilized GST wasenhanced. Immobilized GST showed high efficacy for detoxification in vitro.更多还原