【作者】 时晓磊；

【导师】 张寒琦； 金永日； 李绪文；

【作者基本信息】 吉林大学 ， 分析化学， 2010， 博士

【摘要】 本论文以天然产物中的活性成分作为研究对象,主要包括三部分内容:一、总结了多种天然产物中活性成分的提取方法,包括传统提取方法和近代常用提取方法,重点介绍了基质固相分散法,对基质固相分散法的原理,特点,影响因素以及应用做了一定的描述,尤其是此法在天然产物活性成分的提取方面的应用进行了比较详尽的介绍;总结了天然产物中的活性成分与生物大分子之间相互作用的研究方法,重点介绍了常规荧光光谱法、同步荧光扫描法、三维荧光光谱法、圆二色性光谱法和紫外可见光谱法;二、运用基质固相分散法提取活性成分,采用高效液相色谱法分离测定,对基质固相分散法的各种影响因素,例如分散剂、淋洗剂、净化剂和洗脱剂等进行了选择,在优化的条件下对人参叶中的八种皂苷类成分和桃儿七根中的四种木脂素类成分进行了提取,与传统的回流法相比较,取得了较好的结果;三、对黄酮类化合物与牛血清白蛋白之间的相互作用进行了表征,采用荧光猝灭法,研究了水红花子中花旗松素和狗枣猕猴桃茎叶中的三种黄酮苷与牛血清白蛋白之间的相互作用,对他们的结合常数和结合位点数进行了计算,计算了反应过程中的热力学常数,对其相互作用的作用力类型进行了判断,运用同步荧光扫描法,三维荧光光谱法以及圆二色性光谱法对蛋白质的三维结构进行了研究,对共存离子对黄酮类化合物和牛血清白蛋白之间的相互作用的影响也进行了研究,得到了一些有意义的结果,对黄酮类化合物与血清白蛋白之间的相互作用方法的发展是有益的更多还原

【Abstract】 With the development of science, the study on the Chinese traditional herbals has attracted a lot of attentions in recent years. The studies on the extraction, separation and characterization of the active components of the herbals have become more and more important. In this thesis, the extraction of ginsenosides and lignans, and the interaction between flavonoids and BSA have been studied.The matrix solid phase dispersion (MSPD) was applied to the extraction of the active components from herbals. The optimized conditions of MSPD were selected and the proposed method was applied to the extraction of ginsenosides and lignans. The reflux extraction was used for comparison. When the diatomaceous earth was used as dispersant, the ratio of sample and dispersant was 1:6, 75% methanol was used as elution and the volume was 9 mL, the extraction yields of Rg1,Re,F3,Rc,Rb2,F1,Rd and F2 obtained by MSPD were 14.20,53.07,5.02,5.19,6.57,3.72,12.20 and 3.55 mg·g-1 and those obtained by reflux extraction were 12.88,50.34,4.82,5.42,6.37,3.80,11.47 and 3.48 mg·g-1. The results indicate that except for the extraction yields of Rc and F1, the recoveries are in the range of 80.23-109.49%. In conclusion, the MSPD method is more appropriat than reflux extraction on the extraction of ginsenosides. The extraction yields of the podophyllotoxin, 4’-demethylpodophyllotoxin, podophyllotoxin and isopicropodophylloneas from the roots of Sinopodophyllum emodi Wall. are 45.45,6.30,14.84 and 0.60 mg·g-1 when the diatomaceous earth was used as dispersant and methanol was used as elution solvent. Therefore, the extraction yields for four lignans obtained by reflux extraction are 32.71,4.47,17.55 and 0.38 mg·g-1. So the MSPD method is better than reflux extraction. In the experimental, when hexane was used as washing agent, the interfering substance was not removed and when the clean-up adsorbent was used, the target compounds can be adsorbed. So the hexane and the clean-up adsorbent were not used.The characterization of the interaction between flavonoids and BSA was studied. The interaction between taxifolin and BSA was also studied. The fluorescence quenching for taxifolin and BSA was static quenching. The binding constants and binding sites were also studied, the values of KA are 2.78×104and 3.76×104 L·mol-1 at 295 K and 310 K, respectively. The effect of temperature on the binding constants is not significant. The numbers of binding sites n are 1.01 and 1.04, in the absence of common ions. The values of KA at 295 K decrease in the presence of the ions Cu2+, Mg2+and Al3+, which means that these ions can weaken the binding between taxifolin and BSA. There is no significant effects on the binding in the presence of F-. But Zn2+, NO3ˉand SO42- can enhance the binding. All the ions can enhance the binding at 310 K. According to values of the thermodynamic parameters, hydrophobic force plays a major role in the interaction. The energy transfer parameters were studied in the absence and presence of some common ions,and the experimental results indicate that these ions almost do not affect on the distance between BSA and taxifolin.Three kinds of flavonoids obtained from the leaves of Actinidia kolomikta(Rupr.et Maxim.)Planch, including kaempferol - 3 - O -α- L - rhamnopyranosyl - (1→3) -α- L - rhamnopyranosyl - (1→6) -β-D-galactopyranoside (drug 1),Kaempfol-7-O-rhamnosyl- 3-O-rutinoside(drug 2) and Kaempferide-7-O-(4″-O- acetylrhamnosyl) -3-O-rutinoside (drug 3), are studied. The static quenching occurs in the interaction between flavonoids and BSA. The binding constants and binding sites were also studied. The binding constants were changed accordance to the following orders, drug 1 > drug 3 > drug 2 at 295 K, which means that drug 1 has the strongest ability to bind with BSA and drug 2 is the weakest. However, at 310 K the binding constants ranked in descending order are as follows: drug 3 > drug 2 > drug 1, which means that the complex formed from drug 3 and BSA is most stable in higher temperature, and the complex formed from drug 1 is weakest. All of these have a great relation with the structure of the flavonoids and the interaction forces between falvanoids and BSA. In the BSA-drug 1 system, hydrogen binding and van der Waals force play a major role, which make the banding constant KA of the reaction of BSA and drug 1 be the largest one in the KA values of three reactions. The result is also related to the structure of drug 1. There are three -OH in ring A and B, so the drug 1 has more opportunities to bind with BSA by hydrogen binding. However, in BSA-drug 2 and BSA-drug 3 system hydrophobic interaction plays an important role, so the values of KA for drug 2 and drug 3 are related to the polarity of flavonoids. It is easy for the flavonoids with low polarity to get close to the hydrophobicity of BSA. It is obviously that the polarity of drug 3 is lower than that of drug 2, so KA of BSA-drug 2 is lower than that of BSA-drug 3. Synchronous fluorescence, three-dimensional fluorescence and CD spectrometry were applied to the investigation of the conformation of BSA. The results were shown that the conformation of BSA was changed in the presence of flavonoids.更多还原