葛根、葛花及大豆中异黄酮类化合物的分离提取及体外活性研究

发布时间：2018-04-22 06:03

本文选题：葛根 + 葛花　；参考：《长春师范大学》2017年硕士论文

【摘要】：本文以葛根、葛花和大豆为研究对象,利用高速逆流色谱(High-speed Counter-current Chromatography,HSCCC)和半制备高效液相色谱(Semi-preparative High-performance Liquid Chromatography,SPHPLC)对中药材中的异黄酮类成分进行提取分离,采用超滤及PC12细胞技术对体外活性进行研究。葛根为药食两用植物,是野葛的干燥根。本文应用加热回流技术对葛根提取工艺进行研究,采用单因素实验结合正交实验优化提取工艺。确定最佳提取工艺为:提取溶液为80%乙醇溶液、提取次数2次、料液比为l:20,提取时间120 min,获得了葛根异黄酮总提取物。以大豆苷元的提取量为目标,提取率达到1.263 mg/g。以乳酸脱氢酶作为生物靶分子,以超滤质谱技术作为筛选方法筛选酶抑制剂,确定化合物大豆苷元具有较强的酶抑制活性。以高速逆流色谱技术作为分离方法,以石油醚-乙酸乙酯-甲醇-水(5:5:4:5,v:v:v:v)作为溶剂系统,分离得到的大豆苷元纯度为99.2%。葛花为豆科植物野葛的花。本文以加热回流技术对葛花提取工艺进行研究,采用单因素实验结合正交实验优化提取工艺。确定最佳提取工艺为:提取溶液为70%乙醇溶液、提取次数3次、料液比l:15,提取时间120 min,获得了葛花异黄酮总提取物。以鸢尾苷的提取量为目标,提取率达到5.601 mg/g。以乳酸脱氢酶作为生物靶分子,以超滤质谱技术作为筛选方法筛选酶抑制剂,并通过MTT比色法对分离得到的成分进行体外抗脑卒中活性测定,确定化合物鸢尾黄素-7-O-木糖葡萄糖苷(tectorigenin-7-O-xylosylglucoside)、鸢尾苷(tectoridin)、鸢尾苷元(tectorigenin)具有较强的酶抑制活性。利用半制备高效液相色谱对上述三种乳酸脱氢酶抑制剂进行分离,共分离得到3个化合物,纯度分别为鸢尾黄素-7-O-木糖葡萄糖苷90.1%,鸢尾苷93.4%,鸢尾苷元95.5%。通过MTT实验进一步验证了葛花总提取物可以保护MPP+损伤的PC 12细胞。大豆为豆科大豆属草本植物。本文利用加热回流技术对大豆中的异黄酮类成分进行提取,以乳酸脱氢酶作为生物靶分子,以超滤质谱技术作为筛选方法筛选酶抑制剂,对其活性进行评价。确定大豆中有六种化合物为乳酸脱氢酶抑制剂,其抑制率分别为:乙酰大豆黄素糖苷(48.36%),丙二酰染料木素糖苷(47.14%),大豆苷(41.92%),黄豆黄苷(39.13%),染料木苷(28.41%),乙酰黄豆黄素糖苷(24.21%)。通过MTT实验进一步验证了大豆总提取物对缺血性脑损伤有保护作用。
[Abstract]:In this paper, the isoflavones from Radix Puerariae, Radix Puerariae and Soybean were extracted and separated by High-speed Counter-current Chromatography-HSCCC and semi-preparative High-performance Liquid Chromatography-SPHPLCc. In vitro activity was studied by ultrafiltration and PC12 cell technology. Pueraria lobata is a medicinal and food plant, is the dry root of Pueraria lobata. In this paper, the extraction technology of Pueraria lobata was studied by heating reflux technique, and the extraction process was optimized by single factor experiment and orthogonal experiment. The optimum extraction technology was determined as follows: the extraction solution was 80% ethanol solution, the extraction times were 2 times, the ratio of material to liquid was 1: 20, the extraction time was 120 min, and the total extract of isoflavone from Pueraria lobata was obtained. The extraction rate of daidzein was 1.263 mg / g. Lactate dehydrogenase (LDH) was used as biological target molecule and ultrafiltration mass spectrometry (UF / MS) was used to screen enzyme inhibitors. It was confirmed that daidzein had strong enzyme inhibitory activity. The purity of daidzein was 99.2 using high speed countercurrent chromatography and petroleum ether-ethyl acetate methanol-water 5: 5: 5: 5: 5: 5: v: v: v) as solvent system. Pueraria lobata is the flower of legume. In this paper, the extraction technology of Pueraria lobata was studied by heating reflux technique, and the single factor experiment and orthogonal experiment were used to optimize the extraction process. The optimum extraction technology was determined as follows: the extraction solution was 70% ethanol solution, the extraction times were 3 times, the ratio of material to liquid was 1: 15, the extraction time was 120 min, and the total extract of isoflavone from Pueraria lobata was obtained. The extraction rate of Iris was 5.601 mg / g. Lactate dehydrogenase (LDH) was used as biological target molecule, ultrafiltration mass spectrometry (UF MS) was used as screening method to screen enzyme inhibitors, and the antistroke activity of the isolated components was determined by MTT colorimetry in vitro. It was confirmed that the compounds of Iridin-7-O- xylosylglucoside, tectorigenin-7-O-xylosylglucoside, Iris tectoridinanum and tectorigenin have strong enzyme inhibitory activity. Three kinds of lactate dehydrogenase inhibitors were separated by semi-preparative high performance liquid chromatography. The purity of the three compounds were Iris flavin-7-O-xylose glucoside 90.1, iris 93.4 and iris 95.5 respectively. It was further proved by MTT experiment that total extract of Pueraria lobata could protect PC12 cells damaged by MPP. Soybean is a kind of herbaceous plant of Leguminosae. In this paper, isoflavones from soybean were extracted by heating reflux technique. Lactate dehydrogenase (LDH) was used as biological target molecule and ultrafiltration mass spectrometry (UF MS) was used to screen enzyme inhibitors. Six compounds in soybean were identified as lactate dehydrogenase inhibitors, the inhibition rates were: acetyldaidzein 48.36, malonyl genistein 47.1414, daidzein 41.92, daidzein 39.13, genistein 28.41, acetyl daidzein 24.2121. The inhibitory rates were as follows: acetyldaidzein 48.36, malonyl genistein 47.1421, daidzein 41.92, daidzein 39.13, genistein 28.41, acetyldaidzein 24.2121. The protective effect of total soybean extract on ischemic brain injury was further verified by MTT test.
【学位授予单位】：长春师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284;R285;O652.63

【参考文献】