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新疆药用植物沙枣活性多肽的提取分离与制备工艺研究
艾合米丁·外力
学位类型硕士
导师阿布力米提·伊力、阿吉艾克拜尔·艾萨
2016-06-03
学位授予单位中国科学院大学
学位授予地点北京
学位专业有机化学
关键词沙枣 多肽提取分离 氨基酸序列 生物活性 提取工艺
摘要

沙枣在我国主要分布在西北各省区和内蒙古西部;沙枣,别名:桂香柳、七里香等,拉丁文名Elaeagnus angustifolia L,英文名为Oleaster,维吾尔名为结格代,胡颓子科、胡颓子属落叶乔木或小乔木。据新疆药用植物志记载,中国有狭叶沙枣、准噶尔沙枣、砂生沙枣和尖果沙枣等四个品种;其中准噶尔沙枣和砂生沙枣为新疆特有的品种。沙枣是一种新疆维吾尔族人民喜爱的果品,新疆资源丰富。在维吾尔药中,沙枣果实主要用于治疗脾胃虚弱,头痛,胃病,热性咳嗽,腹泻消化不良,腹泻痢疾等;据《维药志》记载,沙枣花主要用于治疗胸闷气短,胃腹胀痛,咳嗽,食欲不佳以及关节炎和肌无力。目前沙枣在维吾尔医临床上常用于治腹泻痢疾。目前国内对沙枣化学成分研究主要集中在多糖、黄酮类、挥发油、脂肪等。沙枣树种含有树胶,花中含有0.2-0.4%香油,果肉总糖量为66.93%、脂肪2.9%、蛋白质8.5%,干叶中含蛋白质15.7%、脂肪含量为6.5%等。但目前未见蛋白和多肽类成分的研究报道。本论文,以新疆乃至干旱地区特色植物沙枣果实和种子(果仁)为研究对象,以抗菌活性为导向,对其进行系统的多肽类化合物提取分离、纯化和抗菌、抗氧化活性筛选;同时,结合离子交换色谱、双向电泳和液/质联用色谱技术(LC/MS),解释了有效部位的成分,并通过多肽库对比推断了多肽的氨基酸序列,并建立了有效部位的制备工艺。本论文主要研究内容具体如下:1.沙枣果实利用50%乙醇溶液提取,其次利用C18反相硅胶柱(I.D.21.6 x H126, 45 ml)分离SZGS30、SZGS50和SZGS70等三个部位,通过活性筛选和蛋白含量比较,将SZGS50部位进一步使用阴离子交换树脂、HPLC制备色谱分离纯化得到J1、J2和J3等三个多肽部位。活性筛选结果证实,这三个多肽部位具有很好抗菌和清除DPPH自由基的活性;对这三个多肽部位进行清除DPPH自由基活性分析,其结果表明J2多肽部位清除DPPH自由基的作用最强,抑制浓度(IC50)的值为110.7 μg/mL,其余两个多肽部位的IC50值分别为98.3 μg/mL(J3)和87.0μg/mL(J1)。抗菌活性结果显示, J1,J2,J3三个部位都均有Prot. Vulgaris的抑菌作用,抑制圈直径分别为22.0mm(J2)、19.0mm(J1)和20mm(J3),其中J2部位对Prot. Vulgaris抑制作用最好。2.根据沙枣蛋白质的溶解性,沙枣种子(果仁)选用10%醋酸提取溶液提取、丙酮沉淀提取物中的多肽类化合物,测总蛋白含量为72.6%,分别测对白色念珠菌,大肠杆菌和金黄色葡萄球菌的抑制作用,抑菌圈直径分别为7.5mm、8mm和9mm。采用双向电泳和液/质联用色谱技术(LC/MS)相结合的方法分离纯化提取的粗体多肽类物质,共得到了分子量分别为2837Da、4400.5Da、5092.9Da和6551.5Da四个单体多肽,并且根据拟合的二级质谱图成功的鉴定部分肽段序列,序列依次为NH2-YQQEAEGQQR-,NH2-YQEEKQQQR-NH2-VQVVDNYGQSVFDGEVR-和NH2-KQDITITGASTLPGDEVER-。3.最后采用单因素实验(料液比、提取时间、提取次数和乙醇浓度对提取多肽的量的影响)和正交试验,确定了沙枣种子(果仁)多肽的最佳提取工艺路线,通过综合分析正交实验结果,乙醇提取沙枣果仁多肽的最佳工艺条件为提取次数2、提取时间6h、乙醇浓度50%、料液比为1:6。通过对沙枣种子多肽进行抗白色念珠菌,大肠杆菌和金黄色葡萄球菌作用的研究,结果表明利用此提取工艺条件并没有影响所提取的沙枣种子多肽抗菌作用。

其他摘要

Diabetes is an endocrine metabolic disease that threatens human’s health and even life,which was characterized by insulin resistance and accompanied by systemic low-grade inflammation and hyperglycemia, hyperlipidemia, hypertension, atherosclerosis and other symptoms, and mainly caused by obesity. However, anti-inflammatory, hypoglycemic, lipid-lowering and antioxidant are important means of improving insulin resistance.Carthamus tinctorius L. is widely distributed in Xinjiang China. The crop has been well-known as Uighur’s traditional food preservative as well as a remedy for the treatment of gynecological disease. It is also demonstrated that safflower exerts analgesic, anti-inflammatory and antidiabetic activities. The dried flower of the Carthamus tinctorius L. gets its extensive application in traditional Chinese medicine owning to the therapeutic effect on activating blood circulation, eliminating blood stasis and treatment for gynecological disease and cardio-cerebrovascular diseases such as coronary heart disease and hypertension. To research for the water extract of Carthamus tinctorius L. as potent PTP1B inhibitors, the correlation between chromatography fingerprint of Carthamus tinctorius L. and the PTP1B inhibition activity was elaborated and the most effective substances were indicated by this way. The purpose of our research is to observe the effect on the treatment of water extract of Carthamus tinctorius L. to T2DM, and to find the mechanism of water extract of Carthamus tinctorius L..For purpose of further explain the antidiabetic spectrum-effect relationship of the water extract of Xinjiang safflower, the inhibition on PTP1B was tested in vitro, and it was found as an potent PTP1B inhibitors with IC50 in 8.38μg/mL, which could ameliorating the insulin-dependent signaling pathway are potential candidates for the treatment and prevention of diabetes. The correlation between chromatography fingerprint of 10 different Carthamus tinctorius L. and the PTP1B inhibition activity was evaluated to found the most effective compounds in the water extraction, and the content of two marker compounds, which collected in Chinese Pharmacopoeia in Carthamus tinctorius L., were quantitative analysis by HPLC. The efficacy of PTP1B inhibition of the water extract part of Carthamus tinctorius L. resulted in its chemical compositions together. The contribution order for PTP1B inhibition effect was (number of peaks): P5>P6>P3>P1>P7>P4>P8>P2, in which the number of peak 3,6 were identified as 3-O-caffeoylquinic acid,and hydroxysafflor yellow A.In vitro hypoglycemic screening model, the water extract fraction of Carthamus tinctorius L. has strong inhibition effect on PTP1B and α-GAA with IC50 of 2.67 and 365.24 μg/mL, respectively. The water extract part of Carthamus tinctorius L. promoted glucose consumption in 3T3-L1 cells, glucose consumption reached highest in cells when incubating in 25 μg/mL for 15 h. Fed with maximum dose of 26.4 g/kg, no obvious abnormity in Kunming mice, then the water extract part of Carthamus tinctorius L. lavage 4 weeks in diabetes rats induced by feeding high sugar, high fat, diet and injection of low dose streptozotocin (STZ). The water extract part of Carthamus tinctorius L. significantly reduced the blood sugar. It promote the synthesis of glycogen in liver by improving hexokinase activity in liver, and glycogen content increased in liver and muscle of diabetic rats. In addition, the water extract part of Carthamus tinctorius L. lowered the content of free fatty acids in the liver. That means the water extract part of Carthamus tinctorius L. reduced blood sugar, and improve the oxidation activity meantime it can protect the liver from oxidative damage of diabetic rats.Collectively, the water extract part of Carthamus tinctorius L. has obvious hypoglycemic effect in diabetic rats. The possible of mechanism of hypoglycemic could be by inhibit the expression of PTP1B and ERS pathway, improve the liver hexokinase and antioxidant enzymes activity.Key words: diabetes mellitus, insulin resistance, endoplasmic reticulum stress, spectrum-effect relationship, Carthamus tinctorius L.

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条目标识符http://ir.xjipc.cas.cn/handle/365002/4869
专题资源化学研究室
作者单位中国科学院新疆理化技术研究所
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艾合米丁·外力. 新疆药用植物沙枣活性多肽的提取分离与制备工艺研究[D]. 北京. 中国科学院大学,2016.
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