野马追抗炎活性部位筛选及制备工艺研究

发布时间：2018-03-06 15:38

本文选题：野马追　切入点：抗炎活性　出处：《南京中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：江苏省道地药材野马追为菊科泽兰属植物轮叶泽兰(Eupatorium lindleyanun DC.)的干燥地上部分,具有清热解毒、化痰止咳、利尿消肿、降血压等功效,临床上主要用于治疗慢性气管炎、支气管炎、高血压,其糖浆剂为治疗上呼吸道炎症的常用药物。野马追化学成分复杂多样,主要有黄酮、倍半萜、挥发油、香豆素等,但其药效物质基础尚未阐明。为了探索野马追抗炎活性物质基础,本文以《中国药典》收载的野马追为研究对象,对其抗炎活性、化学成分进行深入系统研究,并对其抗炎效应部位的提取制备工艺进行考察,以期为合理有效应用野马追药材提供科学依据。所做工作如下:1.野马追抗炎活性部位的筛选:对野马追药材进行提取及部位分离,通过二甲苯诱导小鼠耳廓肿胀模型和角叉菜胶致小鼠足肿胀模型对野马追水煎液和AB-8大孔树脂不同洗脱部位进行抗炎药效筛选。实验结果表明,野马追水煎液和85%乙醇洗脱部位可明显减少二甲苯诱导的耳廓肿胀小鼠的两耳片重量之差,抑制局部血管扩张;抑制角叉菜胶所致足肿胀小鼠的后足跖肿胀度,减轻急性炎症反应过程中的组织液渗出和炎性肿胀。流出液、25%乙醇洗脱部位及25%乙醇(含0.05%NaOH)洗脱部位未显示出抗炎活性。由此可知野马追水煎液具有优良的抗炎活性,大孔树脂85%乙醇洗脱部位为其主要的抗炎活性部位。2.单体化合物分离和结构鉴定:采用大孔树脂柱色谱、硅胶中压柱色谱、C18中压柱色谱、制备高效液相色谱(PHPLC)等技术从野马追中分离获得了 18个单体化合物,运用NMR,MS等波谱方法并结合已有文献对分离得到的18个化合物进行结构鉴定,分别为:eupalinolid eL(1)、eupalinolide M(2)、eupalinolide A(3)、eupalinolide B(4)、eupalinolide C(5)、3β-acetoxy-8β-(4'-hydroxytigloyloxy)-14-hydroxycostunolide(6)、eupalinolide K(7)、2α-hydroxyeupatolide(8)、eupalinolide H(9)、zhebeiresinol(10)、medioresinol(11)、salicifoliol(12)、5-hydroxy-3,4-dimethy-5-pentyl-2(5H)-furanone(13)、3-(2-hydroxy-4-methylbutyl)-4-methoxyacetophenone(14)、coniferyl alcohol(15)、p-hydroxybenzaldehyde(16)、3-(hydroxymethyl)-1,14,15-trihydroxy-7,11,15-trimethyl-2,6,10-hexadecatrien-13-acetate(17)、3-(hydroxymethyl)-1,13,15-trihydroxy-7,11,15-trimethyl-2,6,10-hexadecatrien-14-acetate(18)。其中化合物1～2为新的倍半萜内酯类化合物,化合物10～16为首次从泽兰属植物中分离得到的化合物。3.野马追化合物抗炎活性研究:采用脂多糖(LPS)诱导小鼠巨噬细胞RAW264.7建立体外炎症模型,并通过ELISA法检测各化合物对肿瘤坏死因子(TNF-α)及白细胞介素6(IL-6)表达的影响。结果发现具有内酯环结构的倍半萜类化合物(1～9)和双环氧木脂素类化合物(10～12)对LPS诱导RAW264.7释放的TNF-α和IL-6具有不同程度的抑制作用,且呈现出一定剂量依赖性,为野马追主要的抗炎活性物质。其中,化合物1对TNF-α的抑制作用最强,化合物5对IL-6的抑制作用最强。4.大孔树脂-CO2超临界萃取联用精制野马追倍半萜内酯的工艺研究:以野马追内酯A、野马追内酯B(EA、EB)为指标,通过建立正交试验表确定了野马追倍半萜内酯的最佳提取方法和条件:以60%乙醇为提取溶剂回流提取2次,第一次加14倍量醇,回流1.5 h,第二次加10倍量醇,回流1 h;通过动态吸附及解吸附实验,考察优选了野马追药材中倍半萜内酯大孔树脂吸附精制工艺:选用AB-8为精制用大孔吸附树脂,上样量(生药量)与干树脂量比为4.2 g/g,洗脱剂为70%乙醇,洗脱体积4 BV;在此基础上,进一步采用响应面法优选了 C02超临界萃取工艺:夹带剂(无水乙醇)用量为30%,萃取压力35 MPa,萃取温度45℃,萃取时间2h,CO2流速40mL/min。以优选的大孔树脂吸附结合超临界CO2萃取工艺制备获得的野马追倍半萜内酯提取物中野马追内酯A、B总含量由药材中0.58%提升到39.07%。该联用方法能有效提升活性成分含量,且工艺简单可行。
[Abstract]:Jiangsu province geoherbs yemazhui for Eupatorium genus Ye Zelan (Eupatorium lindleyanun DC.) round the dry ground part, with detoxification, eliminating phlegm and relieving cough, diuretic swelling, bloodpressur, clinically used to treat chronic tracheitis, bronchitis, hypertension, the syrup is commonly used drugs for the treatment of upper respiratory tract inflammation. Yemazhui chemical composition is complex, mainly flavonoids, sesquiterpene coumarin, volatile oil, etc., but the material basis of its efficacy has not been elucidated. In order to explore the anti-inflammatory activity of yemazhui material basis, based on the China Pharmacopoeia > < wild collected after as the research object, for its anti-inflammatory activity, chemical composition in-depth research system, and extraction system on the anti-inflammatory effect of site preparation were investigated, in order to provide a scientific basis for reasonable application of eupatoriumlindleyanum. The main work is as follows: 1. yemazhui anti-inflammatory activity Department The screening of eupatoriumlindleyanum for extraction and separation of parts, induced mouse ear edema model and carrageenan induced paw swelling model of yemazhui Decoction and AB-8 macroporous resin for different parts. Through the screening of anti-inflammatory efficacy of elution of xylene. The experimental results show that the wild horses after water extract and 85% ethanol elution two parts can significantly reduce the weight of the lug of the xylene induced ear swelling in mice, inhibit local vasodilation; inhibition of carrageenan induced paw edema in mice after swelling, reduce the tissue fluid in the process of acute inflammation infiltration and inflammatory swelling. The outflow of liquid, 25% ethanol elution and 25% ethanol (containing 0.05%NaOH) elution showed no anti-inflammatory activity. Therefore yemazhui decoction has good anti-inflammatory activity, macroporous resin and 85% ethanol elution parts for the main parts of.2. single compound anti-inflammatory activity The separation and structure identification by macroporous resin column chromatography, silica gel column chromatography medium, C18 medium pressure column chromatography, preparative high performance liquid chromatography (PHPLC) technology from yemazhui isolated 18 compounds, the use of NMR, MS and other spectroscopic methods and the combination of 18 compounds obtained from the existing literature on the structures were identified, respectively: eupalinolid eL (1), eupalinolide M (2), eupalinolide A (3), eupalinolide B (4), eupalinolide C (5), 3 beta beta -acetoxy-8 (4'-hydroxytigloyloxy) -14-hydroxycostunolide (6), eupalinolide K (7), 2 (8), alpha -hydroxyeupatolide eupalinolide H (9), zhebeiresinol (10), medioresinol (11), salicifoliol (12), 5-hydroxy-3,4-dimethy-5-pentyl-2 (5H) -furanone (13), 3- (2-hydroxy-4-methylbutyl) -4-methoxyacetophenone (14), coniferyl alcohol (15), p-hydroxybenzaldehyde (16), 3- (hydroxymethyl) -1,14,15-trihydrox Y-7,11,15-trimethyl-2,6,10-hexadecatrien-13-acetate (17), 3- (hydroxymethyl) -1,13,15-trihydroxy-7,11,15-trimethyl-2,6,10-hexadecatrien-14-acetate (18). Compound 1~2 is a sesquiterpene lactone compounds, 10~16 compounds of anti-inflammatory activity of compounds.3. yemazhui were isolated from the plant for the first time: the use of Eupatorium lipopolysaccharide (LPS) induced by RAW264.7 in mouse macrophages in vitro and the inflammatory model. Through the ELISA assay of the compounds on tumor necrosis factor alpha (TNF- alpha) and interleukin 6 (IL-6). The results showed that the expression of sesquiterpene compound having a lactone ring structure (1~9) and double epoxy lignans (10~12) on LPS induced RAW264.7 release of TNF- alpha and IL-6 different degrees of inhibition, and showed a dose dependent anti-inflammatory activity after the main wild horse Quality. Among them, 1 compounds of TNF- alpha had the strongest inhibitory effect, research process combined with refined yemazhui sesquiterpene lactone had the strongest inhibitory effect of.4. macroporous resin -CO2 on compound 5 IL-6 supercritical extraction: yemazhui lactone A yemazhui (EA, EB) B lactone as the index, through the construction of attention orthogonal experiment table to determine the optimum extraction conditions and methods of yemazhui sesquiterpene lactones with 60% ethanol as the extraction solvent to reflux for 2 times, the first time with 14 times the amount of alcohol, refluxing 1.5 h, second times with 10 times the amount of alcohol, refluxing 1 h; the dynamic adsorption and desorption experiments, the optimal inspection purification of sesquiterpene lactone macroporous resin eupatoriumlindleyanum: AB-8 was selected as refined by macroporous adsorption resin, the sample volume (amount) and dry resin volume ratio is 4.2 g/g, eluted with 70% ethanol elution volume 4 BV; on this basis, further optimized by response surface method C02 supercritical extraction Process: modifier (ethanol) was 30%, the extraction pressure of 35 MPa, extraction temperature 45, extraction time 2h, CO2 flow rate of 40mL/min. with macroporous adsorption resin were obtained with the optimization of supercritical CO2 extraction process of yemazhui sesquiterpene lactone in the extract of yemazhui ester A, B content from 0.58% to 39.07%. in traditional Chinese medicine combined with the method can effectively improve the content of active ingredients, and the process is simple and feasible.

【学位授予单位】：南京中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R284

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