本文選題：野馬追　切入點：抗炎活性　出處：《南京中醫(yī)藥大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：江蘇省道地藥材野馬追為菊科澤蘭屬植物輪葉澤蘭(Eupatorium lindleyanun DC.)的干燥地上部分,具有清熱解毒、化痰止咳、利尿消腫、降血壓等功效,臨床上主要用于治療慢性氣管炎、支氣管炎、高血壓,其糖漿劑為治療上呼吸道炎癥的常用藥物。野馬追化學(xué)成分復(fù)雜多樣,主要有黃酮、倍半萜、揮發(fā)油、香豆素等,但其藥效物質(zhì)基礎(chǔ)尚未闡明。為了探索野馬追抗炎活性物質(zhì)基礎(chǔ),本文以《中國藥典》收載的野馬追為研究對象,對其抗炎活性、化學(xué)成分進行深入系統(tǒng)研究,并對其抗炎效應(yīng)部位的提取制備工藝進行考察,以期為合理有效應(yīng)用野馬追藥材提供科學(xué)依據(jù)。所做工作如下:1.野馬追抗炎活性部位的篩選:對野馬追藥材進行提取及部位分離,通過二甲苯誘導(dǎo)小鼠耳廓腫脹模型和角叉菜膠致小鼠足腫脹模型對野馬追水煎液和AB-8大孔樹脂不同洗脫部位進行抗炎藥效篩選。實驗結(jié)果表明,野馬追水煎液和85%乙醇洗脫部位可明顯減少二甲苯誘導(dǎo)的耳廓腫脹小鼠的兩耳片重量之差,抑制局部血管擴張;抑制角叉菜膠所致足腫脹小鼠的后足跖腫脹度,減輕急性炎癥反應(yīng)過程中的組織液滲出和炎性腫脹。流出液、25%乙醇洗脫部位及25%乙醇(含0.05%NaOH)洗脫部位未顯示出抗炎活性。由此可知野馬追水煎液具有優(yōu)良的抗炎活性,大孔樹脂85%乙醇洗脫部位為其主要的抗炎活性部位。2.單體化合物分離和結(jié)構(gòu)鑒定:采用大孔樹脂柱色譜、硅膠中壓柱色譜、C18中壓柱色譜、制備高效液相色譜(PHPLC)等技術(shù)從野馬追中分離獲得了 18個單體化合物,運用NMR,MS等波譜方法并結(jié)合已有文獻對分離得到的18個化合物進行結(jié)構(gòu)鑒定,分別為: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為新的倍半萜內(nèi)酯類化合物,化合物10～16為首次從澤蘭屬植物中分離得到的化合物。3.野馬追化合物抗炎活性研究:采用脂多糖(LPS)誘導(dǎo)小鼠巨噬細胞RAW264.7建立體外炎癥模型,并通過ELISA法檢測各化合物對腫瘤壞死因子(TNF-α)及白細胞介素6(IL-6)表達的影響。結(jié)果發(fā)現(xiàn)具有內(nèi)酯環(huán)結(jié)構(gòu)的倍半萜類化合物(1～9)和雙環(huán)氧木脂素類化合物(10～12)對LPS誘導(dǎo)RAW264.7釋放的TNF-α和IL-6具有不同程度的抑制作用,且呈現(xiàn)出一定劑量依賴性,為野馬追主要的抗炎活性物質(zhì)。其中,化合物1對TNF-α的抑制作用最強,化合物5對IL-6的抑制作用最強。4.大孔樹脂-CO2超臨界萃取聯(lián)用精制野馬追倍半萜內(nèi)酯的工藝研究:以野馬追內(nèi)酯A、野馬追內(nèi)酯B(EA、EB)為指標(biāo),通過建立正交試驗表確定了野馬追倍半萜內(nèi)酯的最佳提取方法和條件:以60%乙醇為提取溶劑回流提取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;通過動態(tài)吸附及解吸附實驗,考察優(yōu)選了野馬追藥材中倍半萜內(nèi)酯大孔樹脂吸附精制工藝:選用AB-8為精制用大孔吸附樹脂,上樣量(生藥量)與干樹脂量比為4.2 g/g,洗脫劑為70%乙醇,洗脫體積4 BV;在此基礎(chǔ)上,進一步采用響應(yīng)面法優(yōu)選了 C02超臨界萃取工藝:夾帶劑(無水乙醇)用量為30%,萃取壓力35 MPa,萃取溫度45℃,萃取時間2h,CO2流速40mL/min。以優(yōu)選的大孔樹脂吸附結(jié)合超臨界CO2萃取工藝制備獲得的野馬追倍半萜內(nèi)酯提取物中野馬追內(nèi)酯A、B總含量由藥材中0.58%提升到39.07%。該聯(lián)用方法能有效提升活性成分含量,且工藝簡單可行。
[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.

【學(xué)位授予單位】：南京中醫(yī)藥大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R284

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