【作者】 张悦；

【导师】 吴继洲；

【作者基本信息】 华中科技大学 ， 药理学， 2011， 博士

【摘要】 龙里冬青Ilex dunniana Levl和猫儿刺Ilex pernyi Franch为冬青科(Aquifoliaceae)冬青属常绿灌木或小乔木,分布于秦岭以南及长江流域以南各省区。冬青属植物中有30多个品种为传统中药材,具有清热解毒、消炎、镇咳祛痰以及治疗心血管疾病的作用。炎症(inflammation)是一种极为常见而又重要的病理过程,许多常见疾病如疖、肺炎、肠炎、外伤感染及各种传染病都是炎症性疾病。炎症是具有血管系统的活体组织对损伤因子所引发的复杂防御反应,其中心环节是血管反应。绝大多数的心脑血管病包括动脉粥样硬化、脑卒中、高血压、心功能不全等都是慢性炎症反应性疾病。随着自由基医学的发展,对氧化应激反应在各种疾病的发生、发展过程中的认识也不断加深。研究证明氧化应激与癌症、心脑血管疾病、糖尿病及衰老的发生等密切相关。因此,我们认为对能用于治疗上述疾病及相关病征的传统中药,在作用机制不明了的情况下,考察其抗炎作用及抗氧化能力对于深入了解它们的药理作用及机制具有重要意义。本课题以龙里冬青和猫儿刺枝叶作为研究对象,开展了如下研究。一.活性部位的筛选通过小鼠耳肿胀急性炎症和大鼠肉芽肿炎症模型考察两种植物90%乙醇提取物的抗炎作用。结果显示,两种植物提取物均具有一定抑制炎症发生的作用,且呈量效关系。在此基础上,将两者的总提取物分别分离成了四个不同部位,考察它们对细胞中炎症及致炎因子的抑制作用,结果表明猫儿刺和龙里冬青两种植物抗炎作用活性最强的部位均为正丁醇提取物。通过总抗氧化能力(T-AOC)以及DPPH自由基和羟自由基清除率指标考察了两种植物总提取物及各个部位的抗氧化能力。结果显示,猫儿刺的乙酸乙酯部位、正丁醇部位和龙里冬青的正丁醇部位比总提取物及其他部位活性更强。二.活性部位化学成分的分离与鉴定基于活性部位筛选结果,本课题针对龙里冬青的正丁醇部位、猫儿刺的乙酸乙酯部位和正丁醇部位进行了进一步的分离工作。1.从龙里冬青中共分离得到并鉴定了18个化合物,其中dunnianaolide A (ID6)、dunnianaolide B (ID12)、dunnianaolide C (ID16)、rotundioic acid 28-O-β-D-glucopyranoside (ID8)和3-O-β-D-glucopyranosyl-rotundic acid 28-O-β-D-glucopyran oside (ID37) 5个化合物为三萜皂苷类化合物；4-[(erythro)2,3-dihydro-3-hydroxy-methyl-5-(3-hydroxypropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenyl-β-D-gluco pyranoside (ID14)、(7S,8R)-dihydrodehydrodiconiferylalcohol-9-β-D-glucopyranoside(ID19)、落叶松脂素-4-O-β-D-吡喃葡萄糖苷(ID33)3个化合物为木脂素类化合物；槲皮素(ID1)、异槲皮苷(ID2)、芦丁(ID3)、山奈酚-3-芸香糖苷(ID4)、山奈酚-3-刺槐糖苷(ID7)、山奈酚-3-葡萄糖苷(ID9)6个化合物为黄酮及黄酮苷类化合物；还有其他类型化合物4个β-谷甾醇(ID5)、β-胡萝卜苷(ID18)、对羟基苯甲酸(ID20)蔗糖(ID21)。上述18个化合物均为首次从龙里冬青中分离得到,ID6、ID12和ID16为新化合物,并得到ID6单晶,提供了ID6 X-ray单晶衍射数据。2.从猫儿刺中共分离得到并鉴定了23个化合物,其中α-香树脂(IP1)、熊果醇(IP2)、熊果酸(IP3-1)、齐墩果酸(IP3-2)、3α,19a-dihydroxyurs-12-en-28-oic acid (IP4)、羽扇豆烯(IP5-1)、白桦脂酸(IP5-2)、1up-20(29)-ene-3β,24-diol (IP6)、3β-hydroxyurs-11-ene-13β-olide(IP7)、β-香树脂酮(IP8-1)、羽扇豆酮(IP8-2)、urs-12(13)-en-3-one(IP8-3)、无羁萜(IP9)、(3β)-3-(β-D-glucopyranosyl oxy)-19,23-dihydroxyurs-12-en-28-oic acid(IP12-1)和(3β)-3-(β-D-glucopyranosyloxy)-hederagenin(IP12-2)14个为三萜及其苷类化合物；槲皮素(IP13),异槲皮苷(IP14),芦丁(IP17)3个为黄酮及黄酮苷类化合物；5,6,11,12-tetramethyltritriaconta-5,11,17,23,29 -pentaenoate (IP10)、十七酸(IP11-1)、十七酸甲酯(IP11-2)3个为脂肪酸类化合物；以及β-谷甾醇(IP15)、β-胡萝卜苷(IP16)2个甾体化合物。其中化合物IP8-1、IP8-2、IP8-3、IP10、IP11-1、IP11-2和IP12-1为首次从该植物中分离得到。三.主要成分活性筛选本课题对分离得到的部分化合物进行了活性筛选。单体化合物对细胞炎症、致炎因子抑制作用实验,结果显示三萜皂苷类化合物ID6、ID8、ID12和ID16对LPS诱导RAW264.7的致炎因子TNF-α、PGE2和IL-1p均有明显的抑制作用；ID16同时还对NO的产生有很好的抑制作用,在剂量较大时对IL-6也有一定的抑制作用。单体化合物的DPPH自由基清除实验显示黄酮类化合物ID1 (EC50 6.5μM)、ID2 (EC50 8.1μM)、ID3(EC507.6μM)和木脂素类化合物ID19 (EC50 16.4μM)、ID33 (EC50 12.9μM)与阳性对照Vc的(EC5016.0μM)比较,作用良好。更多还原

【Abstract】 Ilex dunniana Levl. and Ilex pernyi Franch. are evergreen shrub or small trees of Ilex genus in Aquifoliaceae, widely distributed throughout the southern regions of the Qinling mountains and the provinces northward to the Changjiang River Vally. Many species of Ilex genus have been used as Chinese tradition medicine or beverage to clear away heat and toxic material, to reduce inflammation, to relieve cough and eliminate phlegm or to heal cardiovascular disease.Inflammation is one of the most important pathophysiological factors of many diseases, such as furuncle, pneumonia, intestinal catarrh, infection of external injury and various infectious diseases. Inflammation is the defense reaction of body caused by the damage from outside, in which cardiovascular system is in the central status. In fact, majority of cardio-cerebrovascular diseases including atherosclerosis, stroke, high blood pressure and cardiac insufficiency are chronic inflammatory diseases. With the development of free radical medical study, the important status of oxidation in various diseases was increasingly accepted. Studies demonstrate that oxidative stress is closely involved in many diseases, such as cancer, cardio-cerebrovascular diseases, diabetes, aging, and et al. Consequently, anti-inflammatory and antioxidant activities are key factors deserved to be valued as properties of Chinese traditional medicine which are used to treat diseases mentioned above or related symptoms. In another word, evaluation of the anti-inflammatory and anti-oxidative potential of a plant is an efficient method to set about.1. Screening of active part(s)In this study, anti-inflammatory activities of the 90% EtOH extracts of two Ilex plant leaves were evaluated using mice ear oedama and rats cotton-pellet granuloma tests. The result showed both of the two extract presented the ability of inhibition of inflammation in mice and rats models in dose-dependent mode. Based on this result, two extracts were devided into four portions according to polarity, respectively. Levels of inflammatory and proinflammatory cytokins were valued with two extract and all portions. The results showed that the n-BuOH extracts of I.D. and I.P. were the most active portion. T-AOC, DPPH and hydroxyl radical assays were adopted to evaluate the anti-oxidant and free radical scavenging activities. In these assays, EtOAC and n-BuOH portion of I.P., and n-BuOH portion of I.D. showed better activity than the extracts and the other portions.2. Isolation of compounds from active parts2.1 Compounds from Ilex dunniana Levl.18 compounds were isolated from Ilex dunniana Levl. and they were identified as:dunnianaolide A (ID6), dunnianaolide B (ID12), dunnianaolide C (ID16), rotundioic acid 28-O-β-D-glucopyranoside (ID8), 3-O-β-D-glucopyranosyl-rotundic acid 28-O-β-D-glucopyranoside (ID37), quercetin (ID1), quercetin-3-O-(β-D-glucopyranoside) (ID2), quercetin-3-O-(α-L-rhamnopyranosyl)-(1-6)-β-D-glucopyranoside (ID3), kaempferol-3-O-(α-L-rhamnopyranosyl)-(1-6)-β-D-gluco pyranoside (ID4), kaempferol-3-O-(α-L-rhamnopyranosyl)-(1-6)-β-D-galactose (ID7), kaempferol-4’-ethoxy-3-O-β-D-glucopyranoside (ID9),4-[(erythro)2,3-dihydro-3-hydroxy-methyl-5-(3-hydroxypropyl)-7-methoxy-2-benzofuranyl]-2,6-dimethoxyphenyl-β-D-gluco pyranoside (ID14), (7S,8R)-dihydrodehydrodiconiferylalcohol-9-β-D-glucopyranoside (ID19), lariciresinol-4-O-β-D-glucopyranoside (ID33),β-sitosterol (ID5),β-daucosterol (ID18),p-hydroxybenzoic acid (ID20), sucrose (ID21).All these compounds were isolated from Ilex dunniana Levl. for the first time. ID6, ID12 and ID16 were new compounds. X-ray data of ID6 was provided in this thesis. Most interesting is the fact that it is to our knowledge one of the first X-ray analysis of a saponin, a class of compounds which poorly crystallize.2.2 Compounds from Ilex pernyi Franch.23 compounds were isolated from Ilex pernyi Franch. and they were identified as:a-amyrin (IP1), uvaol (IP2), ursolic acid (IP3-1), Oleanolic acid, (IP3-2),3a,19a-dihydroxyurs-12-en-28-oic acid (IP4), lupeol (IP5-1), betulinic acid (IP5-2), lup-20(29)-ene-3β,24-diol (IP6),3β-hydroxyurs-11-ene-13β-olide (IP7),β-amyrone (IP8-1), lupeone (IP8-2), urs-12(13)-en-3-one (IP8-3), friedelin (IP9), (3β)-3-(β-D-glucopyranosyloxy)-19,23-dihydroxyurs-12-en-28-oic acid (IP12-1), (3β)-3-(β-D-glucopyranosyl oxy)-19,23-dihydroxyolean-12-en-28-oic acid (IP12-2), quercetin (IP13), quercetin-3-O-(β-D-glucopyranoside) (IP14), rutine (IP17), 5,6,11,12-tetramethyl tritriaconta -5,11,17,23,29-pentaenoate (IP10), heptadecanoic acid (IP11-1), methyl heptadecanoate (IP11-2),β-sitosterol (IP15),β-daucosterol (IP16).IP8-1, IP8-2, IP8-3, IP10, IP11-1, IP11-2 and IP12-1 were isolated from Ilex pernyi Franch. for the first time.3. Inflammatory and antioxidative activities of compoundsLevels of NO, TNF-a, PGE2, IL-1βand IL-6 secreted from RAW 264.7 induced by LPS were tested by ELISA kits treated with several compounds isolated. The results showed that ID6, ID8, ID12 and ID16 presented significant inhibition activities of TNF-α, PGE2 and IL-1β; ID16 inhibited NO secreting from cells and at high dose (12μg/ml) ID16 showed mild inhibition activity of IL-6, as well. In DPPH radical assay, three flavonoids, ID1 (EC506.5μM), ID2 (EC50 8.1μM), ID3 (EC50 7.6μM) and two lignens ID19 (EC50 16.4μM), ID33 (EC50 12.9μM) showed good scavenging activity as compared with Vc (EC50 16.0μM).更多还原