【作者】 扈晓佳；

【导师】 张卫东；

【作者基本信息】 上海交通大学 ， 生物医学工程， 2009， 博士

【摘要】 祖师麻为瑞香科Thymelaeaceae瑞香属植物黄瑞香Daphne giraldii. Nitsche的根皮和茎皮,同属植物唐古特瑞香D. tangutica Maxim、凹叶瑞香D. retusa Hemsl.、长白瑞香D. koreana Nakai.和同科结香属植物结香Edgeworthia chrysantha Lindl.均可同等入药。分布在陕西、甘肃、四川、宁夏、青海等地。祖师麻具有祛风除湿、止痛散瘀之功效。民间广泛用于治疗疼痛、跌打损伤、风湿性关节炎和支气管炎等症。其中,凹叶瑞香(D. retusa Hemsl.),又名桂花矮陀陀,杂兰等,为瑞香科瑞香属植物。根及茎皮用来治疗跌打损伤,浙江民间用来治疗慢惊风;长白瑞香(D. koreana Nakai.)是长白山区的特有植物,又名辣根草,为瑞香科瑞香属植物。主要用来治关节酸痛,手足麻木、肢体瘫痪、痛经、胸痛、冠心病,风湿性关节炎、血栓闭塞性脉管炎;结香(Edgeworthia chrysantha Lindl.),又名打结花,为瑞香科结香属植物,根及茎皮入药,主要用来祛风除湿。本文采用抗炎镇痛活性跟踪指导分离的方法研究了作为中药祖师麻入药的三种基原植物凹叶瑞香、长白瑞香和结香的化学成分,并首次报道了凹叶瑞香、结香的抗炎镇痛的药理活性,同时还测试了部分单体化合物的抗炎镇痛活性。本文对凹叶瑞香、长白瑞香、结香的总提物以及各萃取极性部位进行了抗炎和镇痛的活性筛选实验。实验表明凹叶瑞香、长白瑞香、结香的总提物对二甲苯引起的小鼠的耳肿胀具有一定的抑制作用,对完全弗氏佐剂引起的大鼠足肿胀具有一定的抑制作用,同时还对RAW264.7巨噬细胞NO代谢有显著的抑制作用。同时凹叶瑞香、长白瑞香和结香的总提物能够显著减少醋酸引起的小鼠扭体次数,显示了一定的镇痛活性。同时,实验还发现凹叶瑞香、长白瑞香和结香的氯仿部位、乙酸乙酯部位均具有一定的抗炎镇痛的活性。根据药理活性实验结果,应用现代色谱分离技术,我们对凹叶瑞香、长白瑞香和结香的氯仿部位和乙酸乙酯部位进行了系统的化学分离,并分别从中分离纯化得到了65个化合物,运用UV,IR,ESI-MS,1D-NMR,2D-NMR等光谱技术和物理化学方法鉴定了其中的61个,其结构分别为DR-1:对羟基苯甲酸,DR-2:β-谷甾醇,DR-3:胡萝卜苷,DR-4:瑞香素,DR-5:双白瑞香素,DR-6:丁香醛,DR-7:7-羟基-8-甲氧基香豆素,DR-8:芫花素,DR-9:7-甲氧基-8-羟基香豆素,DR-10:伞形花内酯,DR-11:结香苷A,DR-12:结香苷C,DR-13:4’, 5-二羟基-3’, 7-二甲氧基黄酮,DR-14:瑞香新素,DR-15:5’-去甲氧基瑞香新素,DR-16:瑞香醇酮,DR-17:紫丁香苷,DR-18:左旋松脂酚,DR-19:双白瑞香素-7-O-β-D-葡萄糖苷,DR-20 :芫花苷, DR-21 :伞形花内酯-7-O-β-D-葡萄糖苷, DR-28 : [8, 8’-Bi-2H-1-benzopyran]-2, 2’-dione, 7’-(β-D-glucopyranosyloxy)-7-hydroxy-3-[(2-oxo- 2H-1-benzopyran-7-yl)oxy],DK-1:β-谷甾醇,DK-2:十八烷基咖啡酸酯,DK-3:瑞香素,DK-4:伞形花内酯,DK-5:胡萝卜苷,DK-6:秦皮乙素,DK-7:双白瑞香素, DK-8:山柰酚,DK-9:瑞香新素,DK-10:木犀草素,DK-11:芹菜素,DK-12:樱花苷,DK-13:芫花素,DK-14:芫花苷,DK-16:7-甲氧基-木犀草素-5-O-β-D-葡萄糖苷,DK-17:双白瑞香素-7-O-β-D-葡萄糖苷, DK-18:异瑞香新素,DK-19:5′′-去甲氧基瑞香新素,DK-20:紫丁香苷,DK-25:瑞香苷,JX-4:Edgeworin,JX-6:胡萝卜苷,JX-7:伞形花内酯,JX-8:双白瑞香素,JX-9:结香苷A,JX-10:5-羟基-7-甲氧基香豆素-8-O-β-D-葡萄糖苷,JX-11:芹菜素,JX-12:结香苷C,JX-13:结香苷B,JX-14:异双白瑞香素,JX-15:2,6-二甲氧对苯醌,JX-16:伞形花内酯-7-O-β-D-葡萄糖苷,JX-17:紫丁香苷,JX-18:双白瑞香素-7-O-β-D-葡萄糖苷,JX-19:瑞香新素,JX-20:6- hydroxyl-odesmethoxyrutarensin,JX-21:8-(4-(1, 3-benzenediol ) propionic acid methyl ester)-coumarin-7-O-β-D-glucoside,JX-23:Triumbellin。本文对量较大的单体化合物成分进行了抗炎镇痛的活性实验。体内实验发现,JX-4,JX-9,DR-5,DR-16,DK-3能够显著抑制二甲苯引起的小鼠耳肿胀,能够抑制完全弗氏佐剂引起的大鼠关节肿胀,显示了一定的抗炎活性。JX-4,JX-9,JX-12,DR-5,DR-16,DK-4,DK-9还能够显著减少醋酸引起的小鼠扭体次数,显示了较好的镇痛活性。为全面研究祖师麻三种资源药用植物的化学成分,本文对其余部位也进行了系统的研究,从中分离得到了21个化合物,鉴定了其中的14个。其结构分别为:JX-1:二十碳酸单甘油酯,JX-2:对羟基苯甲酸,JX-3:β-谷甾醇,JX-5:肌醇,DR-22:瑞香黄烷A,DR-23:瑞香黄烷B,DR-24:瑞香黄烷C,DR-25:瑞香黄烷D1,DR-26:瑞香黄烷E,DR-27:瑞香黄烷F,DK-21:左旋松脂酚,DK-22:落叶松脂醇,DK-23:左旋松脂酚-4-O-β-D-葡萄糖苷,DK-24:落叶松脂醇-4-O-β-D-葡萄糖苷。所有得到的化合物中,JX-20,JX-21为新化合物, DR-4,5,6,7,9,8,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,28,DK-2,3,4,5,7,8,10,11,13,14,16,17,18,19,21,22,23,24, JX-5,10,11,14,15,17,18,19,16,23为首次从该植物中分离得到。蓼子朴[Inula salsoloides (Turc.) Ostenf.]为菊科旋覆花属植物。花及开花前的全草均可药用,有解热、利尿的功能,全草还为治疗脉管炎主药。在我国的新疆、内蒙古、青海东北部、陕西、河北、山西、辽宁中西部均有广泛的分布。本文运用各种色谱分离手段从中分离得到了32个化合物,运用光谱技术鉴定了其中的25个化合物,结构如下:IS-1:β-谷甾醇,IS-2:cis-1-p-Menthene-3, 6-diol,IS-3:正十二烷,IS-4:对羟基苯甲酸,IS-5:对羟基苯甲酸乙酯,IS-7:3’, 4’, 7-三羟基-5-甲氧基黄酮,IS-8:豆甾醇,IS-9:1-Cyclodecene-2, 5, 6, 9-tetrol, 1, 8-dimethyl-7-(1-methylethyl)-, [(2α, 5β, 6α, 7α, 8β, 9α)],IS-10:沙地旋覆花内酯B,IS-11:Isoalantolactone,IS-12:山柰酚,IS-13:沙地旋覆花内酯,IS-14:堆心菊内酯,IS-15:金合欢素,IS-16:槲皮素,IS-17:大黄素甲醚,IS-18:芹菜素,IS-20:8, 9, 10-Trihydroxythymol,IS-21: 9, 10-Dihydroxythymol,IS-23:4’, 7-二羟基黄酮,IS-24:木犀草素,IS-25:泽兰内酯,IS-26:4α, 5β-Epoxyeupatolide,IS-30:3′, 5′, 5, 7-四羟基黄酮,IS-32:4’, 6, 7-三羟基黄酮。实验发现除了IS-9,IS-20,IS-21三组仅最高剂量组(1μg/mL)有一定的抑制RAW264.7巨噬细胞NO的产生(P<0.01);IS-25组除最低剂量组(0.01μg/mL)没有抑制作用外,其余各剂量组均有一定的抑制作用(P<0.01);其余IS-10,IS-11,IS-13,IS-15,IS-26各组各剂量组对RAW264.7巨噬细胞NO的产生均有一定的抑制作用(P<0.01)。但是在MTT中,各组均显示了细胞毒性,推测对RAW264.7巨噬细胞产生NO的抑制作用的原因可能是细胞毒性的缘故。在对NF-κB激活的抑制作用的实验中,受试样品各组除IS-10,IS-13,IS-14,IS-26有一定的对NF-κB激活的抑制作用外(P<0.05),其他各组均没有表现对NF-κB激活的抑制作用。所得的化合物,IS-9和IS-10为新化合物,IS-1,4,5,7,8,12,14,15,17,18,20,20,23,24,26,30,32为首次从该植物中分离得到。结论:本文从中药祖师麻入药的三种植物中共分离得到了84个化合物,其中2个新化合物,52个首次从该三种植物中分离得到。通过抗炎镇痛的活性筛选实验,发现三种植物的氯仿和乙酸乙酯等部位具有一定的抗炎镇痛活性,单体化合物JX-4,JX-9,DR-5,DR-16,DK-3具有一定的抗炎活性,JX-4,JX-9,JX-12,DR-5,DR-16,DK-4,DK-9和DK-25具有较好的镇痛活性。本文从蓼子朴中共分离得到32个化合物,鉴定了25个,其中2个新化合物,17个首次从该植物中分离得到。更多还原

【Abstract】 The stem and bark of Daphne giraldii Nitsche (Thymelaeaceae), as well as the stem and bark of D. tangutica Maxim, D. retusa Hemsl., D. koreana Nakai., and Edgeworthia chrysantha Lindl., commonly called“Zu Shima”, is widely distributed in Shanxi, Gansu, Ningxia, Sichuan and Qinghai provinces of China. It has been used in Chinese folk medicines to treat aches and rheumatism. In modern pharmacological investigation, it was used to treat toothache, waistache, rheumatoid arthritis, quadriplegia, thrombosis, inflammation, cancer etc. D. retusa, also called Guihua Aituotuo, belongs to the genus of Daphne, Thymelaeaceae family. Its stem and barks are used to treat injuries from fall, and in Zhejiang, it is also treat chronic infantile convulsion in folk; D. koreana belongs to the genus of Daphne and it is only distributed in Changbai Mountain. It is also called La Gencao. It is used for treatment deadlimb, thoracodynia, coronary artery disease, rheumatic arthritis, thromboangiitis obliterans. E. chrysantha Lindl. which collected in JiuJiang, Jiangxi Province in May, 2005, is also called DaJie Hua, belongs to the Edgeworthia genus. The stem and bark has been used to the treatment of injuries from falls, bruisesand faucitis as a folk medicine in China. The flowers of E. chrysantha are also used to treat some diseases in eyes as a supplement of Meng Hua. In this dissertation, we investigate the chemical constituents of the stem and bark of D. retusa Hemsl., D. koreana Nakai. and E. chrysantha Lindl. systematically, and the anti-inflammatory and analgesic activities of the crude extracts, four extractive fractions, and some pure compounds.In anti-inflammatory and analgesic activity tests of all crude extracts, four extractive fractions and three pure compounds from these three plants, the results showed that the crude extracts of D. koreana, D. retusa, and E. chrysantha significantly inhibited the ear edema induced by PX and paw edema induced by complete Freund’s adjuvant. Meanwhile, they also exhibited the inhibitory effects on the NO production in LPS-induced RAW264.7 macrophages in vitro test. Besides, the crude extracts also showed a significant inhibition of writhing induced by acetic acid. We also investigated chloroform and acetoacetate fractions from D. koreana, D. retusa, and E. chrysantha showed some anti-inflammatory and analgesic activities.65 compounds were obtained from chloroform and acetic ether fractions, the active parts of three plants, by a variety of chromatography methods, and their structures were established on the basis of spectroscopic data including UV, IR, ESI-MS, 1D-NMR, 2D-NMR, physical and chemical properties as follow: DR-1: p-hydroxybenzonic acid, DR-2:β-sitosterol, DR-3: daucosterol, DR-4: daphnetin, DR-5: daphnoretin, DR-6: syringaldehyde, DR-7: 7-hydroxy-8-methoxycoumarin, DR-8: yuankanin, DR-9: 7- methoxy-8-hydroxycoumarin, DR-10: umbeliferone, DR-11: edgeworoside A, DR-12: edgeworoside C, DR-13: 4’, 5-dihydroxy-3’, 7-dimethoxyflavone, DR-14: daphneticin, DR-15: 5’’-demethoxydaphneticin, DR-16: daphneolon, DR-17: syringin, DR-18: (?)-pinoresinol, DR-19: daphnoretin-7-O-β-D-glucoside, DR-20: genkwanin, DR-21: umbeliferone-7-O-β-D-glucoside, DR-28: [8, 8’-Bi-2H-1-benzopyran]-2, 2’-dione, 7’-(β- D-glucopyranosyloxy)-7-hydroxy-3-[(2-oxo-2H-1-benzopyran-7-yl)oxy], DK-1:β-sitosterol, DK-2 octadecyl affeate, DK-3: daphnetin, DK-4: umbeliferone, DK-5: daucosterol, DK-6: 6, 7-dihydroxycoumarin, DK-7: daphnoretin, DK-8: kaempferol, DK-9: daphneticin, DK-10: luteolin, DK-11: apigenin, DK-12: sakuranin, DK-13: genkwanin, DK-14: yuankanin, DK-16: luteolin-7-methylether-5-O-β-D-glucoside, DK-17: daphnoretin-7-O-β-D-glucoside, DK-18: isodaphneticin, DK-19: 5′′-demethoxy- daphneticin, DK-20: syringin, DK-25: daphnin, JX-4: edgeworin, JX-6: daucosterol, JX-7: umbeliferone, JX-8: daphnoretin, JX-9: edgeworoside A, JX-10: 5-hydroxy-7- methoxycoumarin-8-O-β-D-glucoside, JX-11: apigenin, JX-12: edgeworoside C, JX-13: edgeworoside B, JX-14: isodaphnoretin, JX-15: 2,6-dimethoxyquinone, JX-16: umbeliferone-7-O-β-D-glucoside, JX-17: syringin, JX-18: daphnoretin-7-O-β-D- glucoside, JX-19: daphneticin, JX-20: 6-hydroxyl-odesmethoxyrutarensin, JX-21: 8-(4-(1, 3-benzenediol)propionic acid methylester)-coumarin-7-O-β-D-glucoside, and JX-23: triumbellin. JX-4, JX-9, DR-5, DR-16 and DK-3 displayed significant inhibition in ear edema induced by PX and paw edema induced by complete Freund′s adjuvant with hydrocortisone and examethasone as a positive control. JX-4, JX-9, JX-12, DR-5, DR-16, DK-4, DK-9 also showed a significant analgesic activity in writhing induced by acetic acid.The chemical investigation of other extracts of the three plants resulted in the isolation of 21 compounds and 14 structures of them were determined as following also counted on the spectral analysis and physic-chemical properties: DR-22: daphnodorin A, DR-23: daphnodorin B, DR-24: daphnodorin C, DR-25: daphnodorin D1, DR-26: daphnodorin E, DR-27: daphnodorin F, DK-21: (?)-pinoresinol, DK-22: lariciresinal, DK-23: (?)-pinoresinol-4-O-β-D-glucopyranoside, DK-24: lariciresinol-4-O-β-D- glucoside, JX-1: glycerol monostearol acid, JX-2: p-hydroxybenzoic acid, JX-3:β-sitosterol, and JX-5: inositol.Among all the compounds mentioned above, JX-20 and JX-21 were new compounds and DR-4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 28 , DK-2, 3, 4, 5, 7, 8, 10, 11, 13, 14, 16, 17, 18, 19, 21, 22, 23, 24, 25, JX-5, 11, 14, 15, 16, 17, 18, 19, and 23 were isolated from these plants for the first time.Inula salsoloides (Turc.) Ostenf. belongs to the genus of Inula, Compositae. Its flowers and the entire herbs before flower can be used fever releasing and uragoga, the plant is also a pricinpal agent to treat angeitis. It has a wide distribution in Xinjiang, Inner Mongolia, Shan′xi, Shanxi and Hebei. The chemical investigation of this plant resulted in the isolation of 32 compounds by a variety of chromatography methods, and 24 compounds were established on the basis of spectroscopic data including UV, IR, ESI-MS, 1D-NMR, 2D-NMR, physical and chemical properties, as follows: IS-1:β-sitosterol, IS-2:cis-1-p-menthene-3, 6-diol,IS-3: n-dodecane, IS-4: p-hydroxybenzonic acid, IS-5: ethyl hydroxybenzoate, IS-7: 3’, 4’, 7-trihydroxy-5- methoxyflavone, IS-8: stigmasterol, IS-9: 1-Cyclodecene-2, 5, 6, 9-tetrol, 1, 8-dimethyl-7-(1-methylethyl)-, [(2α, 5β, 6α, 7α, 8β, 9α)], IS-10: inulasalsolide B, IS-11: isoalantolactone, IS-12: kaempferol, IS-13: inulasalsolide, IS-14: florilenalin, IS-15: acacetin, IS-16: quercetin, IS-17: physcion, IS-18: apigenin, IS-20: 8, 9, 10-trihydroxythymol, IS-21: 9, 10-dihydroxythymol, IS-23: 4’, 7-dihydroxyflavone, IS-24: luteolin, IS-25: eupatolide, IS-26: 4α, 5β-epoxyeupatolide, IS-30: 2-(3, 5-dihydroxy- phenyl)-5, 7-dihydroxyflavone, and IS-32: 4’, 6, 7-trihydroxyflavone.Among all the compounds mentioned above, IS-9 and IS-10 were new compounds and IS-1, 4, 5, 7, 8, 12, 14, 15, 17, 18, 20, 21, 23, 24, 26, 30, and 32 were isolated from this plant for the first time.Conclusion: 84 compounds in total were obtained from three series plant as Zu Shima, including two new compounds and 52 compounds for the first time. 24 compounds, including two new compounds and 16 compounds for the first time were obtained from I. salsoloides (Turc.) Ostenf.. The 75% ethanol extractions, chloroform and acetic ether fractions of three theries of plants as Zu Shima showed significant anti-inflammatory and analgesic effects and on the basic of bioassay, JX-4, JX-9, DR-5, DR-16 and DK-3 showed anti-inflammatory activities, JX-4, JX-9, JX-12, DR-5, DR-16, DK-4, DK-9 and DK-25 exhibited analgesic acitivities.更多还原