中药苦木抗炎活性成分研究

中药苦木抗炎活性成分研究 附件2 作者姓名:焦伟华 题目:中药苦木抗炎活性成分研究 作者简介:焦伟华，男，1982年11月生，2007年9月师从于沈阳药科大学 姚新生教授，于2010年7月获博士学位。 中 文 摘 要 苦木(Picrasma quassioides Bennt)是我国南方民间传统用药，具有清热燥湿、解毒杀虫等功效，是多种清热消炎类中药复方的重要组方中药，在临床上具有广泛的用处。但是，苦木抗炎的作用机制和作用物质基础至今还没有明确的研究报道。在生物活性指导下，本论文对苦木树茎的抗炎活性部位进行了化学成分研究，并对从活性部位中分离得到的化合物进行了抗炎活性，为深入研究苦木的抗炎作用机制奠定了基础。 通过脂多糖诱导的小鼠巨噬细胞RAW264.7释放一氧化氮(NO)抑制活性实验确定了苦木的主要抗炎活性部位为氯仿萃取部位。并运用硅胶柱色谱、Sephadex LH-20柱色谱、ODS柱色谱以及反相高效液相色谱法对苦木氯仿萃取部位的化学成分尤其是生物碱类成分进行了系统的研究，共分离得到48个化合物。通过理化性质以及IR、UV、ESIMS、NMR以及X-ray单晶衍射等多种波谱学方法，确定了这些化合物的结构，包括25个,-卡巴林型(,-carboline)生物碱、6个铁屎米酮型(canthinone)生物碱、11个双分子,-卡巴林型(bis-,-carboline)生物碱、3个木脂素、2个苦味素和1个黄酮醇，其中新化合物20个，分别为3-(1,1-二甲氧基甲基)-,-卡巴林 [3-(1,1-dimethoxylmethyl)-,-carboline] (1)、6,12-二甲氧基-3-(2-羟基乙基)-β-卡巴林 [6,12-dimethoxy-3-(2-hydroxylethyl)-β-carboline] (2)、6,12-二甲氧基-3-(1,2-二羟基乙基)-β-卡巴林 [6,12-dimethoxy-3-(1,2-dihydroxylethyl)-β-carboline] (3)、6,12-二甲氧基-3-(1-羟基乙基)-β-卡巴林 [6,12-dimethoxy-3-(1-hydroxylethyl)-β-carboline] (4)、6-甲氧基-3-(2-羟基-1-乙氧基乙基)-β-卡巴林 [6-methoxy-3-(2-hydroxyl-1-ethoxylethyl)-β-carboline] (5)、3,4-二氢-10-羟基-3-氧代-β-卡巴林 (3,4-dihydro-10-hydroxyl-3-oxo -β-carboline) (6)、6,12-二甲基-3-醛基-β-卡巴林(6,12-dimethoxy-3-formyl-β-carboline) (7)、铁屎米酮-14-丁酸 (canthinone-14-butyric acid) (26)、14,15-二甲氧基-10-羟基-铁屎米酮 (14,15-dimethoxy-10-hydroxy-canthinone) (27)、quassidine A (32)、quassidine I (33)、quassidine F (34)、quassidine G (35)、quassidine C (36)、quassidine B (37)、quassidine D (38)、quassidine H (39)、quassidine E (40)、苦木脂素A (picrasmalignan A) (43) 和2,-异黄楝苦素A (2,-isopicrasin A ) (46)，其中有新生物碱18个，包括1个首次从自然界中发现的两个β-卡巴林母核通过一个罕见的环丁烷结构片段连接在一起 的双分子β-卡巴林型生物碱，以及1个首次从自然界中发现的铁屎米酮母核和β-卡巴林母核通过碳碳单键直接相连的双分子β-卡巴林型生物碱。此外，还包括2个新天然产物，分别为6-甲氧基-12-羟基-3-甲氧甲酰基-β-卡巴林 (6-methoxy-12-hydroxy-3-methoxycarbonyl-β- carboline) (8) 和3,4-二氢-3-氧代-,-卡巴林 (3,4-dihydrogen-3-oxo-,-carboline) (9)，以及4个首次从苦树属植物中分离得到的化合物，分别为9-羟基-铁屎米酮 (9-hydroxy-canthinone) (28)、buddlenol A (44)、buddlenol C (45) 和黄颜木素 (fisetin) (48) 等。 通过对双分子β-卡巴林型生物碱的结构进行分析和归纳，在单分子β-卡巴林型生物碱生物合成途径的基础上，首次对双分子β-卡巴林型生物碱的生物合成途径进行了探讨，并提出了可能的生物合成途径，填补了该类型生物碱生物合成研究的空白。 在对所有化合物进行体外抗炎活性评价过程中，发现化合物1、7、14-16、21-23、27、34、35、40、43-45和48对脂多糖诱导的小鼠巨噬细胞释放一氧化氮(NO)具有很强的抑制活性，化合物3、15、16、22、23、26、27、35、40、43-45和48对脂多糖诱导的小鼠巨噬细胞释放肿瘤坏死因子-,(TNF-,)显示出很强的抑制活性，而化合物14-18、21-23、27、34、35、43-45和48则对脂多糖诱导的小鼠巨噬细胞释放白细胞介素-6(IL-6)显示出很强的抑制活性;但是，所有测试化合物与阳性药咯利普兰(rolipram)相比对环磷酸腺苷(cAMP)特异性磷酸酯酶4(PDE4)都没有显示出明显的抑制活性。这表明这些化合物可能是通过抑制炎症细胞释放过量的一氧化氮、肿瘤坏死因子-,、白细胞介素-6等炎症因子发挥抗炎作用的。 关键词:苦木、生物碱、,-卡巴林、铁屎米酮、双分子,-卡巴林、抗炎活性、生物合成、一氧化氮、肿瘤坏死因子-α、白细胞介素-6、构效关系 Studies on Anti-inflammatory Constituents of Picrasma quassioides Bennt Jiao Weihua ABSTRACT Picrasma quassioides Bennt (Simarubaceae) is a traditional Chinese medicine mainly distributed in South China. As an important folk medicine for many combination Qingrejiedu medicines, it has been used for treatment of clearing heat, dampness, detoxication, and snakebite for a long time. The effective components of P. quassioides and the mechanism of its components preventing and curing diseases, however, has not been reported. Anti-inflammatory activity-guided isolation of the stems of P. quassioides was carried out. 48 compounds were isolated from the anti-inflammatory CHCl3-soluble fraction. Furthermore, these compounds were evaluated for their anti-inflammatory activity in vitro, which lay foundation for the anti-inflammatory mechanism of P.quassioides. The CHCl3-soluble fraction, which showed the most potent inhibitory activity on nitric oxide (NO) production in mouse monocyte-macrophage RAW 264.7 stimulated by LPS, was determined as the key bioactive fraction and was further isolated and purified by silica gel, Sephadex LH-20, MPLC-ODS, reversed phase HPLC, and other chromatographic methods to yield 48 compounds. Their structures were elucidated by IR, UV, ESIMS, HRESIMS, NMR, X-ray diffraction and other spectroscophic methods, including 25 β-carboline alkaloids, 6 canthinone alkaloids, 11 bis-β-carboline alkaloids, 3 lignins, 2 quassinoids, and a flavonol. Among the 48 compounds, 20 new compounds were determined as 3-(1,1-dimethoxylmethyl)-β-carboline (1), 6,12-dimethoxy-3-(2-hydroxylethyl)-β-carboline (2), 6,12-dimethoxy-3-(1,2-dihydroxylethyl)-β-carboline (3), 6,12-dimethoxy-3-(1-hydroxylethyl)-β-carboline (4), 6-methoxy-3-(2-hydroxyl-1-ethoxylethyl)-β-carboline (5), 3,4-dihydro-10-hydroxyl-3-oxo-β-carboline (6), 6,12-dimethoxy-3-formyl-β-carboline (7), canthinone-14-butyric acid (26), 14,15-dimethoxy-10-hydroxy-canthinone (27), quassidine A (32), quassidine I (33), quassidine F (34), quassidine G (35), quassidine C (36), quassidine B (37), quassidine D (38), quassidine H (39), quassidine E (40), picrasmalignan A (43), and 2′-isopicrasin A (46), respectivley. Quassidine A (32) was a new type bis-β-carboline alkaloid from the nature, the two β-carboline moieties in the structure of compound 32 were connected together by a novel cyclobutane moiety; and quassidine E (40) was also a new type bis-β-carboline alkaloid from the nature, the canthinone moiety and the β-carboline moiety in the structure of compound 32 were connected together by a carbon-carbon single bond. Meanwhile, 6-methoxy-12-hydroxy-3-methoxycarbonyl-β-carboline (8) and 3,4-dihydrogen-3-oxo-β-carboline(9) were two new natural products; 9-hydroxy-canthinone (28), buddlenol A (44), buddlenol C (45),and fisetin (48) were four compounds isolated from Picrasma genus for the first time. The plausible biogenetic pathways of bis-β-carboline alkaloids were proposed in this paper for the first time on the basis of detailed analyses of the isolated and reported bis-β-carboline and monomic β-carboline alkaloids. The anti-inflammatory activity evaluation of the 48 compounds from P. quassioides provided several new anti-inflammatory compounds. Compounds 1, 7, 14-16, 21-23, 27, 34, 35, 40, 43-45, and 48 showed potent inhibitory activity on nitric oxide (NO) production in mouse monocyte-macrophage RAW 264.7 stimulated by LPS, compounds 3, 15, 16, 22, 23, 26, 27, 35, 40,43-45, and 48 showed potent inhibitory activity on tumor necrosis factor α (TNF-α) production in mouse monocyte-macrophage RAW 264.7 stimulated by LPS, and compounds 14-18, 21-23, 27, 34,35, 43-45, and 48 showed potent inhibitory activity on interleukin 6 (IL-6) production in mouse monocyte-macrophage RAW 264.7 stimulated by LPS. Inhibitory effects of the 48 compounds on Phosphodiesterase-4 (PDE4) suggested that most of them showed no significant inhibitory activity on PDE4, comparing with the positive control rolipram. The above bioactive evaluation suggested that these compounds isolated from P. quassioides took roles on anti-inflammatory activity by inhibiting inflammatory factors’ production, such as NO, TNF-α, and IL-6, not by inhibiting PDE4. Key words: Picrasma quassioides Bennt;Simarubaceae;alkaloids;β-carboline; canthinone;bis-β-carboline;anti-inflammatory;biogenetic; nitric oxide; tumor necrosis factor α; interleukin 6;structure activity relationship.