【作者】 刘红兵；

【导师】 管华诗； 崔承彬；

【作者基本信息】 中国海洋大学 ， 水产品加工与贮藏， 2004， 博士

【摘要】 东京枫杨Pterocarya tonkinesis(Franch.)Dode.为胡桃科枫杨属植物，是我国云南西双版纳地区河岸季雨林的代表群落类型。该植物在我国部分地区民间作为草药，用于治疗癌症，但其化学成分及药理研究至今尚无报道。我们在筛选近千种中草药抗肿瘤活性的过程中，发现东京枫杨茎皮的乙醇提取物显示很强的细胞凋亡诱导和坏死性细胞毒活性。为了阐明东京枫杨的抗肿瘤活性成分，本文对其进行了研究。 预实验结果表明，东京枫杨的活性部位为氯仿萃取物。取其干燥茎皮粉碎，60％乙醇提取后，浸膏用等量氯仿直接萃取得到活性提取物。利用小鼠乳腺癌tsFT210细胞，以流式细胞术检测结果为分离向导，采用真空柱色谱(VLC)、Sephadex LH-20柱色谱、Pre-HPLC等分离手段，从活性氯仿提取物中跟踪分离得到18个化合物；利用理化及波谱学方法(UV、IR、1D-NMR、2D-NMR、MS)，其结构分别鉴定为：枫杨醌(1)，5-羟基-2-乙氧基-1，4-萘醌(2)，4，8-二羟基-1-四氢萘醌(isosclerone，3)，3，4-二氢-2r，4t，8-三羟基萘-1(2H)-酮(4)，3，4-二氢-2r，4c，8-三羟基萘-1(2H)-酮(5)，枫杨素(6)，myricatomentogenin(7)，枫杨酸(8)，alnincanone(9)，齐墩果酸(10)，2α，3β，23-三羟基齐墩果酸(11)，乌苏酸(12)，白桦脂酸(13)，3-乙酰白桦脂酸(14)，(24S)-4，6，8(14)，22-麦角甾四烯-3-酮(15)，豆甾烷-3，6-二酮(16)，(24R)-5α-豆甾醇(17)和棕榈酸(18)。其中，化合物1，6和8为新化合物(其中1为新骨架化合物)，2和7首次从天然资源中分离获得。化合物1-11，13-18均为首次从该属植物中分离得到。 利用小鼠乳腺癌tsFT210细胞、人白血病K562细胞和人肠癌HCT-15细胞，采用流式细胞术、细胞增殖抑制实验(SRB)及细胞形态的光学显微镜观察等方法，对分离得到的18个化合物进行抗肿瘤活性的初步评价。结果表明，15个化中草药东京枫杨抗肿瘤活性成分的研究合物具有明显抗肿瘤活性。化合物1具有诱导细胞凋亡的活性;化合物6，7，12，15和18具有细胞周期抑制和细胞凋亡诱导的活性;化合物2，3，4，S，8，10，n，13和14对肿瘤细胞的增殖有抑制活性。其中，化合物2的作用最强(IC50<4 .59nmol/mL)。新化合物1，6和8，新天然产物2和7，以及已知化合物3，14和15的抗肿瘤活性系首次报道。蔡醒类和五环三菇酸类化合物的分离产率分别在0.8%和0.6%左右，可能是东京枫杨抗肿瘤的主要活性成分。 综上，本研究初步阐明了东京枫杨抗肿瘤活性的物质基础，确定了活性成分的主要结构类型，发现了具有强抗肿瘤活性的新骨架化合物枫杨醒。以上结果为抗肿瘤药物的研究提供新的化合物结构类型，为民间将东京枫杨用于治疗癌症提供了一定的科学依据，为枫杨属、乃至胡桃科植物的药用研究与开发提供了基础资料。更多还原

【Abstract】 Pterocarya tonkinesis (Franch.) Dode. (Juglandaceae) forms a typical community of riverside season rain forest in Xishuangbanna area of Yunnan province, China. It has been used as a folk medicine for cancer by local people. No report of the chemical constituents and biological activities of this plant have been found up to now. In the course of screening for new anti-tumor agents from natural resources using mouse cdc2 mutant cell line tsFT210, we found that the alcoholic extract of P. tonkinesis significantly induced apoptosis and necrosis in tsFT210 cells. Thus, we have undertaken an investigation on the anti-tumor constituents of the title plant.The pilot experiments showed that the chloroform extract was the active ingredient of P. tonkinesis. The stems / barks of P. tonkinesis were extracted at room temperature with 60% aqueous alcohol to give an alcoholic extract, which was extracted successively with the same volume of chloroform to afford an active chloroform extract. Monitoring by the bioassay-guided isolation, the chloroform fraction was chromatographed on silica gel, Sephadex LH-20 and prepared TLC, and preparative reversed phase HPLC, respectively, to give 18 compounds. Their structures were elucidated on the basis of spectroscopic methods (1D-NMR, 2D-NMR, MS, IR, UV, etc.). The structure of pterocaryquinone (1), which was a new naphthoquinone derivative with a novel skeleton , pterocarine (6), which was a new cyclic diarylheptanoid, and pterocarinic acid (8), which was a new norsesquiterpene were determined, including the relative stereochemistry of 1 and 7 and the absolute configuration of 6 as R. The other compounds were identified as 5-hydroxy-2-ethoxy-1, 4-naphthoquinone (2), 4, 8-dihydroxy-l-tetralone (3), 3, 4-dihydro-2r, 4t, 8-trihydroxynaphthalen-l(2H)-one (4), 3, 4-dihydro-2r, 4c, 8-trihydroxynaphthalen-l (2H)-one (5), , myricatomentogenin (7), , alnincanone (9), oleanolic acid (10),arjunolic acid (11), ursolic acid (12), betulinic acid (13), 3-O- acetyl-betulinic acid (14), (24S)-ergosta-4, 6, 8 (14), 22-tetraen-3-one (15), (24R)-5oc-stigmastane-3, 6-dione (16), stigmasterol (17) and palmitic acid (18), respectively. Among them, compounds 2 and 7 are new natural products and compounds 1-11/13-18, were isolated from the genus Pterocarya for the first time.The anti-tumor activities of compounds 1-18 were evaluated by SRB method and flow cytometry, accompanied with the morphological observation of the cells under light microscope using mouse cdc2 mutant cells tsFT210, human leukemia carcinoma cells K562 and human colon carcinoma cells HCT-15. There were 15 compounds showed potential anti-tumor activities. Compound 1 exhibited apoptosis inducing activity, compounds 6, 7, 12, 15 and 18 showed both cell cycle inhibiting and apoptosis inducing activities, compounds 2, 3, 4, 5, 8, 10, 11, 13 and 14 inhibited the proliferation of the tumor cells. Among them, compound 2 showed the highest activity (ICso<4.59 nmol/mL). The anti-tumor activities of three new compounds 1, 6, 8 and five known compounds 2, 3, 7, 14, 15 were reported for the first time in the present study. Naphthoquinone and triterpenoids might play a main role of anti-tumor constituents of P. tonkinesis on the basis of their contents in the chloroform extract.To our knowledge, it was the first report on anti-tumor constituents of P. tonkinesis, which supported the record of its anti-tumor property. Furthermore, it also provided a clue for the future research and development of plants in Pterocarya.更多还原