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毛果鱼藤中黄酮类化学成分研究

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  [摘要] 综合运用硅胶柱色谱、SephadexLH-20、聚酰胺柱色谱、半制备HPLC以及重结晶等方法对毛果鱼藤中黄酮类化学成分进行分离纯化,并通过质谱、核磁共振谱等方法鉴定各单体化合物的结构。最后从毛果鱼藤的乙酸乙酯部位分离得到9个黄酮类化合物,依次为香叶木素(1),3, 3′-二甲醚-槲皮素(2),阿弗洛莫生(3),6, 3′-dihydroxy-7, 4′-dimethoxyisoflavone(4),飞机草素(5),7, 3′-dihydroxy-8, 4′-dimethoxyisoflavone(6),6, 4′-dihydroxy-7, 3′-dimethoxyisoflavone(7),5, 7, 4′-trihydroxy-3, 3′, 5′-trimethoxyflavone(8),alpinumisoflavone(9)。所有化合物均为首次从该植物中分离得到。运用MTT法进行体外抗肿瘤实验,结果表明化合物2显示一定的抑制细胞增殖活性。
  [关键词] 毛果鱼藤;黄酮类;化学成分;分离纯化
  [收稿日期] 2015-04-07
  [基金项目] 国家自然科学基金项目(81160390)
  [通信作者] *潘卫东,博士,研究员,主要从事天然药物化学研究与开发工作,Tel:(0851) 83805348, E-mail:wdpan@163.com
  [作者简介] 王伦兴,硕士研究生, E-mail:wanglx929@163.com
  Studies on flavonoids from Derris eriocarpa
  WANG Lun-xing1,2, WU Hong-guo2,3, ZHANG Hua4, LOU Hua-yong2, LIANG Guang-yi2, JIANG Wen-wen3,
  YANG Zai-chang3 , PAN Wei-dong2*
  (1.School of Fermentation and Food Engineering, Guizhou University, Guiyang 550025, China;
  2. Key Laboratory of Chemistry for Natural Products of Guizhou Province and Chinese Academy of Sciences, Guiyang 550002, China;
  3. School of Pharmaceutical Science, Guizhou University, Guiyang 550025, China;
  4. Sinopharm A-Think Pharmaceuticals Co., Ltd., Changchun 130012, China)
  [Abstract] Derris eriocarpa, a traditional Chinese medicine belonging to the family of Leguminosae, is widely distributed mainly over Yunnan, Guangxi and Guizhou of China. Modern pharmacological researches on this herb showed that it had extensive bioactivities, such as promoting urination, removing dampness and cough and reducing inspissated mucus and other biological activities. The extensive studies on the chemical constituents of this plant have resulted in the isolation of triterpenoids, steroids, fatty acid and others, but the flavone compounds haven′t reported before. In our further research on the ethyl acetate of this plant, nine flavone compounds were obtained by column chromatography on silica gel, Sephadex LH-20, semi-prep HPLC, polyamide column chromatography and recrystallization for separation and purification. The structures were determined on the basis of extensive spectroscopic analysis, including MS, NMR experiments and comparison with spectroscopic data in the literature, respectively, as diosmetin(1), 3, 3′-di-O-methylquercetin(2), afromosin(3), 6, 3′-dihydroxy-7, 4′-dimethoxyisoflavone(4), odoratin(5), 7, 3′-dihydroxy-8, 4′-dimethoxyisoflavone(6), 6, 4′-dihydroxy-7, 3′-dimethoxyisoflavone(7), 5, 7, 4′-trihydroxy-3, 3′, 5′-trimethoxyflavone(8), and alpinumisoflavone(9). All these compounds were isolated from Derris eriocarpa How for the first time. And the in vitro assays showed that compound 2 possessed moderate inhibitory activity against human cancer cells K562 and HEL.   [Key words] Derris eriocarpa; flavonoids; chemical constituents; separation and purification
  doi:10.4268/cjcmm20151518
  毛果鱼藤Derris eriocarpa How,为豆科Leguminosae鱼藤属Derris植物,攀援状灌木。主要分布在云南、广西、贵州一带,别名为鸡血藤、藤甘草、土甘草、美丽相思子等,其藤茎具有利尿除湿、镇咳化痰的功效,民间用以治疗肾炎、膀胱炎、尿道炎、咳嗽[1]等症。目前毛果鱼藤中已报道的主要化学成分有三萜[2]、甾体、脂肪酸、丁香酸[3]等,而黄酮类化学成分尚未见报道。为了进一步研究毛果鱼藤化学成分特别是其中的黄酮类成分,本文对从毛果鱼藤醇提物的乙酸乙酯萃取部分进行了系统的分离纯化,并从中分离得到9个黄酮类化合物,这些化合物均为首次从该植物中分离得到。
  1 材料
  NMR:INOVA 400 MHz核磁共振测试仪(TMS内标)(Varian,Inc);ESI-MS:HP-5793型气相色谱-质谱联用仪(美国惠普公司);EI:HP-1100 MSD型液相色谱-质谱联用仪(美国惠普公司);柱色谱硅胶(40~80目和300~400目);聚酰胺(300~400目);中压制备色谱仪(S/N3521008001,江苏利穗科技有限公司);Sephadex LH-20(25~100 μm)(Amersham Biosciences,Sweden);半制备HPLC Waters2489(UV/Visible Detector, Waters 600 Controller,美国Waters公司),色谱柱为Waters C18柱(10 mm×150 mm, 10 μm, NO:165138056103)。
  药材采自贵州省黔西南布依族苗族自治州兴义市,由贵阳中医学院陈德媛教授鉴定为毛果鱼藤D. eriocarpa。标本存放于贵州省中国科学院天然产物化学重点实验室。
  2 提取与分离
  40 kg毛果鱼藤干燥全草粉碎后,分别用95%的乙醇充分浸泡并回流提取1次,75%的乙醇回流提取3次,合并提取液,减压蒸馏回收乙醇,浸膏加水分散后分别用石油醚、乙酸乙酯和正丁醇各萃取3次,得到石油醚部位浸膏490 g,乙酸乙酯部位浸膏317 g,正丁醇部位浸膏542 g。
  对乙酸乙酯浸膏进行硅胶柱色谱(200~300目)分离,以石油醚-丙酮(50∶1~0∶1)为洗脱剂进行梯度洗脱,经TLC检测合并相同组分后共得到6个组分(Fr.1~Fr.6)。其中,Fr.4(14.6 g)进一步经过硅胶(300~400目,石油醚-乙酸乙酯20∶1~0∶1)柱色谱梯度洗脱,共得到8段(Fr.4-1~Fr.4-8)不同极性组分,取Fr.4-2(111.4 mg)组分经反复硅胶梯度洗脱(石油醚-丙酮20∶1~0∶1)和聚酰胺柱色谱梯度洗脱(40%~95%乙醇),再由Sephadex LH-20(氯仿-甲醇1∶1)分离纯化,并用丙酮进行重结晶,得到化合物1(8 mg),2(15 mg),3(10 mg)。取Fr.4-3(581.3 mg)组分经反复硅胶(300~400目,石油醚-丙酮4∶1)和Sephadex LH-20(氯仿-甲醇1∶1)柱色谱进行洗脱,再通过半制备HPLC(甲醇-水  51∶49)进行分离纯化,对不同保留时间的单体化合物进行收集,分别得到化合物4(12 mg),5(22 mg),6(14 mg),7(17 mg),8(11 mg),9(13 mg)。
  3 结构鉴定
  化合物1 黄色粉末;EI-MS m/z 300 [M]+・;1H-NMR(C5D5N,400 MHz)δ:13.82(1H,s,5-OH),7.66(1H,dd,J=8.4,2.0 Hz,H-6′),7.63(1H,d,J=2.0 Hz,H-2′),7.30(1H,d,J=8.4 Hz,H-5′),7.00(1H,s,H-3),6.88(1H,d,J=2.0 Hz,H-8),6.79(1H,d,J=2.0 Hz,H-6),3.84(1H,s,-OCH3);13C-NMR(C5D5N,100 MHz) δ:181.7(C-4),164.2(C-2),163.5(C-7),161.4(C-5),157.2(C-9),151.0(C-3′),146.8(C-4′),123.0(C-1′),118.6(C-6′),112.9(C-5′),112.1(C-2′),103.7(C-10),103.5(C-3),98.8(C-6),93.8(C-8),54.0(3′-OCH3)。以上数据与文献[4-5]对照一致,鉴定此化合物1为香叶木素(diosmetin)。
  化合物2 黄色粉末;EI-MS m/z 330 [M]+・; 1H-NMR(C5D5N,400 MHz) δ:13.69(1H,s,5-OH),7.92(1H,d,J=2.0 Hz,H-2′),7.87(1H,dd,J=8.4,2.0 Hz,H-6′),7.33(1H,d,J=8.4 Hz,H-5′),6.84(1H,d,J=2.0 Hz,H-8),6.76(1H,d,J=2.0 Hz,H-6),4.01(3H,s,3-OCH3),3.84(3H,s,3′-OCH3); 13C-NMR(C5D5N,100 MHz) δ:178.0(C-4),164.3(C-7),161.4(C-5),156.5(C-9),155.5(C-2),150.0(C-3′),147.5(C-4′),137.9(C-3),122.4(C-1′),121.0(C-6′),115.7(C-5′),112.3(C-2′),104.4(C-10),98.7(C-8),94.0(C-6),59.9(3-OCH3),55.9(3′-OCH3)。以上数据与文献[6]对照一致,故鉴定化合物2为3,3′-二甲醚-槲皮素(3,3′-di-O-methylquercetin)。   化合物3 白色针状晶体(丙酮);mp 220~222 ℃。ESI-MS m/z 321 [M+Na]+;1H-NMR(C5D5N,400 MHz)δ:8.21(1H,s,H-2),7.80(2H,d,J=8.8 Hz,H-2′,6′),7.49(1H,s,H-5),7.10(2H,d,J=8.8 Hz,H-3′,5′),7.06(1H,s,H-8),3.85(3H,s,6-OCH3),3.67(3H,s,4′-OCH3); 13C-NMR(C5D5N,100 MHz) δ:175.5(C-4),159.9(C-4′),154.4(C-7),152.7(C-2),151.6(C-8a),147.0(C-6),130.8(C-2′,6′),125.7(C-1′),122.9(C-3),119.2(C-4a),114.2(C-3′,5′),109.5(C-5),100.4(C-8),56.3(-OCH3),55.2(-OCH3)。以上数据与文献[7]对照一致,故鉴定化合物3为阿弗洛莫生(afromosin)。
  化合物4 黄色粉末;ESI-MS m/z 315 [M+H]+; 1H-NMR(C5D5N,400 MHz) δ:8.23(1H,s,H-2),7.93(1H,s,H-5),7.87(1H,d,J=2.0 Hz,H-2′),7.38(1H,dd,J=8.4,2.0 Hz,H-6′),7.23(1H,s,H-8),7.07(1H,d,J= 8.4 Hz,H-5′),3.79(3H,s,4′-OCH3),3.73(3H,s,7-OCH3); 13C-NMR(C5D5N,100 MHz) δ:175.5(C-4),154.6(C-6),153.1(C-9),152.6(C-2),148.7(C-4′),148.1(C-3′),147.8(C-7),126.7(C-1′),124.4(C-3),120.5(C-6′),118.0(C-2′),117.6(C-10),112.4(C-5′),105.8(C-5),104.0(C-8),56.0(4′-OCH3),55.9(7-OCH3)。以上数据与文献[8]对照一致,故鉴定化合物4为6,3′-dihydroxy-7,4′-dimethoxyisoflavone。
  化合物5 黄色粉末;ESI-MS m/z 315 [M+H]+; 1H-NMR(CD3OD,400 MHz) δ:8.18(1H,s,H-2),7.48(1H,s,H-5),7.15(1H,d,J=2.0 Hz,H-2′),7.09(1H,s,H-8),6.95(1H,dd,J=8.0 ,2.0 Hz,H-6′),6.84(1H,d,J=8.0 Hz,H-3′),3.99(3H,s,4′-OCH3),3.88(3H,s,7-OCH3); 13C-NMR(CD3OD,100 MHz) δ:175.6(C-4),155.5(C-9),154.7(C-2),153.3(C-7),148.4(C-4′),147.7(C-6),146.0(C-5′),125.5(C-1′),124.9(C-3),122.8(C-2′),116.2(C-10),114.0(C-6′),108.9(C-3′),101.8(C-5),100.7(C-8),56.9(4′-OCH3),56.4(7-OCH3)。以上数据与文献[9]对照一致,故鉴定化合物5为飞机草素(odoratin)。
  化合物6 黄色粉末;EI-MS m/z 314 [M]+・;1H-NMR(C5D5N,400 MHz) δ:8.27(1H,s,H-2),8.20(1H,d,J=7.2 Hz,H-5),7.81(1H,d,J=1.6 Hz,H-2′),7.34(1H,dd,J=1.6,6.8 Hz,H-6′),7.32(1H,d,J=6.8 Hz,H-5′),7.04(1H,d,J=6.8 Hz,H-6),3.96(3H,s,8-OCH3),3.76(3H,s,4′-OCH3);13C-NMR(C5D5N,100 MHz) δ:175.9(C-4),156.4(C-7),152.8(C-2),151.8(C-9),148.8(C-4′),148.1(C-3′),136.0(C-8),126.2(C-1′),124.8(C-3),122.0(C-5),120.5(C-6′),118.9(C-10),117.9(C-6),116.2(C-5′),112.4(C-2′),61.2(8-OCH3),56.0(4′-OCH3)。以上数据与文献[10]对照一致,故鉴定化合物6为7,3′-dihydroxy-8,4′-dimethoxyisoflavone。
  化合物7 黄色粉末;ESI-MS m/z 315 [M+H]+; 1H-NMR(C5D5N,400 MHz) δ:8.27(1H,s,H-2),7.98(1H,s,H-5),7.63(1H,d,J=1.6 Hz,H-2′),7.44(1H,dd,J=8.04,1.6 Hz,H-6′),7.35(1H,d,J=8.04 Hz,H-5′),7.27(1H,s,H-8),3.85(3H,s,3′-OCH3),3.78(3H,s,7-OCH3); 13C-NMR(C5D5N,100 MHz) δ:175.6(C-4),154.6(C-7),153.2(C-8a),152.6(C-2),148.5(C-4′),148.4(C-3′),147.9(C-6),124.6(C-1′),124.5(C-3),122.7(C-6′),117.6(C-4a),116.5(C-5′),114.1(C-2′),105.7(C-5),104.1(C-8),56.0(3′,7-OCH3)。以上数据与文献[11]对照一致,故鉴定化合物7为6,4′-dihydroxy-7,3′-dimethoxyisoflavone。   化合物8 黄色粉末;ESI-MS m/z 361 [M+H]+; 1H-NMR(C5D5N,400 MHz)δ:7.70(2H,s,H-2′,6′),6.88(1H,d,J=2.4 Hz,H-8),6.79(1H,d,J=2.4 Hz,H-6),4.04(3H,s,3-OCH3),3.90(6H,s,3′,5′-OCH3); 13C-NMR(C5D5N,100 MHz) δ:179.1(C-4),166.0(C-7),163.0(C-5),157.6(C-8a),156.4(C-2),149.1(C-3′,5′),141.3(C-4′),139.0(C-3),120.6(C-1′),107.5(C-2′,6′),105.6(C-4a),99.8(C-6), 94.8(C-8),60.1(3-OCH3),56.6(3′,5′-OCH3)。以上数据与文献[12-13]对照一致,故鉴定化合物8为5,7,4′-trihydroxy-3,3′,5′-trimethoxyflavone。
  化合物9 白色粉末;ESI-MS m/z 337 [M+H]+; 1H-NMR(CDCl3,400 MHz) δ:13.12(1H,s,5-OH),7.82(1H,s,H-2),7.37(2H,d,J=8.4 Hz,H-2′,6′),6.87(2H,d,J=8.4 Hz,H-3′,5′),6.72(1H,d,J=10.0 Hz,H-4″),6.34(1H,s,H-8),5.62(1H,d,J=10.0 Hz,H-3″),1.47(6H,s,2″-CH3×2); 13C-NMR(CDCl3,100 MHz) δ:181.0(C-4),159.5(C-7),157.3(C-5),156.8(C-8a),156.0(C-4′),152.6(C-2),130.3(C-2′,C-6′),128.2(C-3″),123.5(C-1′),122.9(C-3),115.6(C-3′,C-5′),115.4(C-4″),106.1(C-4a),105.6(C-6),94.9(C-8),78.1(C-2″),28.3(2″-CH3×2)。以上数据与文献[14]对照一致,故鉴定化合物9为alpinumisoflavone。
  4 结果与讨论
  本研究对毛果鱼藤中黄酮类化学成分进行了研究,从中分离并鉴定了9个黄酮类化合物。这些化合物均首次从该植物中分离得到。
  利用MTT法对所得黄酮类化合物进行了抗肿瘤活性的测试,结果显示,化合物2对人红白血病细胞HEL和人白血病细胞K562具有一定的抑制活性。本研究的结果为进一步开发利用中药毛果鱼藤提供了物质基础和科学依据。
  [致谢] 核磁数据和质谱分别由贵州省中国科学院天然产物化学重点实验室张建新研究员与王道平副研究员测定。
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