陳慶淑 周小雷陳 路江 姍陳譽丹馬國需劉華鋼袁經(jīng)權(quán)*

[1.廣西中醫(yī)藥大學(xué)科學(xué)實驗中心,廣西 南寧 530200;2.海南省人民醫(yī)院(海南醫(yī)學(xué)院附屬海南醫(yī)院),海南 ?？?570311;3.中國醫(yī)學(xué)科學(xué)院藥用植物研究所廣西分所,廣西 南寧 530023]

瑤藥南蛇風(fēng)亦稱南蛇簕,為蘇木科云實屬植物喙莢云實Caesalpinia minax Hance 的藤莖,分布廣西、廣東、福建、臺灣等省區(qū),為我國瑤族地區(qū)104 種老班瑤藥之一,主要用于哈輪(感冒)、泵烈競(尿路感染)、月藏(尿血)、就港蝦(急性腸胃炎)、碰累(痢疾) 等癥,為瑤醫(yī)常用打藥之一[1]。國內(nèi)外學(xué)者研究發(fā)現(xiàn)喙莢云實具有抗流感甲型病毒、抗腫瘤、抗帶狀皰疹、抗蛇毒、鎮(zhèn)痛以及抗炎等藥理作用[2-4]。本課題組前期從喙莢云實種子(苦石蓮) 中分離到一系列新穎卡薩烷二萜成分,具有良好抗腫瘤活性等[5-8]。但國內(nèi)外對瑤藥南蛇風(fēng),即喙莢云實藤莖部位的化學(xué)成分鮮有報道。為進(jìn)一步研究喙莢云實植物,開發(fā)瑤藥,本實驗初步對南蛇風(fēng)進(jìn)行化學(xué)成分研究,從其乙醇提取物中分離鑒定了11 個化合物,其中化合物2～3、5～6、9 為首次從該屬植物中分離得到,化合物1、7 為首次從該植物中分離得到。

1 材料

Trace DSQ 四極桿型質(zhì)譜儀(美國Finnigan 公司)；Dre-600 型核磁共振光譜儀(美國瓦里安公司)；薄層色譜和柱色譜硅膠(青島海洋化工有限公司)。常規(guī)試劑均為分析純。

南蛇風(fēng)采自廣西壯族自治區(qū)南寧市邕寧區(qū)三塘鎮(zhèn),經(jīng)廣西中醫(yī)藥大學(xué)藥用植物教研室韋松基教授鑒定為蘇木科云實屬植物喙莢云實Caesalpinia minax Hance 的藤莖,標(biāo)本保存于廣西中醫(yī)藥大學(xué)科學(xué)實驗中心。

2 提取與分離

將藥材陰干后稱取20 kg,粉碎,加入85%乙醇浸漬滲漉提取,收集合并滲漉液,減壓回收乙醇至基本無乙醇味。總浸膏加入適量水進(jìn)行分散均勻后,依次以石油醚、氯仿、乙酸乙酯、正丁醇進(jìn)行萃取。取氯仿萃取物(90 g) 上硅膠色譜柱,分別在用石油醚-乙酸乙酯(100∶0～0∶100) 洗脫,在石油醚-乙酸乙酯(40∶1) 的洗脫部分得到2 個粗粉末,取其中一粉末經(jīng)硅膠柱、結(jié)晶,得到化合物1 (30 mg)；另一粗粉末經(jīng)石油醚反復(fù)洗除色素,重結(jié)晶,得到化合物2 (160 mg)。在石油醚-乙酸乙酯(10∶1) 的洗脫部分得到黃色膠狀物和黃色粉末,黃色膠狀物經(jīng)硅膠柱、結(jié)晶、重結(jié)晶,得到化合物3 (250 mg)、10 (150 mg)；黃色粉末用氯仿-丙酮(20∶1～1∶1) 洗脫,在乙酸乙酯中析出化合物4 (40 mg)。在石油醚-乙酸乙酯(5∶1) 的洗脫部分得到白色針狀結(jié)晶和白色粉末,白色針狀結(jié)晶經(jīng)硅膠柱、結(jié)晶,得到化合物5 (12 mg)；白色粉末通過石油醚-丙酮(10∶1～1∶1)洗脫,得到化合物6 (15 mg)。用石油醚-乙酸乙酯(1∶1) 洗脫,得到化合物7 (17 mg)。用乙酸乙酯洗脫,得到白色絮狀物和白色粉末,白色絮狀物經(jīng)Sephadex LH-20 柱,得到化合物8 (7 mg)、9(8 mg),白色粉末用甲醇重復(fù)洗除色素,得到化合物11 (35 mg)。

3 結(jié)構(gòu)鑒定

化合物1:白色針狀結(jié)晶,ESI-MS m/z:427.5 [M+H]+。1H-NMR (CDCl3,600 MHz) δ:0.65,0.80,0.88,0.93,0.94,0.98,1.11 (each 3H,s,7×CH3),0.81 (3H,d,J=6.8 Hz,H-4),提示分子式含有8 個甲基,其中7 個為孤立甲基；13C-NMR (CDCl3,150 MHz) δ:21.3 (C-1),40.5 (C-2),212.1 (C-3),57.2 (C-4),41.1(C-5),40.3 (C-6),17.2 (C-7),52.1 (C-8),36.4 (C-9),58.5 (C-10),34.6 (C-11),29.5(C-12),38.7 (C-13),37.3 (C-14),31.4 (C-15),35.0 (C-16),29.0 (C-17),41.8 (C-18),34.3 (C-19),27.2 (C-20),31.8 (C-21),38.2(C-22),5.8 (C-23),13.6 (C-24),16.9 (C-25),19.2 (C-26),17.7 (C-27),31.1 (C-28),34.0 (C-29),30.8 (C-30)。以上數(shù)據(jù)與文獻(xiàn)[9]基本一致,故鑒定為木栓酮。

化合物2:白色粉末,ESI-MS m/z:201.6[M+H]+。1H-NMR (CDCl3,600 MHz) δ:0.88(3H,m,-CH3)；13C-NMR (CDCl3,150 MHz) δ:63.5 (C-OH),32.8,31.9,29.6,29.6,29.6,29.6,29.4,29.3,25.7,24.7,22.6,14.0 (13個脂肪C)。以上數(shù)據(jù)與文獻(xiàn)[10]基本一致,故鑒定為正十三醇。

化合物3:白色針狀結(jié)晶,ESI-MS m/z:457.4 [M+H]+。1H-NMR (CD3OD,600 MHz) δ:4.69 (1H,brs,H-30α),4.57 (1H,brs,H-30β),3.33 (1H,H-3),0.74,0.84,0.93,0.95,0.99,1.68 (6×CH3)；13C-NMR (CD3OD,150 MHz) δ:38.5 (C-1),26.6 (C-2),77.8(C-3),38.6 (C-4),55.4 (C-5),18.0 (C-6),34.2 (C-7),40.5 (C-8),50.5 (C-9),36.9(C-10),20.7 (C-11),25.5 (C-12),38.2 (C-13),42.2 (C-14),30.3 (C-15),31.9 (C-16),56.0 (C-17),47.0 (C-18),48.4 (C-19),150.3 (C-20),29.4 (C-21),36.7 (C-22),27.1 (C-23),14.6 (C-24),15.3 (C-25),15.2(C-26),13.6 (C-27),178.5 (C-28),19.0 (C-29),108.6 (C-30)。以上數(shù)據(jù)與文獻(xiàn)[11]基本一致,并且與白樺脂酸標(biāo)準(zhǔn)對照品通過薄層色譜進(jìn)一步鑒定,在3 種不同的展開系統(tǒng)中比較色譜行為完全一致,故鑒定為白樺脂酸。

化合物4:黃色針狀結(jié)晶,ESI-MS m/z:283.1 [M+H]+。1H-NMR (CD3OD,600 MHz) δ:5.34 (2H,d,J=1.9 Hz,H-2),7.79 (1H,d,J=8.5 Hz,H-5),6.52 (1H,dd,J=8.6,2.2 Hz,H-6),6.29 (1H,d,J=2.1 Hz,H-8),7.71 (1H,brs,H-9),7.33 (2H,d,J=8.1 Hz,H-2′,6′),7.02 (2H,d,J=8.5 Hz,H-3′,5′),3.84 (3H,s,-OCH3)；13C-NMR (CD3OD,150 MHz) δ:67.6 (C-2),128.9 (C-3),181.5(C-4),129.3 (C-5),110.8 (C-6),165.2 (C-7),102.2 (C-8),136.2 (C-9),163.4 (C-10),114.4 (C-11),126.8 (C-1′),131.8 (C-2′),113.9 (C-3′),160.9 (C-4′),113.9 (C-5′),131.8 (C-6′),54.5 (-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[12]基本一致,故鑒定為bonducellin。

化合物5:白色針狀結(jié)晶,ESI-MS m/z:443.2 [M-H ]-,467.3 [M+Na ]+。1H-NMR(CDCl3,600 MHz) δ:7.70 (2H,d,J=7.4 Hz,H-16,20),7.51 (1H,t,J=7.1 Hz,H-18),7.41(2H,t,J=7.6 Hz,H-17,19),2.95 (3H,s,H-12)；13C-NMR (CDCl3,150 MHz) δ:170.4 (C-1,amide),49.6 (C-2),37.6 (C-3),136.8 (C-4),128.8 (C-5),129.0 (C-6),127.0 (C-7),129.0 (C-8),128.8 (C-9),64.8 (C-10),171.0(C-11,ester),21.0 (C-12),55.2 (C-13),167.3(C-14,amide),133.8 (C-15),127.2 (C-16),128.1 (C-17),132.1 (C-18),128.1 (C-19),127.2 (C-20),38.6 (C-21),136.9 (C-22),129.3 (C-23),129.5 (C-24),127.3 (C-25),129.5 (C-26),129.3 (C-27)。以上數(shù)據(jù)與文獻(xiàn)[13]基本一致,故鑒定為金色酰胺醇酯。

化合物6:白色粉末,ESI-MS m/z:457.3[M-H]-。1H-NMR (CD3COCD3,600 MHz) δ:1.91(3H,s,H-29),0.88 (3H,s,H-23),0.83 (3H,s,H-27),0.81 (3H,s,H-26),0.72 (3H,s,H-25),0.62 (3H,s,H-24)；13C-NMR (CD3COCD3,150 MHz) δ:38.6 (C-1),27.4 (C-2),77.6 (C-3),38.7 (C-4),55.4 (C-5),18.2 (C-6),34.2(C-7),40.5 (C-8),50.5 (C-9),37.1 (C-10),20.8 (C-11),27.0 (C-12),37.2 (C-13),42.1(C-14),29.5 (C-15),31.4 (C-16),55.7 (C-17),48.8 (C-18),51.1 (C-19),210.3 (C-20),28.3 (C-21),36.4 (C-22),27.6 (C-23),15.1(C-24),15.7 (C-25),15.5 (C-26),14.1 (C-27),176.5 (C-28),29.6 (C-29)。以上數(shù)據(jù)與文獻(xiàn)[14]基本一致,故鑒定化合物為platanic acid。

化合物7:橙紅色針狀結(jié)晶,ESI-MS m/z:229.1 [M+H]+。1H-NMR (CD3OD,600 MHz) δ:7.33 (2H,d,J=8.2 Hz,H-2′,6′),6.94(1H,d,J=16.2 Hz,β-H),6.78 (1H,d,J=16.2 Hz,α-H),6.75 (2H,d,J=8.3 Hz,H-3′,5′),6.44 (2H,d,J=2.0 Hz,H-2,6),6.15 (1H,s,H-4)；13C-NMR (CD3OD,150 MHz)δ:158.1 (C-3,5),156.9 (C-4′),139.9 (C-1),128.9 (C-1′),128.0 (C-β),127.2 (C-2′,6′),125.4 (C-α),114.5 (C-3′,5′),104.1(C-2,6),101.1 (C-4)。以上數(shù)據(jù)與文獻(xiàn)[15]基本一致,故鑒定為反式白藜蘆醇。

化合物8:白色粉末,ESI-MS m/z:525.3[M+H]+。1H-NMR (CDCl3,600 MHz) δ:4.87(1H,t,J=2.4 Hz,H-1),1.84～1.87 (1H,m,H-2a),1.70～1.72 (1H,m,H-2b),1.73～1.75(1H,m,H-3a),1.04～1.07 (1H,m,H-3b),5.38 (1H,d,J=9.0 Hz,H-6),5.58 (1H,t,J=9.0 Hz,H-7),2.23～2.25 (1H,m,H-8),2.60 (1H,t,J=12.0 Hz,H-9),1.65～1.67(1H,m,H-11a),2.02～2.04 (1H,m,H-11b),5.85 (1H,s,H-15),1.54 (1H,s,H-17),1.05(1H,s,H-18),1.06 (1H,s,H-19),1.10 (1H,s,H-20),1.99 (1-OAc,s),1.90 (6-OCOOH,s),2.08 (7-OCOOH,s)；13C-NMR (CDCl3,150 MHz) δ:71.5 (C-1),21.5 (C-2),31.4 (C-3),37.5 (C-4),78.3 (C-5),74.0 (C-6),74.4(C-7),49.5 (C-8),33.0 (C-9),43.6 (C-10),36.7 (C-11),103.6 (C-12),174.4 (C-13),73.9 (C-14),113.0 (C-15),169.0 (C-16),19.7 (C-17),29.5 (C-18),23.5 (C-19),15.8(C-20),20.3,168.7 (1-OCOOH),20.5,169.4(6-OCOOH),20.3,169.8 (7-OCOOH)。以上數(shù)據(jù)與文獻(xiàn) [16 ]基本一致,故鑒定為neocaesalpin L。

化合物9:白色粉末。1H-NMR (CD3OD,600 MHz) δ:5.37 (1H,brs,H-6),5.08 (1H,d,J=7.7 Hz,H-1′)；13C-NMR (CD3OD,150 MHz)δ:37.5 (C-1),30.2 (C-2),78.7 (C-3),40.0(C-4),140.9 (C-5),123.8 (C-6),34.2 (C-7),32.1 (C-8),50.3 (C-9),37.0 (C-10),21.3(C-11),40.3 (C-12),42.5 (C-13),56.8 (C-14),24.5 (C-15),28.6 (C-16),57.0 (C-17),12.2 (C-18),19.4 (C-19),36.4 (C-20),19.0(C-21),138.9 (C-22),129.5 (C-23),46.1 (C-24),29.5 (C-25),20.0 (C-26),19.2 (C-27),23.4 (C-28),12.0 (C-29),103.0 (C-1′),75.3(C-2′),78.5 (C-3′),71.7 (C-4′),78.1 (C-5′),62.9 (C-6′)。以上數(shù)據(jù)與文獻(xiàn)[17]基本一致,故鑒定為Δ～ (5,22) 豆甾醇-3-O-β-D-吡喃葡萄糖苷。

化合物10:白色固體,TLC 噴灑10% 硫酸乙醇顯紫紅色。與豆甾醇標(biāo)準(zhǔn)對照品共薄層,在3 種不同的展開系統(tǒng)中比較Rf值完全一致,且顯色也相同,故鑒定為豆甾醇。

化合物11:白色粉末。1H-NMR (CD3OD,600 MHz) δ:5.36 (H,m,H-6),5.07 (H,d,J=7.5 Hz,H-1′),4.59～3.94 (m,H-2′～6′),2.73～1.01 (m),0.98～0.82 (m,5 ×-CH3),0.67 (s,-CH3)；13C-NMR (CD3OD,150 MHz)δ:37.5 (C-1),30.3 (C-2),78.5 (C-3),39.4 (C-4),140.9 (C-5),122.0 (C-6),32.2 (C-7),32.1(C-8),50.4 (C-9),37.0 (C-10),21.3 (C-11),40.0 (C-12),42.5 (C-13),56.5 (C-14),24.5(C-15),28.6 (C-16),56.3 (C-17),12.0 (C-18),19.2 (C-19),36.4 (C-20),19.0 (C-21),30.8 (C-22),26.2 (C-23),46.1 (C-24),29.5(C-25),19.5 (C-26),20.0 (C-27),23.4 (C-28),12.2 (C-29),102.6 (C-1′),75.4 (C-2′),78.7 (C-3′),71.7 (C-4′),78.1 (C-5′),62.9 (C-6′)。以上數(shù)據(jù)與文獻(xiàn)[18]基本一致,故鑒定為胡蘿卜苷。

4 討論

前期云實屬植物化學(xué)成分研究的文獻(xiàn)表明,一般是種子富含二萜成分,三萜成分較少；而莖枝正好相反,三萜成分較多,二萜成分較少。本研究分離鑒定的木栓酮、白樺脂酸、platanic acid 為三萜成分,僅neocaesalpin L 為二萜成分,與該屬前期文獻(xiàn)報道基本一致。從南蛇風(fēng)莖中分離得到的白樺脂酸[19]、金色酰胺醇酯[13]、反式白藜蘆醇[20]等成分具有抗炎、抗病毒活性,與其功效相一致,因此對其進(jìn)行化學(xué)成分研究,以期為今后開發(fā)該植物的藥用價值以及臨床療效提供理論基礎(chǔ)。