黃永中,張 偲,黃 升,羅雄明*,王建華*
1重慶大學(xué)生物工程學(xué)院,重慶400044;2中國(guó)科學(xué)院南海海洋研究所,廣州510301
大管Micromelum falcatum(Lour.)Tan.為蕓香科(Rutaceae)小蕓木屬植物,在我國(guó)主要分布于海南島、廣東西南部、廣西合浦至東興一帶和云南東南部。在中國(guó)傳統(tǒng)醫(yī)學(xué)中主要用于治療創(chuàng)傷感染、風(fēng)濕、咳嗽、發(fā)熱等。文獻(xiàn)報(bào)道其主要化學(xué)成分為香豆素、生物堿和苯丙酸類衍生物等[1-4]。本實(shí)驗(yàn)對(duì)大管的化學(xué)成分進(jìn)行了較為系統(tǒng)的研究,分離得到12個(gè)化合物,包括6個(gè)香豆素和6個(gè)芳香類化合物。其中化合物1為新的天然產(chǎn)物;除5和6外,其余化合物均首次從該植物中分離得到。
NMR譜用Bruker DRX-500MHz核磁共振波譜儀測(cè)定,TMS為內(nèi)標(biāo);高效液相色譜儀為半制備型Waters 600(phenomenex OOG-4252-NO,250 mm×10 mm i.d.,5 μm);ESI-MS用島津 API2000 LC/MS/ MS型液相色譜-質(zhì)譜儀聯(lián)用儀測(cè)定;凝膠Sephadex LH-20為Pharmacia公司產(chǎn)品;柱層析用硅膠(200~300目)為青島海洋化工廠產(chǎn)品。高效液相用試劑為色譜純,其他所用試劑均為分析純。
大管莖皮于2009年采集于海南省三亞市,材料由中國(guó)科學(xué)院南海海洋研究所羅雄明助理研究員鑒定為 Micromelun falcatum(Lour.)Tan.,樣品標(biāo)本(No.DAJIAN020)存放于中國(guó)科學(xué)院南海海洋研究所標(biāo)本館。
干燥的大管約10 kg,用95%的乙醇浸提3次,每次7 d。提取液減壓濃縮,用水分散,依次用石油醚、乙酸乙酯、正丁醇萃取。乙酸乙酯部分140 g,首先采用硅膠(200~300目)層析柱,以石油醚-丙酮(9∶1→1∶2)進(jìn)行梯度洗脫,共接收120個(gè)流分,經(jīng)TLC檢測(cè),合并成A-J共10個(gè)粗組分,其中A組分先過(guò)凝膠Sephadex LH-20(氯仿-甲醇1∶1)柱色譜,然后上硅膠柱(200~300目),氯仿-丙酮(60∶1)洗脫,所得部分再用凝膠Sephadex LH-20(甲醇)進(jìn)一步純化,得化合物5(114.8 mg)和6(26.5 mg);B組分先過(guò)凝膠Sephadex LH-20(氯仿-甲醇1∶1),然后上硅膠柱(200~300目),氯仿-丙酮(10∶1~2∶1)梯度洗脫,所得部分再過(guò)凝膠Sephadex LH-20(甲醇),然后用HPLC(MeOH/H2O 45∶55)純化,得到化合物1(12.3 mg)、4(12.9 mg)、10(8.8 mg)、11 (6.2 mg);D組分先過(guò)凝膠Sephadex LH-20(氯仿-甲醇1∶1),然后上硅膠柱,氯仿-丙酮(3∶1~1∶1)梯度洗脫,所得部分用HPLC(MeOH/H2O 45∶55)純化,得到化合物9(11.2 mg)。
正丁醇萃取物42.0 g溶于水中,過(guò)濾,得到水不溶部分10.0 g和水溶解部分30.0 g,后者過(guò)D101大孔吸附樹(shù)脂,依次用水、20%、40%、60%、95%的乙醇洗脫,得五組餾分。經(jīng)TLC檢測(cè),20%和40%乙醇洗脫得到的餾分斑點(diǎn)較相似,故將其合并進(jìn)行分離純化。水不溶部分10.0 g過(guò)硅膠(200~300目)柱分段,依次用石油醚-丙酮(6∶4)、氯仿-丙酮(20∶1)洗脫,所得部分再過(guò)凝膠 Sephadex LH-20 (氯仿-甲醇1∶1)進(jìn)一步純化,得到化合物3(36.3 mg)和12(12.0 mg);20%和40%部分的浸膏合并過(guò)硅膠(200~300目)柱分段,用氯仿-甲醇(8∶2)洗脫,所得部分過(guò)凝膠 Sephadex LH-20(氯仿-甲醇1∶1),再通過(guò)HPLC(MeOH/H2O 50∶50)純化,得到化合物2(7.2 mg)、7(11.7 mg)和8(9.7 mg)。
化合物1:黃色晶體,ESI-MS m/z:257[M+ Na]+,結(jié)合1H NMR譜和ESI-MS數(shù)據(jù)推斷分子式為C12H10O5。1H NMR(500 MHz,DMSO-d6)δ:9.83 (1H,s,5-CHO),7.92(1H,d,J=9.5 Hz,H-4),6.83 (1H,s,H-8),6.36(1H,d,J=9.5Hz,H-3),3.82 (3H,s,6-OMe),3.77(3H,s,7-OMe)。13C NMR(125 MHz,DMSO-d6)δ:193.2(5-CHO),161.6(C-2),148.4(C-6),147.2(C-7),143.9(C-10),143.5(C-4),121.6(C-9),118.8(C-5),113.7(C-3),109.1 (C-8),56.5(6-OMe),56.2(7-OMe)。根據(jù)HMBC譜信號(hào),從δH9.83(s,5-CHO)與δC118.8(C-5)、121.6(C-9)和148.4(C-6)相關(guān),表明醛基在C-5上;從δH3.82(s,6-OMe)與δC148.4(C-6)相關(guān),表明甲氧基在C-6上;從δH6.83(s,H-8)與143.9(C-10)147.2(C-7)和109.1(C-8)相關(guān),表明氫信號(hào)在C-8上;從δH3.77(s,7-OMe)與δC147.2(C-7)相關(guān),表明C-7上有甲氧基。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[5],故鑒定化合物1為5-formyl-6,7-dimethoxycoumarin。
化合物2:白色粉末,ESI-MS m/z:377[M+ Na]+,結(jié)合1H NMR和13C NMR譜數(shù)據(jù)推斷分子式為 C16H18O9。UV(CH3OH):λmax(log ε)226.4 (1.70),288.9(0.75),337.5(1.00)nm;1H NMR (500 MHz,CD3OD)δ:7.91(1H,d,J=9.0 Hz,H-4),7.21(1H,s,H-8),7.19(1H,s,H-5),6.32(1H,d,J =9.5 Hz,H-3),5.08(1H,d,J=7.5 Hz,H-1'),3.94(1H,d,J=2.5 Hz,H-5'),3.92(3H,s,7-OMe),3.72(1H,m,H-6'),3.55(3H,m,H-2',3',6'),3.43(1H,d,J=8.5 Hz,H-4');13C NMR(125 MHz,CD3OD)δ:163.6(C-2),151.8(C-7),150.7 (C-10),148.3(C-4),145.7(C-6),114.6(C-3),114.6(C-9),110.8(C-5),105.3(C-1'),102.1(C-8),78.4(C-5'),77.9(C-3'),74.8(C-2'),71.3(C-4'),62.5(C-6'),57.1(7-OMe)。根據(jù)HMBC譜信號(hào),從δH7.19(s,H-5)與δC110.8(C-5)、114.6(C-9)和145.7(C-6)相關(guān),表明氫在C-5上;從δH5.08 (1H,d,J=7.5 Hz,H-1')與δC145.7(C-6)相關(guān),表明葡萄糖分子連接在C-6上;從δH7.21(s,H-8)與δC151.8(C-7),102.1(C-8)和150.7(C-10)相關(guān),表明C-8上有氫;從δH3.92(s,7-OMe)與δC151.8 (C-7)相關(guān),表明C-7上有甲基。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[6],故鑒定化合物2為isoscoploletin-β-D-glucoside。
化合物3:黃色晶體,ESI-MS m/z:267[M+ Na]+,分子式為 C14H12O4。1H NMR(500 MHz,CDCl3)δ:7.80(1H,d,J=16.5 Hz,H-1'),7.65(2H,dd,J=3.0,9.5 Hz,H-4,H-5),6.84(1H,s,H-8),6.75(1H,d,J=16.5Hz,H-2'),6.30(1H,t,J=3.0,9.5Hz,H-3),3.97(3H,s,7-OMe),2.40(3H,s,H-4');13C NMR(125 MHz,CDCl3)δ:198.6(C-3'),161.1(C-2),160.5(C-7),157.0(C-10),143.1(C-4),136.8(C-1'),128.4(C-2'),127.6(C-5),121.1 (C-6),114.1(C-3),112.5(C-9),99.6(C-8),56.3 (7-OMe),27.4(C-4')。根據(jù)HMBC譜信號(hào),從δH2.40(s,H-4')與δC198.6(C-3')和128.4(C-2')相關(guān),從δH7.80(1H,d,J=16.5 Hz,H-1')與δC127.6 (C-5),121.1(C-6),160.5(C-7),198.6(C-3')和128.4(C-2')相關(guān),表明丁烯支鏈在C-6上。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[7],故鑒定化合物3為6-(trans-1-buten-3-only)-7-methoxycoumarin。
化合物4:黃色晶體,ESI-MS m/z:215[M+ Na]+,結(jié)合1H NMR和13C NMR譜數(shù)據(jù)推斷分子式為C10H8O4。1H NMR(500 MHz,CD3OD)δ:7.86 (1H,d,J=9.0 Hz,H-4),7.11(1H,s,H-8),6.78 (1H,s,H-5),6.21(1H,d,J=9.0 Hz,H-3),3.92 (3H,s,7-OMe)。13C NMR(125 MHz,CD3OD)δ: 161.2(C-2),151.8(C-7),150.5(C-10),148.7(C-4),148.2(C-6),114.5(C-3),113.6(C-9),110.2 (C-5),102.5(C-8),56.4(7-OMe)。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[8],故鑒定化合物4為異莨菪亭(6-羥基-7-甲氧基香豆素,isoscopoletin)。
化合物5:白色粉末,ESI-MS m/z:399[M+ Na]+,分子式為 C20H24O7。1H NMR(500 MHz,CDCl3)δ:7.56(1H,d,J=9.5Hz,H-4),7.35(1H,d,J=8.5Hz,H-5),6.81(1H,d,J=9.0Hz,H-6),6.16(1H,d,J=9.5 Hz,H-3),6.08(1H,m,H-1'),5.58(1H,d,J=5.5 Hz,H-2'),5.26(1H,dd,J= 2.5,3.0 Hz,H-4'),5.20(1H,dd,J=1.5,3.5 Hz,H-4'),4.67(1H,dd,J=2.0 Hz,H-5'),4.56(1H,dd,J=1.5,2.0 Hz,H-5'),3.85(3H,s,7-OMe),2.14(2H,d,J=8.0 Hz,H-2''),2.02(1H,m,H-3''),0.86(6H,d,J=7.0 Hz,3''-CH3);13C NMR (125 MHz,CDCl3)δ:172.8(C-1''),161.0(C-7),160.4(C-2),153.5(C-10),147.0(C-3'),143.7(C-4),129.2(C-5),114.6(C-8),113.1(C-3),113.0 (C-9),108.8(C-4'),108.0(C-6),77.2(C-2'),76.7(C-1'),71.2(C-5'),56.1(C-7-OMe),43.4(C-2''),25.8(C-3''),22.2(C-4''),22.2(C-5'')。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[9],故鑒定化合物5為microfalcatin isovalerate。
化合物6:無(wú)色晶體,紫外燈下有藍(lán)色熒光,ESIMS m/z:311[M+Na]+,分子式為 C15H12O6。1H NMR(500 MHz,CDCl3)δ:7.94(1H,d,J=9.5 Hz,H-4),7.67(1H,s,H-5),7.08(1H,s,H-6),6.26 (1H,d,J=9.5 Hz,H-3),5.55(1H,d,J=6.5 Hz,H-1'),4.03(1H,d,J=6.5 Hz,H-2'),3.92(3H,s,7-OMe),1.67(3H,s,H-5');13C NMR(125 MHz,CDCl3)δ:172.3(C-4'),160.4(C-2),159.9(C-7),156.6(C-10),142.9(C-4),127.6(C-5),120.2 (C-6),114.2(C-3),112.4(C-9),99.8(C-8),77.3(C-1'),63.5(C-2'),57.3(C-3'),56.5(7-OMe),11.2(C-5')。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[10-12],故鑒定化合物6為小蕓木寧micromelin。
化合物7:白色針晶,ESI-MS m/z:395[M+ Na]+,分子式為C17H24O9。UV(CH3OH):λmax(log ε)231.1(2.88),272.3(2.00),337.5(1.42) nm;1H NMR(500 MHz,CD3OD)δ:6.77(2H,s,H-2,6),6.56(1H,d,J=15.5Hz,H-7),6.35(1H,m,H-8),4.90(1H,s,H-1'),4.24(2H,dd,J=1.0,5.5Hz,H-9),3.88(6H,s,3-OMe,5-OMe),3.79 (1H,m,H-6'),3.68(1H,dd,J=5.0,12.0 Hz,H-6'),3.49(1H,m,H-2'),3.43(1H,m,H-3'),3.43 (1H,m,H-4'),3.23(1H,br m,H-5');13C NMR(125 MHz,CD3OD)δ:154.4(C-3,5),135.9(C-4),135.3 (C-1),131.3(C-7),130.1(C-8),105.5(C-2,6),105.4(C-1'),78.4(C-5'),77.9(C-3'),75.8(C-2'),71.4(C-4'),63.6(C-9),62.6(C-6'),57.1(3-OMe,5-OMe)。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[13],故鑒定化合物7為丁香苷。
化合物 8:白色晶體,ESI-MS m/z:365[M+ Na]+,結(jié)合1H NMR和13C NMR譜數(shù)據(jù)推斷分子式為 C16H22O8。UV(CH3OH):λmax(log ε)214.7 (1.45),257.0(1.20)nm;1H NMR(500 MHz,CD3OD)δ:7.20(1H,d,J=14.0 Hz,H-5),7.08 (1H,d,J=8.5 Hz,H-2),6.96(1H,dd,J=2.0,8.5 Hz,H-6),6.56(1H,d,J=16.0 Hz,H-7),6.30(1H,m,H-8),4.90(1H,s,H-1'),4.23(2H,dd,J=1.5,6.0 Hz,H-9),3.88(1H,m,H-6'),3.87(3H,s,3-OMe),3.70(1H,m,H-6'),3.50(2H,m,H-2',5'),3.43(2H,m,H-3',4');13C NMR(125 MHz,CD3OD) δ:150.9(C-4),147.7(C-3),133.7(C-1),131.3(C-7),128.9(C-8),120.8(C-6),118.0(C-5),111.4 (C-2),102.8(C-1'),78.3(C-3'),77.9(C-5'),75.0 (C-2'),71.4(C-4'),63.7(C-9),62.6(C-6'),56.8 (3-OMe)。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[14],故鑒定化合物8為coniferin。
化合物9:褐色針狀晶體,ESI-MS m/z:337[M+ Na]+,分子式為C14H18O8。UV(CH3OH):λmax(log ε)230.0(2.60),286.5(0.90)nm;1H NMR(500 MHz,CD3OD)δ:7.78(1H,t,J=1.0,7.5,8.0 Hz,H-7),7.55(1H,t,J=7.0,7.5 Hz,H-6),7.41(1H,d,J =8.5 Hz,H-4),7.14(1H,t,J=7.5,15.0 Hz,H-5),4.90(1H,d,J=7.5 Hz,H-1'),3.93(1H,m,H-6'),3.91(3H,s,1-OMe),3.74(1H,m,H-6'),3.54(1H,m,H-2'),3.52(1H,m,H-3'),3.50(1H,m,H-4'),3.44(1H,m,H-5');13C NMR(125 MHz,CD3OD)δ: 168.6(C-1),158.8(C-3),135.2(C-5),132.1(C-7),123.7(C-6),122.4(C-2),119.1(C-4),104.1 (C-1'),78.5(C-5'),77.6(C-3'),75.0(C-2'),71.3 (C-4'),62.6(C-6'),52.9(1-OMe)。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[15],故鑒定化合物9為methyl 2-O-β-D-glucopyranosylbenzoate。
化合物10:褐色油狀物,ESI-MS m/z:217[M+ Na]+,結(jié)合1H NMR譜和ESI-MS數(shù)據(jù)推斷分子式為 C10H10O4。UV(CH3OH):λmax(log ε)215.9 (0.85),319.7(0.90)nm;1H NMR(500 MHz,CD3OD)δ:7.59(1H,d,J=15.5 Hz,H-1'),7.19 (1H,d,J=1.5Hz,H-3),7.07(1H,dd,J=1.5,1.5 Hz,H-6),6.82(1H,d,J=8.0 Hz,H-4),6.34(1H,d,J=16.0 Hz,H-2'),3.92(3H,s,5-OMe)。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[16],故鑒定化合物10為2-hydroxy-5-methoxy-trans-cinnamic acid。
化合物11:白色粉末,ESI-MS m/z:205[M+ Na]+,結(jié)合1H NMR譜和ESI-MS數(shù)據(jù)推斷分子式為C9H10O4。1H NMR(500 MHz,CDCl3)δ:9.82(1H,s,H-7),7.15(2H,s,H-2,6),6.09(1H,s,2-OH),3.97(6H,s,3-OMe,5-OMe)。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[17],故鑒定化合物11為3,5-二甲氧基-4-羥基苯甲醛。
化合物12:褐色晶體,ESI-MS m/z:151[M+ H]+,結(jié)合1H NMR和13C NMR譜數(shù)據(jù)推斷分子式為C10H14O。1H NMR(500 MHz,CDCl3)δ:7.15(1H,dd,J=1.5,1.5 Hz,H-6),7.06(1H,ddd,J=6.5,1.5 Hz,H-5),6.91(1H,t,J=8.0,15.5 Hz,H-4),6.75(1H,d,J=8.0 Hz,H-3),2.96(1H,m,H-1'),1.64(2H,m,H-2'),1.24(3H,d,J=7.0 Hz,H-4'),0.87(3H,t,J=7.5,15.0 Hz,H-3');13C NMR(125 MHz,CDCl3)δ:153.0(C-2),133.2(C-1),127.1(C-4),126.6(C-6),120.9(C-5),115.3(C-3),33.9(C-1'),29.8(C-2'),20.5(C-4'),12.2(C-3')。以上波譜數(shù)據(jù)與文獻(xiàn)對(duì)照基本一致[18],故鑒定化合物12為鄰仲丁基苯酚。
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天然產(chǎn)物研究與開(kāi)發(fā)2012年9期