張 悅,魏 華,曹新杰,劉紅玉
(1.北京市平谷區(qū)市場監(jiān)督管理局檢驗檢測中心,北京 101200;2.吉首大學(xué)生物資源與環(huán)境科學(xué)學(xué)院,湖南 吉首 416000;3.吉首大學(xué)植物資源保護(hù)與利用湖南省高校重點實驗室,湖南 吉首 416000)
菊科魚眼草屬(Dichrocephala)植物全球約5~6種,主要分布于亞洲、非洲及大洋洲的熱帶地區(qū).我國有3種,分別是菊葉魚眼草(D.chryaanthemifoliaDC)、魚眼草(D.integrifolia(Linnaeus f.)Kuntze)和小魚眼草(D.benthamiiC.B.Clarke)[1].魚眼草屬植物的全草多作為民間用藥,其中小魚眼草為傣族常用藥物,稱為“帕滾姆”,味苦,具有清熱解毒、祛風(fēng)明目功效,主要用于治療小兒感冒發(fā)燒、肺炎、肝炎、小兒消化不良、痢疾、瘧疾、牙痛、夜盲癥等,外用可治療瘡瘍濕疹、皮炎、子宮托垂和脫肛等病癥[2-4].現(xiàn)代藥理研究表明,小魚眼草提取物具有較強(qiáng)的抑菌、鎮(zhèn)痛、降糖和殺蟲等藥理作用[5].然而,目前有關(guān)小魚眼草化學(xué)成分的研究報道較少,藥效物質(zhì)基礎(chǔ)亟待闡明.基于此,本研究采用各種柱色譜技術(shù)對小魚眼草的乙醇提取物進(jìn)行分離純化,采用波譜技術(shù)結(jié)合化學(xué)方法進(jìn)行結(jié)構(gòu)鑒定,以期探明小魚眼草的化學(xué)成分組成,為后期深入研究小魚眼草的藥用價值,闡明其藥用成分及機(jī)理提供參考.
Bruker Avance Ⅲ 600型核磁共振波譜儀(德國Bruker公司),賽默飛世LTQ-Obitrap XL液質(zhì)聯(lián)用儀(美國Thermo Fisher公司),BYLABUV-Ⅲ紫外燈(北京炳洋科技有限公司),柱色譜用硅膠(青島海洋化工有限公司),薄層色譜用硅膠G、H、GF-254和柱色譜用硅膠(青島海洋化工有限公司),Sephadex LH-20凝膠(Pharmacia公司),MCI(日本三菱化學(xué)公司),常規(guī)試劑均為分析純.
小魚眼草全草采于云南省景洪市,經(jīng)吉首大學(xué)植物資源保護(hù)與利用重點實驗室陳功錫教授鑒定為菊科魚眼草屬小魚眼草D.benthamiiC.B.Clarke.
小魚眼草全草(8.0 kg)干燥后粉碎過80目篩,藥材粉末加10倍量甲醇加熱回流提取2次,每次2 h,合并提取液,減壓回收溶劑,濃縮后得總浸膏541 g.總浸膏用水分散后,依次用石油醚、二氯甲烷、乙酸乙酯、正丁醇各萃取三次,萃取液減壓濃縮至干.最終得到石油醚部位浸膏143.2 g,二氯甲烷部位浸膏86.9 g,乙酸乙酯部位浸膏46.7 g,正丁醇部位浸膏161.8 g.
取二氯甲烷部位浸膏86.9 g經(jīng)硅膠柱色譜(100~200目)分離,石油醚-乙酸乙酯(1∶0~0∶1)梯度洗脫,得到12個餾分(Fr.DB1~Fr.DB12).其中餾分Fr.DB2經(jīng)硅膠柱色譜(300~400目)分離,石油醚-二氯甲烷(1∶0,100∶1,80∶1,70∶1,60∶1)洗脫得到化合物6(5.1 mg),8(5.8 mg),9(7.4 mg)和11(13.6 mg);餾分Fr.DB3經(jīng)硅膠柱色譜(300~400目)分離,石油醚-二氯甲烷(90∶1,80∶1,70∶1,50∶1,40∶1)洗脫得到化合物10(2.9 mg),1(7.1 mg),2(3.6 mg)和4(5.6 mg);Fr.DB4經(jīng)硅膠柱色譜(300~400目)分離,石油醚-丙酮(100∶1,90∶1,70∶1,60∶1,50∶1)洗脫得到化合物3(4.8 mg),5(7.5 mg)和13(8.0 mg);Fr.DB5經(jīng)硅膠柱色譜(300~400目)分離,石油醚-丙酮(90∶1,70∶1,60∶1,50∶1,40∶1)洗脫得到化合物7(2.0 mg)和15(6.2 mg);Fr.DB6經(jīng)硅膠柱色譜(300~400目)分離,二氯甲烷-丙酮(100∶1,90∶1,80∶1,70∶1,60∶1)洗脫后再經(jīng)HPLC純化得到化合物12(1.4 mg),14(3.5 mg)和16(2.0 mg).
化合物1,無色粉末,ESI-MSm/z:308[M+Na]+;1H—NMR(600 MHz,CDCl3)δH:6.42(1H,d,J=14.4 Hz,H-2),7.41(1H,m,H-3),6.72(1H,m,H-4),6.74(1H,d,J=15.6 Hz,H-5),6.96(1H,d,J=1.8 Hz,H-2′),6.75(1H,d,J=8.0 Hz,H-5′),6.87(1H,dd,J=8.0,1.8 Hz,H-6′),5.96(2H,s,OCH2O);13C-NMR(150 MHz,CDCl3)δC:165.4(C-1),119.9(C-2),142.5(C-3),125.3(C-4),138.2(C-5),131.0(C-1′),105.8(C-2′),148.1(C-3′),148.2(C-4′),108.5(C-5′),122.6(C-6′),101.3(—O—CH2—O—),43.5(C-1″),26.3(C-2″),24.7(C-3″),26.2(C-4″),46.8(C-5″).以上數(shù)據(jù)與文獻(xiàn)[6]報道的胡椒堿基本一致.
化合物2,黃色油狀物,ESI-MSm/z:330[M+Na]+;1H—NMR(600 MHz,CD3COCD3)δH:0.87(6H,d,J=6.6 Hz,H-9,10),3.80(3H,s,3′-OCH3),4.26(2H,d,J=6.0 Hz,H-7′),6.70(1H,d,J=7.8 Hz,H-5′),6.72(1H,dd,J=7.8,1.8 Hz,H-6′),6.89(1H,d,J=1.8 Hz,H-2′);13C—NMR(150 MHz,CD3COCD3)δ:172.8(C-1),36.7(C-2),26.3(C-3),30.6(C-4),28.1(C-5),28.0(C-6),39.9(C-7),28.6(C-8),22.9(C-9,10),132.3(C-1′),112.5(C-2′),148.4(C-3′),146.4(C-4′),115.7(C-5′),121.0(C-6′),43.5(C-7′),56.3(OCH3).以上數(shù)據(jù)與文獻(xiàn)[7]報道的辣椒堿基本一致.
化合物3,黃色油狀物,ESI-MSm/z:328[M+Na]+;1H—NMR(600 MHz,CD3COCD3)δH:0.95(6H,d,J=7.2 Hz,H-9,10),3.79(3H,s,3′-OCH3),4.26(2H,d,J=6.0 Hz,H-7′),5.31(1H,m,H-6),5.33(1H,m,H-7),6.73(1H,d,J=8.4 Hz,H-5′),6.75(1H,dd,J=8.4,1.8 Hz,H-6′),6.87(1H,d,J=1.8 Hz,H-2′);13C—NMR(150 MHz,CD3COCD3)δ:172.7(C-1),36.9(C-2),26.3(C-3),30.6(C-4),33.0(C-5),127.9(C-6),138.3(C-7),31.6(C-8),23.1(C-9,10),132.1(C-1′),112.5(C-2′),148.4(C-3′),146.8(C-4′),115.7(C-5′),121.0(C-6′),43.5(C-7′),56.4(OCH3).以上數(shù)據(jù)與文獻(xiàn)[8]報道的二氫辣椒堿基本一致.
化合物4,無色粉末,ESI-MSm/z:212[M+Na]+;1H—NMR(600 MHz,CDCl3)δH:1.43(3H,t,J=7.2 Hz,—OCH2CH3),4.42(2H,m,J=7.2 Hz,—OCH2CH3),7.31(1H,dd,J=7.2,6.6 Hz,H-6),7.42(1H,dd,J=7.8,7.2 Hz,H-7),7.95(1H,d,J=7.8 Hz,H-8),8.02(1H,d,J=1.8 Hz,H-2),8.22(1H,d,J=6.6 Hz,H-5);13C—NMR(150 MHz,CDCl3)δC:100.4(C-3),114.1(C-7),121.8(C-2),122.2(C-6),123.5(C-5),129.1(C-9),151.5(C-8),132.2(C-4),167.4(C-10),59.8(10—OCH2CH3),29.7(10—OCH2CH3).以上數(shù)據(jù)與文獻(xiàn)[9]報道的3-吲哚甲酸乙酯基本一致.
化合物5,無色粉末,ESI-MSm/z:198[M+Na]+;1H—NMR(600 MHz,CDCl3)δH:3.75(3H,s,OCH3),7.39(1H,dd,J=7.2,6.6 Hz,H-6),7.44(1H,dd,J=7.8,7.2 Hz,H-7),7.92(1H,d,J=7.8 Hz,H-8),8.01(1H,d,J=1.8 Hz,H-2),8.18(1H,d,J=6.6 Hz,H-5);13C—NMR(150 MHz,CDCl3)δC:100.4(C-3),114.3(C-7),121.6(C-2),122.4(C-6),123.7(C-5),129.0(C-9),151.7(C-8),132.1(C-4),167.9(C-10),52.4(10—OCH3).以上數(shù)據(jù)與文獻(xiàn)[10]報道的3-吲哚甲酸甲酯基本一致.
化合物6,無色粉末,ESI-MSm/z:168[M+Na]+;1H—NMR(600 MHz,CD3OD)δH:7.21(1H,dd,J=9.0,8.4 Hz,H-5),7.24(1H,dd,J=9.6,8.4 Hz,H-6),7.48(1H,d,J=9.6 Hz,H-7),8.07(1H,s,H-2),8.13(1H,d,J=9.0 Hz,H-4),9.86(1H,s,CHO);13C—NMR(150 MHz,CD3OD)δ:139.2(C-2),122.6(C-3),120.1(C-4),125.0(C-5),123.8(C-6),113.3(C-7),139.9(C-8),125.6(C-9),187.7(CHO).以上數(shù)據(jù)與文獻(xiàn)[11]報道的3-甲酰基吲哚基本一致.
化合物7,白色粉末,分子式C30H48O4.ESI-MSm/z:495[M+Na]+;1H—NMR(600 MHz,pyridine-d5)δH:0.91(3H,s,25—CH3),1.02(3H,s,24—CH3),1.11(3H,s,26—CH3)1.12(3H,d,J=6.6 Hz,30—CH3)1.23(3H,s,23—CH3),1.45(3H,s,29—CH3),1.73(3H,s,27—CH3),3.43(1H,dd,J=15.6 Hz,4.8 Hz,H-3),5.61(1H,t,J=3.6 Hz,H-12);13C—NMR(pyridine-d5,150 MHz)δC:39.5(C-1),28.6(C-2),78.7(C-3),39.9(C-4),56.4(C-5),19.4(C-6),34.1(C-7),40.8(C-8),48.3(C-9),37.8(C-10),24.5(C-11),128.3(C-12),140.4(C-13),42.6(C-14),29.8(C-15),26.9(C-16),48.8(C-17),55.1(C-18),73.2(C-19),42.8(C-20),27.4(C-21),39.0(C-22),17.0(C-23),29.3(C-24),16.0(C-25),17.2(C-26),25.2(C-27),181.2(C-28),27.6(C-29),17.7(C-30).以上數(shù)據(jù)與文獻(xiàn)[12]報道的pomolic acid基本一致.
化合物8,白色粉末,ESI-MSm/z:355[M+Na]+;1H—NMR(600 MHz,CDCl3)δH:0.78(3H,s,H-19),0.85(3H,d,J=6.6 Hz,H-17),1.25(3H,s,H-20),4.77(2H,d,J=6.6 Hz,H-15),6.86(1H,t,J=1.8 Hz,H-3),7.09(1H,s,H-14);13C—NMR(150 MHz,CDCl3)δ:36.2(C-1),35.9(C-2),140.5(C-3),141.4(C-4),39.0(C-5),17.8(C-6),19.7(C-7),36.4(C-8),37.9(C-9),47.1(C-10),27.6(C-11),27.6(C-12),135.3(C-13),143.8(C-14),70.4(C-15),174.6(C-16),16.3(C-17),171.7(C-18),18.5(C-19),20.8(C-20).以上數(shù)據(jù)與文獻(xiàn)[13]報道的floridiolide A基本一致.
化合物9,無色粉末,ESI-MSm/z:385[M+Na]+;1H—NMR(600 MHz,CDCl3)δH:0.81(3H,s,H-19),0.86(3H,d,J=6.0 Hz,H-17),1.29(3H,s,H-20),3.38(3H,s,OCH3),5.88(1H,d,J=4.8 Hz,H-15),6.67(1H,t,J=1.8 Hz,H-3),7.07(1H,d,J=4.8 Hz,H-14);13C—NMR(150 MHz,CDCl3)δ:28.3(C-1),27.9(C-2),137.6(C-3,13),139.1(C-4),40.1(C-5),18.3(C-6),19.8(C-7),37.5(C-8),38.6(C-9),47.9(C-10),36.5(C-11),37.2(C-12),143.7(C-14),103.7(C-15),171.4(C-16),171.2(C-16),16.5(C-17),18.(C-19),21.2(C-20),56.8(OCH3).以上數(shù)據(jù)與文獻(xiàn)[13]報道的16-methoxycleroda-3,13-dien-15,16-olide-18-oic acid基本一致.
化合物10,無色粉末,ESI-MSm/z:399[M+Na]+;1H—NMR(600 MHz,CD3OD)δH:0.79(3H,s,H-19),0.87(3H,d,J=6.6 Hz,H-17),1.26(3H,t,J=6.6 Hz,—OCH2CH3),1.28(3H,s,H-20),3.73(1H,m,—OCH2CH3),3.84(1H,m,—OCH2CH3),5.91(1H,d,J=4.2 Hz,H-15),6.63(1H,t,J=3.6 Hz,H-3),6.97(1H,d,J=4.2 Hz,H-14);13C—NMR(150 MHz,CD3OD)δ:28.6(C-1),28.0(C-2),139.3(C-3,13),139.1(C-13),144.2(C-4),40.4(C-5),18.7(C-6),19.7(C-7),37.6(C-8),38.5(C-9),48.5(C-10),37.4(C-11),37.5(C-12),144.7(C-14),103.5(C-15),173.4(C-16),173.6(C-18),16.5(C-17),18.9(C-19),21.3(C-20),66.7(—OCH2CH3),15.7(OCH2CH3).以上數(shù)據(jù)與文獻(xiàn)[13]報道的16-ethoxycleroda-3,13-dien-15,16-olide-18-oic acid基本一致.
化合物11,白色晶體,mp 212~214 °C,ESI-MSm/z:449[M+Na]+;1H—NMR(600 MHz,CDCl3)δH:0.72(3H,s,CH3),0.88(3H,d,J=6.6 Hz,H-23),0.94(3H,s,CH3),1.00(3H,s,CH3),1.06(3H,s,CH3),1.19(3H,s,CH3);13C—NMR(150 MHz,CDCl3)δC:213.4(C-3),59.6(C-10),58.5(C-4),53.2(C-8),42.8(C-18),42.3(C-5),41.6(C-2),41.4(C-6),39.6(C-13),39.5(C-22),38.4(C-14),37.8(C-9),36.1(C-16),35.5(C-11),35.6(C-19),35.3(C-29),32.9(C-21),32.5(C-15),32.2(C-28),31.8(C-30),30.6(C-12),30.1(C-17),28.4(C-20),22.4(C-1),20.5(C-26),18.8(C-27),18.5(C-7),18.2(C-25),14.8(C-24),7.0(C-23).以上數(shù)據(jù)與文獻(xiàn)[14]報道的木栓酮基本一致.
化合物12,黃色粉末,ESI-MSm/z:309[M+Na]+;1H—NMR(600 MHz,DMSO-d6)δH:12.46(1H,s,5—OH),8.03(2H,d,J=8.4 Hz,H-3′,5′),6.91(2H,d,J=8.4 Hz,H-2′,6′),6.40(1H,d,J=2.4 Hz,H-8),6.16(1H,d,J=2.4 Hz,H-6).以上數(shù)據(jù)與文獻(xiàn)報道[15]的山奈酚基本一致.
化合物13,黃色粉末,ESI-MSm/z:325[M+Na]+;1H—NMR(600 MHz,CD3OD)δH:7.72(1H,d,J=2.4 Hz,H-2′),7.63(1H,dd,J=8.4,2.4 Hz,H-6′),6.87(1H,d,J=8.4 Hz,H-5′),6.38(1H,d,J=1.8 Hz,H-8),6.17(1H,d,J=1.8 Hz,H-6);13C—NMR(150 MHz,CD3OD)δC:176.1(C-4),164.0(C-7),160.9(C-5),156.2(C-9),147.9(C-4′),147.0(C-2),145.1(C-3′),135.8(C-3),122.1(C-1′),120.2(C-6′),116.4(C-5′),115.8(C-2′),103.1(C-10),98.3(C-6),93.6(C-8).以上數(shù)據(jù)與文獻(xiàn)[16]報道的槲皮素基本一致.
化合物14,無色油狀物,ESI-MSm/z:317[M+Na]+;1H—NMR(600 MHz,CD3OD)δH:1.09(6H,d,J=6.6 Hz,H-4′,5′),3.81(6H,s,3,5—OCH3),4.24(2H,dd,J=6.6,1.8 Hz,H-9),6.35(1H,dt,J=15.6,6.6 Hz,H-8),6.57(1H,d,J=15.6 Hz,H-7),6.76(2H,s,H-2,6);13C—NMR(150 MHz,CD3OD)δC:137.5(C-1),104.1(C-2,6),153.8(C-3,5),129.6(C-4),131.2(C-7),130.5(C-8),172.6(C-1′),43.7(C-2′),27.5(C-3′),22.6(C-4′,5′).以上數(shù)據(jù)與文獻(xiàn)[17]報道的syringenin isovalerate基本一致.
化合物15,白色晶體,mp 246~248 °C,ESI-MSm/z:211[M+Na]+;1H—NMR(600 MHz,CD3COCD3)δH:7.15(2H,s,H-2,6);13C—NMR(150 MHz,CD3COCD3)δC:121.8(C-1),110.0(C-2,6),145.7(C-3,5),138.3(C-4),167.9(C-7).以上數(shù)據(jù)與文獻(xiàn)[18]報道的沒食子酸基本一致.
將小魚眼草用甲醇加熱回流提取后,依次使用不同試劑進(jìn)行萃取,得到二氯甲烷部位浸膏,經(jīng)柱色譜分離純化,最終得到15個化合物,包括6個生物堿類,5個萜類,2個黃酮類和3個酚酸類.采用波譜技術(shù)結(jié)合化學(xué)方法進(jìn)行結(jié)構(gòu)鑒定,15個化合物分別為:胡椒堿(1)、辣椒堿(2)、二氫辣椒堿(3)、3-吲哚甲酸乙酯(4)、3-吲哚甲酸甲酯(5)、3-甲?;胚?6)、pomolic acid(7)、floridiolide A(8)、16-methoxycleroda-3,13-dien-15,16-olide-18-oic acid(9)、16-ethoxycleroda-3,13-dien-15,16-olide-18-oic acid(10)、木栓酮(11)、山奈酚(12)、槲皮素(13)、syringenin isovalerate(14)、沒食子酸(15).其中,化合物1~12、14為首次從該植物中分離得到.