劉 艷,陳張林,吳丹丹,周媛媛,匡海學(xué),楊炳友*
(1.黑龍江中醫(yī)藥大學(xué)北藥基礎(chǔ)與應(yīng)用研究教育部重點實驗室,黑龍江 哈爾濱 150040;2.揚子江藥業(yè)集團江蘇龍鳳堂中藥有限公司,江蘇 泰州 225300)
五味子藤莖為木蘭科五味子屬植物五味子Schisandra chinensis(Turcz.)Baill.的干燥藤莖[1],習(xí)稱“山花椒藤”,主要分布于黑龍江、吉林、遼寧等省,自古民間將其用作調(diào)味劑和滋補強壯劑,并用來主治感冒、肺虛咳喘等疾?。?]?,F(xiàn)代研究表明,五味子藤莖具有神經(jīng)細胞保護[3]、預(yù)防治療老年癡呆[4]等生物活性,但它每年被大量剪掉[5],資源浪費嚴重,其化學(xué)成分與北五味子相似,極具研究價值[6]?;诖?,本實驗對五味子藤莖的95%乙醇提取物進行分離純化,獲得13 個萜類和8 個木脂素類化合物,其中化合物1~3、8~10、12、13為首次從五味子屬植物中分離得到。
Bruker-400 超導(dǎo)核磁共振光譜儀(德國Bruker公司);e2695-2998-2424 HPLC 色譜儀(美國Waters 公司);LC-6AD HPLC 色譜儀(日本Shimadzu 公司);Q-TOF(ESI)高分辨質(zhì)譜儀(美國Waters 公司);SunFire C18(4.6 mm×150 mm,5 μm;10 mm×250 mm,5 μm,美國Waters 公司);柱色譜用硅膠(80~100、200~300 目,青島海洋化工廠);柱色譜用ODS(ODS-A-HG,50 μm,日本YMC 公司);Silicagel60 F254薄層色譜硅膠板(德國Merck 公司);Rp-18 薄層色譜反相板(德國Merck 公司)。柱色譜用化學(xué)試劑(分析純,天津富宇精細化工有限公司);甲醇(色譜純,百靈威科技有限公司)。
五味子藤莖采收于黑龍江省饒河林業(yè)局,經(jīng)黑龍江中醫(yī)藥大學(xué)藥學(xué)院藥用植物教研室樊銳鋒副教授鑒定為木蘭科植物五味子Schisandra chinensis(Turcz.)Baill.的干燥藤莖,標(biāo)本(20130985)保存于黑龍江中醫(yī)藥大學(xué)中藥化學(xué)實驗室。
取干燥藥材25 kg,95%乙醇加熱回流提取3次,每次2 h,濾過,減壓回收溶劑,得乙醇提取物4.58 kg,出膏率為18.3%。將乙醇提取物與水均勻混懸,通過HPD-100 型大孔吸附樹脂柱色譜,依次用50%、95%乙醇洗脫,收集各組分洗脫液,回收溶劑,得50%乙醇洗脫組分1.07 kg、95%乙醇洗脫組分1.93 kg。95%乙醇組分(240 g)通過硅膠柱色譜,CH2Cl2-CH3OH(100∶1~0∶1)梯度洗脫,洗脫液反復(fù)經(jīng)過TLC 分析鑒別后合并,最終得到14 個流分,即Fr.A~Fr.N。
Fr.C 經(jīng)硅膠柱以CH2Cl2-CH3OH(50∶1~0∶1)進行洗脫分離,得組分Fr.C-1~Fr.C-10,其中Fr.C-5、Fr.C-7 經(jīng)半制備型HPLC(CH3OHH2O)分離得化合物12(13.1 mg)、10(5.7 mg)。Fr.D 經(jīng)硅膠柱以CH2Cl2-CH3OH(50∶1~0∶1)梯度洗脫,分離得組分Fr.D-1~Fr.D-9,其中Fr.D-4 經(jīng)半制備型HPLC(CH3OH-H2O,56∶44)分離得化合物15(28.0 mg)。Fr.E 經(jīng)硅膠柱以CH2Cl2-CH3OH(50∶1~0∶1)梯度洗脫,分離得組分Fr.E-1~Fr.E-11,其中Fr.E-4 經(jīng)半制備型HPLC 以CH3OH-H2O(52∶48)分離得化合物8(4.8 mg)。Fr.F 經(jīng)硅膠柱以CH2Cl2-CH3OH(30∶1~0∶1)梯度洗脫,分離得組分Fr.F-1~Fr.F-10,其中Fr.F-5 經(jīng)半制備型HPLC,以CH3OH-H2O(49∶ 51)分離得化合物9(3.2 mg),F(xiàn)r.F-6 經(jīng)半制備型HPLC 以CH3OH-H2O(47∶53)分離得化合物14(4.8 mg),F(xiàn)r.F-7 經(jīng)ODS 反相柱以CH3OH-H2O(30∶70~100∶0)梯度洗脫后再經(jīng)半制備型HPLC 以CH3OH-H2O(50∶50)分離得化合物13(5.3 mg)。Fr.G 經(jīng)硅膠柱以CH2Cl2-CH3OH(30∶1~0∶1)梯度洗脫,分離得組分Fr.G-1~Fr.G-8,其中Fr.G-6 經(jīng)半制備型HPLC,以CH3OH-H2O(41∶59)分離得化合物11(5.0 mg)。Fr.H 經(jīng)硅膠柱以CH2Cl2-CH3OH(20∶1~0∶1)梯度洗脫,分離得組分Fr.H-1~Fr.H-11,其中Fr.H-5 經(jīng)半制備型HPLC 以CH3OH-H2O(36∶64)分離得化合物20(4.1 mg),F(xiàn)r.H-8 經(jīng)半制備型HPLC,以CH3OH-H2O(38∶62)分離得化合物21(3.5 mg)。Fr.K 經(jīng)硅膠柱CH2Cl2-CH3OH(20∶1~0∶1)梯度洗脫,分離得組分Fr.K-1~Fr.K-13,其中Fr.K-6 經(jīng)反相ODS 柱CH3OH-H2O(10∶90~1∶0)梯度洗脫分離得化合物7(22.4 mg),F(xiàn)r.K-7 經(jīng)硅膠柱CH2Cl2-CH3OH(20∶1~0∶1)梯度洗脫分離得化合物6(22.0 mg)、5(20.4 mg)。Fr.L 經(jīng)硅膠柱CH2Cl2-CH3OH(20∶1~0∶1)梯度洗脫,分離得組分Fr.L-1~Fr.L-10,其中Fr.L-5 經(jīng)硅膠柱CH2Cl2-CH3OH(20∶1~0∶1)梯度洗脫,分離得化合物4(32.0 mg)、3(22.4 mg),F(xiàn)r.L-7 經(jīng)反相ODS 柱CH3OH-H2O(10∶90~1∶0)梯度洗脫,分離得化合物16(18.0 mg)、17(21.4 mg)。Fr.M 經(jīng)硅膠柱CH2Cl2-CH3OH(20∶1~0∶1)梯度洗脫,分離得組分Fr.M-1~Fr.M-12,其中Fr.M-6經(jīng)反相ODS 柱CH3OH-H2O(10∶90~1∶0)梯度洗脫,分離得化合物18(22.4 mg)。Fr.N 經(jīng)反相ODS 柱以CH3OH-H2O(10∶90~1∶0)梯度洗脫,分離得組分Fr.N-1~Fr.N-8,其中Fr.N-6 經(jīng)反相ODS 柱CH3OH-H2O(10∶90~1∶0)梯度洗脫,分離得化合物2(7.0 mg)、1(6.5 mg),F(xiàn)r.N-8經(jīng)經(jīng)反相ODS 柱CH3OH-H2O(10∶90~1∶0)梯度洗脫,分離得化合物19(4.0 mg)。
化合物1:無色固體(三氯甲烷)。分子式C30H52O,HR-ESI-MS m/z:451.392 0 [M +Na]+。1H-NMR(400 MHz,CDCl3) δ:1.42(1Ha,m,H-1),1.78(1Hb,m,H-1),1.66(1Ha,m,H-2),1.74(1Hb,m,H-2),3.22(1H,dd,J=4.4,11.6,H-3),0.88(1H,m,H-5),1.46(1Ha,m,H-6),1.68(1Hb,m,H-6),1.33(1Ha,m,H-7),1.66(1Hb,m,H-7),2.16(1H,m,H-8),5.20(1H,m,H-11),1.91(1Ha,m,H-12),2.05(1Hb,m,H-12),1.32(1Ha,m,H-15),1.34(1Hb,m,H-15),0.97(1Ha,m,H-16),1.33(1Hb,m,H-16),1.52(1H,m,H-17),1.57(1H,overlapped,H-18),1.61(1Ha,m,H-19),1.63(1Hb,m,H-19),1.13(1Ha,m,H-20),1.34(1Hb,m,H-20),1.12(1Ha,m,H-21),1.13(1Hb,m,H-21),1.45(1H,m,H-22),0.80(3H,s,H-23),0.96(3H,s,H-24),1.01(3H,s,H-25),0.71(3H,s,H-26),0.63(3H,s,H-27),0.85(3H,d,J=6.5,H-28),0.84(3H,d,J=6.0,H-29),0.86(3H,d,J=6.0,H-30);13C-NMR(100 MHz,CDCl3) δ:35.9(C-1),27.8(C-2),78.9(C-3),39.1(C-4),52.5(C-5),20.6(C-6),28.1(C-7),41.8(C-8),148.5(C-9),39.4(C-10),114.9(C-11),37.2(C-12),44.4(C-13),47.0(C-14),33.9(C-15),36.1(C-16),28.0(C-17),50.9(C-18),29.0(C-19),29.7(C-20),39.5(C-21),36.0(C-22),15.7(C-23),28.2(C-24),22.3(C-25),18.5(C-26),14.4(C-27),18.2(C-28),22.2(C-29),22.8(C-30)。以上數(shù)據(jù)與文獻[7] 報道基本一致,故鑒定為retusenol。
化合物2:白色無定形粉末(三氯甲烷)。分子式C30H48O3,HR-ESI-MS m/z:479.350 9 [M+Na]+。1H-NMR(400 MHz,CDCl3) δ:1.87(1Ha,m,H-1),1.01(1Hb,m,H-1),1.94(1Ha,m,H-2),1.64(1Hb,m,H-2),3.30(1H,m,H-3),1.82(1H,m,H-5),1.49(1Ha,m,H-6),0.73(1Hb,m,H-6),1.14(1Ha,m,H-7)1.31(1Hb,m,H-7),1.53(1H,m,H-8),2.00(1Ha,m,H-11),1.13(1Hb,m,H-11),1.64(1Ha,m,H-12),1.63(1Hb,m,H-12),1.28(1Ha,m,H-15),1.28(1Hb,m,H-15),1.28(1Ha,m,H-16),1.09(1Hb,m,H-16),1.58(1H,m,H-17),0.97(3H,s,H-18),0.33(1Ha,d,J=4.2,H-19),0.54(1Hb,d,J=4.2,H-19),1.42(1H,overlapped,H-20),0.89(3H,d,J=6.2,H-21),1.56(1Ha,m,H-22),1.16(1Hb,m,H-22),2.54(1Ha,m,H-23),2.43(1Hb,m,H-23),6.09(1H,t,J=7.2,H-24),1.92(3H,s,H-27),0.98(3H,s,H-28),0.97(3H,s,H-29),0.89(3H,s,H-30);13C-NMR(100 MHz,CDCl3)δ:32.0(C-1),30.4(C-2),78.8(C-3),40.5(C-4),47.1(C-5),21.1(C-6),26.5(C-7),48.0(C-8),20.0(C-9),26.1(C-10),26.9(C-11),32.9(C-12),45.3(C-13),48.8(C-14),35.8(C-15),28.2(C-16),52.2(C-17),18.0(C-18),29.9(C-19),36.1(C-20),18.1(C-21),35.6(C-22),26.0(C-23),147.1(C-24),125.6(C-25),171.8(C-26),20.5(C-27),25.4(C-28),20.5(C-29),19.3(C-30)。以上數(shù)據(jù)與文獻[8] 報道基本一致,故鑒定為schizandrolic acid。
化合物3:白色無定形粉末(三氯甲烷)。分子式C30H48O,HR-ESI-MS m/z:447.360 9 [M+Na]+。1H-NMR(400 MHz,CDCl3) δ:1.55(1Ha,m,H-1),1.85(1Hb,m,H-1),2.28(1Ha,m,H-2),2.70(1Hb,m,H-2),1.67(1H,m,H-5),1.53(1Ha,m,H-6),1.95(1Hb,m,H-6),1.31(1Ha,m,H-7),1.95(1Hb,m,H-7),1.59(1H,m,H-8),1.15(1Ha,m,H-11),2.05(1Hb,m,H-11),1.64(2H,m,H-12),1.12(1Ha,m,H-15),1.04(1Hb,m,H-15),1.31(2H,m,H-16),1.60(1H,m,H-17),1.00(3H,s,H-18),0.58(1H,d,J=4.2,H-19),0.79(1H,d,J=4.2,H-19),1.40(1H,m,H-20),0.89(3H,d,J=7.1,H-21),1.07(1Ha,m,H-22),1.46(1Hb,m,H-22),1.93(1Ha,m,H-23),2.13(1Hb,m,H-23),5.11(1H,m,H-24),1.69(3H,s,H-26),1.61(3H,s,H-27),1.10(3H,s,H-28),1.05(3H,s,H-29),0.91(3H,s,H-30);13C-NMR(100 MHz,CDCl3) δ:33.4(C-1),37.5(C-2),216.6(C-3),50.2(C-4),48.4(C-5),21.5(C-6),28.1(C-7),47.9(C-8),21.1(C-9),26.7(C-10),26.0(C-11),32.8(C-12),45.3(C-13),48.7(C-14),25.9(C-15),35.6(C-16),52.3(C-17),17.6(C-18),29.6(C-19),35.9(C-20),19.3(C-21),36.3(C-22),24.9(C-23),125.2(C-24),130.9(C-25),25.7(C-26),18.2(C-27),20.8(C-28),22.2(C-29),18.1(C-30)。以上數(shù)據(jù)與文獻[9] 報道基本一致,故鑒定為cycloartenone。
化合物4:白色粉末(三氯甲烷)。分子式C31H50O,HR-ESI-MS m/z:461.375 0 [M +Na]+。1H-NMR(400 MHz,CDCl3) δ:1.86(1H,m,H-1),1.54(1H,m,H-1),2.71(1H,m,H-2),2.31(1H,m,H-2),1.72(1H,dd,J=4.4,12.3,H-5),1.56(2H,m,H-6),1.92(1Ha,m,H-7)1.32(1Hb,m,H-7),1.60(1H,m,H-8),1.41(1Ha,m,H-11),1.12(1Hb,m,H-11),1.32(2H,m,H-12),1.67(2H,m,H-15),2.05(1Ha,m,H-16),1.15(1Hb,m,H-16),1.64(1H,m,H-17),1.00(3H,s,H-18),0.58(1H,d,J=4.2,H-19),0.79(1H,d,J=4.2,H-19),1.41(1H,m,H-20),0.90(3H,d,J=7.1,H-21),1.13(2H,m,H-22),2.16(1Ha,m,H-23),1.89(1Hb,m,H-23),2.24(1H,m,H-25),1.03(3H,d,J=6.8,H-26),1.02(3H,d,J=6.8,H-27),1.10(3H,s,H-28),1.05(3H,s,H-29),0.91(3H,s,H-30),4.67(1H,brs,H-31),4.72(1H,br.s,H-31);13C-NMR(100 MHz,CDCl3) δ:33.4(C-1),37.5(C-2),216.6(C-3),50.2(C-4),48.4(C-5),21.5(C-6),28.2(C-7),47.9(C-8),21.1(C-9),26.7(C-10),25.9(C-11),35.6(C-12),45.3(C-13),48.7(C-14),32.8(C-15),26.0(C-16),52.3(C-17),18.3(C-18),29.6(C-19),36.1(C-20),18.1(C-21),35.0(C-22),31.3(C-23),156.9(C-24),33.8(C-25),22.0(C-26),21.9(C-27),20.8(C-28),22.2(C-29),19.3(C-30),106.0(C-31)。以上數(shù)據(jù)與文獻[10] 報道基本一致,故鑒定為24-methylenecycloartenone。
化合物5:白色粉末(三氯甲烷)。分子式C30H46O4,HR-ESI-MS m/z:493.329 0 [M +Na]+。1H-NMR(400 MHz,CDCl3) δ:1.98(1Ha,m,H-1),1.80(1Hb,m,H-1),2.48(1Ha,m,H-2),2.41(1Hb,m,H-2),2.08(1H,m,H-5),1.75(1Ha,m,H-6),1.63(1Hb,m,H-6),1.53(1Ha,m,H-7)1.56(1Hb,m,H-7),2.09(1H,m,H-8),5.36(1H,d,J=5.6,H-11),2.15(1Ha,m,H-12),1.98(1Hb,m,H-12),1.36(2H,m,H-15),1.29(2H,m,H-16),1.61(1H,m,H-17),0.66(3H,s,H-18),1.07(3H,s,H-19),1.42(1H,m,H-20),0.96(3H,d,J=6.2,H-21),1.51(1Ha,m,H-22),1.14(1Hb,m,H-22),2.58(1Ha,m,H-23),2.47(1Hb,m,H-23),6.10(1H,t,J=6.6,H-24),1.94(3H,s,H-27),4.74(1H,s,H-28),4.87(1H,s,H-28),0.74(3H,s,H-29),0.76(3H,s,H-30);13CNMR(100 MHz,CDCl3) δ:37.7(C-1),35.6(C-2),180.9(C-3),147.6(C-4),35.9(C-5),50.5(C-6),33.7(C-7),49.5(C-8),142.3(C-9),47.2(C-10),125.8(C-11),31.9(C-12),44.0(C-13),42.7(C-14),29.0(C-15),27.8(C-16),42.5(C-17),26.4(C-18),23.1(C-19),35.9(C-20),20.5(C-21),27.9(C-22),26.3(C-23),147.1(C-24),118.7(C-25),173.5(C-26),18.3(C-27),111.5(C-28),11.8(C-29),14.6(C-30)。以上數(shù)據(jù)與文獻[11] 報道基本一致,故鑒定為kadsuric acid。
化合物6:白色針狀結(jié)晶(三氯甲烷)。分子式C30H46O4,HR-ESI-MS m/z:493.329 0 [M+Na]+。1H-NMR(400 MHz,CDCl3) δ:2.05(1Ha,m,H-1),1.36(1Hb,m,H-1),2.30(1Ha,m,H-2),2.48(1Hb,m,H-2),2.41(1H,m,H-5),1.28(1Ha,m,H-6),1.94(1Hb,m,H-6),1.31(1Ha,m,H-7)1.12(1Hb,m,H-7),1.56(1H,m,H-8),2.07(1Ha,m,H-11),1.25(1Hb,m,H-11),1.65(2H,m,H-12),1.29(2H,m,H-15),1.10(1Ha,m,H-16),1.52(1Hb,m,H-16),1.61(1H,m,H-17),0.96(3H,s,H-18),0.75(H,overlapped,H-19),0.39(H,overlapped,H-19),1.42(1H,m,H-20),0.90(3H,d,J=6.2,H-21),1.52(1Ha,m,H-22),1.18(1Hb,m,H-22),2.56(1Ha,m,H-23),2.46(1Hb,m,H-23),6.08(1H,t,J=7.2,H-24),1.91(3H,s,H-27),4.72(1H,s,H-28),4.81(1H,s,H-28),1.68(3H,s,H-29),0.94(3H,s,H-30);13C-NMR(100 MHz,CDCl3) δ:28.7(C-1),31.6(C-2),180.3(C-3),149.5(C-4),45.9(C-5),28.0(C-6),24.9(C-7),47.2(C-8),21.3(C-9),27.3(C-10),26.8(C-11),33.0(C-12),45.1(C-13),49.0(C-14),35.5(C-15),27.5(C-16),51.5(C-17),18.1(C-18),29.6(C-19),35.6(C-20),17.9(C-21),35.9(C-22),27.0(C-23),147.2(C-24),125.8(C-25),173.6(C-26),20.5(C-27),113.7(C-28),19.7(C-29),19.3(C-30)。以上數(shù)據(jù)與文獻[12] 報道基本一致,故鑒定為nigranoic acid。
化合物7:白色粉末(三氯甲烷)。分子式C30H46O3,HR-ESI-MS m/z:477.334 8 [M +Na]+。1H-NMR(400 MHz,CDCl3) δ:1.55(1Ha,m,H-1),1.92(1Hb,m,H-1),2.72(1Ha,m,H-2),2.31(1Hb,m,H-2),1.72(1H,m,H-5),1.32(1Ha,m,H-6),1.95(1Hb,m,H-6),1.14(1Ha,m,H-7)1.16(1Hb,m,H-7),1.62(1H,m,H-8),1.45(1Ha,m,H-11),2.05(1Hb,m,H-11),1.66(2H,m,H-12),1.33(2H,m,H-15),1.15(1Ha,m,H-16),1.41(1Hb,m,H-16),1.60(1H,m,H-17),0.91(3H,s,H-18),0.58(1H,overlapped,H-19),0.79(1H,overlapped,H-19),1.40(1H,m,H-20),0.90(3H,d,J=6.4,H-21),1.56(1Ha,m,H-22),1.17(1Hb,m,H-22),2.45(1Ha,m,H-23),2.56(1Hb,m,H-23),6.08(1H,t,J=7.7,H-24),1.92(3H,s,H-27),1.05(3H,s,H-28),1.10(3H,s,H-29),1.00(3H,s,H-30);13CNMR(100 MHz,CDCl3) δ:33.4(C-1),37.5(C-2),216.6(C-3),50.2(C-4),48.4(C-5),28.2(C-6),21.5(C-7),47.9(C-8),21.1(C-9),26.0(C-10),26.9(C-11),32.8(C-12),45.4(C-13),48.8(C-14),35.6(C-15),25.9(C-16),52.2(C-17),18.1(C-18),29.6(C-19),36.0(C-20),18.1(C-21),35.8(C-22),26.7(C-23),147.2(C-24),125.6(C-25),171.8(C-26),20.6(C-27),20.8(C-28),22.2(C-29),19.3(C-30)。以上數(shù)據(jù)與文獻[8] 報道基本一致,故鑒定為schizandronic acid。
化合物8:白色無定形粉末(甲醇)。分子式C21H36O11,HR-ESI-MS m/z:487.214 8 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:5.01(1H,d,J=10.8,H-1),5.23(1H,d,J=17.5,H-1),5.98(1H,dd,J=10.8,17.5,H-2),1.80(1H,m,H-4),1.90(1H,m,H-4),1.80(1H,m,H-5),1.99(1H,m,H-5),4.03(1H,m,H-6),1.20(3H,s,H-8),1.25(3H,s,H-9),1.33(3H,s,H-10),4.50(1H,d,J=7.7,H-1′),3.14(1H,overlapped,H-2′),3.34(1H,m,H-3′),3.24(1H,m,H-4′),3.41(1H,m,H-5′),3.99(1H,dd,J=2.3,11.0,H-6′),3.55(1H,dd,J=6.0,11.0,H-6′),4.93(1H,d,J=1.0,H-1″),3.97(1H,overlapped,H-2″),3.81(1H,m,H-3″),3.97(1H,m,H-4″),3.62(1H,dd,J=5.3,11.8,H-5″),3.72(1H,dd,J=3.3,11.8,H-5″);13C-NMR(100 MHz,CD3OD) δ:112.2(C-1),145.3(C-2),84.6(C-3),38.6(C-4),28.4(C-5),87.0(C-6),80.8(C-7),24.1(C-8),20.9(C-9),26.2(C-10),98.8(C-1′),75.2(C-2′),77.9(C-3′),72.1(C-4′),76.4(C-5′),68.2(C-6′),110.2(C-1″),83.2(C-2″),79.0(C-3″),86.0(C-4″),63.1(C-5″)。以上數(shù)據(jù)與文獻[13-14]報道基本一致,故鑒定為1-methyl-1-(trans-5′-methyl-cis-5′-vinyltetrahydrofuran-r-2′-yl)-ethyl-6-O-α-arabinofuranosyl-β-D-glucopyranoside。
化合物9:白色無定形粉末(甲醇)。分子式C21H36O11,HR-ESI-MS m/z:487.215 0 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:5.01(1Ha,dd,J=10.8,H-1),5.24(1Hb,dd,J=17.4,H-1),5.98(1H,dd,J=10.8,17.4,H-2),1.80(1H,m,H-4),1.90(1H,m,H-4),1.80(1H,m,H-5),1.99(1H,m,H-5),4.05(1H,dd,J=6.8,7.3,H-6),1.28(3H,s,H-8),1.24(3H,s,H-9),1.33(3H,s,H-10),4.50(1H,d,J=7.7,H-1′),3.14(1H,overlapped,H-2′),3.34(1H,m,H-3′),3.24(1H,m,H-4′),3.41(1H,m,H-5′),3.94(1H,dd,J=1.7,11.2,H-6′),3.53(1H,dd,J=6.6,11.2,H-6′),4.96(1H,d,J=2.2,H-1″),3.85(1H,d,J=2.2,H-2″),3.76(1H,d,J=9.5,H-4″),3.95(1H,d,J=9.5,H-4″),3.56(2H,s,H-5″);13C-NMR(100 MHz,CD3OD) δ:112.2(C-1),145.3(C-2),84.6(C-3),38.6(C-4),28.4(C-5),87.0(C-6),80.7(C-7),24.1(C-8),20.9(C-9),26.1(C-10),98.8(C-1′),75.1(C-2′),78.1(C-3′),71.9(C-4′),76.6(C-5′),68.9(C-6′),111.0(C-1″),77.9(C-2″),80.6(C-3″),75.0(C-4″),65.6(C-5″)。以上數(shù)據(jù)與文獻[15] 報道基本一致,故鑒定為( Z)-1-(5-ethenyl-5-methyltetrahydrofuran-2-yl)-1-methylethyl-O-β-Dapiofuranosyl-(1→6)-O-β-D-glucopyranoside。
化合物10:白色無定形粉末(甲醇)。分子式C10H18O3,HR-ESI-MS m/z:209.116 2 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:2.65(1Ha,dd,J=8.1,17.3,H-2),2.40(1Hb,dd,J=11.6,17.3,H-2),2.27(1H,m,H-3),1.44(3H,s,H-5),1.27(3H,s,H-6),1.69(1Ha,m,H-7),1.36(1Hb,m,H-7),1.47(1Ha,m,H-8),1.40(1Hb,m,H-8),3.71(1H,m,H-9),1.16(3H,d,J=6.2,H-10);13C-NMR(100 MHz,CD3OD) δ:178.6(C-1),35.9(C-2),47.1(C-3),88.8(C-4),27.8(C-5),22.0(C-6),27.0(C-7),38.8(C-8),68.5(C-9),23.5(C-10)。以上數(shù)據(jù)與文獻[16] 報道基本一致,故鑒定為4,9-dihydroxy-4-olide。
化合物11:白色無定形粉末(甲醇)。分子式C21H32O10,HR-ESI-MS m/z:467.187 9 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:7.08(1H,d,J=8.1,H-2),7.12(1H,d,J=8.1,H-3),7.08(1H,d,J=8.0,H-5),7.12(1H,d,J=8.0,H-6),2.28(3H,s,H-7),3.03(1H,m,H-8),3.63(1H,dd,J=6.0,9.5,H-9),3.85(1H,dd,J=8.3,9.5,H-9),1.26(3H,d,J=7.0,H-10),4.23(1H,d,J=7.8,H-1′),3.16(1H,overlapped,H-2′),3.30(1H,m,H-3′),3.26(1H,m,H-4′),3.41(1H,m,H-5′),3.62(1H,dd,J=6.2,11.3,H-6′),4.00(1H,dd,J=2.1,11.3,H-6′),4.97(1H,d,J=1.2,H-1″),3.99(1H,overlapped,H-2″),3.82(1H,m,H-3″),3.96(1H,m,H-4″),3.62(1Ha,m,H-5″),3.72(1Hb,dd,J=3.3,11.9,H-5″);13C-NMR(100 MHz,CD3OD) δ:136.9(C-1),130.0(C-2),128.4(C-3),142.5(C-4),128.4(C-5),130.0(C-6),21.1(C-7),40.8(C-8),76.5(C-9),19.1(C-10),104.4(C-1′),75.1(C-2′),78.0(C-3′),72.0(C-4′),76.7(C-5′),68.2(C-6′),110.0(C-1″),83.2(C-2″),79.0(C-3″),86.0(C-4″),63.1(C-5″)。以上數(shù)據(jù)與文獻[17] 報道基本一致,故鑒定為(R)-p-cymen-9-O-α-Darabinofuranosyl-(1→6)-β-D-glucopyranoside。
化合物12:白色無定形粉末(甲醇)。分子式C24H38O11,HR-ESI-MS m/z:525.230 9 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:2.01(1Ha,d,J=16.7,H-2),2.43(1Hb,d,J=16.7,H-2),5.87(1H,s,H-4),3.81(1H,d,J=9.0,H-6),5.62(1H,dd,J=9.0,15.4,H-7),5.73(1H,dd,J=6.4,15.4,H-8),3.35(1H,m,H-9),1.26(3H,d,J=6.3,H-10),1.00(3H,s,H-11),1.03(3H,s,H-12),1.92(3H,s,H-13),4.34(1H,d,J=7.8,H-1′),3.16(1H,overlapped,H-2′),3.32(1H,m,H-3′),3.23(1H,m,H-4′),3.37(1H,m,H-5′),3.95(1Ha,dd,J=2.0,11.3,H-6′),3.56(1Hb,dd,J=5.4,11.3,H-6′),4.95(1H,d,J=1.2,H-1″),3.98(1H,overlapped,H-2″),3.81(1H,m,H-3″),3.87(1H,m,H-4″),3.74(1Ha,dd,J=3.3,11.7,H-5″),3.62(1Hb,dd,J=5.3,11.7,H-5″);13C-NMR(100 MHz,CD3OD) δ:37.2(C-1),48.4(C-2),202.2(C-3),126.1(C-4),166.0(C-5),56.7(C-6),129.1(C-7),138.0(C-8),77.4(C-9),21.3(C-10),27.7(C-11),28.1(C-12),23.8(C-13),102.6(C-1′),75.2(C-2′),78.0(C-3′),71.9(C-4′),76.8(C-5′),68.0(C-6′),109.9(C-1″),83.2(C-2″),79.0(C-3″),86.0(C-4″),63.1(C-5″)。以上數(shù)據(jù)與文獻[18]報道基本一致,故鑒定為ficumegasoside。
化合物13:白色無定形粉末(吡啶)。分子式C15H22O3,HR-ESI-MS m/z:273.145 9 [M +Na]+。1H-NMR(400 MHz,C5D5N) δ:2.53(1H,m,H-1),2.58(1Ha,m,H-2),2.68(1Hb,m,H-2),7.00(1H,s,H-5),2.78(1H,m,H-7)2.11(1Ha,m,H-8),2.57(1Hb,m,H-8),3.89(1H,m,H-9),2.79(1H,m,H-10),2.05(3H,s,H-12),4.99(2H,m,H-13),1.23(3H,d,J=7.0,H-14),1.87(3H,s,H-15);13C-NMR(100 MHz,C5D5N) δ:50.2(C-1),36.5(C-2),198.9(C-3),132.7(C-4),155.2(C-5),72.7(C-6),48.4(C-7),37.1(C-8),70.7(C-9),37.3(C-10),146.8(C-11),22.8(C-12),113.9(C-13),15.3(C-14),15.6(C-15)。以上數(shù)據(jù)與文獻[19] 報道基本一致,故鑒定為 6α,9αdihydroxycadinan-4-en-3-one。
化合物14:黃色膠狀物(甲醇)。分子式C20H24O6,HR-ESI-MS m/z:383.146 6 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:6.97(1H,d,J=1.8,H-2),6.78(1H,d,J=8.1,H-5),6.94(1H,dd,J=1.8,8.1,H-6),5.15(1H,d,J=8.4,H-7),2.35(1H,m,H-8),3.28(1Ha,overlapped,H-9),3.21(1Hb,dd,J=6.5,10.8,H-9),7.07(1H,d,J=1.9,H-2′),6.81(1H,d,J=8.1,H-5′),6.94(1H,dd,J=1.9,8.1,H-6′),4.36(1H,d,J=9.1,H-7′),2.06(1H,m,H-8′),1.12(3H,d,J=6.6,H-9′),3.84(3H,s,3-OCH3),3.88(3H,s,3′-OCH3);13C-NMR(100 MHz,CD3OD) δ:132.4(C-1),116.1(C-2),148.7(C-3),147.0(C-4),111.6(C-5),120.7(C-6),83.0(C-7),54.5(C-8),63.8(C-9),133.1(C-1′),115.8(C-2′),149.1(C-3′),147.6(C-4′),111.9(C-5′),120.7(C-6′),89.3(C-7′),46.5(C-8′),16.8(C-9′),56.4(3-OCH3),56.4(3′-OCH3)。以上數(shù)據(jù)與文獻[20] 報道基本一致,故鑒定為(7R,7′S,8R,8′S)-3,3′-dimethoxy-7,7′-epoxylignan-4,4′,9-triol。
化合物15:白色無定形粉末(甲醇)。分子式C34H48O16,HR-ESI-MS m/z:713.301 2 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:6.91(1H,s,H-4),2.22(1Ha,dd,J=9.4,13.2,H-6),2.07(1Hb,brd,J=13.2,H-6),1.80(1H,m,H-7),1.91(1H,m,H-8),2.65(1Ha,dd,J=7.4,13.5,H-9),2.42(1Hb,brd,J=13.5,H-9),6.89(1H,s,H-11),1.00(3H,d,J=7.0,H-17),0.74(3H,d,J=7.0,H-18),3.49(3H,s,1-OCH3),3.88(3H,s,2-OCH3),3.89(3H,s,13-OCH3),3.46(3H,s,14-OCH3),4.96(H,d,J=7.3,H-1′),3.52(1H,m,H-2′),3.47(1H,overlapped,H-3′),3.40(1H,m,H-4′),3.47(1H,m,H-5′),3.70(1H,dd,J=6.1,12.1,H-6′),3.92(1H,dd,J=2.0,12.1,H-6′),5.00(1H,d,J=7.3,H-1″),3.52(1H,overlapped,H-2″),3.51(1H,m,H-3″),3.46(1H,m,H-4″),3.50(1H,m,H-5″),3.70(1H,dd,J=6.1,12.1,H-6″),3.90(1H,dd,J=2.1,12.1,H-6″);13C-NMR(100 MHz,CD3OD) δ:152.5(C-1),141.6(C-2),152.3(C-3),113.2(C-4),141.2(C-5),36.1(C-6),42.3(C-7),35.3(C-8),39.8(C-9),135.8(C-10),116.2(C-11),150.8(C-12),142.1(C-13),152.6(C-14),126.3(C-15),125.1(C-16),22.1(C-17),12.8(C-18),61.1(1-OCH3),61.9(2-OCH3),61.9(13-OCH3),61.1(14-OCH3),102.5(C-1′),75.1(C-2′),78.2(C-3′),71.6(C-4′),78.3(C-5′),62.6(C-6′),102.7(C-1″),75.0(C-2″),78.1(C-3″),71.3(C-4″),78.0(C-5″),62.4(C-6″)。以上數(shù)據(jù)與文獻[21] 報道基本一致,故鑒定為1,2,13,14-tetramethoxy-dibenzocyclooctadi ene-3,12-O-β-D-diglucopyranoside。
化合物16:無色針晶(三氯甲烷)。分子式C23H28O7,HR-ESI-MS m/z:439.172 1 [M +Na]+。1H-NMR(400 MHz,CDCl3) δ:6.62(1H,s,H-4),2.69(1Ha,d,J=13.4,H-6),2.36(1Hb,d,J=13.4,H-6),1.86(1H,m,H-8),2.34(1Ha,dd,J=7.7,14.3,H-9),2.60(1Hb,dd,J=1.4,14.3,H-9),6.48(1H,s,H-11),1.25(3H,s,H-17),0.82(3H,d,J=7.3,H-18),3.91(3H,s,1-OCH3),3.91(3H,s,2-OCH3),3.52(3H,s,3-OCH3),3.84(3H,s,14-OCH3),5.96(2H,s,-OCH2O-);13C-NMR(100 MHz,CDCl3) δ:152.1(C-1),140.7(C-2),152.3(C-3),110.3(C-4),132.0(C-5),40.5(C-6),71.7(C-7),42.0(C-8),33.7(C-9),132.5(C-10),106.0(C-11),147.9(C-12),134.9(C-13),141.2(C-14),121.9(C-15),124.1(C-16),30.1(C-17),15.8(C-18),60.6(1-OCH3),61.0(2-OCH3),56.0(3-OCH3),59.7(14-OCH3),100.8(-OCH2O-)。以上數(shù)據(jù)與文獻[22] 報道基本一致,故鑒定為gomisin A。
化合物17:白色無定形粉末(三氯甲烷)。分子式C22H26O6,HR-ESI-MS m/z:409.162 1 [M+Na]+。1H-NMR(400 MHz,CDCl3) δ:6.44(1H,s,H-4),2.46(1Ha,dd,J=1.8,13.6,H-6),2.05(1H,brd,J=13.6,H-6),1.94(1H,m,H-7),1.85(1H,m,H-8),2.58(1Ha,dd,J=7.3,13.6,H-9),2.17(1Hb,dd,J=9.4,13.6,H-9),6.65(1H,s,H-11),0.98(3H,d,J=7.1,H-17),0.77(3H,d,J=7.1,H-18),3.57(3H,s,12-OCH3),3.93(3H,s,13-OCH3),3.90(3H,s,14-OCH3),6.02(2H,s,-OCH2O-);13C-NMR(100 MHz,CDCl3) δ:136.8(C-1),133.3(C-2),148.4(C-3),102.1(C-4),138.0(C-5),35.6(C-6),40.6(C-7),33.3(C-8),39.0(C-9),135.7(C-10),112.3(C-11),152.0(C-12),140.4(C-13),150.3(C-14),118.5(C-15),121.5(C-16),21.5(C-17),12.7(C-18),56.0(12-OCH3),61.4(13-OCH3),61.3(14-OCH3),101.3(-OCH2O-)。以上數(shù)據(jù)與文獻[23] 報道基本一致,故鑒定為(+)-gomisin M2。
化合物18:無色晶體(三氯甲烷)。分子式C24H32O6,HR-ESI-MS m/z:439.207 2 [M +Na]+。1H-NMR(400 MHz,CDCl3) δ:6.53(1H,s,H-4),2.58(1H,dd,J=7.4,13.6,H-6),2.58(1H,dd,J=9.5,13.6,H-6),1.85(1H,m,H-7),1.78(1H,m,H-8),2.31(1H,dd,J=1.9,13.6,H-9),2.04(1H,brd,J=13.6,H-9),6.54(1H,s,H-11),0.74(3H,d,J=7.1,H-17),1.00(3H,d,J=7.1,H-18),3.88(3H,s,1-OCH3),3.87(3H,s,14-OCH3),3.90(3H,s,2-OCH3),3.89(3H,s,13-OCH3),3.59(3H,s,3-OCH3),3.59(3H,s,12-OCH3);13C-NMR(100 MHz,CDCl3) δ:151.5(C-1),140.1(C-2),151.4(C-3),107.2(C-4),139.2(C-5),39.1(C-6),33.8(C-7),40.8(C-8),35.6(C-9),133.9(C-10),110.5(C-11),152.9(C-12),139.7(C-13),151.6(C-14),122.3(C-15),123.4(C-16),21.8(C-17),12.7(C-18),60.5(1-OCH3),60.5(14-OCH3),60.9(2-OCH3),60.9(13-OCH3),55.9(3-OCH3),55.9(12-OCH3)。以上數(shù)據(jù)與文獻 [24] 報道基本一致,故鑒定為deoxyschizandrin。
化合物19:無色晶體(三氯甲烷)。分子式C22H24O6,HR-ESI-MS m/z:407.146 5 [M +Na]+。1H-NMR(400 MHz,CDCl3) δ:6.48(1H,s,H-4),2.54(1H,dd,J=7.2,13.6,H-6),2.45(1H,dd,J=9.5,13.6,H-6),1.76(1H,m,H-7),1.87(1H,m,H-8),2.24(1H,dd,J=1.8,13.6,H-9),2.00(1H,brd,J=13.2,H-9),6.48(1H,s,H-11),0.72(3H,d,J=7.2,H-17),0.96(3H,d,J=7.2,H-18),3.83(3H,s,1-OCH3),3.82(3H,s,14-OCH3),5.92(2H,s,-OCH2O-),5.92(2H,s,-OCH2O-);13C-NMR(100 MHz,CDCl3) δ:141.0(C-1),134.7(C-2),148.6(C-3),106.1(C-4),132.7(C-5),38.8(C-6),33.9(C-7),40.7(C-8),35.3(C-9),138.2(C-10),100.7(C-11),147.6(C-12),134.4(C-13),141.2(C-14),122.2(C-15),121.0(C-16),21.7(C-17),12.5(C-18),59.6(1-OCH3),59.6(14-OCH3),100.7(-OCH2O-),100.7(-OCH2O-)。以上數(shù)據(jù)與文獻[25] 報道基本一致,故鑒定為wuweizisu C。
化合物20:白色無定形粉末(甲醇)。分子式C24H32O8,HR-ESI-MS m/z:471.198 3 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:6.74(1H,s,H-4),2.62(1H,d,J=13.9,H-6),2.53(1H,d,J=13.9,H-6),2.97(1H,d,J=13.2,H-9),2.30(1H,d,J=13.2,H-9),6.78(1H,s,H-11),1.23(3H,s,H-17),1.07(3H,s,H-18),3.43(3H,s,1-OCH3),3.89(3H,s,2-OCH3),3.83(3H,s,3-OCH3),3.83(3H,s,12-OCH3),3.89(3H,s,13-OCH3),3.47(3H,s,14-OCH3);13C-NMR(100 MHz,CD3OD) δ:153.4(C-1),141.8(C-2),153.5(C-3),113.0(C-4),134.4(C-5),42.2(C-6),76.4(C-7),73.9(C-8),43.3(C-9),135.7(C-10),112.5(C-11),152.7(C-12),141.8(C-13),152.3(C-14),124.2(C-15),124.4(C-16),27.5(C-17),23.5(C-18),61.0(1-OCH3),61.4(2-OCH3),56.6(3-OCH3),56.4(12-OCH3),61.4(13-OCH3),61.2(14-OCH3)。以上數(shù)據(jù)與文獻[26] 報道基本一致,故鑒定為8(R)-hydroxyl-schizandrin。
化合物21:白色無定形粉末(甲醇)。分子式C23H30O7,HR-ESI-MS m/z:441.187 3 [M +Na]+。1H-NMR(400 MHz,CD3OD) δ:6.42(1H,s,H-4),2.57(1Ha,d,J=13.5,H-6),2.37(1Hb,d,J=13.5,H-6),1.78(1H,m,H-8),2.34(1Ha,dd,J=8.3,14.3,H-9),2.37(1Hb,dd,J=2.4,14.3,H-9),6.73(1H,s,H-11),1.20(3H,s,H-17),0.80(3H,d,J=7.2,H-18),3.85(3H,s,1-OCH3),3.90(3H,s,2-OCH3),3.80(3H,s,3-OCH3),3.55(3H,s,12-OCH3),3.82(3H,s,14-OCH3);13C-NMR(100 MHz,CD3OD) δ:153.6(C-1),141.4(C-2),153.3(C-3),107.8(C-4),135.7(C-5),42.3(C-6),73.8(C-7),42.8(C-8),36.4(C-9),135.1(C-10),112.4(C-11),152.6(C-12),136.2(C-13),148.9(C-14),118.8(C-15),124.8(C-16),29.6(C-17),16.4(C-18),61.2(1-OCH3),61.3(2-OCH3),56.4(3-OCH3),56.3(12-OCH3),61.2(14-OCH3)。以上數(shù)據(jù)與文獻[27] 報道基本一致,故鑒定為13-norschizandrin。
本實驗對五味子藤莖95% 乙醇提取物進行分離純化,獲得13 個萜類,其中,化合物(1~7)為三萜類,化合物(8~11)為單萜類,化合物(12~13)為倍半萜類及化合物(14~21)8 個木脂素類。這些木脂素類被報道具有保護神經(jīng)細胞[3]、肝[6]、腎[3]細胞的藥理作用,同時,在抗炎和免疫調(diào)節(jié)[28]以及抗腫瘤[29]和抗人類免疫缺陷病毒[20]活性等方面也表現(xiàn)出一定的潛力;上述萜類成分亦被報道具有神經(jīng)保護[17]以及抗病毒活性[30-31]等作用。以期該研究內(nèi)容能夠為五味子藤莖的藥效物質(zhì)基礎(chǔ)及生物活性的進一步研究提供參考,為充分利用和擴大五味子藤莖藥用植物資源提供理論依據(jù)。