婁紅波王先宏何麗蓮李富生
(云南農(nóng)業(yè)大學(xué)農(nóng)學(xué)與生物技術(shù)學(xué)院,云南 昆明650201)
甘蔗Saccharum officinarumL.,為禾本科甘蔗屬植物,多分布在熱帶以及亞熱帶地區(qū),我國(guó)的蔗地集中分布于南部及東南片區(qū),如云南、福建、廣西等地[1]。甘蔗除榨糖外,還是一種重要的傳統(tǒng)可再生的藥物資源,全身皆可藥用。莖葉部位味甘、性涼,具有清熱、生津、解酒等功效,常用于治療肺燥、胃痛、便秘等癥狀[2]。甘蔗地上部分(蔗葉、蔗莖)及蔗莖榨糖后產(chǎn)生的蔗渣等副產(chǎn)物產(chǎn)量高,其成分主要為黃酮類、酚類、多糖等[3],具抗氧化、抗腫瘤等多種生物活性[4]。我國(guó)甘蔗資源豐富,甘蔗產(chǎn)糖后的地上部分大多被焚燒,少部分用于牲畜飼料。目前,對(duì)甘蔗化學(xué)成分的研究鮮有報(bào)道,且不系統(tǒng)[5?6]。為充分利用甘蔗副產(chǎn)物,增加附加值,繼續(xù)探索甘蔗莖葉部位的化學(xué)成分,以期從中尋找到量大、安全、有效的活性物質(zhì)。課題組用甲醇提取甘蔗地上部位的莖葉,乙酸乙酯萃取部位分離純化得到14個(gè)化合物,其中化合物5~14為首次從該植物莖葉部位中分離得到。
HC?2500Y304 粉碎機(jī)(中國(guó)武義海納電器公司);填充材料硅膠(100~300 目)、硅膠板GF254(青島海洋化工公司);vg?autospec 3000型質(zhì)譜儀(英國(guó)Msicromass 公司);Bruker avance?600 MHz 核磁共振儀(瑞士Bruker 公司);OSB?2100 旋轉(zhuǎn)蒸發(fā)儀(上海愛(ài)朗儀器有限公司);凝膠Sephadex LH?20(上海伊卡生物技術(shù)有限公司)。
甘蔗莖葉于2018年8 月采自云南農(nóng)業(yè)大學(xué)甘蔗資源圃,經(jīng)云南農(nóng)業(yè)大學(xué)李富生教授鑒定為正品。
取干燥的蔗莖、蔗葉80 kg,粉碎,甲醇浸泡1 d,回流提取,回收甲醇至浸膏狀。浸膏用水溶解,繼用5% HCl 調(diào)pH 3~4,乙酸乙酯連續(xù)萃取3次,回收溶劑,獲得甘蔗粗提物500 g。粗提物用正相硅膠柱,經(jīng)CHCl3?CH3OH?冰醋酸(10∶0.1∶0.02~10∶3.5∶0.02)梯度洗脫,合并色點(diǎn)相同部分共得8個(gè)餾分(Frs.1~8)。Fr.2 過(guò)正相硅膠柱,經(jīng)CHCl3?CH3OH?冰醋酸(10∶0.1∶0.02~10∶0.25∶0.02)梯度洗脫,石油醚?丙酮?冰醋酸(5∶2∶0.02),柱色譜Sephadex LH?20 CHCl3?CH3OH(1∶1)洗脫,經(jīng)過(guò)多次分離純化得化合物1(2.4 g)、2(0.2 g);CHCl3?CH3OH?冰醋酸(10∶0.2∶0.02)洗脫,甲醇結(jié)晶得化合物3(3.7 g)。Fr.3 經(jīng)正相硅膠柱石油醚?乙酸乙酯?冰醋酸(5∶2∶0.02~5∶3.0∶0.02)梯度洗脫,合并色點(diǎn)相同的部分靜置得結(jié)晶化合物4(3.2 g);石油醚?乙酸乙酯?冰醋酸(5∶2∶0.02~5∶3.5∶0.02)、CHCl3?CH3OH?冰醋酸(10∶0.3∶0.02~10∶0.35∶ 0.02)梯度洗脫,Sephadex LH?20 CHCl3?CH3OH(1∶ 1)洗 脫,得化合物5(0.42 g);Fr.4 經(jīng)正相硅膠柱石油醚?丙酮?冰醋酸(5∶4∶0.02~5∶6∶0.02)、CHCl3?CH3OH?冰醋酸(10∶0.3∶0.02~10∶0.5∶0.02)梯度洗脫,得化合物6(1.2 g)、8(0.38 g)、11(0.42 g);CHCl3?CH3OH?冰醋 酸(10∶ 0.3∶ 0.02~10∶0.45∶0.02)、石油醚?乙酸乙酯?冰醋酸(5∶3∶0.02~5∶4.5∶0.02)梯度洗脫,Sephadex LH?20 CHCl3?CH3OH(1∶1)洗脫,得化合物7(0.28 g)、9(0.52 g);CHCl3?CH3OH?冰醋酸(10∶0.3∶0.02~10∶0.4∶0.02)梯度洗脫,甲醇結(jié)晶得化合物10(0.81 g)。Fr.5 過(guò)硅膠柱色譜石油醚?乙酸乙酯?冰醋酸(3∶5∶0.02~1∶5∶0.02),CHCl3?CH3OH?冰醋酸(10∶0.5∶0.02~10∶0.7∶0.02)梯度洗脫,柱色譜Sephadex LH?20 CHCl3?CH3OH(1∶1)洗脫,得化合物13(0.41 g);CHCl3?CH3OH?冰醋 酸(10∶ 0.4∶ 0.02~10∶ 0.75∶0.02)梯度洗脫,石油醚?丙酮?冰醋酸(4∶5∶0.02~2∶5∶0.02)梯度洗脫洗脫,得化合物12(0.39 g)、14(0.28 g)。
化合物1:白色粉末,ESI?MSm/z:189.0[M+H]+,分子式C7H8O6。1H?NMR(600 MHz,CD3OD)δ:6.91(1H,s,H?4),3.83(2H,s,H?2),3.71(3H,s,?OCH3);13C?NMR(150 MHz,CD3OD)δ:173.7(C?1),168.9(C?6),167.3(C?5),142.3(C?3),129.3(C?4),52.4(5?OCH3),33.7(C?2)。以上數(shù)據(jù)與文獻(xiàn)[7]基本一致,故鑒定為烏頭酸C。
化合物2:白色無(wú)定形粉末,ESI?MSm/z:110.0[M+H]+,分子式C6H6O2。1H?NMR(600 MHz,CD3OD)δ:7.82(2H,d,J=8.2 Hz,H?3,4),6.76(2H,d,J=8.3 Hz,H?2,5);13C?NMR(150 MHz,CD3OD)δ:163.4(C?1,6),133.0(C?3,4),116.1(C?2,5)。以上數(shù)據(jù)與文獻(xiàn)[8]基本一致,故鑒定為苯二酚。
化合 物3:白色顆粒,ESI?MSm/z:164.1[M+H]+,分子式C9H8O3。1H?NMR(600 MHz,CD3OD)δ:7.58(1H,d,J=16.2 Hz,H?7),7.44(1H,d,J=8.4 Hz,H?2),7.43(1H,d,J=8.1 Hz,H?6),6.79(1H,d,J=8.4 Hz,H?3),6.68(1H,d,J=8.6 Hz,H?5),6.26(1H,d,J=16.2 Hz,H?8);13C?NMR(150 MHz,CD3OD)δ:171.5(C?9),161.0(C?4),146.3(C?7),131.0(C?2,6),127.1(C?1),116.5(C?3,5),115.6(C?8)。以上數(shù)據(jù)與文獻(xiàn)[9]基本一致,故鑒定為香豆酸。
化合物4:黃色固體,ESI?MSm/z:329.1[M?H]-,分子式 C17H14O7。1H?NMR(600 MHz,CD3OD)δ:7.25(2H,brs,H?2′,6′),6.67(1H,s,H?3),6.49(1H,s,H?6),6.32(1H,s,H?8),3.97(6H,s,2CH3);13C?NMR(150 MHz,CD3OD)δ:183.9(C?4),166.1(C?7,2),163.2(C?5),159.5(C?9),149.7(C?3′,5′),141.0(C?4′),122.7(C?1′),105.4(C?2′,6′),105.3(C?10),104.6(C?3),100.2(C?6),95.1(C?8),57.1(C?3′,5′?OCH3)。以上數(shù)據(jù)與文獻(xiàn)[10]基本一致,故鑒定為5,7,4′?三羥基?3′,5′?二甲氧基黃酮。
化合物5:白色無(wú)定形粉末,ESI?MSm/z:243.2[M?H]-,分子式C13H23O4。1H?NMR(600 MHz,CD3OD)δ:6.06(1H,dd,J=16.1,1.2 Hz,H?7),5.75(1H,dd,J=16.3,6.2 Hz,H?8),4.33(1H,dd,J=6.2,1.1 Hz,H?9),4.04(1H,tt,J=12.3,6.3 Hz,H?3),1.75(1H,ddd,J=12.4,6.2,2.1 Hz,H?4eq),1.73(1H,t,J=12.1 Hz,H?4ax),1.63(1H,t,J=12.2 Hz,H?2ax),1.45(1H,ddd,J=12.0,4.1,2.2 Hz,H?2eq),1.28(3H,d,J=6.3 Hz,H?10),1.25(3H,s,H?13),1.12(3H,s,H?11),0.85(3H,s,H?12);13C?NMR(150 MHz,CD3OD)δ:136.1(C?8),131.2(C?7),78.9(C?6),77.8(C?5),69.6(C?9),65.3(C?3),46.4(C?2),45.7(C?4),40.7(C?1),27.6(C?13),27.0(C?12),26.2(C?11),24.2(C?10)。以上數(shù)據(jù)與文獻(xiàn)[11]基本一致,故鑒定為bridelionol B。
化合物6:黃色無(wú)定形粉末,ESI?MSm/z:283.1[M?H]-,分子式C13H16O7。1H?NMR(600 MHz,CD3OD)δ:7.91(1H,d,J=8.7 Hz,H?2,6),7.06(1H,d,J=8.5 Hz,H?3,5),5.52(1H,d,J=8.4,H?1′),4.00(1H,d,J=1.4 Hz,H?2′),3.83(1H,dd,J=9.5,3.6 Hz,H?3′),3.57(1H,m,H?5′),3.38(1H,t,J=9.3 Hz,H?4′),1.18(3H,d,J=6.1 Hz,H?6′);13C?NMR(150 MHz,CD3OD)δ:161.4(C?7,4),132.7(C?2,6),125.4(C?1),116.9(C?3,5),99.6(C?1′),73.6(C?4′),72.1(C?3′),71.8(C?2′),70.9(C?5′),18.0(C?6′)。以上數(shù)據(jù)與文獻(xiàn)[12]基本一致,故鑒定為4?O?α?L?鼠李糖基苯甲酸。
化合物7:白色粉末,ESI?MSm/z:365.1[M+Na]+,分子式C20H22O5。1H?NMR(600 MHz,CD3OD)δ:6.91(1H,dd,J=8.5,1.3 Hz,H?6),6.87(1H,d,J=1.1 Hz,H?2),6.77(1H,d,J=8.5 Hz,H?5),6.64(1H,d,J=1.1 Hz,H?6′),6.52(1H,d,J=1.4 Hz,H?2′),5.90(1H,ddd,J=16.6,10.2,6.5 Hz,H?8′),5.49(1H,q,J=7.1 Hz,H?8),5.06(1H,dd,J=16.6,1.6 Hz,H?9′a),5.04(1H,dd,J=10.1,1.6 Hz,H?9′b),3.89(2H,m,H?9a),3.81(3H,s,4?OCH3),3.79(3H,s,3?OCH3),3.53(1H,q,J=5.5 Hz,H?8),3.29(1H,brd,J=6.3 Hz,H?7′);13C?NMR(150 MHz,CD3OD)δ:157.1(C?3),147.5(C?4),146.1(C?4′),145.5(C?5′),135.9(C?8′),134.6(C?1′),134.1(C?1),129.3(C?3′),117.0(C?2,6),115.3(C?2′,6′),112.9(C?9′),111.3(C?5),87.8(C?7),65.0(C?9),56.2(3,4?OCH3),51.0(C?8),39.6(C?7′)。以上數(shù)據(jù)與文獻(xiàn)[13]基本一致,故鑒定為miliusin B。
化合物8:白色固體,ESI?MSm/z:381.1[M?H]-,分子式 C18H22O9。1H?NMR(600 MHz,CD3OD)δ:7.69(1H,d,J=15.1 Hz,H?7′),7.20(1H,dd,J=8.5,1.9 Hz,H?2′),7.17(1H,d,J=1.9 Hz,H?6′),6.95(1H,d,J=8.9 Hz;H?5′),6.49(1H,d,J=15.3 Hz;H?8′),4.28(1H,q,J=3.4 Hz;H?3),4.15(1H,ddd,J=10.8,9.7,4.5 Hz,H?5),3.89(3H,s,3′?OCH3),3.84(3H,s,4′?OCH3),3.61(1H,dd,J=9.2,3.3 Hz,H?4),2.57(1H,dd,J=15.6,3.6 Hz,H?2eq),2.49(1H,ddd,J=13.6,4.5,3.4 Hz,H?6eq),2.30(1H,dd,J=15.3,3.6 Hz,H?2ax),1.96(1H,dd,J=13.6,10.7 Hz,H?6ax);13C?NMR(150 MHz,CD3OD)δ:175.6(C?1),167.1(C?9′),149.3(C?3′),148.1(C?4′),145.9(C?7′),128.3(C?1′),116.2(C?6′,8′),115.5(C?2′),115.1(C?5′),80.8(C?1),76.4(C?5),69.3(C?3),65.1(C?4),56.2(3′?OCH3),56.3(4′?OCH3),38.4(C?2),34.6(C?6)。以上數(shù)據(jù)與文獻(xiàn)[14]基本一致,故鑒定為5?O?二甲氧基肉桂酰基奎尼酸。
化合物9:淺棕色無(wú)定形粉末,ESI?MSm/z:387.1[M?H]-,分子式C20H19O8。1H?NMR(600 MHz,CD3OD)δ:7.58(1H,d,J=15.9 Hz,H?3),7.03(1H,d,J=2.1 Hz,H?5),7.01(1H,dd,J=8.1,2.2 Hz,H?9),6.90(1H,d,J=8.0 Hz,H?8),6.79(1H,d,J=8.3 Hz,H?8′),6.77(1H,d,J=2.2 Hz,H?5′),6.70(1H,dd,J=8.2,2.2 Hz,H?9′),6.36(1H,d,J=15.7 Hz,H?2),5.03(1H,d,J=17.4 Hz,H?1′b),4.95(1H,d,J=17.3 Hz,H?1′a),4.77(1H,s,H?3′),3.88(3H,s,7?OCH3),3.36(3H,s,3′?OCH3);13C?NMR(150 MHz,CD3OD)δ:175.4(C?2′),168.2(C?1),150.8(C?7),149.4(C?6),147.4(C?3,6′),145.8(C?7′),127.6(C?4),126.9(C?4′),124.1(C?9),120.0(C?9′),116.5(C?8′),116.4(C?5′),115.6(C?2),115.1(C?5),111.7(C?8),88.7(C?3′),66.9(C?1′),56.4(7?OCH3),56.3(3′?OCH3)。以上數(shù)據(jù)與文獻(xiàn)[15]基本一致,故鑒定為cimiracemate C。
化合物10:白色粉末,ESI?MSm/z:485.3[M+Na]+,分子式C30H38O4。1H?NMR(600 MHz,CD3OD)δ:5.86(2H,s,H?3,3′),5.79(2H,s,H?10,10′),5.77(2H,s,H?8,8′),4.65(2H,brs,?OH),4.30(2H,q,J=6.1 Hz,H?5,5′),2.54(2H,d,J=16.6 Hz,H?1b,1′b),2.16(2H,d,J=16.6 Hz,H?1a,1′a),1.91(6H,s,H?12,12′),1.23(6H,d,J=6.4 Hz,H?13,13′),1.03(6H,s,H?14,14′),1.01(6H,s,H?15,15′);13C?NMR(150 MHz,CD3OD)δ:201.2(C?9,9′),167.4(C?7,7′,11,11′),137.0(C?10),136.9(C?10′),130.1(C?2,2′),129.9(C?8,8′),127.1(C?3,3′),79.9(C?4,4′),68.7(C?5,5′),50.7(C?1,1′),42.4(C?6,6′),24.5(C?14,14′),23.8(C?13,13′),23.5(C?15,15′),19.6(C?12,12′)。以上數(shù)據(jù)與文獻(xiàn)[16]基本一致,故鑒定為conoidol A。
化合物11:黃色無(wú)定形粉末,ESI?MSm/z:493.1[M+H]+,分子式C23H24O12。1H?NMR(600 MHz,CD3OD)δ:12.95(1H,s,5?OH),7.35(2H,s,H?2′,6′),7.06(1H,s,H?3),6.92(1H,d,J=2.1 Hz,H?8),6.45(1H,d,J=2.4 Hz,H?6),5.07(1H,d,J=7.4 Hz,H?1″),3.88(6H,s,3′,5′?OCH3),3.72(1H,m,H?6″a),3.46(2H,m,H?5″,H?6″b),3.32(1H,m,H?3″),3.29(1H,m,H?2″),3.20(1H,t,J=10.7 Hz,H?4″);13C?NMR(150 MHz,CD3OD)δ:182.1(C?4),164.1(C?2,4′),163.0(C?7),161.1(C?9),156.9(C?5),148.2(C?3′,5′),140.3(C?1′),105.5(C?10),104.5(C?3,2′,6′),100.1(C?1″),99.5(C?6),95.3(C?8),77.4(C?5″),76.5(C?3″),73.1(C?2″),69.6(C?4″),60.6(C?6″),56.4(3′,5′?OCH3)。以上數(shù)據(jù)與文獻(xiàn)[17]基本一致,故鑒定為麥黃酮7?O?β?D?吡喃葡萄糖苷。
化合物12:黃色無(wú)定形粉末,ESI?MSm/z:525.2[M?H]-,分子式C27H26O11。1H?NMR(600 MHz,CD3OD)δ:7.21(2H,s,H?2′,6),6.98(1H,d,J=1.6 Hz,H?2″),6.84(1H,dd,J=8.0,1.5,Hz,H?6″),6.73(1H,d,J=8.1 Hz,H?5″),6.57(1H,s,H?3),6.35(1H,s,H?8),6.11(1H,s,H?6),4.91(1H,d,J=5.2 Hz,H?7″),4.43(1H,ddd,J=5.2,5.1,3.5 Hz,H?8″),3.92(1H,dd,J=12.1,5.4 Hz,H?9″a),3.85(6H,s,3′,5′?OCH3),3.82(3H,s,3″?OCH3),3.63(1H,dd,J=12.2,2.9 Hz,H?9″b);13C?NMR(150 MHz,CD3OD)δ:183.7(C?4),166.2(C?7),165.4(C?2),163.3(C?5),159.5(C?9),154.9(C?3′,5′),148.6(C?3″),147.0(C?4″),140.7(C?4′),133.8(C?1″),127.8(C?1′),120.9(C?6″),115.6(C?5″),111.5(C?2″),105.9(C?3),105.5(C?10),105.3(C?2′,6′),100.2(C?6),95.2(C?8),87.5(C?8″),74.2(C?7″),62.1(C?9″),56.7(3′,5′?OCH3),56.3(3″?OCH3)。以上數(shù)據(jù)與文獻(xiàn)[18]基本一致,故鑒定為salcolin B。
化合物13:黃色無(wú)定形粉末,ESI?MSm/z:541.2[M?H]-,分子式C28H28O11。1H?NMR(600 MHz,CD3OD)δ:7.16(2H,s,H?2′,H?6),6.88(1H,d,J=1.6 Hz,H?2″),6.85(1H,dd,J=8.0,1.8 Hz,H?6″),6.74(1H,d,J=8.2 Hz,H?5″),6.52(1H,s,H?3),6.37(1H,s,H?8),6.15(1H,s,H?6),4.44(1H,d,J=5.0 Hz,H?7″),4.43(1H,ddd,J=5.1,5.2,3.2 Hz,H?8″),3.92(1H,dd,J=12.0,4.5 Hz,H?9″a),3.86(6H,s,7″?OCH3),3.83(3H,s,3′,5′?OCH3),3.80(3H,s,3″?OCH3),3.71(1H,dd,J=12.2,3.4 Hz,H?9″b);13C?NMR(150 MHz,CD3OD)δ:183.8(C?4),166.4(C?7),165.4(C?2),163.3(C?5),159.5(C?9),154.8(C?3′),154.7(C?5′),148.8(C?3″),147.4(C?4″),140.1(C?4′),131.1(C?1″),127.7(C?1′),122.1(C?6″),112.2(C?2″),105.8(C?3),105.7(C?10),105.4(C?2′),105.1(C?6′),100.3(C?6),95.2(C?8),86.8(C?8″),83.8(C?7″),62.4(C?9″),57.1(7″?OCH3),56.9(3′,5′?OCH3),56.4(3″?OCH3)。以上數(shù)據(jù)與文獻(xiàn)[19]基本一致,故鑒定為tricin 4′?O?[threo?β?guaiacyl?(7″?O?methyl)?glyceryl]ether。
化合物14:黃色無(wú)定形粉末,ESI?MSm/z:689.2[M+H]+,分子式C33H36O16。1H?NMR(600 MHz,CD3OD)δ:7.23(2H,s,H?2′,6′),6.93(1H,d,J=1.6 Hz,H?2″),6.84(1H,dd,J=8.3,1.7 Hz,H?6″),6.74(1H,d,J=8.2 Hz,H?5″),6.71(1H,s,H?3),6.49(1H,s,H?8),6.21(1H,s,H?6),5.10(1H,d,J=5.2 Hz,H?7″),4.54(1H,ddd,J=5.2,5.2,3.5 Hz,H?8″),3.96(1H,dd,J=12.2,5.4 Hz,H?9″a),3.84(6H,s,3′,5′?OCH3),3.83(3H,s,3″?OCH3),3.69(1H,dd,J=12.2,5.3 Hz,H?9″b);13C?NMR(150 MHz,CD3OD)δ:183.8(C?4),166.3(C?7),165.2(C?2),163.3(C?5),159.5(C?9),154.8(C?3′,5′),148.5(C?3″),147.2(C?4″),140.6(C?4′),131.7(C?1″),128.1(C?1′),121.3(C?6″),115.6(C?5″),112.5(C?2″),106.0(C?3),105.5(C?10),105.2(C?1?),104.9(C?2′,6′),100.3(C?6),95.2(C?8),87.0(C?8″),81.8(C?7″),78.1(C?3?),77.9(C?5?),75.6(C?2?),71.4(C?4?),62.5(C?6?),61.5(C?9″),57.0(3′,5′?OCH3),56.4(3″?OCH3)。以上數(shù)據(jù)與文獻(xiàn)[20]基本一致,故鑒定為tricin4′?O?(erythro?β?guaiacylglyceryl)ether 7?O?β?glucopyranoside。
本實(shí)驗(yàn)從甘蔗莖葉部位分離到14個(gè)化合物,其中5個(gè)黃酮類、4個(gè)酚類、1個(gè)酸類及4個(gè)其他類型,黃酮及酚類為核心成分,與文獻(xiàn)[3,21]報(bào)道一致。黃酮類為自然界一類重要的生物活性成分,如化合物(4)具有很好的抗菌及抑制稻田雜草生長(zhǎng)的作用[22];化合物(14)具有抗炎 功能[23],以期為尋找抗菌和抗炎的活性成分提供了新原料來(lái)源。多酚類成分因其天然、安全、無(wú)毒性等特點(diǎn),備受研究者的重視,目前已運(yùn)用于人工培養(yǎng)酵母、制藥、制酒精、化妝品等化工行業(yè),如化合物(3)具抗癌、抗菌等功能[24],在醫(yī)藥方面利用度高,甘蔗中分離到的香豆素相對(duì)于其他酚類物質(zhì)的量較大,可考慮為產(chǎn)業(yè)化生產(chǎn)的替代資源。甘蔗在我國(guó)是主要的經(jīng)濟(jì)作物,南方各地都有種植,蔗葉、蔗皮、蔗渣皆是加工糖業(yè)過(guò)程中產(chǎn)生的副產(chǎn)物,其質(zhì)量占比達(dá)甘蔗的40% 以上,且價(jià)格低廉。因此,繼續(xù)探索甘蔗莖葉部位的化學(xué)成分,有利于甘蔗資源的循環(huán)利用,變廢為寶,期望從中尋求量大、安全、有效的活性成分,以期豐富其化學(xué)成分,為甘蔗非食用部分的活性開(kāi)發(fā)利用提供參考。