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虎舌红根茎三萜皂苷类成分的研究

2019-10-16凌育赵刘经亮吴晓天刘佩珊

中成药 2019年9期
关键词:鼠李糖木糖浓硫酸

凌育赵, 刘经亮, 吴晓天, 刘佩珊

(1.仲恺农业工程学院,广东 广州510225;2.广东食品药品职业学院,广东 广州510520)

虎舌红Ardisia mamillata Hance,又名红毛毡、老虎舌、红地毡、毛凉伞、金丝红珠等,为紫金牛科紫金牛属植物,原产我国,分布于福建、广西、广东、云南、四川、贵州、江西等地[1],全株具有清热利湿、平肝解郁、活血化淤的功效[2],是民间常用草药。目前从虎舌红植株中已经分离得到三萜类、皂苷类、生物碱类、黄酮类、糖苷类、蒽醌类、酚醚类和甾体类等[3-6]。皂苷类主要为齐墩果烷型五环三萜衍生物,据报道,这类成分具有抗肿瘤、抗HIV 等活性[4]。课题组在前期研究[5-6]的基础上,对虎舌红根茎三萜皂苷类进一步研究,从中分离得到7 个化合物,其中化合物4 ~7 为首次从该植物中分离得到,化合物5 为首次从该属植物中分离得到。

1 材料

IR Prestige-21 型傅立叶变换红外光谱仪(美国Nicolet 公司);Bruker DRX-500 核磁共振仪(德国Bruker 公司);Thermo LCQ FLEET 型电喷雾质谱仪(美国Thermo 公司);Agilent 1100 Series 液相色谱仪(美国Agilent 公司);YMC-Pack ODSAM 色谱柱 (4.6 mm×250 mm,5 μm); 凝胶SephadexLH-20(美国Pharmacia 公司);GF254薄层色谱和柱层析硅胶(青岛海洋化工厂);乙腈为色谱纯,其余试剂均为分析纯。

植物样品采自广州农业科学院白云实验基地,经仲恺农业工程学院植物教研室周俊辉教授鉴定为紫金牛科紫金牛属植物虎舌红Ardisia mamillata Hance 的干燥根茎,标本现存于本实验室。

2 提取与分离

干燥的虎舌红根茎(7.5 kg) 粉碎,用80%乙醇加热回流提取3 次,每次2 h,合并滤液,减压浓缩,得乙醇浸膏1.05 kg,加水混悬,依次用石油醚、乙酸乙酯和正丁醇各萃取3 次,减压浓缩各萃取液,得到乙酸乙酯部分96 g、 正丁醇部分412 g。正丁醇萃取物上D101 大孔吸附树脂,依次用35%、75%、95%乙醇洗脱,收集75%乙醇洗脱部分,回收溶剂后得褐色浸膏21.6 g,将此浸膏进行硅胶柱层析分离,氯仿-甲醇(100 ∶1 ~0 ∶100)梯度洗脱,合并相同流份,得到4 个组分(Fr.1 ~4)。Fr.1 浓缩后析出白色粉末,经重结晶得化合物1(96 mg),经制备HPLC 分离得化合物2(26 mg);Fr.2 经ODS 柱层析和重结晶得化合物3(59 mg),经制备HPLC 分离得化合物4 (57 mg);Fr.3 经反复硅胶柱层析、凝胶层析和制备HPLC分离得到化合物5(29 mg);Fr.4 经Sephadex LH-20 反复柱层析纯化,再用制备HPLC 分离纯化得到化合物6(74 mg)、7(29 mg)。

3 结构鉴定

化合物1:白色粉末,mp 242 ~245 ℃。醋酐-浓硫酸反应呈阳性,Molish 反应显紫红色;完全酸水解TLC 检出阿拉伯糖、葡萄糖和木糖。IRνmax(KBr)cm-1:3 418(OH),2 923,2 856,1 714(-C =0);ESI-MS m/z:921[M+Na]+,899[M+H]+, 分子量898, 分子式C46H74O17。1H-NMR(C5D5N, 400 MHz) δ: 0.86 (3H, s, 24-H),0.97 (3H, s, 25-H), 1.03 (3H, s, 29-H),1.03 (3H, s, 23-H), 1.30 (3H, s, 26-H),1.57(3H, s, 27-H), 1.68 (3H, d, J =6.0,Rha-H-6),3.17(1H,d,J =7.2,28-Ha),3.56(1H,d, J =7.2,28-Hb),3.28(1H, dd, J =4.0,11.4,3-H),4.80(1H,d,J =5.4,Ara-1-H), 5.18 (1H, d, J =5.6, Xyl-1-H), 6.37(1H,d,J =8.0,Glc-1-H),6.33 (1H, s,16-OH),9.64(1H,s,30-CHO);13C-NMR(C5D5N,100 MHz)δ:39.2(C-1),26.7(C-2),89.0(C-3),39.7(C-4),55.7(C-5),18.0(C-6),34.3(C-7),42.5(C-8),50.4(C-9),36.8(C-10),19.2(C-11),32.7(C-12),86.4(C-13),44.6(C-14),36.8(C-l5),76.9(C-16),44.0(C-17),53.4(C-18),33.4(C-19),48.4(C-20),30.5(C-21),32.4(C-22),28.1(C-23),16.7(C-24),16.4(C-25),18.6(C-26),19.8(C-27),77.7(C-28),24.1(C-29),207.6(C-30),107.2(C-ara-1),72.8 (C-ara-2),73.8 (C-ara-3),78.9(C-ara-4),65.3(C-ara-5),105.2(Cglc-1), 78.1 (C-glc-2), 78.9 (C-glc-3), 71.7(C-glc-4), 78.3 (C-glc-5), 62.6 (C-glc-6),108.3(C-xyl-1),76.1 (C-xyl-2),78.5 (C-xyl-3),70.6(C-xyl-4),67.2 (C-xyl-5)。以上数据与文献[7] 基本一致,故鉴定为cyclaminorin。

化合物2:白色结晶,mp 230 ~232 ℃。醋酐-浓硫酸反应呈阳性,Molish 反应显紫红色;完全酸水解TLC 检出阿拉伯糖、木糖、葡萄糖和鼠李糖。IRνmax(KBr)cm-1:3 412(OH),2 926,2 862,1 715(-C =O);ESI-MS m/z:1 094[M +Na]+,分 子 量 1 074, 分 子 式 C53H86O22。1H-NMR(C5D5N, 400 MHz) δ: 0.68 (3H, s, 24-H),0.87 (3H, s, 25-H), 0.87 (3H, s, 29-H),1.03 (3H, s, 23-H), 1.12 (3H, s, 26-H),1.39(3H, s, 27-H), 1.66 (3H, d, J =6.4,Rha-H-6),3.02(1H,dd,J =4.8,11.4,3-H),3.01(1H, d, J =7.6,28-Ha),3.38(1H, d,J =7.8,28-Hb),5.01 (1H,d,J =3.6,Ara-1-H), 5.25 (1H, d, J =7.2, Glc-1-H), 5.13(1H,d,J =7.6,Glc Ⅱ-1-H),6.21 (1H, brs,Rha-1-H),6.27(1H,s,16-OH),9.48(1H,s,30-CHO);13C-NMR(C5D5N,100 MHz) δ:39.4(C-1),26.8(C-2),89.6(C-3),39.9 (C-4),55.7(C-5), 18.2 (C-6), 34.8 (C-7), 42.8(C-8), 50.5 (C-9), 37.1 (C-10), 19.7 (Cl1),32.9(C-12),86.5(C-13),44.9(C-14),37.3(C-15),77.1(C-16),44.3(C-17),53.4(C-18),33.5(C-19),48.6(C-20),30.9(C-21),32.3(C-22),28.3(C-23),16.5(C-24),16.6(C-25),18.5(C-26),20.0(C-27),77.7(C-28), 24.4 (C-29), 207.6 (C-30), 104.7(C-ara-1), 81.2 (C-ara-2), 75.0 (C-ara-3),75.3(C-3-ara-4),63.0 (C-3-ara-5),105.5 (Cglc-1), 76.5 (C-glc-2), 77.4 (C-glc-3), 72.0(C-glc-4), 78.6 (C-glc-5), 63.2 (C-glc-6),102.2(C-rha-1),72.4(C-rha-2),73.0(C-rha-3),75.2(C-rha-4),69.7 (C-rha-5),19.4(Crha-6),102.4(C-glcⅡ-1),78.46(C-glcⅡ-2),79.5(C-glc Ⅱ-3),72.3(C-glc Ⅱ-4),78.7 (Cglc Ⅱ-5),62.6 (C-glc Ⅱ-6)。 以 上 数 据 与 文 献[8-9] 基本一致,故鉴定为ardisicrispin B。

化合物3:白色粉末,mp 280 ~282 ℃。醋酐-浓硫酸反应呈阳性,Molish 反应显紫红色;完全酸水解TLC 检出阿拉伯糖、葡萄糖和鼠李糖。IRνmax(KBr)cm-1:3 403(OH),2 948,2 859,1 706(-C =O),1 651(-C =O);ESI-MS m/z:934[M+Na]+, 分 子 量910, 分 子 式C47H74O17。1H-NMR(C5D5N, 400 MHz) δ: 0.81 (3H, s, 25-H),0.88 (3H, s, 29-H), 0.92 (3H, s, 24-H),1.09(3H,s,27-H),1.21(3H,s,23-H),0.93(3H, s, 24-H), 1.24 (3H, s, 26-H), 1.67(3H,d,J =6.4,Rha-H-6),3.27(1H,dd,J =4.1,11.9, 3-H), 3.32 (1H, d, J =8.2,28-Ha),3.87(1H,d,J =8.6,28-Hb),4.78(1H,d,J =5.6, Ara-1-H),5.16 (1H, d, J =6.8,Glc-1-H),6.30(1H,brs,Rha-1-H),9.51(1H,s,30-CHO);13C-NMR(C5D5N,100 MHz)δ:39.2(C-1),26.6(C-2),88.7 (C-3),39.6(C-4),55.5(C-5),17.8(C-6),33.7(C-7),43.0(C-8),50.1 (C-9),36.5 (C-10),18.7 (C-11),31.5(C-12),86.2(C-13),50.3(C-14),45.7(C-15),212.8(C-16),55.3(C-17),55.7(C-18),33.8(C-19),47.6(C-2O),29.5(C-21),33.8 (C.22), 28.3 (C-23), 16.6 (C-24),16.3(C-25),18.7(C-26),21.9(C-27),74.8(C-28), 23.9 (C-29), 205.8 (C-30), 106.9(C-ara-1), 72.9 (C-ara-2), 73.7 (C-ara-3),78.7(C-ara-4),65.3 (C-ara-5),105.3 (C-glc-1),78.3 (C-glc-2),78.8(C-glc-3),71.5(Cglc-4),78.3 (C-glc-5),62.5 (C-glc-6),102.1(C-rha-1), 72.3 (C-rha-2), 72.5 (C-rha-3),74.9 (C-rha-4),70.1(C-rha-5),18.7 (C-rha-6)。以上数据与文献[9] 基本一致,故鉴定为3β-O-{α-L-吡喃鼠李糖基-(1→3) -β-D-吡喃葡萄糖基-(1→4) -α-L-吡喃阿拉伯糖基} -16-酮-西克拉敏A。

化合物4:白色粉末,mp 284 ~285 ℃。醋酐-浓硫酸反应呈阳性,Molish 反应显紫红色;完全酸水解TLC 检出阿拉伯糖、木糖、葡萄糖和鼠李糖。IRνmax(KBr)cm-1:3 406(OH),2 952,2 859,1 708(-C =O),1 650 (-C =O); ESI-MS m/z:1 065[M +Na]+, 分 子 量 1 072, 分 子 式C53H84O22。1H-NMR(C5D5N, 400 MHz) δ: 0.84(3H, s, 25-H), 0.89 (3H, s, 29-H), 1.02(3H, s, 24-H), 1.08 (3H, s, 27-H), 1.25(3H, s, 23-H), 1.27 (3H, s, 26-H), 1.63(d,J =6.4,Rha-H-6),3.34 (1H,d,J =8.0,28-Ha),3.85 (1H, d, J =8.0, 28-Hb), 4.64(1H,d,J =5.2,Ara-H-1),4.92 (d,J =7.8,Glc-H-1), 4.94 (1H, d, J =8.0, Xyl-H-1),6.01(1H, brs, Rha-H-1), 9.61 (1H, s, 30-CHO);13C-NMR(C5D5N,100 MHz)δ:39.3(C-1), 26.8 (C-2), 88.4 (C-3), 39.5 (C-4),55.7 (C-5), 17.4 (C-6), 33.6 (C-7), 43.2(C-8), 49.8 (C-9), 36.8 (C-10), 18.6 (C-11),31.4(C-12),86.3(C-13),50.3(C-14),45.5 (C-15), 212.4 (C-16), 55.5 (C-17),55.8(C-18),33.7(C-19),48.1(C-20),29.5(C-21),33.5(C-22),28.2 (C-23),16.8 (C-24),16.3(C-25),18.7(C-26),21.6(C-27),74.5 (C-28), 23.7 (C-29), 206.4 (C-30),107.3(C-ara-1),73.5 (C-ara-2),74.5 (C-ara-3),81.7(C-ara-4),66.0(C-ara-5),105.3(Cglc-1), 81.5 (C-glc-2), 86.3 (C-glc-3), 70.2(C-glc-4), 78.2 (C-glc-5), 62.3 (C-glc-6),106.3(C-xyl-1),75.5 (C-xyl-2),78.5 (C-xy1-3), 69.4 (C-xyl-4), 67.1 (C-xy1-5), 103.5(C-rha-1), 72.1 (C-rha-2), 72.6 (C-rha-3),73.9(C-rha-4),70.8 (C-rha-5),18.7 (C-rha-6)。以上数据与文献[10] 基本一致,故鉴定为3β-O-{α-L-吡喃鼠李糖基-(1→3) -[β-D-吡喃木糖基-(1→2) ] -β-D-吡喃葡萄糖基-(1→4) -α-L-吡喃阿拉伯糖基} -16-酮-西克拉敏A。

化合物5:白色粉末,mp 260 ~262 ℃。醋酐-浓硫酸反应呈阳性,Molish 反应显紫红色;完全酸水解TLC 检出阿拉伯糖、木糖、葡萄糖和鼠李糖。IRνmax(KBr)cm-1:3 392,3 271(OH),3 180,2 971,1 693(-C=O),1 631(-C=O);ESI-MS m/z:1 113 [M +Na]+, 分 子 量 1 090, 分 子 式C53H85O23。1H-NMR (C5D5N, 600 MHz) δ: 0.80(3H, s, 25-H), 0.87 (3H, s, 26-H), 1.31(3H, s, 29-H), 1.01 (3H, s, 24-H), 1.81(3H,s,23-H)1.14(3H,s,27-H),1.81(3H,d,J =4.2,Rha-H-6),3.12(1H,d,J =4.2,3-H), 3.54 (1H, d, J = 10.0, 28-Ha), 3.71(1H,d, J =10.0,28-Hb),4.92 (1H, d, J =5.6,Ara-1-H), 5.38 (1H, d, J =7.6, Glc-1-H), 5.27 (1H, d, J =7.8, Xyl-1-H), 6.45(1H, brs, Rha-1-H), 5.61 (1H, brs, 12-H),6.43(1H,brs,16-OH);13C-NMR(C5D5N,150 MHz)δ:38.7(C-1),26.5(C-2),89.0(C-3),39.5 (C-4), 55.6 (C-5), 18.4 (C-6), 33.0(C-7),40.0(C-8),45.9(C-9),36.7(C-10),23.5 (C-11), 123.0 (C-12), 144.6 (C-13),41.6(C-14),34.9(C-15),73.9(C-16),40.1(C-17),43.5(C-18),44.5 (C-19),44.4 (C-20),32.7(C-21),32.5(C-22),27.3(C-23),16.8(C-24),15.6(C-25),16.7(C-26),27.8(C-27),71.5(C-28),28.9(C-29),177.5(C-30),104.5(C-ara-1),80.7(C-ara-2),72.5(Cara-3),75.8 (C-ara-4),70.7 (C-ara-5),102.9(C-glc-1),77.3(C-glc-2),79.5(C-glc-3),71.7(C-glc-4), 78.3 (C-glc-5), 62.8 (C-glc-6),106.3(C-xyl-1),75.5 (C-xyl-2),78.5 (C-xy1-3), 69.4 (C-xyl-4), 67.1 (C-xy1-5), 101.3(C-rha-1), 72.5 (C-rha-2), 72.8 (C-rha-3),74.5 (C-rha-4),68.5 (C-rha-5),18.9(C-rha-6)。以上数据与文献[11-12] 基本一致,故鉴定为3β-O-{α-L-吡喃鼠李糖基-(1→3) -β-D-吡喃木糖糖基-(1→2) -[β-D-吡喃葡萄糖基-(1 →4) ] -α-L-吡喃阿拉伯糖基} -16α,28-二羟基-12-烯-30-齐墩果酸。

化合物6:白色粉末,mp 263 ~265 ℃。醋酐-浓硫酸反应呈阳性,Molish 反应显紫红色;完全酸水解TLC 检出阿拉伯糖、葡萄糖、木糖和鼠李糖。IRνmax(KBr)cm-1:3 417(OH),2 926,2 858,1 711(-C =O);ESI-MS m/z:1 215.619 0[M +Na]+,分子量1 192,分子式C58H96O25。1H-NMR(C5D5N, 400 MHz) δ: 0.82 (3H, s, 25-H),1.00(3H,s,29-H),1.00(3H,s,24-H),1.56(3H, s, 23-H), 1.27 (3H, s, 26-H), 1.54(3H,s,27-H),1.65(3H,d,J =5.6,Rha-H-6),3.15(1H,d,J =7.2,28-Ha),3.51(1H,d,J =7.2, 28-Hb), 3.51 (1H, dd, J =4.0,11.4,3-H),4.83 (1H, d, J =5.6, Ara-1-H),5.41(1H,d,J =7.6,Glc-1-H),4.86(d,J =7.8,GlcⅡ-H-1),5.97(1H,s,J =6.8,Xyl-H-1),5.97(1H,s,Rha-H-1),6.27(1H,s,16-OH);13C-NMR(C5D5N,100 MHz) δ:39.2 (C-1), 26.1 (C-2), 88.6 (C-3), 39.3 (C-4),55.5(C-5), 17.5 (C-6), 34.2 (C-7), 42.1(C-8), 50.3 (C-9), 36.5 (C-10), 19.3 (C-11),32.6(C-12),86.2(C-13),44.5(C-14),36.6(C-15),76.7(C-16),44.5(C-17),52.5(C-18),38.7 (C-19),31.8(C-20),36.6 (C-21),31.9(C-22),27.8(C-23),16.5(C-24),16.3(C-25),18.3(C-26),19.3(C-27),77.7(C-28),33.7(C-29),24.9(C-30),103.9(Cara-1), 79.2 (C-ara-2), 72.3 (C-ara-3), 77.6(C-ara-4), 62.7 (C-ara-5), 104.5 (C-glc-1),76.3(C-glc-2), 77.9 (C-glc-3), 71.6 (C-glc-4), 77.8 (C-glc-5), 63.62 (C-glc-6), 103.4(C-glc Ⅱ-1),81.3(C-glc Ⅱ-2),85.3(C-glc Ⅱ-3),69.3 (C-glc Ⅱ4),77.5 (C-glc Ⅱ-5),62.4(C-glcⅡ-6),105.2(C-xyl-1),74.7 (C-xyl-2),77.8(C-xyl-3), 70.3 (C-xyl-4), 66.5 (C-xyl-5), 103.3 (C-rha-1), 71.9 (C-rha-2), 72.2(C-rha-3), 73.4 (C-rha-4), 70.2 (C-rha-5),18.5(C-rha-6)。以上数据与文献[13] 基本一致,故鉴定为3β-O-{α-L-吡喃鼠李糖基 (1 →3) -[β-D-吡喃木糖基-(1→2) ] -β-D-吡喃葡萄糖基-(1→4) -[β-D-吡喃葡萄糖基-(1→2] -α-L-吡喃阿拉伯糖基} -16α-羟基-13β,28-环氧-齐墩果烷。

化合物7:白色粉末,mp 263 ~265 ℃。醋酐-浓硫酸反应呈阳性,Molish 反应显紫红色;完全酸水解TLC 检出阿拉伯糖、葡萄糖、木糖和鼠李糖。IRνmax(KBr)cm-1:3 417(OH),2 926,2 858,1 711(-C =O),1 661(-C =O); ESI-MS m/z:1 273[M +Na]+, 分 子 量 1 250, 分 子 式C60H98O27。1H-NMR(C5D5N,400 MHz) δ:0.82(3H, s, 25-H), 1.00 (3H, s, 29-H), 1.00(3H, s, 24-H), 1.56 (3H, s, 23-H), 1.27(3H, s, 26-H), 1.54 (3H, s, 27-H), 1.65(3H, d, J =5.6, Rha-H-6), 2.07 (3H, s,-CH3CO),3.15 (1H,d,J =7.2,28-Ha),3.51(1H,d, J =7.2,28-Hb),3.51(1H, dd, J =4.0,11.4,3-H),4.83(1H,d,J =5.6,Ara-1-H),5.41(1H,d,J =7.6,Glc-1-H),4.86(d,J=7.8,GlcⅡ-H-1),5.97(1H,s,J =6.8,Xyl-H-1),5.97(1H,brs,Rha-H-1),6.25(1H,s,16-OH);13C-NMR(C5D5N,100 MHz)δ:39.5(C-1), 26.1 (C-2), 88.7 (C-3), 39.6 (C-4),55.5(C-5), 17.8 (C-6), 34.2 (C-7), 42.3(C-8), 50.3 (C-9), 36.5 (C-10), 18.7 (C-11),32.6(C-12),86.2(C-13),44.4(C-14),36.6(C-15),76.7(C-16),43.6(C-17),51.3(C-18),33.5 (C-19),35.8(C-20),32.6 (C-21),31.9(C-22),27.9(C-23),16.5(C-24),16.1(C-25),18.3(C-26),19.3(C-27),77.3(C-28),28.7(C-29),68.4(C-30),103.8(Cara-1), 79.2 (C-ara-2), 72.3 (C-ara-3), 77.6(C-ara-4), 62.5 (C-ara-5), 104.4 (C-glc-1),75.5(C-glc-2), 77.8 (C-glc-3), 71.6 (C-glc-4),77.5(C-glc-5),62.8(C-glc-6),102.4(CglcⅡ-1),81.7(C-glcⅡ-2),85.5(C-glcⅡ-3),69.3(C-glc Ⅱ4),77.5 (C-glc Ⅱ-5),61.9 (Cglc Ⅱ-6), 104.2 (C-xyl-1), 74.5 (C-xyl-2),77.8(C-xyl-3), 70.5 (C-xyl-4), 65.8 (C-xyl-5), 102.3 (C-rha-1), 71.6 (C-rha-2), 72.6(C-rha-3), 73.1 (C-rha-4), 70.6 (C-rha-5),18.4(C-rha-6)。以上数据与文献[14-15] 基本一致,故鉴定为3β-O-{α-L-吡喃鼠李糖基(1→3) -[β-D-吡喃木糖基-(1→2) ] -β-D-吡喃葡萄糖基-(1→4) -[β-D-吡喃葡萄糖基-(1→2) ] -α-L-吡喃阿拉伯糖基} -16α-羟基-30-乙酰氧基-13,28-环氧-齐墩果烷。

4 讨论

结果表明,从虎舌红分离得到7 个五环三萜类化合物,均为具有3 ~5 个糖的齐墩果烷型三萜皂苷。化合物1 ~2 的苷元结构为13,28-环氧-30-醛基(A 型);化合物3 ~4 的苷元结构为13,28-环氧-16-羰基(B 型);化合物5 的苷元结构为12-烯-30-羰基(C 型);化合物6 ~7 的苷元结构为13,28-环氧醚 (D 型)、13,28-环氧醚-30-羰基 (E型),见图1。

图1 皂苷元的结构类型Fig.1 Structures of aglycones

化合物1 对人卵巢癌细胞A2780 显示出较强的细胞毒性[16]; 化合物2 对肿瘤细胞MCF-7、HepG2 和人红白血病K562、人胃癌KATO-Ⅲ、人卵巢癌OVCA2780 细胞均显示出较强的抑制作用[17-18];化合物4、6 ~7 对人肝癌细胞HepG2 均有较强的抑制细胞增殖的活性[10,13]。可见,三萜皂苷是虎舌红抗肿瘤活性的主要有效成分,以期本研究结果为进一步阐明虎舌红抗肿瘤作用的药效物质提供科学依据。

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