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雀舌黄杨中生物碱成分的研究*

2023-03-08闫玉鑫杨颖陈方圆赵燕沈瑶

关键词:黄杨生物碱硅胶

闫玉鑫, 杨颖, 陈方圆, 赵燕, 沈瑶

(云南师范大学 职业技术教育学院应用化学系,云南 昆明 650092)

雀舌黄杨(Buxusbodinieri),又名匙叶黄杨,为黄杨科黄杨属(Buxus)植物,常绿灌木,叶薄革质,常为匙形,叶面绿色,光亮,叶背苍灰色,中脉两面凸出,侧脉极多,在两面或仅叶面显著.生于海拔400~2 700 m平地或山坡林下,主产于中国云南、四川和贵州等地[1].国内外学者对黄杨属植物的化学成分及药理活性开展了多方面研究,该属植物富含三萜生物碱,在抗肿瘤、抗菌、抗 HIV及抗乙酰胆碱酯酶等方面有很好的生物活性[2].为了开发利用黄杨属植物资源,寻找有药用价值的结构新颖的化合物及生物活性成分,实验研究了雀舌黄杨地上部分的化学成分,从中分离得到10个生物碱,其中化合物1~9为三萜生物碱,化合物10为甾体生物碱,化合物1及3~10为首次从该植物中分离得到.

1 仪器与材料

FABMS在VG Auto Spec-3000质谱仪上测定;NMR在Bruker AM-600和Bruker DRX-500 MHz核磁共振仪上测定;LC-20A高效液相色谱仪(日本岛津公司);N-1100 旋转蒸发仪(日本东京理化株式会社);SHZ-DⅢ循环水真空泵(巩义市予华仪器有限责任公司);DLSB-5/10低温冷却液循环泵(巩义市予华仪器有限责任公司);AL-204电子天平(梅特勒-托利多仪器生产公司);薄层硅胶板F254和柱色谱硅胶(孔径为10~40 μm)为青岛海洋化工厂生产;薄层中性氧化铝板AGF254和碱性氧化铝(孔径为10~40 μm)为上海盛亚化工有限公司生产;凝胶为Sephadex LH-20;显色剂为改良碘化铋钾;其余试剂均为色谱纯或分析纯.

雀舌黄杨地上枝叶于2020年10月采自云南师范大学校内,由中国科学院昆明植物研究所赵玉娟博士鉴定为黄杨科黄杨属雀舌黄杨(Buxusbodinieri).植物标本存放于云南师范大学职业技术教育学院生药材实验室(标本号为YNU-ZJ20201002).

2 提取与分离

称取雀舌黄杨干燥的地上部分15 kg,粉碎后用甲醇加热回流提取3次,合并提取液,浓缩,将浓缩液分散在水中,加0.01 mol/L盐酸调节溶液至pH≈4,分别用4 L乙酸乙酯萃取3次,浓缩得到非生物碱部分(1.4 kg).酸水液用2%NaOH碱化至pH≈10,用4 L氯仿萃取3次得到总生物碱部分(260 g).总生物碱部分进行硅胶柱色谱分离,氯仿-甲醇 (体积比分别为100∶0、50∶1、20∶1和5∶1)洗脱并浓缩各流分,分别得到组分Fr.1~4.Fr.2 (70 g) 用中性氧化铝柱色谱分离,以石油醚-乙酸乙酯 (体积比分别为50∶1、40∶1、20∶1、10∶1)洗脱,对石油醚-乙酸乙酯 (体积比40∶1)分离出来的组分,用硅胶柱色谱以石油醚-乙酸乙酯 (体积比40∶1)洗脱,再经半制备高效液相色谱分离(色谱柱:Agilent XDB-C18 9.4 mm×250 mm;甲醇∶0.5%NH3·H2O体积比为60∶40~100∶0,洗脱时间70 min;波长为225 nm;流速为4.0 mL/min),得到化合物1 (14 mg,tR=26.5)、5 (12 mg,tR=28.9)、6 (21 mg,tR=41.3)和9 (9 mg,tR=31.1).Fr.3 (20 g) 用氧化铝柱色谱,经氯仿-甲醇(体积比25∶1~10∶1)系统洗脱,分为3个流分Fr.3a~3c,对Fr.3a部分(10 g) 用SephadexLH-20 (甲醇),硅胶柱色谱以氯仿-甲醇 (体积比25∶1)洗脱,得到化合物2 (85 mg).Fr.3b用中性氧化铝柱色谱以氯仿-甲醇 (体积比30∶1)洗脱,得到化合物3 (180 mg)和4 (35 mg).Fr.4 (30 g) 用硅胶柱色谱,以氯仿-甲醇(体积比10∶1~5∶1)系统洗脱,再用氧化铝柱层析,乙酸乙酯洗脱去大部分色素后,经反相制备HPLC(甲醇∶0.5%NH3·H2O,体积比60∶40~100∶0,洗脱时间40 min),得到化合物7 (20 mg,tR=14.9)、8 (9 mg,tR=16.7)和10 (12 mg,tR=22.1).

3 结构鉴定

化合物1:无色结晶,改良碘化铋钾反应呈阳性,C26H41NO,EI-MS:m/z383[M]+.1H NMR(ppm,CDCl3,500 MHz)δ:6.74(d,J=10.5,H-1),5.89(d,J=10.4,H-2),0.70,1.26(d,J=4.4,H-19),0.79(d,J=6.0,21-CH3),0.88(s,18-CH3),2.14 (s,Nb(CH3)2);13C NMR (ppm,CDCl3,125 MHz)δ:153.8(d,C-1),126.7(d,C-2),205.1(s,C-3),45.3(s,C-4),44.2(d,C-5),29.0(t,C-6),27.7(t,C-7),43.3(d,C-8),24.6(s,C-9),30.1(s,C-10),23.2(t,C-11),19.4(t,C-12),44.3(s,C-13),49.3(s,C-14),24.6(t,C-15),34.2(t,C-16),50.4(d,C-17),19.1(q,18-CH3),32.0(t,C-19),61.2(d,C-20),9.5(q,21-CH3),18.5(q,C-30),21.4(q,C-31),17.0(q,C-32),39.9(q,Nb(CH3)2).以上数据与文献[3]报道基本一致,故鉴定为cyclobuxoviridine.

化合物2:无色结晶,改良碘化铋钾反应呈阳性,C26H41NO2,EI-MS:m/z399[M]+.1H NMR(ppm,CDCl3,500 MHz)δ:6.81(d,J=10.6,H-1),5.98(d,J=10.4,H-2),2.18(dd,J=12.6,4.0,H-5),4.11(m,H-16),0.82,1.32(d,J=4.4,H-19),1.00(s,18-CH3),1.15(s,32-CH3),2.20 (s,Nb(CH3)2);13C NMR (ppm,CDCl3,125 MHz) δ:153.6(d,C-1),126.6(d,C-2),205.1(s,C-3),45.9(s,C-4),44.5(d,C-5),31.0(t,C-6),27.3(t,C-7),44.2(d,C-8),23.9(s,C-9),29.8(s,C-10),23.7(t,C-11),19.6(t,C-12),45.9(s,C-13),47.6(s,C-14),43.4(t,C-15),78.6 (d,C-16),56.7(d,C-17),19.0(q,18-CH3),30.4(t,C-19),62.0(d,C-20),9.6(q,21-CH3),20.2(q,C-30),21.3(q,C-31),17.9(q,C-32),44.5(q,Nb(CH3)2).以上数据与文献[4]报道基本一致,故鉴定为N-dimethylcycloxobuxovircine.

化合物3:无色晶体,改良碘化铋钾反应呈阳性,C27H48N2O,ESI-MS:m/z417[M+H]+.1H NMR (ppm,CDCl3,500 MHz)δ:2.60(m,H-3),4.01(t,J=7.5,H-16),0.28,0.52 (d,J= 4.2,H-19),0.85(d,J=6.0,H-21);13C NMR(ppm,CDCl3,125 MHz)δ:31.5(t,C-1),32.6(t,C-2),62.4(d,C-3),39.9(s,C-4),47.2(d,C-5),21.3(t,C-6),26.7(t,C-7),48.0(d,C-8),19.4(s,C-9),26.1(s,C-10),26.1(t,C-11),35.3(t,C-12),44.8(s,C-13),47.2(s,C-14),44.6(t,C-15),79.0(d,C-16),68.5(d,C-17),19.0(q,18-CH3),30.3(t,C-19),56.9(d,C-20),9.5(q,21-CH3),18.9 (q,C-30),25.7(q,C-31),15.0(q,C-32),35.6 (q,N-CH3).以上数据与文献[5]报道基本一致,故鉴定为cyclovirobuxine C.

化合物4:无色结晶,改良碘化铋钾反应呈阳性,C28H50N2O, ESI-MS:m/z431[M+H]+.1H NMR (ppm,CDCl3,500 MHz)δ:2.41(dd,J=3.0,12.5,H-3),0.28,0.54(d,J=4.0,H-19),0.86(s,18-CH3),1.02(s,32-CH3),2.17(s,N-CH3),0.98(d,J=6.4,21-CH3).13C NMR(ppm,CDCl3,125 MHz)δ:32.8(t,C-1),25.0(t,C-2),53.6 (d,C-3),40.3(s,C-4),45.7(d,C-5),21.6(t,C-6),27.0(t,C-7),46.7(d,C-8),19.5(s,C-9),24.9(s,C-10),28.5(t,C-11),33.2(t,C-12),43.9(s,C-13),47.2(s,C-14),29.5(t,C-15),78.4(d,C-16),58.2(d,C-17),11.1(q,18-CH3),30.2(t,C-19),56.3(d,C-20),18.8(q,21-CH3),25.2(q,C-30),9.6(q,C-31),20.9(q,C-32),41.2(q,N-CH3).以上数据与文献[6]报道基本一致,故鉴定为cyclovirobuxine A.

化合物5:白色针晶,改良碘化铋钾反应呈阳性,C27H48N2, ESI-MS:m/z401[M+H]+.1H NMR (ppm,CDCl3,500 MHz)δ:0.28,0.51(d,J=5.0,H-19),0.85(d,J=6.0,H-21),2.29 (s,Nb(CH3)2),2.52(s,Na-CH3).13C NMR(ppm,CDCl3,125 MHz)δ:31.2(t,C-1),32.6(t,C-2),68.9(d,C-3),39.9(s,C-4),48.0(d,C-5),21.3(t,C-6),26.3(t,C-7),48.4(d,C-8),19.4(s,C-9),26.0(s,C-10),26.0(t,C-11),35.3 (t,C-12),44.3(s,C-13),48.9(s,C-14),32.5(t,C-15),26.3(t,C-16),50.6(d,C-17),18.3(q,18-CH3),29.7(t,C-19),61.6(d,C-20),9.3(q,21-CH3),19.4(q,C-30),25.8(q,C-31),15.1(q,C-32),39.9(q,N-CH3).以上数据与文献[5]报道基本一致,故鉴定为cycloprotobuxine C.

化合物6:无色晶体,改良碘化铋钾反应呈阳性,C28H50N2,ESI-MS:m/z415[M+H]+.1H NMR(ppm,CDCl3,500 MHz)δ:2.50(brs,H-3),0.28,0.53(d,J=4.1,H-19).13C NMR(ppm,CDCl3,125 MHz)δ:31.4(t,C-1),32.6(t,C-2),77.0(d,C-3),39.9(s,C-4),47.2(d,C-5),21.3(t,C-6),26.7(t,C-7),48.0(d,C-8),19.4(s,C-9),26.1(s,C-10),27.1(t,C-11),35.3(t,C-12),44.8(s,C-13),47.2(s,C-14),32.6(t,C-15),19.0(t,C-16),50.6(d,C-17),19.0(q,18-CH3),30.3(t,C-19),56.2(d,C-20),9.3(q,21-CH3),19.0(q,C-30),25.7(q,C-31),15.0(q,C-32),35.3(q,N(CH3)2).以上数据与文献[7]报道基本一致,故鉴定为cycloprotobuxine A.

化合物7:无色结晶,改良碘化铋钾反应呈阳性,C27H46N2O2, ESI-MS:m/z431[M+H]+.1H NMR(ppm,CDCl3,500 MHz)δ:2.58 (dd,J=3.5,12.0,H-3),0.33,0.59(d,J=4.1,H-19),0.96(s,18-CH3),1.12(s,32-CH3),1.88(s,N-CH3),0.89(d,J=6.4,21-CH3),3.26,3.76(dd,J=11.0,29-CH2),4.26,4.61(dd,J=10.4,31-CH3).13C NMR (ppm,CDCl3,125 MHz)δ:33.0(t,C-1),25.9(t,C-2),63.5(d,C-3),38.2(s,C-4),45.1(d,C-5),19.1(t,C-6),25.3(t,C-7),47.3(d,C-8),20.9(s,C-9),25.8(s,C-10),27.4(t,C-11),30.6(t,C-12),44.6(s,C-13),47.2(s,C-14),44.9(t,C-15),80.4(d,C-16),57.0(d,C-17),11.3(q,18-CH3),31.6(t,C-19),57.0(d,C-20),18.8(q,21-CH3),76.9(q,C-30),9.8(q,C-31),19.7(q,C-32),42.2(q,N-CH3),80.1(t,C-33).以上数据与文献[8]报道基本一致,故鉴定为cyclobuxoxazine.

化合物8:无色结晶,改良碘化铋钾反应呈阳性,C28H48N2O2,ESI-MS:m/z445[M+H]+.1H NMR(ppm,CDCl3,500 MHz)δ:2.71(m,H-3),0.34,0.58(d,J=4.4,H-19),0.90(d,J=6.0,21-CH3),3.14,3.58(dd,J=11.0,30-CH2),3.72,4.42(dd,J=10.4,31-CH3),2.09(s,N-CH3).13C NMR(ppm,CDCl3,125 MHz)δ:31.6(t,C-1),23.8(t,C-2),71.8(d,C-3),38.7(s,C-4),44.7(d,C-5),20.1(t,C-6),25.3(t,C-7),47.3(d,C-8),18.9(s,C-9),25.6(s,C-10),5.8(t,C-11),31.6(t,C-12),44.9(s,C-13),47.3(s,C-14),44.6(t,C-15),78.3(d,C-16),62.7(d,C-17),18.8(q,18-CH3),30.6(t,C-19),56.9(d,C-20),9.8(q,21-CH3), 78.3(t,C-30),13.7(q,C-31),20.8(q,C-32),88.7(t,C-33),36.5(q,N-CH3).以上数据与文献[5]报道基本一致,故鉴定为cyclobuxoxazine A.

化合物9:无色结晶,改良碘化铋钾反应呈阳性,C25H40NO,ESI-MSm/z:370[M+H]+.δ:2.85 (m,H-3),-0.04,0.50(d,J=4.1,H-19),0.85(s,18-CH3),1.08(s,21-CH3),4.61,4.85(s,29-CH2),0.90(s,30-CH3),2.50(s,N-CH3).13C NMR(ppm,CDCl3,125 MHz)δ:31.0(t,C-1),25.7(t,C-2),63.6(d,C-3),153.3(s,C-4),41(d,C-5),21.2(t,C-6),26.2(t,C-7),42.4(d,C-8),23.3(s,C-9),32.0(s,C-10),21.4(t,C-11),33.3(t,C-12),42.7(s,C-13),46.0(s,C-14),22.4(t,C-15),34.2(d,C-16),46.8 (d,C-17),18.3(q,18-CH3),22.0(t,C-19),212.9(d,C-20),17.8(q,21-CH3),101.5(t,C-28),11.5(q,C-30),34.5(q,N-CH3).以上数据与文献[9]报道基本一致,故鉴定为buxruguline B.

图1 雀舌黄杨中得到的化合物1~10的结构

化合物10:无色结晶,改良碘化铋钾反应呈阳性,C23H39NO,EI-MS:m/z345[M+H]+.1H NMR(ppm,CDCl3,500 MHz)δ:5.36(1H,d,J=4.2,H-6),3.50(m,H-3),0.88(s,J=6.4,21-CH3),0.68(s,18-CH3),0.97(s,19-CH3),2.14(s,N-CH3);13C NMR (ppm,CDCl3,125 MHz)δ:31.8(t,C-1),39.5(t,C-2),71.7(d,C-3),42.2(t,C-4),140.7(s,C-5),121.6(d,C-6),37.2(t,C-7),50.1(d,C-8),31.8(d,C-9),36.5(s,C-10),21.0(t,C-11),31.6(t,C-12),41.4(s,C-13),54.7(d,C-14),24.1(t,C-15),27.6(t,C-16),56.8(d,C-17),12.1(q,18-CH3),19.4(q,C-19),61.1(d,C-20),9.9(q,21-CH3),39.8(q,N-CH3).以上数据与文献[10]报道基本一致,故鉴定为irehine.

4 讨论

研究发现黄杨中含有生物碱类、黄酮类、甾醇、香豆素、木脂素和酸类化合物等多种化学成分,其中生物碱成分类型最为丰富.研究从雀舌黄杨的甲醇提取物中分离得到10个黄杨生物碱类化合物.其中化合物1~9为三萜生物碱,化合物10为甾体生物碱,化合物1和3~10为首次从该植物中分离得到.已有的研究表明化合物5、6和7对HL-60、SMMC-7721、A-549、MCF-7和SW480肿瘤细胞株有较好的抗肿瘤活性[5].研究为雀舌黄杨的资源开发和利用提供参考,同时为其主要药效活性物质的深入研究提供数据支持.

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