海滨大戟化学成分研究

2021-11-11 10:33段瑞军袁靖喆戴好富梅文莉许凤清黄圣卓
安徽中医药大学学报 2021年6期

赵 焕,段瑞军,于 淼,曾 军,袁靖喆,戴好富,梅文莉,孙 磊,许凤清,黄圣卓

(1.安徽中医药大学药学院,安徽 合肥 230012;2.中国热带农业科学院热带生物技术研究所海南省黎药资源天然产物研究与利用重点实验室,海南 海口 571101;3.海南热带农业资源研究院,海南 海口 571101)

海滨大戟

Euphorbia

atoto

Forst.为大戟科(Euphorbiaceae)大戟属

Euphorbia

Linn.多年生草本植物,别名林氏大戟、滨大戟等,主产于广东(南部沿海)、海南和台湾等地。农业农村部“南锋专项”考察团队调查发现,海滨大戟为西沙群岛特色植物,并且是永乐群岛和宣德群岛的主要海岸带优势植物。大戟属植物化学成分结构类型多样,富含三萜、二萜等成分,且该属中有三萜及甾醇类化合物具有多种生物活性。民间有人将海滨大戟作为抗炎、抗菌药物使用,可治疗痢疾、泄泻、白喉等症。本研究以海滨大戟为研究对象,综合利用各种色谱技术对海滨大戟乙醇提取物中乙酸乙酯萃取部位化学成分的分离和鉴定进行了相关研究工作,现报道如下。

1 仪器与材料

1.1 仪器 Bruker AV-500型超导核磁仪:德国Bruker公司;质谱仪:Bruker amazon SL公司;CA-1111 冷却水循环装置:上海爱朗仪器有限公司;BSA-100A 自动部分收集器:上海青浦沪西仪器厂;旋转蒸发仪:德国HeidolphLaborota;METTLER TOLEDO ME204精密和分析天平(万分之一):梅特勒-托力多仪器(上海)公司;色谱硅胶板G,柱色谱硅胶(200~300目):青岛海洋化工厂;Rp-18(20~45 μm):日本富士硅化学株式会社;Sephadex LH-20:GE Healthcare公司。

1.2 材料 海滨大戟样品(10.0 kg),采集于海南省三沙市宣德群岛赵述岛,经鉴定为大戟科大戟属多年生草本海滨大戟

Euphorbia

atoto

Forst.。凭证标本(编号 HUANG20200055)存放于中国热带农业科学院热带生物技术研究所。

2 提取与分离

取新鲜海滨大戟地上部分10 kg,晒干、粉碎,用95%乙醇回流,提取3次,每次3 h,合并提取液,减压浓缩回收溶剂,得乙醇浸膏。浸膏加适量水混悬,用乙酸乙酯萃取3次,得乙酸乙酯萃取物347 g。取乙酸乙酯部分347 g,经硅胶柱色谱,石油醚-丙酮(30∶1→0∶1)梯度洗脱,得12个流分Fr.1—Fr.12。Fr.3(37.0 g)经Rp-18反相柱色谱梯度洗脱得4个流分Fr.3.1—Fr.3.4。Fr.3.1(2.3 g)流经Sephadex LH-20(甲醇)洗脱得10个流分Fr.3.1.1—3.1.10。过凝胶过程中有结晶析出,洗出结晶,经硅胶柱色谱石油醚-乙酸乙酯(9∶1→ 4∶1)洗脱纯化后得化合物12(6.8 mg)和13(6.4 mg)。Fr.3.1.3(51.3 mg)经硅胶柱色谱石油醚-丙酮(15∶1)洗脱得化合物2(3.4 mg)。Fr.3.1.5(70.0 mg)经硅胶柱色谱石油醚-丙酮(9∶1)洗脱得化合物8(1.8 mg)。Fr.3.3(383.4 mg)经反复硅胶柱色谱石油醚-丙酮(40∶1)洗脱,再经Sephadex LH-20(甲醇)分离纯化得化合物7(2.6 mg)和9(3.5 mg)。Fr.4(42.0 g)经Rp-18反相柱色谱梯度洗脱得7个流分Fr.4.1—4.7。Fr.4.2(6.7 g)经反复硅胶柱色谱,石油醚-丙酮(100∶1)、石油醚-丙酮(30∶1)、石油醚-丙酮(15∶1)洗脱,再经Sephadex LH-20(甲醇)分离纯化得化合物1(2.1 mg)、3(1.3 mg)和11(1.0 mg)。

Fr.5(29.0 g)经Rp-18反相柱色谱梯度洗脱得3个流分Fr.5.1—5.3。Fr.5.1(6.7 g)经反复硅胶柱色谱,石油醚-丙酮(30∶1)、石油醚-丙酮(50∶1)、石油醚-丙酮(20∶1)洗脱,再经Sephadex LH-20(甲醇)分离纯化得化合物4(2.9 mg)和5(3.7 mg)。

Fr.6(5.8 g)经Rp-18反相柱色谱梯度洗脱得5个流分Fr.6.1—6.5。Fr.6.2(1.3 g)经硅胶柱色谱,石油醚-丙酮(10∶1)洗脱,再经Sephadex LH-20(甲醇),重结晶得到化合物6(2.9 mg)。

Fr.8(3.1 g)经Rp-18反相柱色谱梯度洗脱得6个流分Fr.8.1—8.6。Fr.8.3(40.3 mg)经硅胶柱色谱,石油醚-丙酮(10∶1)洗脱得化合物10(2.8 mg)和14(1.6 mg)。

3 结构鉴定

化合物1:白色固体。ESI-MS

m/z

:533[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:5.31-5.45(6H,m),3.69(1H,s),1.20-3.00(46H,m),1.00(3H,d,

J

=7.5 Hz),0.90(3H,d,

J

=6.2 Hz);C-NMR(125 MHz,CDCl)

δ

:180.2(s,C-1),132.1(d,C-9),128.4(d,C-10),51.6(d,C-11),127.9(d,C-13),127.3(d,C-15),14.3(q,C-18),173.9(s,C-1′),20.7-34.3(23C,t,C-28,14,17,2′-15′),130.4(d,C-11′),128.4(d,C-15′),14.4(q,C-18′)。以上数据与文献[6]报道一致,故鉴定化合物1为mayolene-16。检索相关文献发现,目前都是通过一定的合成方法得到该化合物,本次为首次从海滨大戟植物中分离得到,故化合物1为新的天然产物。化合物2:无色针晶。ESI-MS

m/z

:279[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:6.29(1H,d,

J

=2.6 Hz,H-3),6.24(1H,d,

J

=2.6 Hz,H-5),5.16(1H,m,H-8′),3.27(1H,t,

J

=12.0 Hz,H-1′a),2.49(1H,dt,

J

=5.8,12.0 Hz,H-1′b),1.38(3H,d,

J

=6.3 Hz,H-9′);C-NMR(125 MHz,CDCl)

δ

:105.7(s,C-1),165.6(s,C-2),101.5(d,C-3),160.2(s,C-4),110.8(d,C-5),149.6(s,C-6),172.0(s,C-7),33.7(t,C-1′),30.8(t,C-2′),27.4(t,C-3′),21.3(t,C-4′),24.3(t,C-5′),24.8(t,C-6′),31.2(t,C-7′),75.2(d,C-8′),20.3(q,C-9′)。以上数据与文献[7]报道一致,故鉴定化合物2为(+)-(

R

)-脱-

O

-甲基拉西奥地丁。化合物3:白色针晶。ESI-MS

m/z

:427[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:1.19(3H,s,H-2),3.21(1H,dd,

J

=9.1,3.6 Hz,H-3),5.56(1H,t,

J

=3.5 Hz,H-12),1.15(3H,s),1.12(3H,s),0.99(3H,s),0.88(3H,d,

J

=6.3 Hz),0.91(3H,s),0.83(3H,s),0.82(3H,d,

J

=6.8 Hz);C-NMR(125 MHz,CDCl)

δ

:38.9(t,C-1),27.2(t,C-2),78.7(d,C-3),38.9(s,C-4),54.8(d,C-5),18.4(t,C-6),33.2(t,C-7),39.1(d,C-8),47.5(d,C-9),38.9(s,C-10),22.6(t,C-11),130.3(d,C-12),140.3(s,C-13),43.5(s,C-14),29.0(t,C-15),26.3(t,C-16),33.8(s,C-17),58.9(d,C-18),39.0(d,C-19),38.9(d,C-20),31.8(t,C-21),40.9(t,C-22),28.9(q,C-23),15.5(q,C-24),14.0(q,C-25),16.4(q,C-26),23.3(q,C-27),28.0(q,C-28),17.4(q,C-29),23.5(q,C-30)。以上数据与文献[8-9]报道一致,故鉴定化合物3为熊果烷。化合物4:白色针晶。EI-MS

m/z

:427[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:3.23(1H,m,H-3),5.72(1H,m,H-12),1.16(3H,s,H-23),0.78(3H,s,H-24),0.87(3H,s,H-25),0.86(3H,s,H-26),1.16(3H,s,H-27),0.99(3H,s,H-28),1.27(6H,s,H-29,30);C-NMR(125 MHz,CDCl)

δ

:39.2(t,C-1),27.0(t,C-2),78.0(d,C-3),38.0(s,C-4),55.1(d,C-5),18.3(t,C-6),32.6(t,C-7),40.0(s,C-8),47.4(d,C-9),37.3(s,C-10),25.1(t,C-11),125.5(d,C-12),142.1(s,C-13),41.9(s,C-14),25.8(t,C-15),26.6(t,C-16),32.3(s,C-17),45.3(d,C-18),44.2(d,C-19),31.7(d,C-20),35.7(t,C-21),38.0(t,C-22),25.7(q,C-23),15.5(q,C-24),15.6(q,C-25),16.6(q,C-26),26.1(q,C-27),27.7(q,C-28),33.2(q,C-29),25.0(q,C-30)。以上数据与文献[10]报道基本一致,故鉴定化合物4为

α

-香树脂醇。化合物5:白色固体。EI-MS

m/z

:427[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:3.25(1H,m,H-3),5.25(1H,d,

J

=7.8 Hz,H-21),0.96(3H,s,H-23),0.86(3H,s,H-24),0.83(3H,s,H-25),1.10(3H,s,H-26),0.90(3H,s,H-27),1.72(3H,s,H-28),0.80(3H,d,

J

=6.0 Hz,H-29),0.80(3H,s,H-30);C-NMR(125 MHz,CDCl)

δ

:39.1(t,C-1),27.4(t,C-2),79.1(d,C-3),38.9(s,C-4),55.2(d,C-5),18.2(t,C-6),34.1(t,C-7),40.8(s,C-8),50.7(d,C-9),37.3(s,C-10),21.7(t,C-11),27.7(t,C-12),39.1(d,C-13),42.6(s,C-14),27.4(t,C-15),36.8(t,C-16),34.1(s,C-17),49.1(d,C-18),36.2(d,C-19),140.0(s,C-20),117.9(d,C-21),42.5(t,C-22),28.0(q,C-23),15.5(q,C-24),16.6(q,C-25),16.1(q,C-26),14.8(q,C-27),17.7(q,C-28),22.8(q,C-29),21.6(q,C-30)。以上数据与文献[11]报道基本一致,故鉴定化合物5为伪蒲公英甾醇。化合物6:白色针晶。ESI-MS

m/z

:443[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:3.08(1H,dd,

J

=11.4,4.0 Hz,H-3),5.15(1H,m,H-7),0.77(3H,d,

J

=6.0 Hz,H-21),3.90(1H,t,

J

=6.2 Hz,H-24),4.83,4.73(1H each,s,H-26),0.86(9H,s,H-18,H-19,H-28),0.76,0.71,0.64(3H each,s);C-NMR(125 MHz,CDCl)

δ

:37.3(t,C-1),27.8(t,C-2),79.4(d,C-3),39.1(s,C-4),50.8(d,C-5),24.1(t,C-6),118.0(d,C-7),146.0(s,C-8),49.1(d,C-9),35.1(s,C-10),18.3(t,C-11),33.9(t,C-12),43.7(s,C-13),51.3(s,C-14),34.1(t,C-15),28.3(t,C-16),53.00(d,C-17),22.1(q,C-18),13.3(q,C-19),36.2(d,C-20),18.6(t,C-21),31.0(t,C-22),31.9(t,C-23),75.5(d,C-24),147.9(s,C-25),111.1(t,C-26),17.8(q,C-27),27.8(q,C-28),14.9(q,C-29),27.4(q,C-30)。以上数据与文献[12]报道一致,故鉴定化合物6为(3

β

,24

R

)-大戟烷-7,25-二烯-3,24-二醇。化合物7:白色针晶。ESI-MS

m/z

:427[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:2.03(1H,m,H-1),2.40(2H,m,H-2),1.87(1H,m,H-9),1.84(1H,t,

J

=11.5 Hz,H-13),2.58(1H,td,

J

=11.2,6.0 Hz,H-17),2.15(3H,s,H-29),2.18,1.09,1.07,1.04,1.00,0.94,0.80(3H each,s);C-NMR(125 MHz,CDCl)

δ

:39.9(t,C-1),34.2(t,C-2),218.3(s,C-3),47.4(s,C-4),54.9(d,C-5),19.8(t,C-6),33.6(t,C-7),40.8(s,C-8),49.7(d,C-9),37.0(s,C-10),21.5(t,C-11),27.3(t,C-12),49.6(d,C-13),42.9(s,C-14),35.0(t,C-15),27.8(t,C-16),52.7(d,C-17),43.2(s,C-18),39.7(t,C-19),27.4(t,C-20),37.3(d,C-21),213.0(s,C-22),26.9(q,C-23),21.2(q,C-24),15.8(q,C-25),16.1(q,C-26),18.1(q,C-27),14.5(q,C-28),29.4(q,C-30)。以上数据与文献[13]报道一致,故鉴定化合物7为29-nor-21-α-H-hopane-3,22-dione。化合物8:白色针晶。ESI-MS

m/z

:359[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:0.91,0.97,1.05,1.06,1.11(3H each,s),2.14(3H,s,H-21);C-NMR(125 MHz,CDCl)

δ

:40.2(t,C-1),34.3(t,C-2),218.8(s,C-3),48.4(s,C-4),55.6(d,C-5),19.8(t,C-6),35.0(t,C-7),40.6(d,C-8),50.2(d,C-9),37.1(s,C-10),21.9(t,C-11),25.8(t,C-12),45.3(d,C-13),50.2(s,C-14),31.7(t,C-15),26.2(t,C-16),54.3(d,C-17),15.9(q,C-18),15.5(q,C-19),211.7(s,C-20),30.2(t,C-21),26.9(q,C-28),21.2(q,C-29),16.2(q,C-30)。以上数据与文献[14]报道一致,故鉴定化合物8为hollongdione。化合物9:白色针晶。ESI-MS

m/z

:429[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:0.71(3H,s,H-18),0.96(3H,s,H-19),0.94(3H,d,

J

=6.4 Hz,H-21),0.84(3H,d,

J

=4.9 Hz,H-26),0.82(3H,d,

J

=1.4 Hz,H-27),0.98(3H,H-29);C-NMR(125 MHz,CDCl)

δ

:37.6(t,C-1),37.2(t,C-2),209.4(s,C-3),38.3(t,C-4),57.7(d,C-5),211.5(s,C-6),46.8(t,C-7),38.2(d,C-8),53.7(d,C-9),41.5(s,C-10),21.9(t,C-11),39.6(t,C-12),43.2(s,C-13),56.8(d,C-14),24.2(t,C-15),28.2(t,C-16),56.2(d,C-17),12.2(q,C-18),12.8(q,C-19),36.2(d,C-20),18.9(q,C-21),34.0(t,C-22),26.2(t,C-23),45.9(d,C-24),29.3(d,C-25),19.2(q,C-26),20.0(q,C-27),23.2(t,C-28),12.2(q,C-29)。以上数据与文献[15]报道一致,故鉴定化合物9为5

α

-豆甾醇-3,6-二酮。化合物10:白色针晶。ESI-MS

m/z

:431[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:3.08(1H,dd,

J

=11.4,4.0 Hz,H-4),0.77(3H,d,

J

=6.0 Hz,H-21),5.15(1H,m,H-23),3.90(1H,t,

J

=6.2 Hz,H-24),4.83,4.73(1H each,s,H-26),0.86(9H,s),076,0.71,0.64(3H each,s);C-NMR(125 MHz,CDCl,)

δ

:33.1(t,C-1),31.7(C-2),67.9(d,C-3),39.3(t,C-4),76.1(s,C-5),73.8(d,C-6),117.7(d,C-7),144.2(s,C-8),43.6(d,C-9),37.3(s,C-10),22.2(t,C-11),39.6(t,C-12),43.9(s,C-13),54.9(d,C-14),22.8(t,C-15),28.1(t,C-16),56.1(d,C-17),14.3(q,C-18),17.7(q,C-19),40.5(d,C-20),21.2(q,C-21),135.5(d,C-22),132.3(d,C-23),43.0(d,C-24),33.2(d,C-25),19.8(q,C-26),20.0(q,C-27),19.0(q,C-28)。以上数据与文献[16]报道一致,故鉴定化合物10为22

E

,24

R

-麦角甾烷-7,22-二烯-3

β

,5

α

,6

β

-三醇。化合物11:白色针状晶体。ESI-MS

m/z

:401[M+H],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:3.51(1H,m,H-3),5.35(1H,t,

J

=6.5 Hz,H-6),1.00(3H,s,H-18),0.91(3H,d,

J

=7.0 Hz,H-19),1.47(3H,s,H-21),0.85(3H,d,

J

=6.5 Hz,H-26),0.87(3H,d,

J

=6.8 Hz,H-27),0.68(3H,s,H-28);C-NMR(125 MHz,CDCl)

δ

:36.1(t,C-1),28.4(t,C-2),71.9(d,C-3),42.4(t,C-4),141.4(d,C-5),121.84(d,C-6),29.2(t,C-7),24.4(d,C-8),50.2(d,C-9),36.6(s,C-10),22.4(t,C-11),37.3(t,C-12),45.9(s,C-13),56.9(d,C-14),23.8(t,C-15),23.8(t,C-16),56.4(d,C-17),12.0(q,C-18),19.5(q,C-19),34.0(d,C-20),18.9(t,C-21),29.8(t,C-22),31.8(d,C-23),39.9(d,C-24),32.0(q,C-25),21.2(q,C-26),21.2(q,C-27),19.1(q,C-28)。以上数据与文献[17]报道基本一致,故鉴定化合物11为菜油甾醇。化合物12:无色针晶。ESI-MS

m/z

:435[M+Na],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:3.51(1H,m,H-3),5.37(1H,dd,

J

=2.7,2.1 Hz,H-6),5.15(1H,dd,

J

=15.0,8.7 Hz,H-22),5.01(1H,dd,

J

=15.0,8.8 Hz,H-23);C-NMR(125 MHz,CDCl)

δ

:37.4(t,C-1),31.8(t,C-2),71.9(d,C-3),42.4(t,C-4),140.9(s,C-5),121.8(d,C-6),32.0(t,C-7),32.1(d,C-8),50.3(d,C-9),37.4(s,C-10),21.4(t,C-11),39.8(t,C-12),42.4(s,C-13),57.0(d,C-14),25.6(t,C-15),28.4(t,C-16),56.1(d,C-17),12.4(q,C-18),19.1(q,C-19),40.6(d,C-20),21.2(q,C-21),138.5(d,C-22),129.4(d,C-23),51.4(d,C-24),29.1(d,C-25),19.9(t,C-26),19.9(q,C-27),24.5(q,C-28),12.2(q,C-29)。以上数据与文献[18]报道一致,故鉴定化合物12为豆甾醇。化合物13:无色针晶。ESI-MS

m/z

:437[M+Na],分子式为CHO。H-NMR(500 MHz,CDCl)

δ

:3.53(1H,m,H-3),5.37(1H,dd,

J

=2.7,2.1 Hz,H-6);C-NMR(125 MHz,CDCl)

δ

:37.4(t,C-1),31.8(t,C-2),71.9(d,C-3),42.4(t,C-4),140.9(s,C-5),121.9(d,C-6),32.0(t,C-7),32.1(d,C-8),50.3(d,C-9),37.4(s,C-10),21.2(t,C-11),39.9(t,C-12),42.5(s,C-13),56.9(d,C-14),24.5(t,C-15),28.4(t,C-16),56.2(d,C-17),12.0(q,C-18),19.2(q,C-19),36.3(d,C-20),18.9(q,C-21),34.1(t,C-22),26.2(t,C-23),45.9(d,C-24),29.3(d,C-25),19.6(t,C-26),19.6(q,C-27),23.2(q,C-28),12.1(q,C-29)。以上数据与文献[19]报道一致,故鉴定化合物13为

β

-谷甾醇。化合物14:黄色粉末。ESI-MS

m/z

:269[M-H],分子式为CHO。H-NMR(500 MHz,acetone-d6)

δ

:6.66(1H,s,H-3),6.26(1H,d,

J

=2.1 Hz,H-6),6.55(1H,d,

J

=2.1 Hz,H-8),7.03(2H,d,

J

=8.8 Hz,H-3′,-5′),7.95(2H,

J

=8.8 Hz,H-2′,-6′);C-NMR(125 MHz,acetone-d6)

δ

:162.4(s,C-2),103.2(d,C-3),182.2(s,C-4),161.5(s,C-5),99.8(d,C-6),164.0(s,C-7),93.8(d,C-8),157.9(s,C-9),104.1(s,C-10),122.4(s,C-1′),128.4(d,C-2′,-6′),115.9(d,C-3′,-5′),161.5(s,C-4′)。以上数据与文献[20]报道基本一致,故鉴定化合物14为芹菜素。

4 讨论

海滨大戟作为南海岛礁和海岸带优势植物,在民间常被用以入药,目前尚未检索到其化学成分相关研究报道。本研究首次对海滨大戟地上部分的化学成分进行了研究,从海滨大戟的地上部分分离并鉴定出14个单体化合物,为海滨大戟的药效物质基础提供参考依据。

据文献[21]报道,化合物(+)-(

R

)-脱-

O

-甲基拉西奥地丁(2)具有抗菌活性,且对K-562细胞具有体外细胞毒活性。化合物22

E

,24

R

-麦角甾烷-7,22-二烯-3

β

,5

α

,6

β

-三醇(10)在诱导MC3T3-E1细胞分化活性中起重要作用,且能抑制诱导MC3T3-E1细胞凋亡。化合物

α

-香树脂醇(4)具有一定的皮肤刺激性。化合物芹菜素(14)具有抗肿瘤、抗炎、降血压、抗动脉硬化和血栓、抗焦虑、抗菌、抗病毒以及抗氧化作用。海滨大戟中的单体化合物生物活性丰富,为海滨大戟药用提供了一定的科学依据,也为海滨大戟的药理研究、产品开发等奠定了基础。

(致谢:化合物的波谱数据由中国热带农业科学院热带生物技术研究所海南省黎药资源天然产物研究与利用重点实验室测试中心测定)