冬凌草的化學(xué)成分研究

李珊珊，張琦，李鑫，許婧，郭遠(yuǎn)強(qiáng)

(1.哈爾濱商業(yè)大學(xué)藥學(xué)院，黑龍江 哈爾濱 150076； 2.南開大學(xué)藥學(xué)院，天津 300350)

冬凌草 [Rabdosiarubescens(Hemsl.) Hara]為唇形科香茶菜屬植物，學(xué)名碎米椏，主要分布于河南及黃河流域以南[1]。冬凌草味甘苦、性微寒，具有清熱解毒、消炎止痛及抗腫瘤等作用[2]。臨床上主要用于治療咽喉腫痛、扁桃體炎、蛇蟲咬傷及食道癌等疾病[3]。近年來，對(duì)冬凌草的化學(xué)成分研究表明，其主要成分為二萜類化合物，另外還含有一些黃酮、三萜、生物堿、多糖和揮發(fā)性成分[4-6]。為了充分開發(fā)冬凌草的藥用植物資源，本實(shí)驗(yàn)對(duì)冬凌草的化學(xué)成分及其抗腫瘤活性進(jìn)行了研究，從中分離鑒定了6個(gè)化合物，分別為：rabdosianin A(1)、parvifoline G(2)、rabdoternin C(3)、betulin(4)、betulinic acid(5)、古柯二醇(6)，結(jié)構(gòu)見圖1，其中1～3為二萜類化合物，4～6為三萜類化合物。經(jīng)文獻(xiàn)檢索，化合物6為首次從香茶菜屬中分離得到，化合物1、2、4、5為首次從冬凌草中分離得到。

1 儀器與材料

1.1 儀器 半制備高效液相色譜儀 (北京創(chuàng)新通恒科技有限公司，LC 3000)，色譜柱為YMC-Pack ODS-AM(250 mm × 20 mm)；德國Bruker AV-400型核磁共振波譜儀。

1.2 材料 柱層析硅膠(100～200目，煙臺(tái)江友硅膠開發(fā)有限公司)；ODS 中壓柱色譜填料(50 μm，日本 YMC公司)；顯色試劑為10%的硫酸乙醇溶液；有機(jī)溶劑購于天津化學(xué)試劑公司，均為分析純，水為蒸餾水。冬凌草藥材于2017年購于河北安國藥材市場，由南開大學(xué)藥學(xué)院郭遠(yuǎn)強(qiáng)教授鑒定。藥材標(biāo)本保存于南開大學(xué)藥學(xué)院天然藥物研究室。

圖1 化合物1～6的結(jié)構(gòu)

2 提取和分離

干燥的冬凌草(12.0 kg)，用甲醇加熱回流提取3次(首次2 h，后兩次各1.5 h)，合并甲醇提取液，過濾并減壓濃縮得浸膏1.0 kg。浸膏混懸于水中，經(jīng)乙酸乙酯萃取，直至萃取到乙酸乙酯層顏色清淺，合并濃縮得乙酸乙酯萃取物205.5 g。乙酸乙酯萃取物經(jīng)硅膠柱色譜分離，石油醚-丙酮梯度洗脫(100∶0～100∶25)，薄層色譜(TLC)檢測并合并得到6個(gè)粗分組分Fr.1～Fr.6。組分Fr.5經(jīng)中壓液相色譜(MPLC)(60%～90% 甲醇/水)梯度洗脫，得到8個(gè)亞組分Fr.5-1～Fr.5-8，亞組分Fr.5-4經(jīng)半制備高效液相色譜 (HPLC)(70% 甲醇/水)分離純化，得到化合物1(7.1 mg)，亞組分Fr.5-6經(jīng)半制備高效液相色譜 (HPLC)(83% 甲醇/水)分離純化，得到化合物3(55.9 mg)。組分Fr.4經(jīng)同上分離方法得到化合物2(6.9 mg)。組分Fr.2得到化合物4(5.6 mg)、化合物5(4.4 mg)、化合物6(4.2 mg)。

3 結(jié)構(gòu)鑒定

3.1 化合物1 白色固體。1H-NMR (CDCl3,400 MHz)δ:4.87 (1H,m,H-1),1.76 (1H,m,H-2),1.33 (1H,m,H-2),1.37 (1H,m,H-3),1.48 (1H,m,H-3),1.68 (1H,m,H-5),3.95 (1H,d,J=8.1 Hz,H-6),2.14 (1H,d,J=3.4 Hz,H-9),4.82 (1H,m,H-11),1.63 (1H,m,H-12),2.32 (1H,dd,J=9.3,16.4 Hz,H-12),2.67 (1H,m,H-13),1.62 (1H,m,H-14),2.48 (1H,d,J=12.3 Hz,H-14),5.62 (1H,t,J=2.5 Hz,H-15),5.04 (1H,d,J=2.3 Hz,H-17),4.86 (1H,brs,H-17),1.08 (3H,s,H-18),1.13 (3H,s,H-19),4.12 (1H,d,J=9.0 Hz,H-20),4.50 (1H,dd,J=1.8,9.1 Hz,H-20),2.09 (3H,s,H-2′),2.11 (3H,s,H-2″),1.94 (3H,s,H-2?)。13C-NMR (CDCl3,100 MHz)δ:76.7 (C-1),25.2 (C-2),38.7 (C-3),33.2 (C-4),53.1 (C-5),74.0 (C-6),96.3 (C-7),50.4 (C-8),48.2 (C-9),40.5 (C-10),69.5 (C-11),39.8 (C-12),34.9 (C-13),26.6 (C-14),74.6 (C-15),156.6 (C-16),109.5 (C-17),33.7 (C-18),22.6 (C-19),65.5 (C-20),171.4 (C-1′),22.2 (C-2′),170.6 (C-1″),21.9 (C-2″),169.9 (C-1?),21.8 (C-2?)。以上數(shù)據(jù)與文獻(xiàn)[7]報(bào)道的化合物rabdosianin A的數(shù)據(jù)基本一致，因此鑒定化合物1為rabdosianin A。

3.2 化合物2 無色晶體(甲醇)。1H-NMR (CDCl3,400 MHz)δ:4.94 (1H,m,H-1),1.76 (1H,m,H-2),1.80 (1H,m,H-2),1.43 (1H,m,H-3),1.52 (1H,m,H-3),2.43 (1H,s,H-5),2.08 (1H,m,H-9),4.91 (1H,m,H-11),1.71 (1H,m,H-12),2.35 (1H,dd,J=8.8,15.6 Hz,H-12),2.78 (1H,dd,J=4.7,8.9 Hz,H-13),1.49 (1H,m,H-14),2.56 (1H,d,J=12.3 Hz,H-14),5.41 (1H,t,J=2.9 Hz,H-15),5.08 (1H,dd,J=1.3,1.6 Hz,H-17,4.83 (1H,brs,H-17),1.40 (3H,s,H-18),1.08 (3H,s,H-19),4.82 (1H,d,J=8.7 Hz,H-20),4.24 (1H,d,J=9.2 Hz,H-20),2.13 (3H,s,H-2′),1.96 (3H,s,H-2″),2.04 (3H,s,H-2?)。13C-NMR (CDCl3,100 MHz)δ:75.8 (C-1),24.9 (C-2),38.8 (C-3),34.4 (C-4),61.8 (C-5),206.2 (C-6),92.2 (C-7),48.4 (C-8),47.1 (C-9),42.8 (C-10),69.4 (C-11),41.0 (C-12),35.0 (C-13),24.9 (C-14),76.5 (C-15),153.9 (C-16),110.1 (C-17),34.4 (C-18),21.8 (C-19),65.4 (C-20),170.5 (C-1′),22.2 (C-2′),169.7 (C-1″),21.7 (C-2″),169.4 (C-1?),20.7 (C-2?)。以上數(shù)據(jù)與文獻(xiàn)[8]報(bào)道的化合物parvifoline G的數(shù)據(jù)基本一致，因此鑒定化合物2為parvifoline G。

3.3 化合物3 白色粉末。1H-NMR (CDCl3,400 MHz)δ:1.42 (1H,m,H-1),1.08 (1H,m,H-1),1.26 (1H,m,H-2),1.50 (1H,m,H-2),1.46 (1H,m,H-3),1.12 (1H,m,H-3),1.59 (1H,d,J=8.1 Hz,H-5),5.28 (1H,d,J=7.6 Hz,H-6),2.30 (1H,m,H-9),1.46 (1H,m,H-11),1.29 (1H,m,H-11),2.38 (1H,m,H-12),1.54 (1H,m,H-12),2.58 (1H,d,J=9.3 Hz,H-13),4.61 (1H,s,H-14),6.04 (1H,s,H-15),5.09 (1H,s,H-17),5.16 (1H,s,H-17),0.83 (3H,s,H-18),1.13 (3H,s,H-19),4.15 (1H,d,J=9.6 Hz,H-20),3.84 (1H,d,J=9.9 Hz,H-20),2.20 (3H,s,H-2′),2.04 (3H,s,H-2″)。13C-NMR (CDCl3,100 MHz)δ:31.5 (C-1),14.8 (C-2),41.2 (C-3),33.4 (C-4),53.3 (C-5),74.1 (C-6),97.7 (C-7),51.9 (C-8),46.5 (C-9),35.8 (C-10),18.5 (C-11),31.2 (C-12),44.4 (C-13),75.2 (C-14),73.2 (C-15),156.9 (C-16),111.7 (C-17),33.0 (C-18),22.5 (C-19),66.9 (C-20),172.3 (C-1′),22.0 (C-2′),170.8 (C-1″),21.2 (C-2″)。以上數(shù)據(jù)與文獻(xiàn)[3]報(bào)道的化合物rabdoternin C的數(shù)據(jù)基本一致，因此鑒定化合物3為rabdoternin C。

3.4 化合物4 白色固體。1H-NMR (CDCl3,400 MHz)δ:3.19 (1H,dd,J=5.2,11.2 Hz,H-3),0.69 (1H,d,J=9.4 Hz,H-5),1.26 (1H,m,H-9),1.60 (1H,m,H-13),2.38 (1H,m,H-18),1.59 (1H,m,H-19),0.97 (3H,s,H-23),0.76 (3H,s,H-24),0.82 (3H,s,H-25),1.02 (3H,s,H-26),0.98 (3H,s,H-27),3.33 (1H,d,J=10.4 Hz,H-28),3.80 (1H,d,J=10.8Hz,H-28),4.58 (1H,s,H-29),4.68 (1H,d,J=2.0 Hz,H-29),1.68 (3H,s,H-30)。13C-NMR (CDCl3,100 MHz)δ:38.9 (C-1),27.4 (C-2),79.0 (C-3),38.7 (C-4),55.3 (C-5),18.3 (C-6),34.2 (C-7),40.9 (C-8),50.4 (C-9),37.3 (C-10),20.8 (C-11),25.2 (C-12),37.1 (C-13),42.7 (C-14),27.0 (C-15),29.2 (C-16),47.8 (C-17),47.8 (C-18),48.7 (C-19),150.5 (C-20),29.7 (C-21),34.0 (C-22),28.0 (C-23),15.4 (C-24),16.1 (C-25),16.0 (C-26),14.8 (C-27),60.5 (C-28),109.7 (C-29),19.1 (C-30)。以上數(shù)據(jù)與文獻(xiàn)[9]報(bào)道的化合物betulin的數(shù)據(jù)基本一致，因此鑒定化合物4為betulin。

3.5 化合物5 白色固體。1H-NMR (CDCl3,400 MHz)δ:3.19 (1H,dd,J=4.8,11.1 Hz,H-3),0.68 (1H,d,J=9.4 Hz,H-5),1.26 (1H,m,H-9),2.18 (1H,m,H-13),1.63 (1H,m,H-18),2.99 (1H,m,H-19),0.96 (3H,s,H-23),0.75 (3H,s,H-24),0.82 (3H,s,H-25),0.93 (3H,s,H-26),0.98 (3H,s,H-27),4.60 (1H,s,H-29),4.74 (1H,s,H-29),1.69 (3H,s,H-30)。13C-NMR (CDCl3,100 MHz)δ:38.9 (C-1),27.4 (C-2),79.0 (C-3),38.7 (C-4),55.3 (C-5),18.3 (C-6),34.3 (C-7),40.7 (C-8),50.5 (C-9),37.2 (C-10),20.8 (C-11),25.5 (C-12),38.4 (C-13),42.4 (C-14),30.5 (C-15),32.1 (C-16),56.3 (C-17),46.9 (C-18),49.2 (C-19),150.4 (C-20),29.7 (C-21),37.0 (C-22),28.0 (C-23),15.4 (C-24),16.1 (C-25),16.0 (C-26),14.7 (C-27),180.5 (C-28),109.7 (C-29),19.4 (C-30)。以上數(shù)據(jù)與文獻(xiàn)[9]報(bào)道的化合物betulic acid的數(shù)據(jù)基本一致，因此鑒定化合物5為betulic acid。

3.6 化合物6 白色固體。1H-NMR (CDCl3,400 MHz)δ:3.23 (1H,m,H-3),0.75 (1H,m,H-5),1.53 (1H,m,H-9),5.19 (1H,t,J=3.7 Hz,H-12),1.98 (1H,m,H-18),1.00 (3H,s,H-23),0.78 (3H,s,H-24),0.93 (3H,s,H-25),0.94 (3H,s,H-26),1.17 (3H,s,H-27),3.21 (1H,m,H-28),3.56 (1H,d,J=11.0 Hz,H-28),0.89 (3H,s,H-29),0.87 (3H,s,H-30)。13C-NMR (CDCl3,100 MHz)δ:38.6 (C-1),27.2 (C-2),79.0 (C-3),38.8 (C-4),55.1 (C-5),18.3 (C-6),32.6 (C-7),39.8 (C-8),47.6 (C-9),36.9 (C-10),23.5 (C-11),122.4 (C-12),144.2 (C-13),41.8 (C-14),25.5 (C-15),22.0 (C-16),47.6 (C-17),42.3 (C-18),46.4 (C-19),31.0 (C-20),34.0 (C-21),31.0 (C-22),28.1 (C-23),15.6 (C-24),15.5 (C-25),16.7 (C-26),26.0 (C-27),69.8 (C-28),33.2 (C-29),23.6 (C-30)。以上數(shù)據(jù)與文獻(xiàn)[10]報(bào)道的化合物古柯二醇的數(shù)據(jù)基本一致，因此鑒定化合物6為古柯二醇。

4 體外抗腫瘤活性

采用噻唑藍(lán)(MTT)法，以依托泊苷為陽性對(duì)照，檢測化合物1～6對(duì)人白血病細(xì)胞K562的體外抗腫瘤活性。實(shí)驗(yàn)結(jié)果表明，陽性對(duì)照依托泊苷的IC50值為2.57 μmol·L-1，化合物5表現(xiàn)出高于陽性對(duì)照的抑制作用，其IC50值為1.45 μmol·L-1，其余化合物均無活性。