竹靈消化學(xué)成分的研究

司盈盈，孫彥君，薛貴民，趙珍珠，馮衛(wèi)生*

(1.河南中醫(yī)藥大學(xué)藥學(xué)院，河南 鄭州 450046；2.河南省中藥開發(fā)工程技術(shù)研究中心，河南 鄭州 450046)

竹靈消Cynanchuminamoenum(Maxim.) Loes.隸屬蘿藦科鵝絨藤屬，別名雪里蟠桃、老君須、婆婆針線包等。產(chǎn)于遼寧、河北、河南、山東、山西、安徽、浙江、湖北、湖南、陜西、甘肅、貴州、四川、西藏，生長于海拔100～3 500 m的山地疏林、灌木叢中或山頂、山坡草地上，朝鮮和日本也有分布[1]。根可藥用，具有清熱涼血、利膽、解毒的功效，主治陰虛發(fā)熱、虛勞久咳、咯血、脅肋脹痛、嘔惡、瀉痢、產(chǎn)后虛煩、無名腫毒、蛇蟲或瘋狗咬傷。民間用作治婦女血厥、產(chǎn)后虛煩、妊娠遺尿、疥瘡及淋巴炎等[2]。關(guān)于竹靈消的化學(xué)成分研究報道很少，目前僅從中分離得到四十余個化合物[3-4]，且研究均集中在根部，其他部位的化學(xué)成分則未見報道。鵝絨藤屬植物的主要化學(xué)成分為C21甾體及其苷類[5-7],而竹靈消作為鵝絨藤屬植物中的一種，主要化學(xué)成分也是C21甾體類化合物，除此之外，還含有少量苯乙醇衍生物、生物堿、甘油酯、三萜、脂肪酸等其他類型成分[3-4]。本實驗對竹靈消中的化學(xué)成分進(jìn)一步研究，從中分離得到了14個化合物，化合物2、4～6、8、10、12為首次從該屬植物中分離得到，化合物3、7、9、11、13～14為首次從該植物中分離得到。

1 材料

ARX-400 MHz 核磁共振儀、Bruker maxis HD型飛行時間質(zhì)譜儀(德國Bruker公司)；FLEXA HP50型中壓制備系統(tǒng)(天津博納艾杰爾公司)；LC-8A型高效液相色譜儀(日本島津公司)；柱層析Sephadex LH-20(瑞士Parmacia Biotech 公司)；柱層析硅膠(200～300目，青島海洋化工廠)。所用分析純和色譜純試劑(北京化工廠和天津第三化學(xué)試劑廠)。

竹靈消全株采自河南省伏牛山，經(jīng)沈陽藥科大學(xué)中藥學(xué)院賈景明教授鑒定為鵝絨藤屬植物竹靈消Cynanchuminamoenum(Maxim.) Loes.。

2 提取與分離

將采集的竹靈消(濕質(zhì)量6.5 kg)粉碎，用95%乙醇回流提取3次，每次1.5 h，合并提取液，減壓濃縮至無醇味得到總提物。將總提物用2 L水分散，分別加入等體積的石油醚、二氯甲烷、乙酸乙酯、正丁醇依次萃取，萃取液減壓濃縮后得到相應(yīng)的萃取部位。取乙酸乙酯部位(37 g)過硅膠柱，以二氯甲烷-甲醇(0∶1～1∶0)梯度洗脫，得到16個流分(A-P)。流分O過凝膠柱得到5個亞流分O1～O5，O2過中壓柱色譜，80%甲醇水洗脫部分經(jīng)HPLC甲醇-水(68∶32)純化得化合物1(12.3 mg)；O3過中壓柱色譜，40%甲醇水洗脫部分經(jīng)HPLC甲醇-水(34∶66)純化得化合物4(9.5 mg)；O5過中壓柱色譜，50%甲醇水洗脫部分經(jīng)HPLC純化甲醇-水(42∶58)得化合物5(8.7 mg)。流分M過凝膠柱得化合物2(2.6 mg)。流分N過中壓柱色譜，40%甲醇洗脫部分過凝膠，得化合物3(16.3 mg)。流分L過凝膠得到4個亞流分L1～L4，L1經(jīng)HPLC純化得化合物7(10.3 mg)。流分J過中壓柱色譜，40%甲醇洗脫部分過凝膠，得化合物9(11.8 mg)。流分B和流分C析出白色粉末，經(jīng)洗滌、重結(jié)晶后分別得化合物12(24 mg)、13(37 mg)。流分A過凝膠柱，在洗脫的過程中析出無色針狀結(jié)晶，經(jīng)重結(jié)晶得化合物14(8.6 mg)。取正丁醇萃取部位(23 g)過硅膠柱，以二氯甲烷-甲醇(0∶1～1∶0)梯度洗脫，得到5個流分Fr.1～Fr.5。Fr.3過中壓柱色譜，60%甲醇洗脫部分過凝膠得化合物6(8.2 mg)；40%甲醇洗脫部分過凝膠得化合物8(4.4 mg)、10(5.6 mg)。Fr.4過中壓柱色譜，60%甲醇洗脫部分過凝膠得化合物11(3.8 mg)。

3 結(jié)構(gòu)鑒定

化合物1：無色粉末，ESI-MSm/z:505.2[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:6.39 (1H,brs,H-18),5.37 (1H,d,J=4.2 Hz,H-6),5.26 (1H,dd,J=16.8,7.5 Hz,H-16),5.04 (1H,d,J=5.5 Hz,4′-OH),4.59 (1H,dd,J=9.5,1.4 Hz,H-1′),4.15 (1H,dd,J=8.3,7.2 Hz,H-15α),3.62 (1H,t,J=9.1 Hz,H-15β),3.44 (1H,m,H-3),3.36 (1H,d,J=7.8 Hz,H-17),3.30 (3H,s,3′-OCH3),1.42 (3H,s,H-21),1.15 (3H,d,J=6.1 Hz,H-6′),0.85 (3H,s,H-19);13C-NMR (100 MHz,DMSO-d6)δ:174.8 (C-14),143.3 (C-18),139.9 (C-5),120.0 (C-6),117.7 (C-13),113.5 (C-20),96.9 (C-1′),80.0 (C-3′),76.5 (C-3),75.1 (C-4′),74.7 (C-16),71.6 (C-5′),66.8 (C-15),56.4 (3′-OCH3),55.3 (C-17),52.4 (C-8),39.8 (C-9),38.0 (C-10),38.0 (C-4),36.5 (C-1),35.8 (C-2′),29.3 (C-2),29.2 (C-7),27.5 (C-12),24.4 (C-21),23.3 (C-11),18.1 (C-19),17.5 (C-6′)。以上數(shù)據(jù)與文獻(xiàn)[8]基本一致，故鑒定為白薇苷甲。

化合物2：黃色針晶，ESI-MSm/z:455.1[M+Na]+，433.1[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:8.14 (2H,d,J=8.0 Hz,H-2′,6′),6.91 (2H,d,J=8.0 Hz,H-3′,5′),6.81 (1H,s,H-8),6.40 (1H,s,H-6),5.54 (1H,s,H-1″),3.84～3.31 (4H,m,H-2″-5″),1.12 (3H,d,J=6.1 Hz,H-6″);13C-NMR (100 MHz,DMSO-d6)δ:176.1 (C-4),161.4 (C-7),160.4 (C-5),159.4 (C-4′),155.7 (C-9),147.5 (C-2),132.1 (C-3),129.6 (C-2′,6′),121.6 (C-1′),115.5 (C-3′,5′),104.7 (C-10),98.8 (C-6),98.4 (C-1″),94.3 (C-8),71.6 (C-4″),70.3 (C-3″),70.1 (C-2″),69.9 (C-5″),17.9 (C-6″)。以上數(shù)據(jù)與文獻(xiàn)[9]基本一致，故鑒定為山柰酚-7-O-α-L-鼠李糖苷。

化合物3：黃色針晶，ESI-MSm/z:579.2[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:12.61 (1H,s,5-OH),10.27 (1H,s,4′-OH),7.79 (2H,d,J=8.8 Hz,H-2′,6′),6.92 (2H,d,J=8.8 Hz,H-3′,5′),6.79 (1H,d,J=2.0 Hz,H-8),6.46 (1H,d,J=2.0 Hz,H-6),5.55 (1H,d,J=1.6 Hz,H-1″),5.30 (1H,d,J=1.1 Hz,H-1?),1.13 (3H,d,J=6.1 Hz,7-rha,H-6″),0.80 (3H,d,J=5.5 Hz,3-rha,H-6?);13C-NMR (100 MHz,DMSO-d6)δ:178.0 (C-4),161.7 (C-7),161.0 (C-5),160.2 (C-4′),157.8 (C-2),156.1 (C-9),134.5 (C-3),130.7 (C-2′),130.7 (C-6′),120.3 (C-1′),115.5 (C-3′),115.5 (C-5′),105.8 (C-10),101.9 (C-1″),99.5 (C-1?),98.4 (C-6),94.6 (C-8),71.6 (C-4″),71.1 (C-4?),70.7 (C-3″),70.3 (C-2?),70.2 (C-2″),70.1 (C-5″),70.1 (C-3?),69.8 (C-5?),17.9 (C-6″),17.5 (C-6?)。以上數(shù)據(jù)與文獻(xiàn)[10]基本一致，故鑒定為山柰酚-3,7-O-α-L-二鼠李糖苷。

化合物4：黃色針晶，ESI-MSm/z:565.2[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:12.61 (1H,s,5-OH),10.26 (1H,s,7-OH),8.13 (2H,d,J=8.8 Hz,H-2′,6′),6.89 (2H,d,J=8.8 Hz,H-3′,5′),6.83 (1H,d,J=2.0 Hz,H-8),6.45 (1H,d,J=2.0 Hz,H-6),5.56 (1H,brs,H-1?),5.36 (1H,d,J=5.1 Hz,H-1″),3.84 (1H,brs,H-2?),3.75 (1H,dd,J=11.7,5.0 Hz,H-2″),3.66 (1H,m,H-4″),3.63 (1H,m,H-3?),3.58 (1H,m,H-5″),3.52 (1H,m,H-3″),3.41 (1H,dt,J=12.3,6.1 Hz,H-5?),3.30 (1H,m,H-4?),3.21 (1H,d,J=9.3 Hz,H-5″),1.12 (3H,d,J=6.1 Hz,H-6?);13C-NMR (100 MHz,DMSO-d6)δ:177.7 (C-4),161.6 (C-7),160.9 (C-5),160.2 (C-2),156.8 (C-4′),155.9 (C-9),133.8 (C-3),131.1 (C-6′),131.1 (C-2′),120.5 (C-1′),115.3 (C-3′),115.3 (C-5′),105.6 (C-10),101.2 (C-1″),99.4 (C-6),98.3 (C-1?),94.6 (C-8),71.6 (C-4?),71.5 (C-3″),70.8 (C-2″),70.3 (C-3?),70.1 (C-5?),69.8 (C-2?),66.0 (C-4″),64.3 (C-5″),17.9 (C-6?)。以上數(shù)據(jù)與文獻(xiàn)[11]基本一致，故鑒定為山柰酚-3-O-α-L-阿拉伯糖-7-O-α-L-鼠李糖苷。

化合物5：黃色針晶，ESI-MSm/z:595.2[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:7.34 (1H,d,J=1.3 Hz,H-2′),7.29 (1H,dd,J=8.3,1.3 Hz,H-6′),6.88 (1H,d,J=8.3 Hz,H-5′),6.76 (1H,d,J=1.4 Hz,H-8),6.45 (1H,d,J=1.4 Hz,H-6),5.55 (1H,s,H-1?),5.26 (1H,s,H-1″),3.98 (1H,brs,H-2?),3.84 (1H,brs,H-2″),3.64 (1H,d,J=8.8 Hz,H-3?),3.52 (1H,d,J=7.8 Hz,H-3″),3.45 (1H,m,H-5?),3.40 (1H,m,H-4?),3.22 (1H,m,H-5″),3.15 (1H,m,H-4″),1.13 (3H,d,J=6.0 Hz,H-6?),0.83 (3H,d,J=6.0 Hz,H-6″);13C-NMR (100 MHz,DMSO-d6)δ:178.0 (C-4),161.7 (C-5),160.9 (C-7),157.9 (C-2),156.1 (C-9),148.6 (C-4′),145.3 (C-3′),134.5 (C-3),121.2 (C-1′),120.6 (C-6′),115.7 (C-5′),115.5 (C-2′),105.7 (C-10),101.9 (C-6),99.5 (C-1″),98.4 (C-1?),94.5 (C-8),71.6 (C-4″),71.2 (C-4?),70.6 (C-3″),70.4 (C-3?),70.2 (C-2″),70.1 (C-2?),70.0 (C-5″),69.8 (C-5?),17.9 (C-6″),17.5 (C-6?)。以上數(shù)據(jù)與文獻(xiàn)[12]基本一致，故鑒定為槲皮素-3,7-O-α-L-二鼠李糖苷。

化合物6：淡黃色粉末，ESI-MSm/z:411.2[M+H]+。1H-NMR (400 MHz,DMSO-d6)δ:7.38 (1H,d,J=15.9 Hz,H-7′),7.02 (1H,brs,H-5′),6.96 (1H,br.d,J=8.1 Hz,H-6′),6.76 (1H,d,J=8.1 Hz,H-2′),6.10 (1H,d,J=15.9 Hz,H-8′),5.65 (1H,s,1-OH),5.01 (1H,brs,H-5),3.97 (1H,m,H-3),3.88 (2H,m,H-8),3.57 (1H,brs,H-4),2.11 (2H,m,H-2),1.91 (1H,dd,J=13.2,2.4 Hz,H-6α),1.75 (1H,dd,J=12.0,9.9 Hz,H-6β),1.48 (2H,dq,J=13.6,6.9 Hz,H-9),1.23 (2H,dq,J=14.6,7.2 Hz,H-10),0.79 (3H,t,J=7.4 Hz,H-11);13C-NMR (100 MHz,DMSO-d6)δ:173.2 (C-7),165.4 (C-1′),148.5 (C-7′),145.6 (C-3′),145.1 (C-6′),125.4 (C-4′),121.3 (C-9′),115.8 (C-8′),114.6 (C-2′),113.8 (C-5′),73.1 (C-1),71.1 (C-4),69.4 (C-5),66.9 (C-3),64.1 (C-8),30.0 (C-9),18.5 (C-10),13.5 (C-11)。以上數(shù)據(jù)與文獻(xiàn)[13]基本一致，故鑒定為5-O-咖啡酰基奎寧酸丁酯。

化合物9：白色粉末，ESI-MSm/z:193.1[M-H]-。1H-NMR (400 MHz,DMSO-d6)δ:7.48 (1H,d,J=15.9 Hz,H-7),7.27 (1H,d,J=1.6 Hz,H-2),7.08 (1H,dd,J=8.2,1.6 Hz,H-6),6.79 (1H,d,J=8.2 Hz,H-5),6.36 (1H,d,J=15.9 Hz,H-8),3.81 (3H,s,3-OCH3)。以上數(shù)據(jù)與文獻(xiàn)[16]基本一致，故鑒定為阿魏酸。

化合物10：白色粉末，ESI-MSm/z:163.0[M-H]-。1H-NMR (400 MHz,CD3OD)δ:7.60 (1H,d,J=15.9 Hz,H-7),7.44 (2H,d,J=8.6 Hz,H-2,6),6.80 (2H,d,J=8.6 Hz,H-3,5),6.28 (1H,d,J=15.9 Hz,H-8)。以上數(shù)據(jù)與文獻(xiàn)[17]基本一致，故鑒定為對羥基桂皮酸。

化合物12：白色粉末，ESI-MSm/z:427.4[M+H]+。1H-NMR(400 MHz,CDCl3)δ:5.63 (1H,d,J=6.0 Hz,H-6),3.48 (1H,t,J=5.4 Hz,H-3),1.16 (3H,s,H-23),1.14 (3H,s,H-24),1.09 (3H,s,H-25),1.04 (3H,s,H-26),1.00 (3H,s,H-27),0.99 (3H,s,H-28),0.95 (3H,s,H-29),0.85 (3H,s,H-30);13C-NMR (100 MHz,CDCl3)δ:141.7 (C-5),122.2 (C-6),76.5 (C-3),49.8 (C-10),47.6 (C-8),43.2 (C-18),41.0 (C-4),39.4 (C-22),39.1 (C-13),38.0 (C-14),36.2 (C-16),35.2 (C-19),35.0 (C-9),34.7 (C-21),34.7 (C-29),33.2 (C-11),32.5 (C-30),32.2 (C-15),32.2 (C-28),30.5 (C-12),30.5 (C-17),29.1 (C-24),28.4 (C-20),28.0 (C-2),25.6 (C-23),23.8 (C-7),19.8 (C-26),18.6 (C-27),18.4 (C-1),16.4 (C-25)。以上數(shù)據(jù)與文獻(xiàn)[19]基本一致，故鑒定為粘霉烯醇。

化合物13：白色粉末，ESI-MSm/z:427.4[M+H]+。1H-NMR (400 MHz,C5D5N)δ:5.64 (1H,dd,J=8.2,3.1 Hz,H-15),3.46 (1H,dd,J=10.5,5.7 Hz,H-3),1.27 (3H,s,H-23),1.14 (3H,s,H-24),1.10 (3H,s,H-25),1.03 (3H,s,H-26),1.01 (3H,s,H-27),1.01 (3H,s,H-28),0.98 (3H,s,H-29),0.92 (3H,s,H-30);13C-NMR (100 MHz,C5D5N)δ:158.8 (C-14),117.5 (C-15),78.5 (C-3),56.4 (C-5),49.9 (C-18),49.5 (C-9),42.0 (C-19),39.8 (C-4),39.7 (C-8),38.7 (C-10),38.6 (C-1),38.3 (C-16),38.2 (C-13),37.3 (C-17),36.4 (C-7),35.7 (C-12),34.4 (C-21),33.8 (C-22),33.8 (C-29),30.4 (C-26),30.4 (C-28),29.4 (C-20),29.1 (C-23),28.5 (C-2),26.6 (C-27),21.9 (C-30),19.6 (C-6),18.2 (C-11),16.8 (C-25),16.1 (C-24)。以上數(shù)據(jù)與文獻(xiàn)[20]基本一致，故鑒定為蒲公英賽醇。

化合物14：無色針晶，ESI-MSm/z:427.4[M+H]+。1H-NMR (400 MHz,CDCl3)δ:1.17 (3H,s,H-28),1.04 (3H,s,H-27),1.00 (6H,s,H-26,30),0.95 (3H,s,H-29),0.86 (6H,s,H-23,25),0.72 (3H,s,H-24);13C-NMR (100 MHz,CDCl3)δ:213.4 (C-3),59.6 (C-10),58.4 (C-4),53.2 (C-8),42.9 (C-18),42.3 (C-5),41.7 (C-2),41.4 (C-6),39.8 (C-13),39.4 (C-22),38.4 (C-14),37.6 (C-9),36.1 (C-16),35.8 (C-11),35.5 (C-19),35.2 (C-29),32.9 (C-21),32.6 (C-15),32.2 (C-28),31.9 (C-30),30.6 (C-12),30.1 (C-17),28.3 (C-20),22.4 (C-1),20.4 (C-26),18.8 (C-27),18.4 (C-7),18.1 (C-25),14.8 (C-24),7.0 (C-23)。以上數(shù)據(jù)與文獻(xiàn)[21]基本一致，故鑒定為木栓酮。

4 討論

竹靈消作為鵝絨藤屬植物中的一種，主要化學(xué)成分同樣也是該屬植物的特征性成分——C21甾體類，本研究除了得到了1個C21甾體外，還分離出了13個其他類型的化合物，包括4個黃酮苷類、3個簡單苯丙素及其衍生物、2個雙四氫呋喃型木脂素、1個生物堿和3個五環(huán)三萜。在這些結(jié)構(gòu)類型中，C21甾體、生物堿和三萜已被報道過，而本研究首次從竹靈消中分離得到黃酮苷類、簡單苯丙素類和雙四氫呋喃型木脂素類化合物，豐富竹靈消中化學(xué)成分的結(jié)構(gòu)類型，為竹靈消的進(jìn)一步開發(fā)提供參考。