苦地丁化學(xué)成分研究

肖 揚，楊春娟，鐘明亮，蔣學(xué)春，劉高峰

1哈爾濱醫(yī)科大學(xué)藥學(xué)院，哈爾濱150081;2 中國醫(yī)學(xué)科學(xué)院北京協(xié)和醫(yī)學(xué)院藥用植物研究所，北京100193;3哈爾濱醫(yī)科大學(xué)附屬第二醫(yī)院藥學(xué)部，哈爾濱150086

Introduction

Corydalis bungeana Turcz. belongs to family Papaveraceae and is a perennial herb with violet to pink flowers distributing in the northern and eastern parts of China，the southeast of Mongolia，the northern part of the Korean peninsula and the far east of Russia［1］.The dried whole plant of Corydalis bungeana Turcz. were used as a folk medicine in China for treatment of influenza，upper respiratory tract infections，bronchitis，tonsillitis，acute nephritis，and pyelonephritis［1］. Previous phytochemical investigations indicated that alkaloids were its main components，such as corynoline，12-hydroxycorynoline，sanguinarine，acetylcorynoline，protopine and so on［2］. Modern pharmacological studies showed that alkaloids as corynoline，acetylcorynoline，and protopine could significantly impede liver damage caused by CCl4in mice［3］. Some isoquinoline alkaloids isolated from Corydalis bungeana Turcz.also had bacteriostatic activity［4］. In this paper，ten compounds were isolated from the 95% ethanol extract of Corydalis bungeana Turcz，and were identified as corynoline (1)，acetylcorynoline (2)，protopine (3)，8-oxocorynoline(4)，neoechinulin A (5)，7'-(3'，4'-dihydroxyphenyl)-N-［(4-methoxyphenyl) ethyl］ propenamide(6)，kaempferol (7)，n-trans-p-coumaroyltyramine(8)，quercetin (9)，tianshic acid (10)，respectively.To the best of our knowledge，compounds 5，6，8，10 were obtained from the genus for the first time，and compounds 4，7 and 9 were isolated from this plant for the first time.

Experimental

General

Silica gel (H，200-300 mesh，Qingdao Haiyang Chemical Co. Ltd. Qingdao，China)and silica gel (GF254，Qingdao Haiyang Chemical Co. Ltd. Qingdao，China)were used for column chromatography (CC)and TLC，respectively.Sephedax LH-20 (Pharmacia Fine Chemical Co.Ltd.Sweden)and ODS (71-154 mesh，F(xiàn)uji Silysia Chemical Ltd.Japan)were used during the isolation.Preparative HPLC analysis was carried out on Agilent 1260 series HPLC with a YMC-Pack ODS-A column (20 mm× 250 mm，5 μm). D-101 Macroporous Resin (Tianjin big Jun Ltd.China)was used to isolate 95% EtOH crude extract. NMR spectra were recorded on Bruker Avance Ⅲ600 (600 MHz for1H NMR and 150 MHz for13C NMR)with TMS as internal standard，the chemical shift values (δ)were reported in ppm and coupling constants (J)in Hz.Mass data were obtained on Agilent 6400 QQQ-LC/MS mass spectrometer.

Plant materials

Corydalis bungeana Turcz. was collected from Tianyi medicinal materials Sci-Tech Co. Ltd. of Harbin，Heilongjiang Province，China，in August，2010 and identified by Professor Zhenyue Wang in School of Heilongjiang University of Chinese Medicine. A voucher specimen was deposited in the herbarium of Harbin Medical University，Harbin，China.

Extraction and isolation

Corydalis bungeana Turcz. (10 kg)，was cut into pieces and then extracted for three times with 95%EtOH under reflux，3 h for each time. The combined filtrate was concentrated under vacuum at 50 ℃using a rotary evaporator to afford a residue as 95% EtOH crude extract (800 g).95% EtOH crude extract was then subjected to D-101 Macroporous Resin column chromatography，eluting with 30% EtOH，60% EtOH and 95% EtOH，namely fraction A，fraction B，and fraction C. Fraction C was then subjected to column chromatography on a silica gel，eluting with dichloromethane-methanol (from 30∶1 to 1∶1，v/v)，and was fractionated into sixty fractions (Fr.1→60).Sixty fractions (Fr.1→60)were collected based on TLC analysis.Fr.9，F(xiàn)r.22 were purified by recrystallization from dichloromethane-methanol (30 ∶1，v/v)to give compound 1 (500 mg)and compound 2 (30 mg)，and Fr.45 was purified by recrystallization from dichloromethane-methanol (20 ∶1，v/v)to give compound 3 (22 mg)，respectively.

Fraction B was separated on an ODS open tube column using a gradient eluent of MeOH-H2O (10∶90-100∶0，v/v).Ten subfractions (subFr.1→10)were collected based on TLC analysis.SubFr.2 was subjected to preparative HPLC on a YMC-PACK ODS-A C18column(20 × 250 mm，5 μm)eluting with MeOH-H2O (45∶55，v/v)to give compound 5 (tR= 23.0 min，15 mg)and compound 4 (tR= 40.0 min，11 mg). SubFr.3 was purified by Sephadex LH-20 column chromatography eluting with MeOH to give subFr.3-1 and subFr.3-2. SubFr.3-1 was successively purified on a Sephedax LH-20 column，eluting with MeOH to afford compound 7 (10.2 mg). SubFr.3-2 was purified by recrystallization from MeOH to give compound 6 (8.3 mg). SubFr.4 was subjected to preparative HPLC on a YMC-PACK ODS-A C18column (20 × 250 mm，5 μm)eluting with MeOH-H2O (45 ∶55，v/v)to give compound 8 (tR= 20.0 min，13 mg)and compound 9(tR= 33.0 min，18 mg). SubFr.6 was successively purified on a Sephedax LH-20 column，eluting with MeOH to afford compound 10 (16.7 mg).

Identification

Corynoline (1) C21H21NO5，colorless granular crystal;ESI-MS m/z:368.2［M+H］+;1H NMR (CDCl3，600 MHz)δ:6.92 (1H，d，J =7.8 Hz，H-10)，6.79(1H，d，J =7.8 Hz，H-9)，6.66 (1H，s，H-4)，6.64(1H，s，H-1)，5.90-6.00 (4H，m，2 ×-O-CH2-O-，2，3-，7，8-)，4.04，3.44 (2H，d，J = 15.6 Hz，H-6)，3.95 (1H，dd，J =4.2，1.8 Hz，H-11)，3.30 (1H，d，J=1.8 Hz，H-14)，3.16 (1H，d，J = 17.2 Hz，H-12α)，3.09 (1H，dd，J =17.2，4.2 Hz，H-12β)，2.21(3H，s，5-NCH3)，1.14 (3H，s，CH3-13).13C NMR(CDCl3，125 MHz，)δ:107.8 (C-1)，145.4 (C-2)，148.1 (C-3)，101.1 (2-O-CH2-O-3)，112.8 (C-4)，128.0 (C-4a)，54.4 (C-6)，117.0 (C-6a)，142.9(C-7)，145.2 (C-8)，101.4 (7-O-CH2-O-8)，109.5(C-9)，118.7 (C-10)，136.2 (C-10a)，76.3 (C-11)，36.9 (C-12)，125.4 (C-12a)，41.0 (C-13)，69.9 (C-14)，43.3 (C-5-NCH3)，23.5 (CH3-13). The NMR and MS data were in accordance with those reported in the literature［5］，and identified 1 as corynoline.

Acetylcorynoline (2) C23H23NO6，white crystal;ESI-MS m/z:410.2［M+H］+;1H NMR (MeOD，600 MHz)δ:6.96 (1H，d，J=8.4 Hz，H-10)，6.67 (1H，d，J =8.4 Hz，H-9)，6.88 (1H，s，H-4)，6.58 (1H，s，H-1)，5.90-5.93 (4H，m，2 ×-O-CH2-O-，2，3-，7，8-)，5.18 (1H，dd，J = 7.8，6.6 Hz，H-11)，3.88，3.51 (2H，d，J = 16.2 Hz，H-6)，3.53 (1H，s，H-14)，2.96 (1H，dd，J =15.6，7.8 Hz，H-12α)，2.89(1H，dd，J =15.6，6.6 Hz，H-12β)，2.39 (3H，s，5-NCH3)，1.78 (3H，s，COCH3)，1.23 (3H，s，CH3-13).13C NMR (MeOD，125 MHz)δ:107.5 (C-1)，150.8 (C-2)，152.0 (C-3)，102.4 (2-O-CH2-O-3)，111.1 (C-4)，129.0 (C-4a)，51.4 (C-6)，114.5 (C-6a)，148.2 (C-7)，148.7 (C-8)，102.6 (7-O-CH2-O-8)， 109.5 (C-9)，121.7 (C-10)，134.7 (C-10a)，77.1 (C-11)，34.0 (C-12)，126.3 (C-12a)，43.7(C-13)，71.6 (C-14)，43.8 (C-5-NCH3)，28.3(CH3-13)，172.5，21.2 (OAc). The NMR and MS data were in accordance with those reported in the literature［6］，and identified 2 as acetylcorynoline.

Protopine (3) C20H19NO5，white amorphous powder;ESI-MS m/z:354.4 ［M + H］+;1H NMR(CDCl3，600 MHz)δ:6.90 (1H，s，H-1)，6.64 (1H，s，H-4)，6.67 (1H，d，H-12)，6.68 (1H，d，H-11)，5.95 (2H，s，H-15)，5.92 (2H，s，H-16)，1.92 (3H，s，7-NCH3).13C NMR (CDCl3，125 MHz)δ:108.5(C-1)，146.7 (C-2)，148.4 (C-3)，110.8 (C-4)，136.6 (C-4a)，32.2 (C-6)，41.8 (C-7-NCH3)，51.2(C-8)，146.4 (C-9)，146.2 (C-10)，107.1 (C-11)，125.5 (C-12)，129.4 (C-12a)，46.9 (C-13)，195.3(C-14)，133.1 (C-14a)，101.2 (C-15). The NMR and MS data were in accordance with those reported in the literature［7］，and identified 3 as protopine.

8-Oxocorynoline (4) C21H19NO6，white amorphous powder;ESI-MS m/z:382.1 ［M + H］+，1H NMR(MeOD，600 MHz)δ:7.61 (1H，d，J =8.4 Hz，H-11)，6.41 (1H，d，J =8.4 Hz，H-12)，6.54 (1H，s，H-1)，6.41 (1H，s，H-4)，6.00，5.88 (1H×2，s，9-OCH2-O-10)，5.84，5.81 (1H ×2，s，2-O-CH2-O-3)，4.29 (1H，s，H-14)，4.11 (1H，dd，J=11.4，7.2 Hz，H-6)，3.45 (3H，s，5-NCH3)，2.97 (2H，m，H-5)，1.47 (3H，s，CH3-13).13C NMR (MeOD，125 MHz)δ:106.6(C-1)，148.3 (C-2)，148.7 (C-3)，102.5(2-O-CH2-O-3)，109.3 (C-4)，131.0 (C-4a)，36.4(C-5)，74.6 (C-6)，164.9 (C-8)，149.0 (C-9)，149.1 (C-10)，103.3 (7-O-CH2-O-8)，112.0 (C-11)，120.7 (C-12)，137.1 (C-12a)，45.1 (C-13)，70.0 (C-14)，38.6 (C-7-NCH3)，25.0 (CH3-13).The NMR and MS data were in accordance with those reported in the literature［8］，and identified 4 as 8-oxocorynoline.

Neoechinulin A (5) C19H21N3O2，light yellow amorphous powder;ESI-MS m/z:322.2 ［M－H］－，1H NMR (DMSO，600 MHz)δ:11.04 (1H，s，NH-1)，8.65 (1H，s，NH-14)，8.32 (1H，s，NH-11)，7.41(1H，d，J =7.8 Hz，H-7)，7.19 (1H，d，J =7.8 Hz，H-4)，7.08 (1H，s，H-6)，7.01 (1H，s，H-5)，6.89(1H，s，H-8)，6.08 (1H，dd，J =17.2，10.8 Hz，H-16)，5.02 (2H，dd，J =16.8，10.8Hz，H-17)，4.15(1H，q，J =7.2 Hz，H-12)，1.47 (6H，s，2 × CH3-15)，1.37 (3H，d，J = 7.2 Hz，CH3-12).13C NMR(DMSO，125 MHz)δ:143.9 (C-2)，103.3 (C-3)，125.9 (C-3a)，118.8 (C-4)，119.5 (C-5)，120.7(C-6)，111.5 (C-7)，135.1 (C-7a)，110.0 (C-8)，124.9 (C-9)，159.8 (C-10)，50.5 (C-12)，166.3(C-13)，39.1 (CH3-15)，145.1 (C-16)，111.5 (C-17)，27.4 (C-18)，27.4 (C-19)，19.6 (CH3-12).The NMR and MS data were in accordance with those reported in the literature［9］，and identified 5 as neoechinulin A.

7'-(3'，4'-Dihydroxyphenyl)-N-［(4-methoxyphenyl)ethyl］propenamide (6) C18H19NO4，light yellow amorphous powder;ESI-MS m/z:314.2 ［M +H］+，1H NMR (DMSO，600 MHz)δ:7.97 (1H，t，J=6.0 Hz，NH)，7.31 (1H，d，J =15.6 Hz，H-7')，7.11 (1H，d，J=1.8 Hz，H-2')，7.01 (2H，d，J=7.8 Hz，H-2，6)，6.98 (1H，dd，J =7.8，1.8 Hz，H-6')，6.78 (1H，d，J=7.8 Hz，H-5')，6.68 (2H，d，J=7.8 Hz，H-3，5)，6.43 (1H，d，J =15.6 Hz，H-8')，3.80(3H，s，OMe)，3.32 (2H，t，J =7.2 Hz，H-8)，2.64(2H，t，J = 7.2 Hz，H-7).13C NMR (DMSO，125 MHz)δ:126.4 (C-1)，129.4 (C-2)，115.1 (C-3)，155.6 (C-4)，115.1 (C-5)，129.4 (C-6)，34.4 (C-7)，40.6 (C-8)，129.5 (C-1')，121.4 (C-2')，147.7(C-3')，148.1 (C-4')，119.0 (C-5')，110.8 (C-6')，115.6 (C-7')，138.8 (C-8')，165.3 (C-9').The NMR and MS data were in accordance with those reported in the literature［10］，and identified 6 as 7'-(3'，4'-dihydroxyphenyl)-N-［(4-methoxyphenyl)ethyl］propenamide.

Kaempferol (7) C15H10O6，yellow powder;ESI-MS m/z:287.2［M +H］+，1H NMR (MeOD，600 MHz)δ:8.07 (2H，d，J=8.4 Hz，H-3'，5')，6.90 (2H，d，J=8.4 Hz，H-2'，6')，6.38 (1H，d，J =1.2 Hz，H-8)，6.17 (1H，d，J =1.2 Hz，H-6).13C NMR (MeOD，125 MHz)δ:148.3 (C-2)，137.3 (C-3)，177.6 (C-4)，162.7 (C-5)，99.5 (C-6)，165.7 (C-7)，94.7(C-8)，158.5 (C-9)，104.8 (C-10)，123.9 (C-1')，130.9 (C-2')，117.8 (C-3')，160.7 (C-4')，117.8(C-5')，130.9 (C-6'). The NMR and MS data were in accordance with those reported in the literature［11］，and identified 7 as kaempferol.

N-trans-p-coumaroyltyramine (8) C17H17NO3，white solid;ESI-MS m/z:284.3 ［M +H］+，1H NMR(MeOD，600 MHz)δ:7.44 (1H，d，J =15.6 Hz，H-7')，7.39 (2H，d，J =7.8 Hz，H-2'，6')，7.05 (2H，d，J =7.8 Hz，H-2，6)，6.79 (2H，d，J =7.8 Hz，H-3'，5')，6.72 (2H，d，J=7.8 Hz，H-3，5)，6.38 (1H，d，J =15.6 Hz，H-8')，3.46 (2H，t，J =7.2 Hz，H-8)，2.75 (2H，t，J=7.2 Hz，H-7).13C NMR (MeOD，125 MHz)δ:128.0 (C-1)，131.6 (C-2)，116.2 (C-3)，157.1 (C-4)，116.2 (C-5)，131.6 (C-6)，36.0(C-7)，42.7 (C-8)，132.7 (C-1')，131.6 (C-2')，116.7 (C-3')，160.7 (C-4')，116.7 (C-5')，131.6(C-6')，142.0 (C-7')，118.7 (C-8')，169.5 (C-9'). The NMR and MS data were in accordance with those reported in the literature［12］，and identified 8 as Ntrans-p-coumaroyltyramine.

Quercetin (9) C15H10O7，yellow powder;ESI-MS m/z:303.2［M +H］+，1H NMR (MeOD，600 MHz)δ:7.73 (1H，d，J =2.4 Hz，H-2')，7.63 (1H，dd，J =8.4，2.4 Hz，H-6')，6.88 (1H，d，J =8.4 Hz，H-5')，6.38 (1H，d，J=1.8 Hz，H-8)，6.18 (1H，d，J =1.8 Hz，H-6).13C NMR (MeOD，125 MHz)δ:146.4 (C-2)，137.4 (C-3)，177.5 (C-4)，162.7 (C-5)，99.5(C-6)，165.8 (C-7)，94.6 (C-8)，158.4 (C-9)，104.7 (C-10)，124.4 (C-1')，121.9 (C-2')，149.0(C-3')，148.2 (C-4')，116.5 (C-5')，116.2 (C-6').The NMR and MS data were in accordance with those reported in the literature［13］，and identified 9 as quercetin.

Tianshic acid (10) C18H34O5，white powder;ESIMS m/z:329.2 ［M－H］－，1H NMR (MeOD，600 MHz)δ:5.73 (1H，dd，J = 15.6，6.0 Hz，H-10)，5.67 (1H，dd，J =15.6，5.4 Hz，H-9)，4.05 (1H，q，J=6.0 Hz，H-8)，3.90 (1H，t，J =6.0 Hz，H-11)，3.41 (1H，m，H-12)，2.28 (2H，t，J =7.2 Hz，H-2)，0.91 (3H，t，J =7.2 Hz，H-18).13C NMR (MeOD，125 MHz)δ:177.9 (C-1)，35.2 (C-2)，26.2 (C-3)，30.4 (C-4)，30.6 (C-5)，26.6 (C-6)，38.5 (C-7)，73.2 (C-8)，131.3 (C-9)，136.8 (C-10)，76.7(C-11)，76.0 (C-12)，33.8 (C-13)，26.8 (C-14)，30.7 (C-15)，33.3 (C-16)，23.9 (C-17)，14.6 (C-18). The NMR and MS data were in accordance with those reported in the literature［14］，and identified 10 as tianshic acid.

1 Xie C，Veitch NC，Houghton PJ，et al. Flavonoid glycosides and isoquinolinone alkaloids from Corydalis bungeana.Phytochemistry，2004，65:3041-3047.

2 Niu LL，Xie ZS，Cai TX，et al. Preparative isolation of alkaloids from Corydalis bungeana Turcz.by high-speed countercurrent chromatography using stepwise elution. J Sep Sci，2011，34:987-994.

3 Wei HL，Liu GT. Protective action of corynoline，acetylcorynoline and protopine against experimental liver injury in mice.Acta Pharmaceutica Sinica，1997，32:331-336.

4 Liu XJ，Zhang HL，Tan ZC，et al.Microcalorimetric study on the bacteriostatic activity of isoquinoline alkaloids. J Therm Anal Calorim，2007，89:907-911.

5 Ma WG，F(xiàn)ukushi Y，Tahara S. Fungitoxic alkaloids from Hokkaido Corydalis species.Fitoterapia，1999，70:258-265.

6 Zeng WG，Liang WZ，Tu GS. Chemical study of alkaloids from Corydalis bungeana.Planta Med，1987，53:418-420.

7 Xu W，Song QS，Wang P，et al.Chemical constituents of the leaves and twigs of Ficushispida.Nat Prod Res Dev(天然產(chǎn)物研究與開發(fā))，2010，22:1003-1005.

8 Zhang GL，Pan WE，Peng SL，et al. Studies on the medical isoquinoline alkaloidsⅠ. Alkaloids of corydalis trachycarpa maxim.Nat Prod Res Dev(天然產(chǎn)物研究與開發(fā))，1989，2:1-5.

9 Li Y，Li XF，Kim SK，et al.Golmaenone，a new diketopiperazine alkaloid from the Marine-Derived fungus Aspergillus sp..Chem Pharm Bull，2004，52:375-376.

10 Anis E，Anis I，Ahmed S，et al.α-Glucosidase inhibitory constituents from Cuscuta reflexa. Chem Pharm Bull，2002，50:112-114.

11 Liao LP，Li P.Compounds from leaf of Ilex purpurea Hassk.J China Pharm Univ，2004，35:205-206.

12 Duan CL，Yong YJ，Jiang Y，et al.Liposoluble chemical constituents from the fibrous root of Ophiopogon japonicas. J Chin Pharm Sci，2009，18:236-239.

13 Wang DY，Liu EG，F(xiàn)eng YJ.Study on the Flavonoid Constituents from the Bark of Myrica ruba.Lishizhen Med Mater Med Res，2008，19:1149-1150.

14 Sang SM，Lao AN，Wang YS，et al. Antifungal constituents from the seeds of Allium fistulosum L..J Agric Food Chem，2002，50:6318-6321.