刺楸中酚性成分研究

么焕开，李 岩，段静雨，王健慧，段宏泉

1徐州医学院药学院，徐州221004;2天津医科大学药学院，天津医科大学基础医学研究中心，天津300070

Introduction

Kalopanax septemlobus(Thunb.)Koidz.，a deciduous tree of the family Araliaceae，grows in Japan，Korea，China，and Russia.The stem bark of this plant has been used for the treatment of rheumatoidal arthritis，neurotic pain and diabetes mellitus in traditional Chinese medicine［1］.Several phytochemical investigations of this plant showed that the main constituents of Kalopanax were triterpene saponins，lignans and simple phenolic constituents［2-4］.In our investigation of chemical constituents from Kalopanax septemlobus (Thunb.) Koidz.，6 phenolic compounds were isolated from the roots of that plant.Their structures were identified as ethyl orsellinate(1)，2-hydroxy-4-methoxy-3，6-dimethylbenzoic acid(2)，vanillin(3)，protocatechuic aldehyde(4)，protocatechuic acid(5)，and 3-methoxybenzaldehyde(6)on the basis of spectral data and physical and chemical properties.All compounds were obtained from this genus for the first time.

Experimental

General

NMR spectra were recorded on Bruker AM-300 and DRX-400 NMR spectrometers.The chemical shift(δ) values were given in ppm with TMS as internal standard，and coupling constants(J)were in Hz.ESI-MS spectra were obtained on an Agilent 1100 LC-MS spectrometer with ESI ionization in positive model.Toyopearl HW-40C was produced by Tosoh Corporation.Silica gel(300-400 mesh)for open column chromatography was produced by Qingdao Marine Chemical Industrials as well as silica gel GF254for TLC.TLC and preparative TLC were carried out on plates coated with silica gel GF254，and the TLC spots were viewed at 254 nm and then heated after sprayed with cerous sulfate solution.Semi-preparative HPLC was carried out on a JASCO HPLC system equipped with a PU-2089 pump，a RI-2031 detector and a UV-2075 detector.Columns for semi-preparative HPLC were ODS-A SH-343-5(20 mm×250 mm，5 μm，YMC-Pack)，SIL-06(20 mm× 250 mm，5 μm，YMC-Pack)and GPC GS-31020G(20 mm×500 mm，5 μm，Shodex).All solvents for HPLC purification were redistilled from commercial HPLC grade solvent.

Plant Material

The roots of Kalopanax septemlobus(Thunb.)Koidz.were collected in Tianshui，Gansu Province in September 2004 and identified by Professor Zhigang Li，Gansu Institute for Drug Control.A voucher specimen (D20040901)has been deposited in School of Pharmaceutical Sciences，Tianjin Medical University.

Extraction and Isolation

The air-dried roots of Kalopanax septemlobus (Thunb.)Koidz.(6.0 kg)were ground and extracted three times with 95%EtOH(30 L)for 4 h under reflux condition.The extract was evaporated in vacuo to give black syrup.The black syrup was suspended in water and then partitioned successively with petroleum ether(6.0 L)，EtOAc(6.0 L)and n-BuOH(6.0 L) to give fractions PE(50 g)，EtOAc(95 g)，and n-BuOH(170 g)，respectively.The EtOAc extract was subjected to column chromatography over silica gel and eluted with PE/EtOAc(from 8∶2 to 2∶8，v/v)and then EtOAc/MeOH(from 5∶95 to 20∶80，v/v)to yield 27 fractions(E1-E27).E2 was separated over Toyopearl HW-40C eluted with CH2Cl2/MeOH(2∶1，v/v)to afford 4 fractions(E2a-E2d)and then E2d was further purified by PTLC to afford 1(5 mg)and 2 (5 mg).E3 was separated over Toyopearl HW-40C eluted with CH2Cl2/MeOH(2∶1，v/v)to give 4 fractions(E3a-E3d)，and then E3b-E3d were further separated by HPLC(YMC-Pack ODS-A，MeOH/ H2O，9∶1)and purified by HPLC(YMC-Pack SIL-06，PE/EtOAc，2∶1)to afford 3(27 mg).E5 was also subjected to a Toyopearl HW-40C column to afford 4 fractions(E5a-E5e)and E5d was further purified by PTLC to give 4(5 mg)as well as E5e by recrystallization to afford 5(113 mg).

The PE extract was also subjected to column chromatography over silica gel and eluted with PE/EtOAc (from 8∶2 to 2∶8，v/v)and EtOAc/MeOH(from 5∶95 to 20∶80，v/v)to yield 39 fractions(P1-P39).P9 was subjected to column chromatography over Toyopearl HW-40C eluted with CH2Cl2/MeOH(2∶1，v/v)to afford 5 fractions(P9a-P9e).P9e was further purified by HPLC(GPC GS-31020G)eluted with MeOH and then recrystallized to give 6(7 mg).

Identification

Ethyl orsellinate(1) Yellowish needles(CH2Cl2)，ESI-MS m/z:197［M+H］+.1H NMR(CDCl3，400 MHz)δ:6.42(2H，q，J=7.1 Hz，OCH2)，1.43(3H，t，J=7.1 Hz，CH3)，2.52(3H，s，2-CH3)，6.30(1H，d，J=2.4 Hz，H-3)，6.25(1H，d，J=2.2 Hz，H-5);13C NMR(CDCl3，100 MHz)δ:171.7(s，1-CO2)，165.4(s，C-6)，160.2(s，C-4)，144.0(s，C-2)，111.3 (d，C-3)，105.8(s，C-1)，101.3(d，C-5)，61.3(t，OCH2)，24.3(q，2-CH3)，14.2(q，CH3).The data above showed to be identical with ethyl orsellinate［5］by direct comparison.

2-Hydroxy-4-methoxy-3，6-dimethylbenzoic acid (2) Colorless needles(CH2Cl2)，ESI-MS m/z:197［M+H］+.1H NMR(CDCl3，400 MHz)δ:2.12(3H，s，3-CH3)，2.47(3H，s，6-CH3)，3.94(3H，s，4-OCH3)，5.15(1H，br s，2-OH)，6.22(1H，s，H-5)，12.06(1H，s，1-CO2H);13C NMR(CDCl3，100 MHz) δ:172.4(s，1-CO2H)，163.0(s，C-4)，158.0(s，C-2)，140.1(s，C-6)，110.2(d，C-5)，108.4(s，C-3)，105.2(s，C-1)，51.8(q，4-OCH3)，24.1(q，3-CH3)，7.6(q，6-CH3).The data above showed to be identical with 2-hydroxy-4-methoxy-3，6-dimethylbenzoic acid［6］by direct comparison.

Vanillin(3) Colorless needles(EtOH)，ESI-MS m/ z:153［M+H］+.1H NMR(DMSO-d6，300 MHz)δ: 9.75(1H，s，1-CHO)，7.40(1H，dd，J=1.9，8.0 Hz，H-6)，7.36(1H，d，J=1.9 Hz，H-2)，6.94(1H，d，J =8.0 Hz，H-5)，3.83(3H，s，3-OCH3).The data above showed to be identical with vanillin［7］by direct comparison.

Protocatechuicaldehyde (4) Brown crystals (EtOH)，ESI-MS m/z:139［M+H］+，437［3M+ Na］+.1H NMR(DMSO-d6，400 MHz)δ:9.70(1H，s，CHO)，7.28(1H，br d，J=8.1 Hz，H-6)，7.24(1H，br s，H-2)，6.92(1H，d，J=8.1 Hz，H-5).The data above showed to be identical with protocatechuic aldehyde［8］by direct comparison.

Protocatechuic acid (5) Colorless crystals (EtOH)，ESI-MS m/z:155［M+H］+.1H NMR(DMSO-d6，300 MHz)δ:7.34(1H，d，J=1.7 Hz，H-2)，7.24(1H，dd，J=1.7，8.1 Hz，H-6)，6.69(1H，d，J =8.1 Hz，H-5)，9.71(2H，br s，OH)，12.13(1H，br s，OH).The data above showed to be identical with protocatechuic acid［9］by direct comparison.

3-Methoxybenzaldehyde(6) Colorless needles (EtOH)，ESI-MS m/z:137［M+H］+.1H NMR (DMSO-d6，300 MHz)δ:9.84(1H，s，1-CHO)，6.22 (1H，br s，H-2)，7.06(1H，dd，J=3.1，5.3 Hz，H-5)，7.44(2H，m，H-4，6)，3.98(3H，s，3-OCH3).The data above showed to be identical with 3-methoxybenzaldehyde［10］by direct comparison.

1 Nanjing University of Chinese Medicine(南京中医药大学).Dictionary of Chinese Materia Medica(中药大辞典).2nd Edition，Vol 1.Shanghai Science and Technology Press，2006.1848-1849.

2 Shao CJ，Kasai R，Xu J，et al.Saponins from roots of Kalopanax septemlobus(Thunb.)Koidz.，Ciqiu:structures of kalopanaxsaponins C，D，E and F.Chem Pham Bull，1989，37，311-314.

3 Sano K，Sanada S，Ida Y，et al.Studies on the constituents of the bark of Kalopanax pictus Nakai.Chem Pham Bull，1991，39:865-870.

4 Hong SS，Han XH，Hwang JS，et al.Lignans from the Stem Barks of Kalopanax septemlobus.Nat Prod Sci，2006，12:201-204.

5 Liu DL(刘岱琳)，Pang FG(庞发根)，Zhang JX(张家欣)，et al.Studies on the chemical constituents of Bulbophyllum odoratissimum Lindl.Chin J Med Chem(中国药物化学杂志)，2005，15:1422-1424.

6 Fang ZF(方振峰)，Li ZL(李占林)，Wang Y(王宇)，et al.Studies on chemical constituents from Euonymus alatusⅡ.Chin J Chin Mater Med(中国中药杂志)，2008，33:1422-1424.

7 Ito J，Chang F，Wang H，et al.Anti-AIDS agents.48.anti-HIV activity of moronic acid derivatives and the new melliferone-related triterpenoid isolated from Brazilian propolis.J Nat Prod，2001，64:1278-1281.

8 Prachayasittikul S，Buraparuangsang P，Worachartcheewan A，et al.Antimicrobial and antioxidative activities of bioactive constituents from Hydnophytum formicarum Jack.Molecules，2008，13:904-921.

9 Lin Y，Wang W，Kuo Y，et al.Nonsteroidal constituents from Solanum incanum L..J Chin Chem Soc，2000，47:247-251.

10 Master HE，Khanb SI，Poojaria KA.Design and synthesis of low molecular weight compounds with complement inhibition activity.Bioorg Med Chem，2005，13:4891-4899.