2. 天津国际生物医药联合研究院中药新药研发中心, 天津 300457
2. Research and Development Center of Traditional Chinese Medicine, Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China
Nardostachys chinensis Batal. (NCB),a member of the genus Nardostachys (Valerianaceae),is distributed mainly in Sichuan,Gansu,and Qinghai provinces in China. The rhizome and root of NCB,also called “Gansong or Gansongxiang”,was used as an herbal drug in traditional Chinese medicine for centuries to elicit the stomachic and sedative effects[1]. “Gansong” is now also a well-known medicinal spice used in Chinese hotpot,especially numb and spicy hotpot originates in Songpan County,Sichuan Province in China[2].
In order to exploit natural 5-hydroxytryptamine transporter (SERT) regulators from traditional Chinese medical herbs,we’ve promoted a systematical study on the bioactive chemical constituents of NCB. In this report,isolation and purification of the phenolic compounds (Figure1),recently isolated from NCB,as well as the structural elucidation of these compounds based on their spectroscopic data and physicochemical properties was summarized.
Compound 1 was obtained as a white amorphous powder. The molecular formula of this compound was assigned to be C20H22O9,as deduced from its quasi- molecular ion peak at (-)-ESI-MS m/z 405.73 [M-H]- and NMR spectroscopic data (Table1).
In the 1H NMR spectrum of compound 1,the signals at δH 6.26 (1H,d,J = 16.2 Hz) and 7.46 (1H,d,J = 16.2 Hz) indicated the existence of a trans-form double bond in 1; the signals at δH 7.00 (1H,dd,J = 8.4,1.8 Hz),6.76 (1H,d,J = 8.4 Hz) and 7.04 (1H,br s),and the signals at δH 6.61 (2H,s) suggested respectively the presences of a 1,3,4-trisubstituted and a 1,3,4,5- tetrasubstituted benzene rings; and the signal at δH 3.73 (6H,s) indicated two magnetic equivalent methoxy groups of 1. The 13C NMR spectrum of compound 1 confirmed the above deduction and showed another one oxygenated methylene (δC 65.7) and two oxygenated methines (δC 73.6,72.9) in the more upfield area. Further analysis of the HSQC spectrum disclosed that δH 4.95 (1H,d,4.8),5.23 (1H,d,4.2),9.59 (1H,br s),8.17 (1H,br s) and 9.15 (1H,br s) were five active hydroxyl proton signals. The HMBC correlations (Figure2) from δH 7.46 (H-7) to δC 114.8 (C-2),121.3 (C-6),and 166.5 (C-9),from δH 4.46 (H-7') to δC 132.6 (C-1'),104.2 (C-2'/6') and 72.9 (C-8'),and from δH 3.73 to δC 147.5,indicated the presences of (E)-caffeoyl andsyringylglyceryl groups in the structure of compound 1. As was expected,the two groups came to forming an ester,confirmed by the HMBC correlation from δH 3.84 (H-9') to δC 166.5 (C-9). Considering the coupling constant between H-7' and H-8' was 4.3 Hz,the configuration between H-7' and H-8' should prefer an erythro-form[3, 4]. Thus,the structure of compound 1 was finally confirmed as (E)-erythro-syringylglyceryl caffeate.
Considering that the optical rotation ( -1.22 (c 0.60,MeOH) and CD spectra [λ (Δε): 203 (+7.32) nm,c 0.53,MeOH] of 1 was observed not significant and unstable,while the NMR spectra indicated there was no threo-conformer mixed in it,therefore,compound 1was deduced as a racemic mixture of two erythro- isomers with contrast absolute configurations. So,compound 1was finally confirmed as a mixture of (E)-7'R,8'R-syringylglyceryl caffeate and (E)-7'S,8'S- syringylglyceryl caffeate.
Another nine known compounds were elucidated as (+)-licarin A (2)[5],naringenin 4',7-dimethyl ether (3)[6],pinoresinol-4-O-β-D-glucoside (4)[7],caraphenol A (5)[8],Z-miyabenol C (6)[9],protocatechuic acid (7)[10],caffeic acid (8)[11],gallic acid (9)[8] and vanillic acid (10)[12] by comparing their spectroscopic data (including 1D/2D NMR,ORD and CD experiments) and physicochemical properties with the reported data. Furthermore,the relative configurations of the two resveratrol oligomers 5and 6 were confirmed on the basis of their NOESY/ ROESY spectra (Figure3 and 4) and ORD/CD properties. Among these nine known compounds,compounds 2,5 and 6 were isolated from Nardostachys genus for the first time.
All reagents were of HPLC or analytical grade. Column chromatography (CC) was performed over silica gel (SiO2; 200- 300 mesh),Sephadex LH-20,polyamide (80-100 mesh) and D101 macroporous resin; TLC was conducted on precoated silica gel plates GF254 (SiO2; 400-500 mesh). Preparative HPLC was performed using an Agilent Zorbax SB-C18 ODS column (21.2 mm × 250 mm,7 μm) at 10 mL·min-1. NMR spectra were recorded on Bruker AV-IIIspectrometer using TMS as the internal standard. UV and CD were recorded with a Jasco J-815 Circular Dichroism (CD) spectropolarimeter. Specific optical rotation values () were recorded with a Rudolph AUTOPOL V polarimeter. IR spectra were obtained on a Perkin Elmer Spectrum 65 FT-IR spectrometer. ESI-MS were measured on a Waters Quattro Premier XE mass spectrometer.
Plant materialThe dry roots and rhizomes of Nardostachys chinensis Batal. were purchased from Anhui Jiren Pharmacy Co.,Ltd.,P. R. China,in July,2011. The plant material was authenticated by Prof. Tian-xiang Li,Tianjin University of Traditional ChineseMedicine,P. R. China. A voucher specimen (No. B20604126) was deposited in the Traditional Chinese Medicine Research and Development Center,Tianjin University of Traditional Chinese Medicine.
Extraction and isolationThe air-dried underground parts (20 kg) of Nardostachys chinensis Batal. were extracted successively with cold and hot 70% EtOH. The 70% EtOH extract (3.4 kg) combined was then evaporated under reduced pressure and distributed in water before being partitioned with petroleum ether (PE),ethyl acetate (EA),and n-butanol (BU) successively. The BU fraction,combined with the fraction afforded by alcohol precipitation of the rest water solution,was chromatographed on D101 macroporous resin column,gradiently eluted with EtOH-H2O (0∶100- 95∶5) to obtain 5 fractions. Among them,the 95% EtOH fraction was added to the EA fraction.
The PE extract (320 g) was chromatographed on silica gel column,gradiently eluted with PE-EA (100∶0-0∶100) to obtain 22 fractions (PE.1-PE.22). Fractions PE.6-9 were then subjected to silica gel column gradiently eluted with PE-EA (100∶0-100∶30) respectively,and afforded compounds 3 (11.0 mg) and 2 (7.0 mg).
The EA extract (1.2 kg) was separated into 15 fractions (EA.1-EA.15) on a normal-phase silica gel column eluted with CHCl3-MeOH (from 100∶0 to 0∶100,v/v) solvent. Fraction EA.8 (38.8 g) was further purified by subjecting to polyamide column,Sephadex LH-20 column and HPLC chromatography to yield compound 7 (MeOH-H2O = 30∶70,tR = 9.18 min,350.0 mg) and 8 (MeOH-H2O = 22∶78,tR = 28.88 min,256.0 mg). Fraction EA.9 (10.8 g) was separated into seven subfractions (EA.9-1-EA.9-7) by polyamide column chromatographywith a stepwise gradient elution of MeOH-H2O (0∶100-100∶0). Subfraction EA.9-2 (0.9 g) was subjected to preparative HPLC (MeOH- H2O-HCOOH = 40∶60∶0.1) to yield compound 4 (tR = 20.11 min,25.0 mg). Subfraction EA.9-4 and EA.9-7 was then further isolated to yield compound 1 (tR = 60.30 min,9.4 mg),compound 5 (tR = 40.35 min,7.1 mg),compound 6 (tR = 20.11 min,7.4 mg) and compound 9(240.0 mg).
The 30% EtOH fraction (190 g) was chromatographed on D101 macroporous resin column,gradiently eluted with MeOH-H2O (10∶90-100∶0) to obtain 5 subfractions. The 10% MeOH subfraction (20 g) was then further isolated by combined Sephadex LH-20 (MeOH-H2O = 1∶1) column and preparative HPLC (MeOH-H2O = 8∶92) chromatography method to afford compound 10 (tR = 96.30 min,20.0 mg).
(E)-erythro-Syringylglyceryl caffeate (1): white amorphous powder; C20H22O9; -1.22 (c 0.60,MeOH); UV (MeOH) λmax (logε): 209 (3.13),331 (2.83) nm; CD (c 0.53,MeOH) λ (Δε): 203 (+7.32) nm; IR (KBr) νmax: 2 960,2 927,2 854,1 695,1 602,1 516,1 494,1 453,1 375,1 326,1 269,1 184,1 046,1 024,990,907,823,759 cm-1; 1H and 13C NMR see Table1; (-)-ESI-MS m/z 405.73 [M-H]-.
Caraphenol A (5): yellow amorphous powder; C42H28O9; +148.76 (c 0.51,MeOH); UV (MeOH) λmax (logε): 210 (3.33),267 (2.91),287 (3.20),308 (2.57),329 (2.55) nm; CD (c 0.55,MeOH) λ(Δε): 199 (+7.27),214 (+8.45),247 (+0.42),265 (+4.47),282 (+3.28),300 (+5.71) nm. 1H NMR (acetone-d6,400 MHz) δH: 7.25 (2H,d,J = 7.4 Hz,H-2c/6c),7.23 (2H,d,J = 7.4 Hz,H-2a/6a),7.05 (2H,d,J = 8.5 Hz,H-2b/6b),6.93 (1H,d,J = 1.6 Hz,H-14b),6.80 (2H,d,J = 7.4 Hz,H-3a/5a),6.79 (1H,J = 1.6 Hz,H-12b),6.74 (2H,d,J = 7.4 Hz,H-3c/5c),6.69 (2H,d,J = 8.5 Hz,H-3b/5b),6.54 (1H,br d,J = 1.6 Hz,H-14a),6.49 (1H,d,J = 2.0 Hz,H-12c),6.31 (1H,d,J = 2.0 Hz,H-14c),6.24 (1H,d,J = 1.6 Hz,H-12a),5.91 (2H,br s,H-7a/7b),4.85 (1H,br s,H-8b),4.33 (1H,br s,H-8a); 13C NMR (acetone-d6,100 MHz) δC: 163.6 (C-11c),160.6 (C-13c),160.0 (C-11a),159.1 (C-13a),158.3 (C-4c),158.11 (C-4b),158.05 (C- 4a),157.1 (C-13b),155.3 (C-11b),149.6 (C-7c),141.1 (C-9a),139.8 (C-9b),135.4 (C-9c),133.6 (C-1a),132.8 (C-1b),128.4 (C-2b/6b),128.3 (C-2c/6c),127.4 (C-2a/ 6a),122.94 (C-10a),122.87 (C-1c),120.7 (C-10b),119.2 (C-10c),116.2 (C-3c/5c),116.0 (C-3a/5a),115.8 (C-3b/ 5b),114.6 (C-8c),109.7 (C-14c),108.71 (C-14a),108.69 (C-14b),98.4 (C-12c),97.6 (C-12a),96.4 (C-12b),95.2 (C-7a),88.0 (C-7b),54.1 (C-8a),46.0 (C-8b). The key HMBC and NOESY correlations were showed in Figure3. (-)-ESI-MS m/z 675.25 [M-H]-.
Z-Miyabenol C (6): yellow amorphous powder; C42H32O9; +82.22 (c 0.45,MeOH); UV (MeOH) λmax (logε): 208 (3.23),286 (2.71) nm; CD (c 0.67,MeOH) λ (Δε): 220 (+1.81),234 (-0.44),251 (+3.53),273 (-0.29),286 (+0.30),312 (-0.56),342 (+0.15) nm. 1H NMR (DMSO-d6,600 MHz) δH: 7.00 (2H,d,J = 8.6 Hz,H-2a/6a),6.72 (2H,d,J = 8.6 Hz,H-3a/5a),6.69 (2H,d,J = 8.6 Hz,H-2c/6c),6.47 (2H,d,J = 8.6 Hz,H-3c/5c),6.46 (2H,d,J = 8.6 Hz,H-3b/5b),6.31 (2H,d,J = 8.6 Hz,H-2b/6b),6.28 (1H,d,J = 1.9 Hz,H-12c),6.16 (1H,d,J = 2.1 Hz,H-12b),6.10 (1H,t,J = 2.1 Hz,H-12a),6.06 (1H,d,J = 2.1 Hz,H-14b),5.99 (1H,d,J = 1.9 Hz,H-14c),5.88 (2H,d,J = 2.1 Hz,H-10a/14a),5.74 (2H,d,J = 2.0 Hz,H-7c,8c),5.31 (1H,J = 1.9 Hz,H-7b),5.23 (1H,d,J = 3.2 Hz,H-7a),4.22 (1H,d,J = 3.2 Hz,H-8a),3.78 (1H,d,J = 1.9 Hz,H-8b); 13C NMR (DMSO-d6,150 MHz) δC: 160.7 (C-11b),160.5 (C-11c),159.8 (C-13b),159.6 (C-11a/13a),158.6 (C-13c),157.7 (C-4a),157.2 (C-4c),156.8 (C-4b),147.0 (C-9a),142.4 (C-9b),135.9 (C-9c),133.2 (C-1a),132.2 (C-1b),130.8 (C-7c),130.2 (C-2c/6c),127.2 (C-1c),127.0 (C-2a/6a),125.9 (C-2b/6b),124.7 (C-8c),120.8 (C-10c),118.1 (C-10b),115.6 (C-3a/5a),115.3 (C-3b/5b/3c/5c),107.6 (C-14c),106.1 (C-14b),105.7 (C-10a/14a),101.5 (C-12a),96.5 (C-12c),95.6 (C-12b),92.7 (C-7a),90.9 (C-7b),55.8 (C-8a),51.6 (C-8b). The key HMBC and ROESY correlations were shown in Figure4. (-)-ESI-MS m/z 679.15 [M-H]-.
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