Liparis nervosa (Thunb. ex A. Murray) Lindl. is widely used in folk medicine for detoxicating and hemostatic function . Plants belonging to the genus Liparis,are herbaceous plants widely distributed in China. Previous phytochemical and biological investigations of L. nervosa led to the isolation of a series of pyrrolizidine alkaloids,nervogenic acid derivatives,triterpenoids,steroids and flavonoids [2, 3, 4, 5, 6, 7]. Notably,numerous pyrrolizidine alkaloids with significant anti-inflammatory activity were isolated .
In our ongoing study to find active natural products,chemical study was carried out on the ethanol extract of L. nervosa,and obtained three new pyrrolizidine alkaloids,nervosine Ⅶ (1),nervosine Ⅷ (2) and nervosine Ⅸ (3) (Fig. 1). Their structures and absolute configurations were elucidated by use of various spectral methods (IR,HR-ESI-MS,1D and 2D NMR) and chemical methods. All the compounds were evaluated for their cytotoxic activity against A549,MCF-7 and H460 human cancer cell lines. Herein,we report the isolation and structural elucidation of these compounds,as well as their cytotoxicities.
2. Experimental 2.1. General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. 1D and 2D NMR spectra were recorded on a Bruker AV 600 NMR spectrometer,and IR spectra on a ThermoFisher Nicolet 6700 spectrometer (KBr discs,cm-1 [2TD$DIF]). HR-ESI-MS were carried out on a Q-TOF micro mass spectrometer (Waters,USA). Silica gel (Qingdao Haiyang Chemical Co.,Ltd.,China). TLC plates precoated with silica gel GF254 (Qingdao Haiyang Chemical Co.,Ltd.,China) were visualized under a UV lamp at 254 nm or by spraying the Dragendorff’s reagent or by iodine.2.2. Plant material
The whole plants of L. nervosa were collected in Zunyi,Guizhou Province,China in July 2014. The plant was identified by Professor Liang-Ke Song in School of Life Science and Engineering,Southwest Jiaotong University,Sichuan,China,where a voucher specimen is deposited (No. ZN361520140801).2.3. Extraction and isolation
The whole plants of L. nervosa (14 kg) were extracted with 95% ethanol at room temperature (50 L × 3,each 7 days). After removal of the solvent by evaporation,the ethanol extract (750 g) was recovered. The extract was then suspended in H2O (2 L) and extracted successively with petroleum ether (60-90 ℃) (1 L × 4),EtOAc (1 L × 4),and n-butanol (1 L × 4) to obtain the petroleum ether extract (260 g),EtOAc extract (160 g) and n-butanol extract (180 g) successively.
The EtOAc extract (120 g) was subjected to a silica gel (100- 200 mesh) column eluted in a step gradient manner with CH2Cl2:CH3OH (50:1-0:1) to afford fractions (A-F) based on TLC analysis. Fraction E (7 g) was subjected to silica gel column,eluted with petroleum ether:Me2CO:Et2N (20:1:1) to yield compounds 1 (80 mg),2 (50 mg) and 3 (3 mg).
Nervosine Ⅶ (1): Amorphous solid; [α]D20 +23.0 (c 0.860 CHCl3); IR (KBr,cm-1): νmax 3411,2966,2921,1709,1600,1452,1383,1309,1258,1245,1184,1102,992,770. 1H NMR and 13C NMR data see Table 1. HR-ESI-MS at m/z 398.2710 [M + H]+ (calcd. for C25H36NO3,398.2695).
Nervosine Ⅷ (2): Amorphous solid; [α]D20 -7.6 (c 0.696 CHCl3); IR (KBr,cm-1): νmax 3403,2964,2918,2951,1711,1601,1452,1375,1308,1284,1245,1184,1098,905,770. 1H NMR and 13C NMR data see Table 1. HR-ESI-MS at m/z 398.2703 [M + H]+ (calcd. for C25H36NO3,398.2695).
Nervosine Ⅸ (3): Amorphous solid; [α]D20 -13.6 (c0.174 CHCl3); IR (KBr,cm-1): νmax 3418,2960,2918,2950,1714,1641,1602,1463,1375,1361,1310,1284,1247,1193,1166,1124,1101,1056,991,953,905,769,726. 1H NMR and 13C NMR data see Table 1. HR-ESI-MS atm/z 396.2536 [M + H]+ (calcd. for C25H34NO3,396.2539).
Lindelofidine: [α]D20 +70.2 (c 0.600,EtOH); HR-ESI-MS at m/z 142.1223 [M + H]+ (calcd. for C8H16NO,142.1232).
Laburnine: [α]D20+12.3 (c 0.025,EtOH); HR-ESI-MS at m/z 142.1248 [M + H]+ (calcd. for C8H16NO,142.1232).2.4. Alkaline hydrolysis of compounds 1 and 2
A solution of compound 1 (30 mg) in 0.5 mL of MeOH was refluxed with 1 mol/L NaOH (0.5 mL) for 1 h. After cooling,the reaction mixture was acidified with 1 mol/L HCl and extracted with CHCl3 (3 × 4 mL). The aqueous solution was made alkaline with 1 mol/L NaOH and extracted with CHCl3 (3 ×4mL),evaporation of the organic phase yielded lindelofidine (8.0 mg) . The process was repeated for compound 2 (40 mg) to get laburnine (10 mg).2.5. Cell lines and cell culture and cytotoxicity assay
A549 (non-small-cell lung cancer,NSCLC),MCF-7 (human breast cancer),H460 (human lung adenocarcinoma) were obtained from ATCC. Proliferation inhibition of compounds 1-3 against A549,MCF-7 and H460 cells was measured by the MTS method [4, 5]. Cells were plated in 96-well plates 24 h before treatment and continuously exposed to different concentrations of compounds for 72 h. DMSO (0.1% v/v) were used as negative controls. Adriamycin was used as the positive control.3. Results and discussion
Compound 1 was amorphous solid. Its molecular formula was determined as C25H35NO3 by analysis of its HR-ESI-MS spectrum (m/z 398.2710 [M + H]+,calcd. for 398.2695). The IR spectrum of 1 indicated the presence of hydroxy group (3411 cm-1) and carbonyl group (1709 cm-1) and an aromatic ring (1600 and 770 cm-1). The 1H NMR spectrum (Table 1) displayed a singlet at δ d 7.64 (s,2H) integrating for two aromatic protons. In addition,signals due to two prenyl groups were present,namely,δ 5.30 (t,2H,J = 6.8 Hz),δ 3.34 (d,4H,J = 6.8 Hz),and two singlets at δ 1.73 (s,6H) and δ 1.74 (s,6H) integrating together for 12 protons . In the 13C NMR spectrum (Table 1),the signal at δ 121.4 (s,C-1'),129.3 (d,C-2' and 6'),127.9 (s,C-3' and 5'),157.9 (s,C-4') and 166.8 (s,C-7') showed the presence of a nervogenic acid unit [4, 5, 6]. The 13C NMR (DEPT) spectra displayed the rest eight carbon signals at δ 40.7 (d,C-1),27.2 (t,C-2),54.0 (t,C-3),55.9 (t,C-5),26.6 (t,C-6),26.5 (t,C-7),66.2 (d,C-8),64.8 (t,C-9). Comparison of the 1H NMR and 13C NMR data (Table 1) of 1 with those reported for nervosine I ,suggested that 1 possessed a lindelofidine moiety. The NOESY cross-peak between H-2α/H-9 and H-7α/H-9,indicating the cisconfiguration for these protons. Alkaline hydrolysis gave the necine base,its molecular formula is determined as C8H15NO by HR-ESI-MS atm/z 142.1223 ([M + H]+,calcd. for 142.1232),and the optical rotation [α]D20 + 70.2 (c 0.348,EtOH),which was in good agreement with that of lindelofidine [α]D20+ 75.0 (c 3.2,EtOH) . In addition,HMBC correlations (Fig. 2) from H-9 to the carbonyl carbon (C-7') and to C-1,C-2,and C-8 indicated the linkage of nervogenic acid and lindelofidine moieties at C-9. Thus,structure 1 was confirmed as nervogenic acid ester of lindelofidine,named nervosine Ⅶ.
Compound 2,an amorphous solid,showed the same molecular formula as compound 1. The NMR spectroscopic data (Table 1) of compound 2 were comparable to those of 1,which means compound 2 also possessed one nervogenic acid unit and one 1- hydroxymethylpyrrolizidine unit. In the 1H NMR spectrum (Table 1) of 2,the signal of H-1 was shifted upfield about △δ 0.57. In the 13C NMR spectrum,the signal of C-1,C-2,C-7,C-8 and C-9 were shifted downfield about △δ 4.6,3.7,5.4,2.2 and 2.1,respectively. The major difference between compounds 1 and 2 was the absolute configuration of 1-hydroxymethylpyrrolizidine unit. The alkaloid base was estimated as laburnine by comparison of the NMR data of the pyrrolizidine part of 2 with nervosine Ⅱ  and the positive NOEs between the protons at H-9/H-8,H-1/H-7α,indicating the α-orientation of H-1,and the β-orientation of H-8 . Confirmatory evidence was provided by alkaline hydrolysis to get the necine base,its molecular formula was determined as C8H15NO by HR-ESI-MS at m/z 142.1248 (calcd. for C8H16NO,142.1232). Its optical rotation [α]D20 + 12.3 (c 0.435,EtOH) was in good agreement with that of laburnine [α]D20+ 14.6 (c 3.2,EtOH) [8, 9]. In addition,the position of the ester attachment at C-9 was determined by the HMBC signal between the proton H-9 to the carbon atom C-7' (Fig. 2). Thus,structure 2 was confirmed as nervogenic acid ester of laburnine,named nervosine Ⅷ.
Compound 3 was assigned the molecular formula of C25H33NO3 from the pseudomolecular ion peak at m/z 396.2536 [M + H]+ (calcd. for C25H34NO3,396.2539) in HR-ESI-MS. Its 1H NMR spectrum (Table 1) showed signals of two aromatic protons [δ 7.67 (br.s,1H),δ 7.52 (br.s,1H)],two cis-coupled olefinic protons [δ 6.35 (d,1H,J = 9.6 Hz),δ 5.64 (d,1H,J = 9.6 Hz)],one prenyl group [δ 5.27 (t,1H,J = 7.8 Hz),δ 3.28 (d,2H,J = 7.8 Hz),δ 1.73 (s,6H)] and two quaternary methyl groups δ 1.44 (6H,s). The 13C NMR spectrum of 3 (Table 1) gave the signals due to one carboxyl carbon (δ 166.5),one oxygenated aromatic carbon δ 154.8),five carbons for a prenyl group (d 132.5,122.0,28.1,25.8,17.9),two olefinic carbons (δ 131.0,122.1),one oxygenated carbon (δ 77.2). Comparison of the NMR data (Table 1) of compound 3 and nervosine Ⅵ  revealed that 3 also possessed one 2,2-dimethyl-6- carboxyl-8-prenylchromene unit and one 1-hydroxymethylpyrrolizidine unit,the main difference between two compounds was the absolute configuration of 1-hydroxymethylpyrrolizidine unit. The alkaloid base was estimated as laburnine by comparison of the NMR data of the pyrrolizidine part of 3 with those of compound 2 and NOEs between the protons at H-9/H-8,and H-1/H-7α. Thus,the structure of compound 3 was elucidated as 2,2-dimethyl-6- carboxyl-8-prenylchromene ester of laburnine,named nervosine Ⅸ.
Compounds 1-3 were tested for their in vitro cytotoxicities against A549,MCF-7 and H460 human cell lines by the MTS method,as described previously in the literature [4, 5]. However,all compounds did not show significant inhibitory activity against the tumor cells used (IC50 > 100 μmol/L,n = 3).4. Conclusions
Three new pyrrolizidine alkaloids,nervosine Ⅶ (1),nervosine Ⅷ (2) and nervosine Ⅸ (3) were isolated from the whole plant extract of L. nervosa. Their structures were elucidated by extensive spectroscopic examinations. In a cytotoxity assay,all tested compounds displayed no cytotoxicity against three cancer cell lines (A549,MCF-7 and H460).
|||Y.X. Hua, S.F. Liu, Z.Q. Yang. . The Chinese Herbal, vol. 12 (1999) p997|
|||K. Nishikawa, Y. Hirata. Chemotaxonomical alkaloid studies. I. Structure of nervosine. Tetrahedron Lett. 27 (1967) 2591–2596|
|||K. Nishikawa, M. Miyamura, Y. Hirata. Chemotaxonomical alkaloid studies structures of Liparis alkaloids. Tetrahedron 25 (1969) 2723–2741|
|||S. Huang, X.L. Zhou, C.J. Wang, H.Y. Wang, Y.S. Wang, L.H. Shan, J. Weng. New nervogenic acid derivatives from Liparis nervosa. Planta Med. 79 (2013) 281–287|
|||S. Huang, X.L. Zhou, C.J. Wang, Y.S. Wang, X. Feng, L.H. Shan, Z.Y. Guo, J. Weng. yrrolizidine alkaloids from Liparis nervosa with inhibitory activities against LPSinduced NO production in RAW264.7 macrophages,. Phytochemistry 93 (2013) 154–161|
|||S. Huang, M.F. Pan, X.L. Zhou, Z.L. Zhou, C.J. Wang, L.H. Shan, J. Weng. Five new nervogenic acid derivatives from Liparis nervosa. Chin. Chem. Lett. 24 (2013) 734–736|
|||Y. Zhao, S.N. Hu, C.H. Wang, Y.M. Zheng, X.Y. Xu. Chemical constituents from Liparis nervosa. Chin. Tradit. Herb. Drugs 44 (2013) 2955–2959|
|||S. Mohanraj, P.S. Subramanian, W. Herz. Minor alkaloids of Heliotropium curassavicum. Phytochemistry 21 (1982) 1775–1779|
|||S. Ravi, A.J. Lakshmanan. Neo coramandaline, a pyrrolizidine alkaloid from Cynoglossum furcatum,. Indian J. Chem. Sect. B. 39B (2000) 80–82|