English

• 1.   Introduction

  Nicotiana tabacum tobacco is a stout herbaceous plant,and it is cultivated worldwide as the primary commercial source of tobacco ^{[1, 2]}. In addition,N. tabacum is also used as insecticide,anesthetic,diaphoretic,sedative,and emetic agents in Chinese folklore medicine because it contains many useful chemical compounds ^{[2, 3, 4]}. Previous phytochemical studies on this plants have shown the presence of sesquiterpene ^{[5, 6]},alkaloids ^{[7, 8]},lignans ^{[9, 10]},flavonoids ^{[11, 12, 13]},phenylpropanoids ^{[14, 15]},chromanones ^{[16, 17]},biphenyls ^[18],isocoumarins ^[19],and the homologous compounds. Therefore,the multipurpose utilization of this plant is an interesting topic and attracts more and more attention. Motivated by the possibility of the existence of more bioactive metabolites from this plant,an investigation on the chemical constituents of the tobacco leaves of K-326 (a variety of N. tabacum) was carried out. As a result,two new (1 and 2) and three known (3-6) sesquiterpenes were isolated from this plant. Compounds 1 and 2 are the first naturally occurring pterosin-type sesquiterpene bearing an isopropyl moiety. In addition,the anti-tobacco mosaic virus (anti-TMV) activity of compounds 1-6 was evaluated. This article describes the isolation,structural elucidation and the anti-TMV activity of these sesquiterpenes.

  2.   Experimental

  Optical rotations were measured with a Horiba SEPA-300 polarimeter; UV spectra were obtained using a Shimadzu UV- 2401A spectrophotometer. ECD spectra were measured on a JASCO J-810 spectropolarimeter. A Tenor 27 spectrophotometer was used for scanning IR spectra with KBr pellets. 1D and 2D NMR spectra were recorded on a DRX-500 NMR spectrometer using Trimethylsilane (TMS) as an internal standard. Chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HRESIMS was performed on a VG Autospec-3000 spectrometer. Semipreparative HPLCwas performed on a Shimadzu LC-8A preparative liquid chromatography machine using Zorbax PrepHT GF (21.2 mm × 25 cm) or Venusil MP C18 (20 mm × 25 cm) columns. Column chromatography (CC) was performed using silica gel (200-300 mesh,Qingdao Marine Chemical,Inc.,China),Lichroprep RP-18 gel (40-63 μm,Merck,Germany),and MCI gel (75-150 μm,Mitsubishi Chemical Co.,Japan). Fractions were monitored by TLC analysis,and spots were visualized by heating silica gel plates sprayed with 5% H₂SO₄ in EtOH.

  2.1   Plant material

  The leaves of N. tabacum L (tobacco leaves) were collected from Honghe County,Yunnan Province,China,in September 2013. The tobacco is K326,which is widely cultivated in China. The identification of the plant material was verified by Prof. H. W. Yang (School of Tobacco,Yunnan Agriculture University).

  2.2   Extraction and isolation

  The air-dried and powdered leaves of N. tabacum (4.8 kg) were extracted with 70% aqueous acetone (3 h × 8 L × 4) at room temperature and filtered. The extract (242.5 g) was applied to silica gel (200-300 mesh) column chromatography,eluted with a CHCl₃-CH₃OH gradient system (10:0,9:1,8:2,7:3,6:4,5:5),to give six fractions A-F. Further separation of fraction B (9:1,28.4 g) by silica gel column chromatography,eluting with CHCl₃ -(CH₃)₂CO (9:1-2:1),yielded mixtures B1-B6. Fraction B2 (8:2,4.86 g) was subjected to silica gel column chromatography using petroleum ether-acetone and semi-preparative HPLC (50-56% MeOH-H2O,flow rate 12 mL/min) to give 1 (12.4 mg),2 (10.8 mg),3 (15.2 mg),4 (11.7 mg),5 (14.6 mg) and 6 (16.4 mg).

  Compounds 1 and 2 were identified as new compounds. The known compounds,by comparing with the published literature,were identified as glutinosone (3) ^[20],capsidiol (4) ^[21],1-β- α-cyperone (5) ^[22],and arundinol B (6) ^[23]. The structures of the compounds 1-6 were shown in Fig. 1,and the ¹H NMR and ¹³C NMR data of 1 and 2 were listed in Table 1.

  图 1

  

  图 1  The structures of sesquiterpenes from Nicotiana tabacum.

  Figure 1.  The structures of sesquiterpenes from Nicotiana tabacum.

  下载: 全尺寸图片 幻灯片

  表 1

  

  表 1  ¹H NMR and ¹³C NMR data for compounds 1 and 2 (500 and 125 MHz, in CD₃OD).

  Table 1.  ¹H NMR and ¹³C NMR data for compounds 1 and 2 (500 and 125 MHz, in CD₃OD).

  下载: 导出CSV

  Nicosesquiterpene A (1): Pale yellow gum; obtained as light yellow gum,[α]_D^25:8 + 14.8 (c 0.20,MeOH); UV (MeOH) λmax (log ε): 285 (3.20),248 (3.68),210 (4.18) nm; CD (MeOH,c 0.020) λ_max (△ε) 326.8 (+18.2) nm; IR (KBr,cm^-1): ν_max 3368,1685,1600,1536,1472,1358,1203,1070,891,562; ¹³C NMR (125 MHz,CDCl₃,Fig. S1 in Supporting information) and ¹H NMR (500 MHz,CDCl₃,Fig. S2 in Supporting information) data,see Table 1; negative ESIMS m/z 231 [M - H]^-; negative HRESIMS m/z 231.1379 [M - H]^- (calcd. for C₁₅H₁₉O₂ [M-H]^-,231.1385) (Fig. S5 in Supporting information).

  Nicosesquiterpene B (2): Light yellow gum,[α]_D^25.2+ 16.5 (c 0.20,MeOH); UV (MeOH) λ_max (log ε): 283 (3.25),248 (3.59),210 (4.07) nm; CD (MeOH,c 0.020) λ_max (△ε) 327.6 (+19.6) nm; IR (KBr) ν_max 3360,1683,1600,1542,1465,1347,1215,1058,890,575 cm^-1 ;¹³C NMR (125 MHz,CDCl₃,Fig. S7 in Supporting information) and ¹H NMR (500 MHz,CDCl₃,Fig. S8 in Supporting information) data,see Table 1; negative ESIMS m/z 231 [M-H]^-; negative HRESIMS m/z 231.1393 [M-H]^- (calcd. for C₁₅H₁₉O₂ [M-H]^-,231.1385) (Fig. S10 in Supporting information).

  2.3   Anti-TMV assays

  TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry of Yunnan Province,Yunnan Academy of Tobacco Science,China. The virus was multiplied in Nicotiana tabacum cv. K326 and purified as described. ^[24] The concentration of TMV was determined as 20 mg/mL with a UV spectrophotometer [virus concentration = (A₂₆₀×dilution ratio)/E_1cm^0.1% 260 nm]. The purified virus was kept at -20 ℃ and was diluted to 32 μg/mL with 0.01 mol/L PBS before use.

  Nicotiana glutinosa plants were cultivated in an insect-free greenhouse. N. glutinosa was used as a local lesion host. The experiments were conducted when the plants grew to the 5/6-leaf stage. The tested compounds were dissolved in DMSO and diluted with distilled H₂O to the required concentrations. A solution of equal concentration of DMSO was used as a negative control. The commercial antiviral agent ningnanmycin (purity >98%) was used as a positive control.

  For the half-leaf method ^[25],the virus was inhibited by mixing with the solution of the compounds. After 30 min,the mixture was inoculated on the left side of the leaves of N. glutinosa,whereas the right side of the leaves was inoculated with the mixture of DMSO solution and the virus as a control. The local lesion numbers were recorded 3-4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula:

  $ {\rm{Inhibition}}\;{\rm{rate}}\left( \% \right) = \left[{\frac{{C - T}}{C}} \right]{\rm{ \times }}100{\rm{\% }} $

  where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment.

  3.   Results and discussion

  3.1   Structure elucidation

  Compound 1 was obtained as a light yellow gum. The negative mode HRESIMS data showed a quasimolecular ion peak at m/z 231.1379 [M-H]^-,suggesting a molecular formula of C₁₅H₂₀O₂,and indicating the presence of 6 degrees of unsaturation in the molecule. The IR spectrum showed major absorption bands at 3368 cm^-1 (-OH),1685 cm^-1 (-C＝O),and 1600 cm^-1 (aromatic - C＝C-). The NMR data in Table 1 and spectra of HSQC (Fig. S3 in Supporting information) and HMBC (Fig. S4 in Supporting information) indicated that 1 has a pentasubstituted benzene ring,a methine,an oxidated methine,a carbonyl group,a isopropyl group,and three methyl group. The HMBC correlations (Fig. 2) of H-2 with C-1,C-8,C-13,C-9,and C-3; H-3 with C-1,C-2,C-4,C-8,C- 9,and C-13; H-4 with C-3; H-13 with C-1,C-2,and C-3,indicated that 1 should be a pterosin-type sesquiterpene.^[26] In addition,the ¹H NMR and ¹³C NMR data of 1 were similar to those of a known compound,(2R,3S)-pterosin C at C-1-C-9 and C-13-C-15. ^[26] The chemical shift differences resulted from the disappearance of a 2-hydroxyethyl signal and appearance of isopropyl group in 1. This indicated that the 2-hydroxyethyl group in (2R,3S)-pterosin C was converted into an isopropyl group in 1. The isopropyl group located at C-6 was supported by the HMBC correlations of H₆-11,12 with C-6,and of H-10 with C-5,C-6,and C-7. Moreover,three methyl groups located at C-2,C-5,and C-7 was supported by the HMBC correlations of H-4 with V-14; of H₃-13 with C-1,C-2,and C-3; of H₃-14 with C-4,C-5,and C-6; of H₃-15 with C-6,C-7,and C-8. The ROESY correlations (Fig. 3) of H-2/H-3,H-3/H-4,H-4/H3-14,H-10/ H₃-14,H-10/H₃-15 also supported the substituents positions in compound 1. Based on the above information,the planar structure of compound 1 was deduced.

  图 2

  

  图 2  The Key HMBC correlations of compounds 1 and 2.

  Figure 2.  The Key HMBC correlations of compounds 1 and 2.

  下载: 全尺寸图片 幻灯片

  图 3

  

  图 3  The Key ROESY correlations of compound 1.

  Figure 3.  The Key ROESY correlations of compound 1.

  下载: 全尺寸图片 幻灯片

  The configurations of the pterosin-type sesquiterpenes were obtained from the CD spectrum (Fig. S6 in Supporting informaion) ^[27]. The signs of the CD spectra for these types of compounds dependmainly on the configuration at the C-3position,regardless of the configuration at C-2 and the solvent ^[28]. Thus,3S and 3R pterosin sesquiterpenes exhibit positive and negative Cotton effects,respectively. A positive Cotton effect (△ε + 18.2)) for the CDdata of 1 at 326.8 nm indicated that the C-3 position is S. Therefore,the configuration of C-2 and C-3 in compounds 1 was deduced as 2R,3S. Thus,the structure of nicosesquiterpene A (1) was established.

  Nicosesquiterpene A (2) was also obtained as a light yellow gum,and assigned a molecular formula of C₁₅H₂₀O₂ by HRESIMS at m/z 231.1393 [M - H]^- (Fig. S10 in Supporting information),which indicated 6 degrees of unsaturation. The ¹H NMR and ¹³C NMR spectra of 2 (Table 1) display a pentasubstituted benzene ring,a methine,an oxidated methine,a carbonyl group,an isopropyl group,and three methyls,and these data were very similar to those of compound 1. The major differences were due to the substituents position variation on the benzene ring. The isopropyl group was located at C-6,and the two methyls were located at C-4 and C-7,respectively,by further analysis of its HMBC correlations (Fig. S9 in Supporting informaion). In addition,the 2R,3S-configurations of C-2 and C-3 in compound 2 were also determined by the comparison of its specific rotation,NMR and CD (Fig. S11 in Supporting information) data with those of known compounds ^[28]. The structure of 2 is therefore determined.

  3.2   Anti-TMV assay

  Compounds 1-6 were tested for their anti-TMV activity. The inhibitory activity of compounds 1-6 against TMV replication was tested using the half-leaf method ^[25]. Ningnanmycin (a commercial product for plant disease in China),with an inhibition rate of 31.5%,was used as a positive control. The results showed that compounds 1 and 2 exhibited high anti-TMV activity with inhibition rates of 36.7% and 45.6%,respectively. These rates are higher than that of the positive control. The other compounds also showed potential activity with inhibition rates in the range of 22.7%-29.2% (Table 2).

  表 2

  

  表 2  TMV Infection inhibition activity of 1–6.

  Table 2.  TMV Infection inhibition activity of 1–6.

  下载: 导出CSV

  4.   Conclusion

  In this paper,two new sesquiterpenes,nicosesquiterpene A and B (1 and 2),along with four known sesquiterpenes derivatives (3-6) were isolated from the leaves of K-326 (a variety of Nicotiana tabacum). Compounds 1 and 2 are the first naturally occurring pterosin-type sesquiterpene bearing an isopropyl moiety because of their structural were elucidated by HRMS,IR,UV,1D and 2D NMR spectroscopy. In addition,the anti-tobacco mosaic virus (anti-TMV) activity of compounds 1-6 was evaluated. This article describes the isolation,structural elucidation and the anti-TMV activity of these sesquiterpenes.

  Appendix A. Supplementary data

  Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2016.01.048.

• 1. [1]

     Hu T.W., Mao Z., Ong M., Yurekli A.. China at the crossroads:the economics of tobacco and health[J]. Tob. Control, 2006, 15:  i37-i41.

  2. [2]

     Kuang K.R., Lu A.M.. Flora of China[J]. Beijing Science and Technology Press, 2005, :  .

  3. [3]

     Rodgman A., Perfetti T.A.. The Chemical Components of Tobacco and Tobacco Smoke, CRC Press, Taylor and Francis Group Boca Raton[J]. Florida, 2008, :  .

  4. [4]

     Miao M.M., Li L., Shen Q.P.. Anti-TMV activity flavones from the leaves of Yunnan local air cured tobacco[J]. Fitoterapia, 2015, 103:  260-264.

  5. [5]

     Chen Y.K., Meng C.Y., Su Z.B.. A new sesquiterpene from Nicotiana tabacum and its cytotoxicity[J]. Asian J. Chem., 2014, 22:  2246-2248.

  6. [6]

     Yang C.Y., Geng C.A., Huang X.Y.. Noreudesmane sesquiterpenoids from the leaves of Nicotiana tabacum[J]. Fitoterapia, 2014, 96:  81-87.

  7. [7]

     Wei X.C., Sumithran S.C., Deaciuc A.G.. Identification and synthesis of novel alkaloids from the root system of Nicotiana tabacum:affinity for neuronal nicotinic acetylcholine receptors[J]. Life Sci., 2005, 78:  495-505.

  8. [8]

     Shang S.Z., Duan Y.X., Zhang X.. Phenolic amides from the leaves of Nicotiana tabacum and their anti-tobacco mosaic virus activities[J]. Phytochem. Lett., 2014, 7:  413-416.

  9. [9]

     Chen Y.K., Li X.S., Yang G.Y.. Phenolic compounds from Nicotiana tabacum and their biological activities[J]. J. Asian Nat. Prod. Res., 2012, 14:  450-456.

  10. [10]

      Gao X.M., Li X.S., Yang X.Z.. Lignan derivatives from the leaves Nicotiana tabacum and their activities[J]. Heterocycles, 2012, 85:  147-153.

  11. [11]

      Chen J.X., Leng H.Q., Duan Y.X.. Three new flavonoids from the leaves of oriental tobacco and their cytotoxicity[J]. Phytochem. Lett., 2013, 6:  144-147.

  12. [12]

      Chen Z.Y., Tan J.L., Yang G.Y.. Isoflavones from the roots and stems of Nicotiana tabacum and their anti-tobacco mosaic virus activities[J]. Phytochem. Lett., 2012, 5:  233-235.

  13. [13]

      Zhao W., Li L., Lei P.. Three newflavones from the roots and stems of Tiandeng tobacco and their anti-TMV activities[J]. Phytochem. Lett., 2015, 12:  125-129.

  14. [14]

      Leng H.Q., Chen J.X., Hang Y.. Two new phenylpropanoids from the leaves of oriental tobacco and their cytotoxicity[J]. Chem. Nat. Compd., 2014, 49:  1028-1290.

  15. [15]

      Tan J.L., Yang G.Y., Miao M.M.. Two new benzofuranylpropanoids from Nicotiana tabacum and their anti-tobacco nosaic virus activities[J]. Heterocycles, 2011, 83:  2381-2385.

  16. [16]

      Mou D.R., Zhao W., Zhang T.. Two new chromanone derivatives from the roots and stems of Nicotiana tabacum and their cytotoxicity[J]. Heterocycles, 2012, 85:  2485-2490.

  17. [17]

      Yang G.Y., Zhao W., Zhang T.. Chromone derivatives from the leaves of Nicotiana tabacum and their anti-tobacco mosaic virus activities[J]. Heterocycles, 2014, 89:  183-188.

  18. [18]

      Shang S.Z., Xu W.X., Lei P.. Biphenyls from Nicotiana tabacum and their antitobacco mosaic virus[J]. Fitoterapia, 2014, 99:  35-39.

  19. [19]

      Shang S.Z., Xu W.X., Li L.. Phenolic amides from the leaves of Nicotiana tabacum and their anti-tobacco mosaic virus activities[J]. Phytochem. Lett., 2015, 11:  53-56.

  20. [20]

      Burden R.S., Bailey J.A., Vincent G.G.. A new antifungal sesquiterpene from Nicotiana glutinosa infected with tobacco mosaic virus[J]. Phytochemistry, 1975, 14:  221-223.

  21. [21]

      Bailey J.A., Burden R.S., Vincent G.G.. Capsidiol:an antifungal compound produced in Nicotiana tabacum and Nicotiana clevelandii following infection with tobacco necrosis virus,[J]. Phytochemistry, 1975, 14:  597,582.

  22. [22]

      Sanz J., Marco J.. Sesquiterpene lactones from Artemisia caerulescens Subsp, Gargantae[J]. Phytochemistry, 1990, 29:  2913-2917.

  23. [23]

      Luo J., Liu X.Y., Li E.W.. Arundinols A-C and arundinones A and B from the plant endophytic fungus Microsphaeropsis arundinis[J]. J. Nat. Prod., 2013, 76:  107-112.

  24. [24]

      Zhou M., Miao M.M., Du G., Aspeverin X.N.. Aspergillines A-E, highly oxygenated hexacyclic indole-tetrahydrofuran-tetramic acid derivatives from Aspergillus versicolor[J]. Org. Lett., 2014, 16:  5016-5019.

  25. [25]

      Hu Q.F., Zhou B., Huang J.M.. Antiviral phenolic compounds from Arundina gramnifolia[J]. J. Nat. Prod., 2013, 76:  292-296.

  26. [26]

      Fukuoka M., Yoshihira K., Natori S.. C-13 nuclear magnetic-resonance spectra of pterosin-sesquiterpenes and related indan-1-one derivatives[J]. Chem. Pharm. Bull., 1983, 31:  3113-3128.

  27. [27]

      Kuroyanagi M., Fukuoka M., Yoshihira K.. Further characterization of pterosins and pterosides, sesquiterpenes and the glucosides having 1-indanone skeleton, from the rhizomes[J]. Chem. Pharm. Bull., 1979, 27:  592-601.

  28. [28]

      Kuroyanagi M., Fukuoka M., Yoshihira K.. Circular-dichroism and conformations of pterosins, 1-indanone derivatives from bracken[J]. Chem. Pharm. Bull., 1979, 27:  731-741.

• 

  Figure 1  The structures of sesquiterpenes from Nicotiana tabacum.

  下载: 全尺寸图片 幻灯片
  

  Figure 2  The Key HMBC correlations of compounds 1 and 2.

  下载: 全尺寸图片 幻灯片
  

  Figure 3  The Key ROESY correlations of compound 1.

  下载: 全尺寸图片 幻灯片

  Table 1.  ¹H NMR and ¹³C NMR data for compounds 1 and 2 (500 and 125 MHz, in CD₃OD).

  下载: 导出CSV

  Table 2.  TMV Infection inhibition activity of 1–6.

  下载: 导出CSV
•