HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 12, 2014, pp. 2771-2776
Received, 16th October, 2014, Accepted, 6th November, 2014, Published online, 17th November, 2014.
DOI: 10.3987/COM-14-13108

■ Flavonoids from the Leaves of Nicotiana tabacum and Their Anti-Tobacco Mosaic Virus Activity

Yinke Li, Yingliang Zhao, Nengjun Xiang, Liu Yang, Fei Wang, Guangyu Yang, and Zhengyin Wang*

College of Resources and Environmental, Southwest University, NO.2 Rd Tiansheng, Beibei District,Chongqing, 400716, China

Abstract

Three new flavonoids, 8-formyl-4'-hydroxy-7-methoxy-6-methyl- flavone (1), 8-formyl-4',7-dimethoxy-6-methylflavone (2), 4',6-dihydroxy- 7-methoxy-8-methoxycarbonylflavone (3) together with two known flavonoids (4 and 5) were isolated from the leaves of Nicotiana tabacum. Their structures were determined by means of HRESIMS and extensive 1D and 2D NMR spectroscopic studies. Compounds 1-3 were tested for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compounds 1-3 exhibited inhibition rates (%) of 18.4, 22.1 and 16.4, respectively.

Nicotiana tabacum L. is the most commonly grown of all plants in the Nicotiana genus, and its leaves are commercially grown in many countries to be processed into tobacco.1,2 In addition, N. tabacum is also used as insecticide, anesthetic, diaphoretic, sedative, and emetic agents in Chinese folklore medicine because of it containing many useful chemical compounds.1,3-5 In previous work, a number of bioactive compounds, such as terpenoids,6-8 alkaloids,9,10 lignans,11,12 flavonoids,13 phenylpropanoids,14 and the homologous, were isolated from this plant. Therefore, the multipurpose utilization of flue-cured tobacco is an interesting topical and attracts more and more attentions.15,16 Motivated by search for more bioactive metabolites from this plant, an investigation on the chemical constituents of the leaves of Yunyan-200 (a variant of N. tabacum) was carried out. As a result, three new (1-3) and two known flavonoids (4 and 5) were isolated from this plant. In addition, the anti-tobacco mosaic virus (anti-TMV) activities of compounds 1–3 were evaluated. This article deals with the isolation, structural elucidation and biological activities of the new flavonoids.
A 70% aq. methanol extract prepared from the leaves of tobacco was subjected repeatedly to column chromatography on silica gel and preparative HPLC to afford three new flavonoids, 8-formyl-4'-hydroxy-7-methoxy-6-methylflavone (1), 8-formyl-4',7-dimethoxy-6-methylflavone (2), 4',6-dihydroxy-7-methoxy-8-methoxycarbonylflavone (3), and two known flavonoids (4 and 5). The structures of the compounds 1-5 were as shown in Figure 1, and the 1H and 13C NMR data of 1-3 were listed in Table 1. The known compounds, compared with literature, were identified as kaempferol (4)17 and quercetin (5).17

Compound 1 was obtained as an orange-yellow gum. It has the molecular formula C18H14O5 from HRESIMS (m/z: 333.0732 [M+Na]+, calcd 333.0739). The 1H and 13C NMR spectra of 1 (Table 1) along with analysis of the DEPT spectra displayed 18 carbon signals and 8 hydrogen signals for 14 protons, respectively, corresponding to a pentasubstituted benzene ring (δC 137.0 d, 120.2 s, 166.1 s, 113.8 s, 154.0 s, and 119.0 s; δH 7.69 s), a 1,4-disubstitued benzene ring [δC 122.8 s, 131.3 d (2C), 116.4 d (2C), and 158.2 s; δH 7.87 (d) and 6.88 (d)], a pair of double bond (δC 163.5 s and 107.0 d; δH 6.58 s), a carbonyl group (δC 178.8), one aldehyde group (δC 191.1; δH 10.23), one methyl group (δC 16.2; δH 2.32), one methoxy group (δC 61.0; δH 3.83), and one low-field hydroxy proton (δH 11.09). These NMR data together with the HMBC correlations (Figure 2) of H-3 (δH 6.58 s) with C-4 (δC 178.8), C-10 (δC 119.0), and C-1′ (δC 122.8) suggested that 1 should be a flavone.17

The HMBC of correlations of the hydroxy proton signal (δH 11.09) with C-3′,5′ (δC 116.4) and C-4′ (δC 158.2), suggested the attachment position of the hydroxy group at C-4′. Since the substituents on ring C were evident, the surplus substituents (one aldehyde group, one methyl group, and one methoxy group) should be located at ring B. The HMBC correlations of aldehyde proton signal (δH 11.09) with C-7 (δC 166.1), C-8 (δC 113.8), and C-9 (δC 154.0) suggested the placement of the aldehyde group at C-8. The HMBC correlations of methyl proton signal (δH 2.32) with C-5 (137.0), C-6 (120.2), and C-7 (166.1) suggested that the methyl group located at C-6. The methoxy group located at C-7 was also supported by the HMBC correlation of the methoxy proton signal (δH 3.83) with C-7 (δC 166.1). Thus, the structure of 1 was established as 8-formyl-4'-hydroxy- 7-methoxy- 6-methylflavone.

Compounds 2 was obtained as orange-yellow gum, and showed a quasi-molecular ion peak at m/z 347.0888 [M+Na]+ in the HRESIMS (calcd m/z 347.0895), corresponding to the molecular formula of C19H16O5. The 1H and 13C NMR spectra of 2 were very similar to these of 1. The only difference was due to a hydroxy group in 1 was substituted by a methoxy group in 2 on the aromatic rings. This was supported by the disappearance of a phenolic proton signal and appearance of methoxy group signals in NMR spectrum of 2. The HMBC correlations of two methoxy proton signals (3.83 s and 3.81 s) with C-4′ (δC 161.0) and C-7 (δC 166.4) indicated that two methoxy groups were located at C-4′ and C-7 respectively. The down-shift of 13C NMR signal of C-4′ from δC 158.2 ppm to δC 161.0 ppm also suggested that the substituent group should be varied at C-4′. Therefore, the structure of 8-formyl-4',7- dimethoxy-6-methylflavone (2) was assigned as shown.
Compound 3 was also obtained as an orange-yellow gum. It was assigned the molecular formula C18H14O7 by its HRESIMS at m/z 365.0637 [M+Na]+. The 1H and 13C NMR spectra of 3 are also similar to those of compound 1. Analysis of the 1H and 13C NMR spectral data of 3 with those of 1 suggested that the difference was due to the disappearance of aldehyde group signals (δC 191.1; δH 10.23), methyl group signal (δC 16.2 s; δH 2.32 s) and appearance of methoxycarbonyl group signals (δC 168.7, 52.8; δH 3.97), and a phenolic hydroxy group signal (δH 11.29 s) in 1. These indicated that the aldehyde group at C-8 and methyl group at C-6 in 1 were substituted by a methoxycarbonyl and a phenolic hydroxy group in 3. In HMBC spectrum, the correlations of one phenolic hydroxy signal (δH 11.29 s) with C-5, C-6, and C-7, and the correlations of the other phenolic hydroxy signal (δH 11.07) with C-3′,5′ and C-4′ indicated that two phenolic hydroxy groups were located at C-6 and C-4′ respectively. A methoxy group located at C-7 was supported by the HMBC correlation of methoxy proton signal (δH 3.97 s) with C-7. The observation of a HMBC correlation of H-5 with C-4 suggested the methoxycarbonyl group should be located at C-8. Accordingly, the structure of 4',6-dihydroxy-7-methoxy-8-methoxycarbonylflavone (3) was established.
Since certain of the flavonoid derivatives exhibit potential anti-tobacco mosaic virus (anti-TMV) activities,18-21 compounds 1 and 2 were tested for their anti-tobacco mosaic virus activity. The anti-TMV activities were tested using the half-leaf method.22 Ningnanmycin (2% water solution), a commercial product for plant disease in China, was used as a positive control. The results showed that compounds 1-3 exhibited inhibition rates (%) of 18.4, 22.1 and 16.4, respectively.

EXPERIMENTAL
General. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. A Tenor 27 spectrophotometer was used for scanning IR spectroscopy with KBr pellets. 1D and 2D NMR spectra were recorded on DRX-500 spectrometers with TMS as internal standard, and the chemical shifts (δ) were expressed in ppm. HRESIMS was performed on an API QSTAR time-of-flight spectrometer and a VG Autospec-3000 spectrometer, respectively. Preparative HPLC was performed on a Shimadzu LC-8A preparative liquid chromatograph with a ZORBAX PrepHT GF (21.2 mm × 25 cm, 7 µm) column or a Venusil MP C18 (20 mm × 25 cm, 5 µm) column. Column chromatography was performed with Si gel (200–300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China). The fractions were monitored by TLC, and spots were visualized by heating Si gel plates sprayed with 5% H2SO4 in EtOH.
Plant material. The tobacco leaves were collected in Yuxi Prefecture, Yunnan Province, People’s Republic of China, in September 2013. The identification of the plant material was verified by Prof. Chen Y. J (Yunnan University of Nationalities).
Extraction and Isolation. The air-dried and powdered tobacco leaves (4.2 kg) were extracted three times with 70% aqueous MeOH (3 × 5.0 L) at room temperature and filtered to yield a filtrate, which was successively evaporated under reduced pressure to obtained a crude extract (410 g). This crude extract was applied to Si gel (200-300 mesh) column chromatography eluting with a CHCl3-MeOH gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5) to give six fractions A–F. The separation of fraction B (28.3 g) was subjected to Si gel column chromatography eluting with CHCl3-(Me)2CO and then run on preparative HPLC (50-55% MeOH-H2O, flow rate 12 mL/min) to yield compounds 1 (11.8 mg), 2 (12.6 mg), and 3 (14.5 mg). Fraction C (24.6 g) was subjected to Si gel column chromatography eluting with CHCl3- (Me)2CO and then run on preparative HPLC (40-45% MeOH-H2O, flow rate 12 mL/min) to yield compounds 4 (11.7 mg) and 5 (15.0 mg).
Anti-TMV Assays. The anti-TMV activities were tested using the half-leaf method,22 and Ningnanmycin (2% water solution), a commercial product for plant disease in China, was used as a positive control.
8-Formyl-4'-hydroxy-7-methoxy-6-methylflavone (1): C18H14O5, orange-yellow gum; UV (MeOH), λmax (log ε) 210 (4.28), 255 (3.89), 287 (3.48), 368 (3.67) nm; IR (KBr) νmax 3460, 1685, 1655, 1618, 1534, 1472, 1455, 1126, 1074, 985, 862 cm-1; 1H NMR and 13C NMR data (C5D5N, 500 and 125 MHz) see Table 1; HRESIMS m/z 333.0732 [M+Na]+ (calcd for C18H14NaO5, 333.0739).
8-Formyl-4',7-dimethoxy-6-methylflavone (2): C19H16O5, orange-yellow gum; UV (MeOH), λmax (log ε) 210 (4.28), 255 (3.89), 287 (3.48), 368 (3.67) nm; IR (KBr) νmax 3452, 1689, 1658, 1615, 1546, 1489, 1448, 1134, 1078, 986, 874 cm-1; 1H NMR and 13C NMR data (C5D5N, 500 and 125 MHz) see Table 1; HRESIMS m/z 347.0888 [M+Na]+ (calcd for C19H16NaO5, 347.0895).
4',6-Dihydroxy-7-methoxy-8-methoxycarbonylflavone (3): C18H14O7, orange gum; UV (MeOH), λmax (log ε) 210 (4.28), 258 (3.86), 372 (3.74), nm; IR (KBr) νmax 3418, 1708, 1659, 1612, 1547, 1483, 1446, 1292, 1165, 1058, 864, 785 cm-1; 13C NMR and 1H NMR data (in C5D5N, 500 and 125 MHz) see Table 1; HRESIMS m/z 365.0637 [M+Na]+ (calcd for C18H14NaO7, 365.0637).

ACKNOWLEDGEMENTS
This research was supported by the National Natural Science Foundation of China (No. 31360081), the Applied Fundamental Foundation of Yunnan Province (No. 2013FB097), and the Basic Research Foundation of Yunnan Tobacco Industry Co. Ltd (2012JC01).

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