Anti-tobacco mosaic virus phenylpropanoids from the stems of Nicotiana tabacum

Phytochemistry Letters 14 (2015) 230–233

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Anti-tobacco mosaic virus phenylpropanoids from the stems of Nicotiana tabacum Guang-Hui Konga,b , Yu-Ping Wua , Jun-Li Shia , Neng-Jun Xiangb , Ling-Xuang Liub , Guo-Rong Yangb , Yin-ke Lic, Xiu-Ping Lua , Qiang Liub,* , Qiu-Fen Hua,c,* a

Yunnan Academy of Tobacco Agricultural Sciences, Kunming, Yunnan 650031, PR China Key Laboratory of Tobacco Chemistry of Yunnan Province, China Tobacco Yunnan Industrial Co., Ltd., Kunming 650231, PR China Key Laboratory of Chemistry in Ethnic Medicinal Resources, State Ethnic Affairs Commission & Ministry of Education, Yunnan Minzu University, Kunming 650031, PR China b c

A R T I C L E I N F O

A B S T R A C T

Article history: Received 27 July 2015 Received in revised form 24 October 2015 Accepted 27 October 2015 Available online xxx

Three new phenylpropanoids, 3-(6-methoxy-3-oxo-1, 3-dihydroisobenzofuran-5-yl)-3-oxopropyl acetate (1), 3-hydroxy-1-(6-methoxy-1, 3-dihydroisobenzofuran-5-yl)propan-1-one (2), and 3-hydroxy-1(6- methyl-1, 3-dihydroisobenzofuran-5-yl)propan-1-one (3), together with three known phenylpropanoids (4–6) were isolated from the stems of Nicotiana tabacum. Their structures were examined using different spectroscopic techniques, including extensive 1D- and 2D NMR. Compounds 1–6 were also evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The anti-TMV results demonstrated that compounds 1 and 6 exhibited high anti-TMV activities, with inhibition rates of 34.6 and 35.4%, both of which were higher than the positive control (32.5%). The other compounds (2–5) also had anti-TMV activities, with inhibition rates between 16.8 and 28.6%. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

Keywords: Nicotiana tabacum Phenylpropanoids Anti-tobacco mosaic virus activities

1. Introduction Nicotiana tabacum, a plant belonging to the Nicotiana genus of the Solanaceace family, is an economically important crop that is well known in the tobacco industry (Hu et al., 2006; Kuang and Lu, 2005). In addition to the leaves, the aerial part of the plant contains many active chemical compounds and is used for sedative, diaphoretic, anesthetic and emetic purposes (Kuang and Lu, 2005; Rodgman and Perfetti, 2008). Previous phytochemical investigations revealed that Nicotiana plants were rich in sesquiterpenes (Chen et al., 2014; Yang et al., 2013), alkaloids (Braumann et al., 1990; Wei et al., 2005), lignans (Chen et al., 2012a; Gao et al., 2012), flavonoids (Li et al., 2015; Miao et al., 2015; Chen et al., 2013, 2012b), phenylpropanoids (Leng et al., 2014; Tan et al., 2011), chromanones (Mou et al., 2012; Yang et al., 2014), biphenyls (Shang et al., 2014a), phenolic amides (Shang et al., 2014b), isocoumarins (Shang et al., 2015), and homologs. The roots and stems of N. tabacumare considered to be a byproduct of tobacco farming and are frequently used as organic fertilizers; the utilization of byproducts, such as roots and stems, is a topic that is

* Corresponding author. E-mail addresses: [email protected] (L.-X. Liu), [email protected] (Q.-F. Hu).

receiving more attention (Chen et al., 2012b; Mou et al., 2012; Shang et al., 2015). Our investigations into the chemical constituents of the tobacco stems from the K-326 variety of N. tabacum have led to the isolation several bioactive metabolites, including three new (1–3) and three known (4–6) phenylpropanoids. The isolation, structural elucidation, and anti-TMV activities of these six compounds are described in this manuscript. 2. Results and discussion A 70% aqueous acetone extract prepared from the stems of N. tabacum was repeatedly applied to chromatography columns containing Si gel, RP-18 and preparative HPLC to purify the three new phenylpropanoids, 3-(6-methoxy-3-oxo-1, 3-dihydroisobenzofuran-5-yl)-3-oxopropyl acetate (1), 3-hydroxy-1-(6-methoxy1, 3-dihydroisobenzofuran-5-yl) propan-1-one (2), and 3-hydroxy1-(6-methyl-1, 3-dihydroisobenzofuran- 5-yl) propan-1-one (3), together with the three known phenylpropanoids (4–6). The structures of compounds 1–6 are shown in Fig. 1, and the 1H and 13 C NMR data from compounds 1–3 are presented in Table 1. The three known compounds were identified as 3-hydroxy-1-(4hydroxy-3, 5-dimethoxyphenyl)-propan-1-one (4) (Jones et al., 2000), lanceolune B (5) (Hu et al., 2012a), and nicotpanoid A (Leng et al., 2014).

http://dx.doi.org/10.1016/j.phytol.2015.10.023 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

G.-H. Kong et al. / Phytochemistry Letters 14 (2015) 230–233

O

O O

9 8

7

MeO

1

5

2'

O 2

4

3

O

O

O

6

HO

HO

O

O

MeO

1'

1

2

O

HO

O O

HO OH OMe 4

3

COOH

O

OMe

HO

231

HO

O

O OH

MeO 5

6

OH

Fig. 1. Phenylpropanoids from the stems of N. tabacum.

Table 1 1 H NMR and NO.

13

C NMR data (in C5D5N) of compounds 1–3. 1

2

dC (m) 1 2 3 4 5 6 7 8 9 10 20 Me OAc OMe

dH (m, J, Hz)

121.2 s 166.3 s 113.0 d 151.8 s 116.2 s 131.0 d 198.3 s 40.7 t 60.9 t 68.3 t 169.2 s

6.88 s

8.39 s 3.44 (t) 6.8 4.57 (t) 6.8 5.38 s

21.4 q, 169.9 s 56.1 q

1.97 s 3.85 s

121.2 s 158.6 s 113.2 d 143.8 s 132.3 s 127.2 d 198.6 s 43.2 t 58.8 t 74.2 t 73.6 t

56.0 q

Compound 1 is a pale-yellow gum with a molecular formula of C14H14O6 and contains 8 degrees of unsaturation, as determined by HRESIMS (a measured m/z of 301.0682 [M + Na]+ compared with a theoretical MW of 301.0688 for C14H14NaO6). The 1H, 13C and DEPT NMR data for compound 1 (Table 1) contained 14 carbon and 14 proton signals resulting from the following structural elements: a benzolactone nucleus (C-1–C-6, C-10, and C-20 ; H-3, H-6, and H10 ) (Wu et al., 2015), a 3-oxopropan-1-one [C(O)CH2CH2O] moiety (C-7–C-9; H-8 and H-9) (Hu et al., 2012b), a methoxy group (dC 56.1; dH 3.85), and an acetoxy group (dC 21.4 q, 169.9 s; dC 1.97 s). The benzolactone nucleus was also identified from the HMBC correlations H-10 /C-20 , C-3, C-4, C-5; H-3/C-10, and H-6/C-20 (Fig. 2). The position of the 3-oxopropan-1-one moiety at the C-1 position was deduced based on the HMBC correlations from H-8/C-1 and H6/C-7. The HMBC signals from the methoxy protons (dH 3.85) to C-2 (dC 166.3) suggest that the methoxy group is present at C-2. The acetoxy group located at C-9 was identified based on the HMBC correlation data for H-9 (dH 4.57) and the acetyl carbonyl (dC 169.2). Based on these data, the structure of compound 1 is defined as 3(6-methoxy- 3-oxo-1, 3-dihydroisobenzofuran-5-yl)-3-oxopropyl acetate. 3-Hydroxy-1-(6-methoxy-1, 3-dihydroisobenzofuran-5-yl) propan-1-one (compound 2) is a pale-yellow gum with a

O

O O

O O

MeO Fig. 2. Key HMBC (

) correlations of 1.

3

dC (m)

dH (m, J, Hz)

6.74 s

7.79 s 3.28 (t) 6.8 4.34 (t) 6.8 5.37 s 5.41 s

dC (m) 133.2 s 138.5 s 128.3 d 145.5 s 136.7 s 125.8 d 198.4 s 42.2 t 58.5 t 74.0 t 73.6 t 18.4 q

dH (m, J, Hz)

6.91 s

7.76 s 3.16 (t) 6.8 4.30 (t) 6.8 5.35 s 5.40 s 2.20 s

3.81 s

[M + Na]+ peak at m/z245.0782, which is consistent with the molecular formula of C12H14O4. The 1H and 13C NMR data for compound 2 were similar to those of compound 1; the major differences between the two data sets are the lack of an acetoxy group and ester carbonyl carbon signals and the inclusion of an oxidized methylene carbon signal (dC 73.6 s; dH 5.41) in compound 2. These differences suggest that the acetoxy group at C-9 and the ester carbonyl carbon at C-2 in compound 1 were replaced by a hydroxy group and a methylene carbon in compound 2. The HMBC correlation data for compound 2 supports the proposed structure. The molecular formula of compound 3 is C12H14O3 based on the HRESIMS data (m/z 229.0850 [M + Na]+). The 1H and 13C NMR data closely resembled to those of compound 2, with the presence of a methyl group (dC 18.4 q; dH 2.20 s) in compound 3 instead of the methoxy group that is present in compound 2 (dC 56.0 q; dH 3.81 s). The HMBC correlation data for the methyl protons (dH 2.20) to C-1 (dC 133.2), C-2 (dC 138.5) and C-3 (dC 128.3) suggest that the methyl group is located at C-2. Accordingly, the structure of 3-hydroxy-1(6-methyl-1, 3-dihydroisobenzofuran-5-yl) propan-1-one (3) is proposed. Compounds 1–6 were tested for their anti-TMV properties. The inhibitory activities of compounds 1-6 against TMV replication were tested using the half-leaf method (Hu et al., 2013; Zhou et al., 2015) at a concentration of 20 mM. Ningnanmycin, a commercial product for treating plant disease that is commercially available in China, was used as a positive control and had an inhibition rate of 30.4%. The anti-TMV results demonstrated that compounds 1 and 6 exhibited high anti-TMV activities, with inhibition rates of 34.6 and 35.4%, both of which are higher than that of the positive control (32.5%). The other four compounds also showed some anti-TMV activities with inhibition rates in the range of 16.8 to 28.6%.

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G.-H. Kong et al. / Phytochemistry Letters 14 (2015) 230–233

3. Experimental 3.1. 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 using DRX-500 spectrometers with TMS as internal standard. Unless otherwise specified, chemical shifts (d) were expressed in ppm with reference to the solvent signals. HRESIMS was performed on an API QSTAR time-of-flight spectrometer, or 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 mm) column or a Venusil MP C18 (20 mm  25 cm, 5 mm) column. Column chromatography was performed on Si gel (200–300 mesh, Qing-dao Marine Chemical, Inc., Qingdao, China), Lichroprep RP-18 gel (40–63 mm, Merck, Darmstadt, Germany) and MCI gel (75– 150 mm, Mitsubishi Chemical Corporation, Tokyo, Japan). The fractions were monitored by TLC, and spots were visualized by heating Si gel plates sprayed with 5% H2SO4 in EtOH.

Table 2 TMV infection inhibition activities of 1–6. Compounds

Inhibition rate (%)

Compounds

Inhibition rates (%)

1 2 3 4

25.2  3.1 28.6  3.0 34.6  3.2 16.8  2.8

5 6 Ningnanmycin

22.97  3.0 35.4  3.2 32.5  3.3

All results are expressed as mean  SD; n = 3 for all groups.

[M+Na]+; HRESIMS (positive mode) m/z 245.0782 [M + Na]+ (calcd 245.0790 for C12H14NaO4). 3.3.3. 3-Hydroxy-1-(6-methyl-1,3- dihydroisobenzofuran-5-yl) propan-1-one (3) C12H14O3, obtained as pale-yellow gum; UV (MeOH), lmax (log e) 278 (3.62), 235 (3.82), 210 (4.08) nm; IR (KBr) lmax 3325, 3108, 2932, 1654, 1610, 1576, 1453, 1340, 1262, 1140, 1022, 892 cm1; 1H NMR and 13C NMR data (C5D5N, 500 and 125 MHz, respectively), Table 1; ESIMS (positive ion mode) m/z229 [M + Na]+; HRESIMS (positive mode) m/z [M + Na]+ 229.0850 (calcd 229.0841 for C12H14NaO3) (Table 2).

3.2. Plant Material 3.4. Anti-TMV assay The stems of N. tabacum L. (tobacco leaves) was collected from Yuxi County, Yunnan Province, P.R. China, in September 2014. The tobacco variety is K326, which had been widely cultivated in China. 3.3. 2 Extraction and isolation The air-dried and powdered stems of N.tabacum (5.8 kg) were extracted four times with 70% aqueous acetone (3  8 L) at room temperature and filtered. The extract (386 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 C (8:2, 55.1 g) by silica gel column chromatography, eluted with CHCl3–(CH3)2CO (8:2– 2:1), yielded mixtures C1–C6. Fraction C2 (7:3, 5.52 g) was subjected to silica gel column chromatography using petroleum ether–acetone and semi-preparative HPLC (40% MeOH–H2O, flow rate 12 mL/min) to give 1 (12.2 mg). Fraction C3 (6:4, 6.18 g) was subjected to silica gel column chromatography using petroleum ether–acetone and semi-preparative HPLC (34% MeOH–H2O, flow rate 12 mL/min) to give 2 (14.8 mg), 3 (9.26 mg), 4 (13.6 mg), and 5 (15.8 mg). Fraction C4 (1:1, 3.92 g) was subjected to silica gel column chromatography using petroleum ether–acetone and semi-preparative HPLC (30% MeOH–H2O, flow rate 12 mL/min) to give 6 (12.9 mg). 3.3.1. 3-(6-Methoxy-3-oxo-1,3-dihydroisobenzofuran-5-yl)-3oxopropyl acetate (1) C14H14O6, obtained as pale-yellow gum; UV (MeOH), lmax (log e) 315 (2.78), 280 (3.57), 210 (4.03) nm; IR (KBr) lmax 3115, 2927, 1754, 1728, 1655, 1600, 1543, 1480, 1282, 1136, 1065, 956, 847 cm1; 1 H NMR and 13C NMR data (C5D5N, 500 and 125 MHz, respectively), Table 1; ESIMS (positive ion mode) m/z301 [M + Na]+; HRESIMS (positive mode) m/z 301.0682 [M + Na]+ (calcd 301.0688 for C14H14NaO6). 3.3.2. 3-hydroxy-1-(6-methoxy-1,3-dihydroisobenzofuran-5-yl) propan-1-one (2) C12H14O4, obtained as pale-yellow gum; UV (MeOH), lmax (log e) 286 (3.57), 238 (3.86), 210 (4.15) nm; IR (KBr) lmax 3418, 3096, 2921, 1658, 1605, 1562, 1483, 1426, 1357, 1275, 1140, 1058, 958, 865 cm1; 1H NMR and 13C NMR data (C5D5N, 500 and 125 MHz, respectively), Table 1; ESIMS (positive ion mode) m/z245

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