Antiviral sesquiterpenes from leaves of Nicotiana tabacum Shan-Zhai Shang, Wei Zhao, Jian-Guo Tang, Xing-Meng Xu, Han-Dong Sun, Jian-Xin Pu, Zhi-Hua Liu, Ming-Ming Miao, Yong-Kuan Chen, Guang-Yu Yang PII: DOI: Reference:

S0367-326X(15)30111-8 doi: 10.1016/j.fitote.2015.11.004 FITOTE 3290

To appear in:

Fitoterapia

Received date: Revised date: Accepted date:

11 October 2015 9 November 2015 11 November 2015

Please cite this article as: Shan-Zhai Shang, Wei Zhao, Jian-Guo Tang, Xing-Meng Xu, Han-Dong Sun, Jian-Xin Pu, Zhi-Hua Liu, Ming-Ming Miao, Yong-Kuan Chen, GuangYu Yang, Antiviral sesquiterpenes from leaves of Nicotiana tabacum, Fitoterapia (2015), doi: 10.1016/j.fitote.2015.11.004

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Antiviral Sesquiterpenes from leaves of Nicotiana tabacum

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Shan-Zhai Shang a,b, Wei Zhao a,b, Jian-Guo Tanga, Xing-Meng Xua, Han-Dong Sun b,

Key Laboratory of Tobacco Chemistry of Yunnan Province, China tobacco yunnan industrial

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a

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Jian-Xin Pu b, Zhi-Hua Liua, Ming-Ming Miaoa, Yong-Kuan Chen,a,* and Guang-Yu Yang a,*

Co., Ltd, Kunming 650231, P.R. China. State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming

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b

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Institute of Botany, Chinese Academy of Sciences, Kunming 650204, P. R. China.

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. (Tel: 0086+871+68315280; Fax: 0086+871+68315280; [email protected];

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[email protected])

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Corresponding author; E-Mail address: [email protected], [email protected].

ACCEPTED MANUSCRIPT ABSTRACT Three unreported sesquiterpenes possessing two new skeletons, tabasesquiterpenes A−C

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(1−3), together with three known sesquiterpenes (3−6) were isolated from the leaves of

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Nicotiana tabacum. Their structures were determined mainly by spectroscopic methods, including extensive 1D- and 2D- NMR techniques. Compounds 1−6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 2

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exhibited high anti-TMV activity with inhibition rate of 35.2%, which were higher than that

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of positive control (ningnanmycin). The other compounds also showed potential anti-TMV activity with inhibition rates in the range of 20.5  28.6%.

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Sesquiterpenes

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Nicotiana tabacum

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Key words:

Tabasesquiterpenes A−C

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Anti-tobacco mosaic virus activity

ACCEPTED MANUSCRIPT 1. Introduction Nicotiana tabacum, tobacco, is a stout herbaceous plant in the Solanaceae (nightshade

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family) that originated in the tropical Americas (South America, Mexico, and the West Indies)

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and now cultivated worldwide as the primary commercial source of tobacco, which is smoked or chewed as a drug for its mild stimulant effects [1-2]. N. tabacum is a kind of plant containing most complex secondary metabolites in nature. It was reported that more than

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2549 kinds of chemical compositions has been identified in N. tabacum, while up to 8700

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kinds of chemical compositions were found in tobacco and tobacco substitutes and cigarette smoke (including combustion products) [3]. Our previous investigation of this species led to

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the discovery of a number of new compounds that showed various bioactivities, such as

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anti-HIV-1, anti-TMV, and cytotoxicity by our groups [4-9]. In continuing efforts to utilize N.

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tabacum and identify bioactive natural products, the phytochemistry investigation of the leaves of Yunyan 201 (a variety of N. tabacum) led to the isolation of three new (1−3) and

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three known (4−6) sesquiterpenes, of which possessed two unprecedented skeletons (one skeleton for compounds 1 and 2 while the other for compound 3). This paper deals with the isolation, structural elucidation, and their anti-TMV activity of these compounds.

2. Experimental 2.1. General experimental procedures 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 2DNMR spectra were recorded on DRX-500 spectrometers with TMS as internal standard.

ACCEPTED MANUSCRIPT Unless otherwise specified, chemical shifts (δ) were expressed in ppm with reference to the solvent signals. HRESIMS was performed on an API QSTAR time-of-flight spectrometer, or

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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 ×

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25 cm, 7 m) column or a Venusil MP C18 (20 mm × 25 cm, 5 m) column. Column chromatography was performed with Si gel (200–300 mesh, Qing-dao Marine Chemical, Inc.,

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Qingdao, China). The fractions were monitored by TLC, and spots were visualized by heating

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Si gel plates sprayed with 5% H2SO4 in EtOH.

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2.2. Plant material

The leaves of tobacco (Yunyan 201, a variety of N. tabacum L widely planted in Yunnan)

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were collected from Yuxi County, Yunnan Province, P. R. China, in September 2014.

2.3. Extraction and isolation The air-dried and powdered leaves of N. tabacum (6.0 kg) were extracted four times with 70% aqueous acetone (3 × 12 L) at room temperature and filtered. The solvent was evaporated in vacuo, and the crude extract was dissolved in H2O and partitioned with EtOAc. The EtOAc partition (220 g) was applied to silica gel (200–300 mesh) column chromatography, eluting with a CHCl3- (CH3)2CO gradient system (10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 0:10), to give six fractions A–F. Further separation of fraction B (9:1, 25.4 g) by silica gel column chromatography, eluted with CHCl3 -(CH3)2CO (14:1 – 1:1), yielded mixtures B1–B6.

ACCEPTED MANUSCRIPT Fraction B2 (11:1, 4.1 g) was subjected to silica gel column chromatography using petroleum ether-acetone and semi-preparative HPLC (40% Acetonitrile-H2O, flow rate 12 mL/min) to

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give 1 (11.2 mg), 2 (8.5 mg), and 3 (9.7 mg). Fraction B2 (9:1, 5.1 g) was subjected to silica

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gel column chromatography using petroleum ether-acetone and semi-preparative HPLC (37% Acetonitrile-H2O, flow rate 12 mL/min) to give 4 (10.8 mg), 5 (12.4), and 6 (14.4).

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Tabasesquiterpene A (1) : Obtained as pale-yellow gum; UV (MeOH), λmax (log ε) 210 (4.17), 252 (3.56) nm; IR (KBr) νmax 3386, 2926, 1605, 1538, 1446, 1142, 1057, 857 cm–1; 1H

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NMR and 13C NMR data (C5D5N, 500 and 125 MHz, respectively), Table 1; ESIMS (negative

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ion mode) m/z 233 [M − H]−; HRESIMS (negative ion mode) m/z 233.1549 [M − H]− (calcd

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233.1542 for C15H21O2).

Tabasesquiterpene B (2): Obtained as pale-yellow gum; UV (MeOH), λmax (log ε) 210

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(4.22), 257 (3.63) nm; IR (KBr) νmax 3386, 2928, 1657, 1600, 1546, 1433, 1158, 1064, 936 cm-1; 1H NMR and 13C NMR data (C5D5N, 500 and 125 MHz, respectively), Table 1; ESIMS (negative ion mode) m/z 247 [M − H]−; HRESIMS (negative ion mode) m/z 247.1327 [M − H]− (calcd 247.1334 for C15H20O3).

Tabasesquiterpene (3): Obtained as pale-yellow gum; UV (MeOH), λmax (log ε) 210 (4.09), 250 (3.50) nm; IR (KBr) νmax 3386, 2926130, 1602, 1535, 1450, 1136, 1063, 865 cm-1; 1

H NMR and

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C NMR data (C5D5N, 500 and 125 MHz, respectively), Table 1; ESIMS

ACCEPTED MANUSCRIPT (negative ion mode) m/z 233 [M − H]−; HRESIMS (negative ion mode) m/z 233.1546 [M −

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H]− (calcd 233.1542 for C15H21O2).

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2.4. Anti-MTV Assay.

TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry of

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Yunnan Province, YunnanAcademy of Tobacco Science, P. R. China. The virus was multiplied in Nicotiana tabacum cv. K326 and purified as described [10]. The concentration of TMV was

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determined as 20 mg/mL with a UV spectrophotometer [virus concentration = (A260 × dilution ratio)/E 10.m1%，260nm ]. The purified virus was kept at − 20 °C and was diluted to 32 μg/mL with

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0.01 M PBS before use.

Nicotiana glutinosa plants were cultivated in an insect-free greenhouse. N. glutinosa was

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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

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the required concentrations. A solution of equal concentration of DMSO was used as a negative control. The commercial antiviral agent ningnanmycin was used as a positive control. For the half-leaf method [11], the virus was inhibited by mixing with the solution of compound. 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 control. The local lesion numbers were recorded 3 or 4 days after inoculation. Three repetitions were conducted for each compound. The inhibition rates were calculated according to the formula

ACCEPTED MANUSCRIPT inhibition rate (%) = [(C — T)/C] × 100% where C is the average number of local lesions of the control and T is the average

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number of local lesions of the treatment.

3. Results and Discussion

A 70% aq. acetone extract prepared from the leaves of N. tabacum was partitioned

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between EtOAc and H2O. The EtOAc layer was subjected repeatedly to column

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chromatography on Si gel and preparative HPLC to afford compounds 1−6. Compounds 1−3 were identified as new compounds, and being named as tabasesquiterpenes A−C (1−3). Their

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structures are shown in Figure 1, and the 1H and 13C NMR data of compounds 1−3 are listed

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in Table 1. The known compounds, comparing with the published literatures, were identified

[14].

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as balsamiferine B (4) [12], samboginone (5) [13], and ent-4(15)-eudesmen-1α,11-diol (6)

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Compound 1, obtained as pale-yellow gum, had a molecular formula C15H22O2 as established by the quasimolecular ion peak observed using HRESIMS measurement at m/z 233.1549 [M − H]− (calcd for 233.1542), suggesting 5 degrees of unsaturation. The UV spectrum showed absorption maxima at 210 and 252 nm, and the IR spectrum showed absorption bands at 3386, 1605, and 1538 cm-1, indicating the presence of hydroxy group(s), and an aromatic ring. The HMQC data of 1 showed a pentasubstituted aromatic ring [δC 152.6 (s, C-4), 124.8 (s, C-5), 134.4 (s, C-6), 144.2 (s, C-8), 132.7 (s, C-9), and δC 122.3 (d, C-7), δH 6.80 (s, H-7)], three methyls [δH 1.17 (s, H3-13 and H3-14), 2.20 (s, H3-15)], and five methylene groups (including an oxygenated one) [δH 2.90 (s, H2-1), 2.82 (s, H2-3), 2.64 (t,

ACCEPTED MANUSCRIPT H2-10), 1.83 (m, H2-11), 3.54 (t, H2-12) ] (Table 1). The 1H−1H COSY correlations of H2-10/H2-11/H2-12, which showed the connection pattern of C-10—C-11—C-12, together

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with the HMBC correlations of H-10 with C-4, C-5, and C-6; of H-11 with C-5, determined

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the presence of a 3-hydroxypropyl group being located at C-5 (Fig. 2). The HMBC correlations of H-15 with C-5 (δC 131.9), C-6 (δC 113.2), and C-7 (δC 122.2); of H-7 with C-5 and C-6, suggested that C-15 was connected with the aromatic ring at C-6. The phenolic

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hydroxyl group (δH 10.3, s) was located at C-4 on the basis of its correlation with C-4. The

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stong HMBC correlations from H-13, H-14 to C-1, C-2, C-3 indicated that the C-13 and C-14 were connected with the quaternary carbon C-2 (δc 40.1, s); Further more, the weak

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correlations from H-13, H-14 to C-8 and C-9, as well as the correlations from H-3 to C-4, C-8,

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and C-9; from H-1 to C-8, C-9, and C-7, suggested that C-2 was connected with the aromatic

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ring by the linkages C-2—C-3—C-9 and C-2—C-1—C-8. Therefore, the structure of 1 was established and named as tabasesquiterpene A.

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Compound 2 gave an [M − H]− peak at m/z 247.1327 in the HRESIMS (negative ion mode), consistent with a molecular formula of C15H20O3. The 1D NMR data of 1 showed a close relationship to those of 2, which indicated that they should possess the same skeleton (Table 1). It was found that the major differences of their NMR data were the disappearance of a methylene group and the addition of a carbonyl group. The reason for the different NMR data of 1 with those of 2 could be rationalized to the oxidation of C-10 in 1, which was further testified by the HMBC correlations from H-11 to C-10 and C-5. Thus, compound 2 was elucidated as tabasesquiterpene B. Compound 3 was assigned the same molecular formula of C15H22O2 as that of compound 1,

ACCEPTED MANUSCRIPT supported by the HRESIMS (m/z 233.1546 [M − H]−). Detailed analysis of the 1D NMR data of 1 and 2 suggested they were quite compatible with each other, implying that they could

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possess the similar skeleton. The HMBC correlations of H2-10 with C-4 (δc 126.0, s), C-5 (δc

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132.8, s), and C-6 (δc 119.7, d); of H3-15 with C-4, C-5, and C-9 (δc 147.5, s), suggested that C-15 was located at C-4 in 3 instead of at C-6 in 1; Besides, the location of the phenolic hydroxyl group was changed from C-4 to C-7 on the basis of the correlations in HMBC

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of 3 was formulated as tabasesquiterpene C.

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spectum from the atoms in hydroxyl group to C-7. Based on the above findings, the structure

Moreover, it was worth mentioning that the three compounds possessed two unprecedented

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skeletons, of those were both comportible with that of illudalane sesquiterpenoids skeleton

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being a class of compounds rare in nature and only being isolated from the fungal subdivision

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of Basidiomycotina1 and from the fern family of Pteridaceae [15]. The major defferences of these three skeletons were that two methyl groups were located at C-4, and C-5, and one ethyl

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group was located at C-5 in the illudalane sesquiterpenoids skeleton, respectively; while the two skeletons for compounds 1−3 both had a propyl group at C-5 and each possessed one methy group at C-4 and C-6, respectively (Fig. 3). It was assumed that these two skeletons were assembled through a biosynthetic pathway similar to that for illudalane sesquiterpenoids skeleton (Fig. 4). In this pathway, all these three skeletons were derived from the same precusor farnesyl pyrophosphate, and shared the key step in the formation process of the unstabled precusor a. Their major differences were showed as belows: precusor a were biosyntherized to the two skeletons by series steps including two different ring enlarging and ring opening steps, respectively, while illudalane sesquiterpenoids skeleton was formed

ACCEPTED MANUSCRIPT through the formation of the precusor b possessing a three-membered ring. Compounds 1−6 were tested for their anti-TMV activities. The inhibitory activities of

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compounds 1−6 against TMV replication were tested using the half-leaf method [11, 16].

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Ningnanmycin, a commercial product for plant disease in China, was used as a positive control. The antiviral inhibition rates of compounds 1−6 at the concentration of 20 μM were listed Table 2. The results showed that compound 2 exhibited high anti-TMV activity with

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inhibition rate of 35.2%, which were higher than that of positive control (ningnanmycin). The

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other compounds also showed potential anti-TMV activity with inhibition rates in the range of

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Acknowledgments

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20.5  28.6%.

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This research was supported by the National Natural Science Foundation of China (No. 21562049, No. 31360081 and No. 31400303) and Natural Science Foundation of Yunnan

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Province (No. 2014FB163, No. 2015FB162).

References

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ACCEPTED MANUSCRIPT [4] Tan JL.; Chen ZY, Yang GY, Miao MM, Chen YK, Li TF. Two new benzofuranylpropanoids from Nicotiana tabacum and their anti-tobacco mosaic virus

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activities. Heterocycles 2011; 83: 2381-5.

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[5] Chen YK, Li XS, Yang GY, Chen ZY, Hu QF, Miao MM. Phenolic compounds from Nicotiana tabacum and their biological activities. J Asian Nat Prod Res 2012; 14: 450-6. [6] Gao XM, Li XS, Yang XZ, Mu HX, Chen YK, Yang GY, Hu QF. Lignan derivatives from

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the leaves Nicotiana tabacum and their activities. Heterocycles 2012; 85: 147-53.

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[7] Chen ZY, Tan JN, Yang GY, Miao MM, Chen YK, Li TF. Isoflavones from the roots and stems of Nicotiana Tabacum and their anti-tobacco mosaic virus activities. Phytochem

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Lett 2012; 5: 233-5.

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[8] Chen JX, Leng HQ, Duan YX, Zhao W, Yang GY, Guo YD, Chen YK, Hu QF. Three new

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flavonoids from the leaves of oriental tobacco and their cytotoxicity. Phytochem Lett 2013; 6: 144-7.

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[9] Mou DR, Zhao W, Zhang T, Wan L, Yang GY, Chen YK, Hu QF, Miao MM. Two new chromanone derivatives from the roots and stems of Nicotiana tabacum and their cytotoxicity. Heterocycles 2012; 85: 2485-90. [10] Gooding, GV, Hebert TT. A simple technique for purification of tobacco mosaic virus in large quanities. Phytopathol 1967; 57: 1285-9. [11] Hu QF, Zhou B, Huang JM, Gao XM, Shu LD, Yang GY, Che CT. Antiviral phenolic compounds from Arundina gramnifolia. J Nat Prod, 2013; 76: 292-296.

ACCEPTED MANUSCRIPT [12] Xu J, Jin DQ, Liu CZ, Xie CF, Guo YQ, Fang LZ. Isolation, Characterization, and NO inhibitory activities of sesquiterpenes from Blumea balsamifera. J Agric Food Chem,

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2012, 60: 8051−8058.

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[13] Shirota O, Oribello JM, Sekita S, Satake M. Sesquiterpenes from Blumea balsamifera. J

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Nat Prod, 2011, 74: 470−476.

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13199. J Nat Prod, 2009, 72, 1720-1722.

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and ent-eudesmane sesquiterpenes from the Endophytic Fungus Eutypella sp. BCC

[15] Wolf-Rainer, A. Bioactive sesquiterpenes produced by fungi: are they useful for humans

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as well. Curr. Med. Chem. 2001, 8, 583-606.

[16] Yan XH, Chen J, Di YT, Fang X, Dong JH, Sang P, Wang YH, He HP, Zhang ZK, Hao

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XJ. Anti-tobacco mosaic virus (TMV) quassinoids from Brucea javanica (L.) Merr. J

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Agric Food Chem 2010; 58: 1572-7

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OH

8

6 7

OH

15

9

2 14

5

1

8

6 7

10

11

3

13

12

4

3

13

OH 14

5 9

2 1

O

6

8 7

OH 3 OH

OH

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OH OH

11

15

2

1

12

10

4

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1

11

O 5

O 4

OH

OH

6

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Fig. 1 The structures of sesquiterpenes from Nicotiana tabacum

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14

5

9

2

12

10

4

3

13

OH O

OH

ACCEPTED MANUSCRIPT OH

OH O OH

OH

OH OH 3

) and 1H-1H COSY (

) correlations of 1−3

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Fig. 2 Key HMBC (

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15 3

13

9

12

10

4

5

14

1

8

6 7

3

13 11

9

5

14

1

8

9

10

4 5

11

2

6

6 14

7

1

12

8 7

illudalane skeleton

2

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1

3

13

11

2

15

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4

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Fig. 3 The two skeletons of compounds 1−3 and the skeletons of illudalane

OPP

CH3 CH3

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H3C

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X

H3C

illudalane skeleton

X CH3 CH3 CH3

CH3 CH3

H3C

CH3

CH3

CH3 CH3

H3C

1

CH3

H

CH3 CH3

H3C

CH3

H3C

CH3

CH3 CH3

H3C

CH3

HO H

CH3

CH3 CH3 CH3

H3C

H3C

a

H b

X

CH3

CH3

H2C

CH3 CH3

CH3 CH3

CH3 CH3 CH3

CH3 CH3

H

Fig. 4 Hypothetical biosynthesis route of skeletons 1, 2 and illudalane

2

ACCEPTED MANUSCRIPT Table 1. H and 13C NMR data for compounds 1−3 (500 and 125 MHz, in CD3OD) 2

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H (m, J, Hz)

C

H (m, J, Hz)

1

38.3 t

2.90 s

38.7 t

2

40.1 s

3

43.7 t

4

152.6 s

5

124.8 s

6

134.4 s

7

122.3 d

8

144.2 s

9

132.7 s

10

27.4 t

41.0 s 43.9 t

2.80 s

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2.82 s

2.99 s

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159.0 s

H (m, J, Hz)

38.4 t

2.90 s

41.3 s 44.7 t

132.8 s

136.2 s

119.7 d

121.7 d

6.69 s

2.86 s

126.0 s

117.6 s

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6.80 s

3

C

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1

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1

6.61 s

152.0 s 133.4 s

130.9 s

147.5 s

2.64 t (7.8)

198.1 s

32.9 t

2.54 t (7.8)

32.2 t

1.83 m

41.8 t

3.40 t (6.6)

35.8 t

1.91 m

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150.8 s

62.6 t

3.54 t (6.5)

58.6 t

4.34 t (6.6)

62.7 t

3.55t (6.5)

13,14

28.5 q

1.17 s

28.2 q

1.21 s

28.2 q

1.1723 s

15

23.5 q

2.20 s

23.1 q

2.19 s

21.1 q

2.12 s

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ACCEPTED MANUSCRIPT Table 2. TMV infection inhibition activity of compounds 1−6

1

26.4 ± 3.1

5

2

35.2 ± 3.4

3

28.6 ± 2.8

4

22.1 ± 2.5

26.4 ± 3.0

ningnamycin

33.2 ± 3.2

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20.5 ± 2.6

6

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

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Inhibition rates (%)

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Compounds

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Inhibition rates (%)

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Compounds

ACCEPTED MANUSCRIPT Graphical Abstract Three unreported sesquiterpenes possessing two new skeletons, tabasesquiterpenes A−C

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(1−3), together with three known sesquiterpenes (4−6) were isolated from the leaves of

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Nicotiana tabacum. Compound 2 exhibited high anti-TMV activity with inhibition rate of

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35.2%, which were higher than that of positive control (ningnanmycin).

15

OH 9 6 7

OH

14

15

2

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1

1

12

10 5

9

2

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8

OH

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1

11

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2 14

5

4

3

13

12

10

4

3

13

OH O

11 6

8

7

OH 3

OH