HETEROCYCLES

Short Paper | Regular issue | Vol. 91, No. 2, 2015, pp. 375-380
Received, 18th November, 2014, Accepted, 24th December, 2014, Published online, 26th December, 2014.
DOI: 10.3987/COM-14-13130

■ Isoprenylated Flavones from Garcinia bracteata and Their Anti-Tobacco Mosaic Virus Activity

Yuehong Yang,1 Limei Li,2 Jie Lou,2 Yang Cheng,1 Yede Wang,2 Duntao Shu,1 Lei Shi,1 Xue-Mei Gao,2 Ping Ning,1,* and Qiufen Hu2,*

1 Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming 650500, China; 2 Key Laboratory of Chemistry in Ethnic Medicinal Resources, Yunnan University of Nationalities, Kunming 650500, P. R. China

Abstract

Two new isoprenylated flavones, bracteflavones A and B (1 and 2), together with four known isoprenylated flavones (3-6) were isolated from the twigs of Garcinia bracteata. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D- NMR techniques. The anti-tobacco mosaic virus (anti-TMV) activities of compounds 1-6 were evaluated, and the results revealed that 1-6 showed potential anti-TMV activity with inhibition rates in the ranges of 17.6-24.3%, respectively.

The genus Garcinia (Clusiaceae) is commonly distributed in tropical and subtropical countries of South East Asia, West and East Africa, and Central and South America. This genus is known to produce xanthones and benzophenones,1-5 and many of these compounds show interesting biological activities including anti-microbial, anti-HIV and anti-oxidant activities.
The G. bracteata, a plant of genus Garcinia, is distributed in the south of Yunnan and Guangxi Province of China.6 In the published literatures, some xanthones and benzophenones were isolated from this plant,7-11 and these compounds exhibited various activities. Motivated by a search for new bioactive metabolites from genus Garcinia, the chemical constituents of the twigs of G. bracteata were reinvestigated by our group. As a result, two new (1 and 2) and four known (3-6) isoprenylated flavones were isolated from this plants. This paper deals with the isolation and structural characterization of these compounds, and their anti-tobacco mosaic virus (anti-TMV) activity.

The twigs of G. bracteata were extracted with 70% aqueous acetone. The extract was subjected repeatedly to column chromatography on silica gel, sephadex LH-20, RP-18 and preparative HPLC to afford compounds 1-6, including two new isoprenylated flavones, bracteflavones A and B (1 and 2), together with four known isoprenylated flavones, artocarmin D (3),12 6-prenylapigenin (4),13 cycloartocarpesin (5),14 and artochamin C (6).15 The structures of the compounds 1-6 were shown in Figure 1, and the 1H and 13C NMR data of 1 and 2 were listed in Table 1.

Compound 1 was obtained as a pale yellow gum. It gave an [M+Na]+ ion peak at m/z 387 in the ESIMS, and was shown to possess the molecular formula C22H20O5 by HRESIMS (m/z 387.1202 [M+Na]+). The UV absorptions at 362, 257, and 210 nm showed an extended chromophore and a substituted benzene ring. Its IR spectral data showed the presence of carbonyl groups (1680 and 1658 cm-1) and phenyl groups (1602, 1561, and 1474 cm-1). The 1H and 13C NMR spectrum of 1 showed 22 carbons and 20 protons, corresponding to a 1,4-disubstituted benzene ring [δC 123.0 s, 130.8 d (2C), 115.8 d (2C), and 161.1 s; δH 7.73 (d) J = 8.8 (2H) and 6.80 (d) J = 8.8 (2H)], a 1,2,3,4-tetrasubstituted benzene ring [δC 124.1 d, 127.9 d, 133.6 s, 155.2 s, 150.7 s, and 122.1 s; δH 7.43 (d) J = 8.2 and 6.71 (d) J = 8.2], one 2-oxo-3-methylbut-3-enyl group (δC 37.9 t, 200.7 s, 144.8 s, 123.4 t, and 18.7 q; δH 4.63 s, 5.85, 6.11 s, and 1.90 s),16 two methoxy groups (δC 61.4 q and 56.0 q; δH 3.80 s and 3.84 s, 3H each), a pair of olefin proton signal (δC 163.6 s and 105.3 d; δH 6.42 s, 1H), and a carbonyl carbon (δC 181.5). These spectral data indicated that compound 1 should be a flavone derivative bearing a 2-oxo-3-methylbut-3-enyl group and two methoxy groups.17 The HMBC correlations (Figure 2) of one methoxy proton signals (δH 3.80) with C-4′ (δC 162.0) suggested the position of this methoxy group at C-4′, this was also supported by the fact of the typical protons signals at [δC 7.73 (d) J = 8.8 (2H) and 6.80 (d) J = 8.8 (2H)]. Since the substituents on ring B were evident, the surplus substituents (one 2-oxo-3-methylbut-3-enyl group and one methoxy) should be located at ring A. The HMBC correlations of H-1′′ (δC 4.63) with C-6 (δC 127.9), C-7 (δC 133.6), and C-8 (δC 155.2), and of H-6 (δH 6.71) with C-1′′ (δC 37.9), suggested the 2-oxo-3-methylbut-3-enyl group should be located at C-7. The other methoxy group located at C-8 was supported by the HMBC correlation of the methoxy proton signal (δH 3.84) with C-8 (δC 155.2). Accordingly, the structure of 1 was established, and gives the trivial name of bracteflavones A.

Bracteflavones B (2) was also obtained as yellow gum. It HRESIMS at m/z 387.1215 [M+Na]+ revealed that compounds 1 and 2 had the same molecular formula. The 1H and 13C NMR spectra of 2 (Table 1) were very similar to those of 1. The obvious chemical shift differences came from the variations of the NMR data for ring A of the two compounds. This indicated the substituents position on the ring A should be varied. The 2-oxo-3-methylbut-3-enyl group located at C-6 was supported by the HMBC correlations of H-1′′ (δC 4.65) with C-5 (δC 124.5), C-6 (δC 132.2), and C-7 (δC 118.8), and of H-5 (δH 7.04) and H-7 (δH 6.66) with C-1′′ (δC 38.1).

The other precise subsituents positions, two methoxy group located at C-8 and C-4′, respectively, were also conducted by further analysis of its HMBC correlations. The structure of 2 is therefore determined.
Since certain of the flavonoids exhibit potential anti-TMV activity,18-21 compounds 1-6 were tested for their anti-TMV activity. The anti-TMV activity were tested using the half-leaf method.22 Ningnanmycin (a commercial product for plant disease in China), was used as a positive control. Their antiviral inhibition rates at the concentration of 20 μM were listed in Table 2. Compounds 1-6 showed potential anti-TMV activity with inhibition rates in the ranges of 17.6-24.3%, respectively.

EXPERIMENTAL
General. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained in KBr disc on a Bio-Rad Win infrared spectrophotometer. ESI-MS were measured on a VG Auto Spec-3000 MS spectrometer. 1H, 13C and 2D NMR spectra were recorded on Bruker 500 instrument with TMS as internal standard. Column chromatography was performed on silica gel (200-300 mesh), or on silica gel H (10 ∼ 40 µm, Qingdao Marine Chemical Inc., China). Second separate was used an Agilent 1100 HPLC equipped with ZORBAX-C18 (21.2 mm × 250 mm, 7.0 µm) column and DAD detector.
Plant material. The twigs of Garcinia bracteata C. Y. Wu ex Y. H. Li were collected in Pu′er Prefecture, Yunnan Province, People’s Republic of China, in September 2012. The identification of the plant material was verified by Prof. Ren P. Y (Xishuangbanna Botanical Garden). A voucher specimen (YNNI-2012-88) has been deposited in our laboratory.
Anti-TMV Assays. TMV (U1 strain) was obtained from the Key Laboratory of Tobacco Chemistry of Yunnan Province, Yunnan Academy of Tobacco Science, P. R. China. The virus was multiplied in Nicotiana tabacum cv. K326 and purified as described. The concentration of TMV was determined as 20 mg/mL with a UV spectrophotometer [virus concentration = (A260 × dilution ratio)/E0.1%, 260 nm (1cm)]. The purified virus was kept at -20 °C and was diluted to 32 μg/mL with 0.01 M 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 was used as a positive control.
For the half-leaf method, 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:
inhibition rate (%) = [(C − T)/C] × 100%
where C is the average number of local lesions of the control and T is the average number of local lesions of the treatment.
Extraction and isolation. The air-dried and powdered twigs of G. bracteata (2.5 kg) were extracted four times with 70% acetone (4 × 3.0 L) at room temperature and filtered. The filtrate was concentrated and successively partitioned with CH2Cl2 and EtOAc. The EtOAc fraction (68 g) was submitted to silica gel (200–300 mesh) column chromatography, eluting with a CHCl3-acetone gradient system (20:1, 9:1, 8:2, 7:3, 6:4, 5:5), to give six fractions A–F. The further separation of fraction B (9:1, 20 g) by silica gel column chromatography, eluted with petroleum ether-EtOAc (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures B1–B5. Fraction B2 (8:2, 1.6 g) was subjected to preparative HPLC (65% MeOH, flow rate 12 mL/min) to give 1 (13.2 mg) and 2 (10.6 mg). The further separation of fraction B3 (7:3, 1.2 g) by silica gel column chromatography, and preparative HPLC (58% MeOH, flow rate 12 mL/min) to give 3 (14.0 mg), 4 (16.7 mg), 5 (18.5 mg), and 6 (15.8 mg).
Bracteflavones A (1): C22H20O5, Pale yellow gum; UV (MeOH) λmax (log ε) 362 (3.60), 257 (3.72), 210 (4.28) nm; IR (KBr): νmax 2947, 2832, 1680, 1658, 1602, 1561, 1474, 1338, 1285, 1142, 1071, 845, 762 cm–1; 1H and 13C NMR data (in CDCl3, 500 and 125 MHz), see Table 1; ESIMS m/z 387; HRESIMS m/z 387.1202 [M+Na]+ (calcd C22H20O5 for 387.1208).
Bracteflavones B (1): C22H20O5, Pale yellow gum; UV (MeOH) λmax (log ε) 365 (3.68), 262 (3.75), 210 (4.32) nm; IR (KBr): νmax 2958, 2826, 1685, 1654, 1608, 1576, 1461, 1327, 1276, 1157, 1064, 869, 753 cm–1; 1H and 13C NMR data (in CDCl3, 500 and 125 MHz), see Table 1; ESIMS m/z 387; HRESIMS m/z 387.1215 [M+Na]+ (calcd C22H20O5 for 387.1208).

ACKNOWLEDGMENT
This research was supported by the National Natural Science Foundation of China (No. 21362044), the the program for Innovative Research Team (in Science and Technology) in University of Yunnan Provinve (NO. IRTSTYN 2014-11), and start-up funds of Yunnan University of Nationalities.

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