HETEROCYCLES

Short Paper | Regular issue | Vol. 87, No. 1, 2013, pp. 125-131
Received, 6th September, 2012, Accepted, 31st October, 2012, Published online, 8th November, 2012.
DOI: 10.3987/COM-12-12580

■ Isoaurones from the Stem of Cassia siamea and Their Anti-Tobacco Mosaic Virus (Anti-TMV) Activity

Xue-mei Gao, Li-dan Shu, Li-ying Yang, Yan-qiong Shen, Ming-zhu Cui, Xue-mei Li,* and Qiu-fen Hu*

Key Laboratory of Ethnic Medicine Resource Chemistry, State Ethnic Affairs Commission & Ministry of Education, School of Chemistry and Biotechnology, Yunnan University of Nationalities, Kunming 650031, China

Abstract

Two new isoaurones, siamaurones A and B (1-2), together with three known isoaurones (3-5) were isolated from the stems of Cassia siamea. The structures of 1-5 were elucidated by spectroscopic methods including 1D- and 2D-NMR techniques. Compounds 1-5 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activity. As a result, compound 2 showed high anti-TMV activity with inhibition rate of 31.8%, which is higher than that of Ningnanmycin (26.5%). Compounds 1, 3, and 5 showed modest anti-TMV activity with inhibition rate of 15.2%, 16.4%, and 12.8%, respectively.

Cassia siamea Lam. (Leguminosae) is a common tree in the southern part of China. It has been used widely in traditional medicine, particularly for treatment of periodic fever, alaria, aperient, antiarthritic and swellings.1,2 In previous work, a number of bioactive compounds, such as anthraquinone,3,4 alkaloids,5,6 triperpenoids,7,8 steroids,8,9 chromones,10-12 and their homologous, were isolated from this plant. Motivated by a search for new bioactive metabolites from this plant, our group has investigated the chemical constituents of the stems of C. siamea, which led to the isolation and characterization of two new isoaurones (1-2), along with three known isoaurones (3-5). The structures of the isolated compounds were established by means of spectroscopic methods including extensive 1D and 2D NMR techniques. This paper deals with the isolation, structural characterization, and anti-tobacco mosaic virus (anti-TMV) activity of these compounds.

The stems of C. siamea were extracted with 95% aqueous methanol. The extract was subjected repeatedly to column chromatography on silica gel, Sephadex LH-20, RP-18, and semi-preparative RP-HPLC separation to afford two new isoaurones, siamaurones A and B (1-2), together with three known isoaurones (3-5). The structures of the compounds 1-5 were as shown in Figure 1, and the 1H and 13C NMR data of compounds 1 and 2 were listed in Table 1. Compared with literature data, the known compounds were identified as: 4′,6-dihydroxy-4-methoxyisoaurone (3),13 isoaurone A (4),14 and 6-methoxyisoaurone (6).14

Compound 1 was obtained as a yellow solid. It gives a parent ion by HR-ESIMS at m/z 445.1139 [M-H]- (calcd for 445.1135) corresponding to a molecular formula of C22H22O10, requiring twelve degrees of unsaturation. The 1H NMR spectrum of 1 showed the presence of an AA′BB′ aromatic system at δH 8.12 (2H, d, J=8.9 Hz, H-2′,6′) and 6.90 (2H, d, J=8.8 Hz, H-3′,5′), two meta-coupled aromatic protons at δH 6.16 (1H, d, J=1.8 Hz, H-7) and 6.26 (1H, d, J=1.8 Hz, H-5), an isolated olefinic proton at δH 7.80 (1H, s, H-10), a methoxy group proton (δH 3.88 s), and a glucosyl moiety [δH 5.30 (1H, d, J=7.3, H-1′′); δH 3.22 ∼ 3.70 (6H, m, H-2′′, H-3′′, H-4′′, H-5′′, H-6′′)]. The 13C NMR spectrum of 1 revealed the presence of an isoaurone nucleus13 (δC 170.2 s, 119.0 s, 158.8 s, 96.2 d, 159.4 s, 93.0 d, 155.0 s, 105.1 s, 142.1 d, 128.1 s, 135.4 d (2C), 116.1 d (2C), 161.7 s), a glucosyl moiety (δC 103.5 d, 75.7 d, 78.5 d, 71.3 d, 78.0 d, 62.6 t), and a methoxy group (δC 56.1 q). The HMBC correlations of 1 (Figure 2) showing the deshielded H-10 (δH 7.80) coupled to C-2 (δC 170.2) suggested that the structure of 1 could be an isoaurone too.15,16 The AA′BB′ spin system in the 1H NMR spectrum indicated one hydroxy group attached to C-4′ (δC 161.7). In the class of C6-C3-C6 compounds possessing hydroxy substituents at C-5 and C-7, the chemical shift of C-6 (or C-5 in the case of 1) appears at lower field than C-8 (or C-7 in the case of 1);14 therefore, the signals at δC 96.2 and 93.0 were assigned to C-5 and C-7, respectively. The HSQC spectrum indicated that the meta-coupled aromatic protons at δH 6.27 and 6.18 associated with C-5 and C-7, respectively. The HMBC spectrum revealed the coupling from the methoxy group (δH 3.94) to the carbon atom at δC 158.8, which further showed the 1H-1H COSY correlation with H-5 (δH 6.27) but not with H-7 (δH 6.18). This indicated that the methoxy group was located at C-4. The long-range correlations in the HMBC spectrum between H-1′′ (δH 5.30 d) and C-6 (δC 159.4 s) indicated the glucosyl was linked to C-6, and the coupling constant value of H-1′′ (J=7.3 Hz) indicated that the glucosyl moiety was connected to the aglycone by a β-linkage.17,18 The configuration of E- and Z-isoaurones is determined on the basis of the chemical shift of H-10, which is anisotropically and diamagnetically affected by the C-2 carbonyl group. It is known that the chemical shift of H-10 in Z-isoaurone resonates at higher field (7.4∼7.5 ppm) than in the E-isomer (7.8∼8.0 ppm).16,19 The differences in 13C shifts of the olefinic carbon between E (169 ppm) and Z (166 ppm) isomers were reportedly too small to be useful.16 Therefore, the H-10 chemical shift at δH 7.80 suggested that 1 is E-isoaurone. This conclusion was supported by the cross-peak between the methoxy group (δC 3.88) and H-2′,6′ (δH 8.12) in a NOESY experiment. Thus, compound 1 was evident, and given the name as siamaurone A.
Compound 2 was obtained as pale yellow solid, and showed quasi molecular ion at m/z 269.0443 [M-H]- in the HRESIMS (calcd m/z 269.0450), corresponding to the molecular formula of C15H10O5. The 1H and 13C NMR signals revealed that compound 2 also have isoaurone skeleton with an AA′BB′ spin system. By the comparison NMR data of compounds 1 and 2, the obvious chemical shift differences resulted from the substituent group variations in the aromatic ring. In the class of C6-C3-C6 compounds possessing the substituents at C-5 and C-6, the chemical shift of C-4 appears at lower field than C-7;14,19 therefore, the carbons signals at δC 109.4 and 104.2 were assigned to C-4 and C-7, and the HSQC spectrum indicated that two singlets δH 6.61 and 6.36 was associated with C-4 and C-7, respectively. Since no other substituents signals were obverved in its 1H and 13C spectrometry, all substituents in 2 should be hydrogen. On the basis of the above evidence, the structure of 2 was established as shown, and given the name as siamaurone B.

The compounds 1-5 were tested for their anti-TMV activity. The inhibitory activities of compounds 1-5 against TMV replication were tested using the half-leaf method.20,21 Ningnanmycin, a commercial product for plant disease in China, was used as a positive control.22 The antiviral inhibition rates of compounds 1-5 at the concentration of 20 μM were listed in Table 2. The results showed that compound 2 showed high anti-TMV activity with inhibition rate of 31.8%, which is higher than that of Ningnanmycin (26.5%). Compounds 1, 3, and 5 showed modest anti-TMV activity with inhibition rate of 15.2%, 16.4%, and 12.8%, respectively. Compound 4 also showed weak anti-TMV activity with inhibition rate of 8.25%.

EXPERIMENTAL
General. UV spectra were obtained using a Shimadzu UV-2401A spectrophotometer. IR spectra were obtained in KBr disc on a Bio-Rad Wininfmred spectrophotometer. ESI-MS were measured on a VG Auto Spec-3000 MS spectrometer. 1H and 13C NMR spectra were recorded on Bruker DRX-400 instrument with TMS as internal standard. 2D NMR spectra were recorded on Bruker DRX-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). Preparative HPLC 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 stems of C. siamea were collected on Dehong Prefecture, Yunnan Province, People’s Republic of China, in September 2011. The identification of the plant material was verified by Dr. Yuan. N, of Kunming Institute of Botany, Chinese Academy of Sciences. A voucher specimen (YNNU 11-9-32) has been deposited in our Laboratory.
Extraction and Isolation. The air-dried and powdered stems of C. siamea (5.0 kg) were extracted four times with 90% aq. EtOH (4 × 50 L) at room temperature and filtered. The crude extract (321 g) was applied 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 C (8:2, 8.26 g) by silica gel column chromatography, eluted with CHCl3-MeOH (9:1, 8:2, 7:3, 6:4, 1:1), yielded mixtures C1–C5. Fraction C1 (9:1, 1.57 g) was subjected to preparative HPLC (40% MeOH, flow rate 12 mL/min) to give 3 (34.5 mg), 4 (16.7 mg), and 5 (19.2 mg). The further separation of fraction D (7:3, 22.6 g) by silica gel column chromatography, and preparative HPLC (32% MeOH, flow rate 12 mL/min) to give 1 (28.6) and 2 (17.8 mg).
Siamaurone A (1). Obtained as pale yellow gum; UV (MeOH) max (log ε) 205 (4.22), 262 (3.87), 390 (3.64) nm; IR (KBr) νmax 3420, 2910, 2854, 1688, 1624, 1548, 1430, 1356, 1135, 922, 846 cm-1; 1H NMR and 13C NMR data (CD3OD, 400 and 100 MHz), see Table 1; negative ESIMS m/z 445 [M-H]-; negative HRESIMS m/z 445.1139 [M-H]- (calcd for C22H21O10, 445.1135).
Siamaurone B (2). Obtained as yellow solid; UV (MeOH) max (log ε) 210 (4.31), 260 (3.90), 385 (3.68) nm; IR (KBr) νmax 3434, 2915, 2862, 1668, 1624, 1462, 1375, 1126, 943, 835 cm-1; 1H NMR and 13C NMR data (CD3OD, 400 and 100 MHz), see Table 1; negative ESIMS m/z 269 [M-H]-; negative HRESIMS m/z 269.0443 [M-H]- (calcd for C15H9O5, 269.0450).
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 in literature.23 The concentration of TMV was determined as 20 mg/mL with an ultraviolet spectrophotometer [virus concentration = (A260 × dilution ratio) / E 0.1%, 260 nm 1 cm]. The purified virus was kept at -20 ºC 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 could be conducted when the plants grow to the 5-6-leaf stage. The tested compounds were dissolved in DMSO and diluted with distilled H2O to the required concentrations. The solution of equal concentration of DMSO was used as negative control. The commercial antiviral agent ningnanmycin was used as a positive control.
For Half-Leaf Method,20 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 3-4 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.

ACKNOWLEDGMENT
This project was supported financially by the Excellent Scientific and Technological Team of Yunnan High School (2010CI08), the Yunnan University of Nationalities Green Chemistry and Functional Materials Research for Provincial Innovation Team (2011HC008), and Open Research Fund Program of Key Laboratory of Ethnic Medicine Resource Chemistry (Yunnan University of Nationalities) (2010XY08).

References

1. X. M. Hu, W. K. Zhang, and Q. S. Zhu, Zhong Hua Beng Cao (Encyclopaedia of Chinese Medical Herbs). Shanghai Scientific Technological Press, Shanghai, 1999 edition, p. 3105.
2. S. F. Mbatchi, B. Mbatchi, J. T. Banzouzi, T. Bansimba, G. F. Nsonde, J. M. Ouamba, A. Berry, and F. Benoit-Vical, J. Ethnopharmacol., 2006, 104, 168. CrossRef
3. J. Koyama, I. Morita, K. Tagahara, and M. Aqil, Phytochemistry, 2001, 56, 849. CrossRef
4. J. Koyama, Y. Nisino, I. Morita, N. Kobayashi, T. Osakai, and H. Tokuda, Bioorg. Med. Chem. Lett., 2008, 18, 4106. CrossRef
5. S. Oshimi, J. Deguchi, Y. Hirasawa, W. Ekasari, A. Widyawaruyanti, T. S. Wahyuni, N. C. Zaini, and H. Morita, J. Nat. Prod., 2009, 72, 1899. CrossRef
6. H. Morita, S. Oshimi, Y. Hirasawa, K. Koyama, T. Honda, W. Ekasari, G. Indrayanto, and N. C. Zaini, Org. Lett., 2007, 9, 3691. CrossRef
7. H. J. Singh and B. Agawal, Int. J. Pharmacogn., 1994, 32, 65. CrossRef
8. S. V. T. Sob, H. K. Wabo, A. T. Tchinda, P. Tane, B. T. Ngadjui, and Y. Ye, Biochem. System. Ecol., 2010, 38, 342. CrossRef
9. C. Srivastava, I. R. Siddiqui, and R. Singh, J. Indian Chem. Soc., 1992, 69, 111.
10. S. Oshimi, Y. Tomizawa, Y. Hirasawa, T. Honda, W. Ekasari, A. Widyawaruyanti, M. Rudyanto, and H. Morita, Bioorg. Med. Chem. Lett., 2008, 18, 3761. CrossRef
11. K. M. Biswas and H. Mallik, Phytochemistry, 1986, 25, 1727. CrossRef
12. S. Thadaniti, W. Archakunakorn, P. Tuntiwachwuttikul, and J. B. Bremner, J. Sci. Soc. Thailand, 1994, 20, 183. CrossRef
13. N. T. Dat, X. J. Jin, Y. S. Hong, and J. J. Lee, J. Nat. Prod., 2010, 73, 1167. CrossRef
14. D. Q. Yu and J. S. Yang, Handbook of Analytical Chemistry (7th volume), Nuclear Magnetic Resonance Spectroscopy, Chemical Industry Press, Beijing, 2nd Ed, 1999, p. 831.
15. K. Suzuki, S. Yahara, K. Maehata, and M. Uyeda, J. Nat. Prod., 2001, 64, 204. CrossRef
16. S. Venkateswarlu, G. K. Panchagnula, M. B. Guraiah, and G. V. Subbaraju, Tetrahedron, 2006, 62, 9855. CrossRef
17. O. Fulvia, P. Francesca, B. Barbara, and M. Giuliana, Carbohydr. Res., 1997, 301, 95. CrossRef
18. S. Sianne, B. N. Zhou, E. G. Thomas, L. S. Jessica, and G. I. K. David, J. Nat. Prod., 2000, 63, 457. CrossRef
19. X. M. Gao, L. Y. Yang, L. D. Shu, Y. Q. Shen, Y. J. Zhang, and Q. F. Hu, Heterocycles, 2012, 85, 1925. CrossRef
20. X. H. Yan, J. Chen, Y. T. Di, X. Fang, J. H. Dong, P. Sang, Y. H. Wang, H. P. He, Z. K. Zhang, and X. J. Hao, J. Agric. Food Chem., 2010, 58, 1572. CrossRef
21. B. A. Song, H. P. Zhang, H. Wang, S. Yang, L. H. Jin, D. Y. Hu, L. L. Pang, and W. Xue, J. Agric. Food Chem., 2005, 53, 7886. CrossRef
22. G. B. Deng, B. Wan, H. Z. Hu, J. R. Chen, and M. Q. Yu, Chinese J. Appl. Environ. Biol., 2004, 10, 695.
23. G. V. Gooding and T. T. Hebert, Phytopathology, 1967, 57, 1285.