HETEROCYCLES

Short Paper | Regular issue | Vol. 89, No. 2, 2014, pp. 481-486
Received, 18th November, 2013, Accepted, 18th December, 2013, Published online, 20th December, 2013.
DOI: 10.3987/COM-13-12889

■ Anthraquinones from Cassia fistula and Their Anti-tobacco Mosaic Virus Activity

Yinke Li, Yuchun Yang, Ying Qin, Yanlin Meng, Yanqing Ye, Haiyin Yang, Xuemei Gao,* and Qiufen 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, Jingming South Road, Chenggong New District, Kunming, Yunnan 650500, China

Abstract

Two new anthraquinones, 9,11-dihydroxy-2-(hydroxymethyl)-5-methyl-4H-naphtho[2,3-h]chromene-4,7,12-trione (1) and 9,11-dihydroxy-2,5-dimethyl-4H-naphtho[2,3-h]chromene-4,7,12-trione (2), together with five known anthraquinones (3-7) were isolated from the stems of Cassia fistula. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D NMR techniques. Compounds 1-7 were tested for their anti-tobacco mosaic virus activity. The results showed that compound 7 exhibited high anti-TMV activity with inhibition rate of 35.2%. The inhibition rate is high than that of positive control. The other compounds also showed potential anti-TMV activities with inhibition rates in the range of 15.2 –24.8%, respectively.

Cassia fistula L., (Leguminosae) is an ornamental tree with beautiful yellow flowers.1 In China, it has been used as traditional Chinese medicine by people of Dai nationality, who lived in Xishuangbanna, Yunnan province for treatment of diarrhea, gastritis, ringworm, and fungal skin infections.2,3 Previous phytochemical studies of C. fistula have shown the presence of anthraquinones,4,5 steroids,6 chromones,7,8 and flavonoids.9 Flavonoids possessing anti-tobacco mosaic virus (anti-TMV) have been isolated from C. fistula grown in De Hong Prefecture by our group.9 Motivated by a search for new bioactive metabolites from local plants, our group investigated the chemical constituents of the stems of C. fistula growing in Xishuangbanna Prefecture, which led to the isolation and characterization of two new (1 and 2) and five known (3-7) anthraquinones derivatives. This paper deals with the isolation, structural characterization, and the anti-tobacco mosaic virus (anti-TMV) activities of these compounds.

The air-dried and powdered stems of C. fistula (4.8 kg) was extracted with 70% aqueous acetone (4 × 5.0 L) at room temperature and filtered to yield a filtrate, which was successively evaporated under reduced pressure to obtain a crude extract (258 g). This crude extract was subjected repeatedly to column chromatography on Silica gel, Sephadex LH-20, RP-18 and preparative HPLC to afford compounds 1-7. Their structures were shown in Figure 1. The 1H- and 13C NMR data of the compounds 1 and 2 were listed in Table 1. By compared with the literature, the known compounds were identified as lupinacidin A (3),10 (2S)-7,9-dihydroxy-2-(prop-1-en-2-yl)-1,2-dihydroanthra[2,1-b]furan-6,11-dione (4),11 austrocortirubin (5),12 4,6,11-trihydroxy-9- propyltetracene-5,12-dione (6),13 fusarnaphthoquinone B (7).14

Compound 1 was isolated as a yellow powder. High-resolution ESIMS analysis gave a quasi-molecular ion at m/z 351.0501 [M-H]-, consistent with a molecular formula of C19H12O7, which indicated 14 degrees of unsaturation. The UV spectrum of 1 exhibited absorption bands at 364, 276, 254, and 210 nm, highly suggesting the existence of aromatic chromophore.15 Strong absorption bands accounting for hydroxy (3392 cm-1), carbonyl (1693 and 1650 cm-1), and aromatic groups (1605, 1564, and 1483 cm-1) could also be observed in its IR spectrum. The 1H NMR spectrum of 1 (Table 1) showed the presence of two phenolic hydroxy proton (δH 11.83 and 12.20), four singlet aromatic protons (δH 7.62, 7.07, 6.90, and 6.52), and two aliphatic protons contributed by one methyl singlet (δH 2.05), one O-methylene singlet (δH 4.43). In the 13C NMR spectrum of 1 (Table 1), 14 sp2 carbon signals, including three oxygenated quaternary sp2 carbon signals (δC 155.0, 161.3, and 164.0), and two carbonyl carbon signals (δC 184.4 and 182.0) were observed, which highly suggested the presence of anthraquinone core.15 The additional carbons account for the remaining substituents, a hydroxymethyl chromone ring (δC 182.9 s, 108.2 d, 168.1 s, 62.0 t),16 and a methyl carbon (δC 18.8) on the anthraquinone ring. The substituents and their location on the anthraquinone ring were established by analysis of the HMBC spectra of 1 (Figure 2). The HMBC correlations from a methyl singlet (δH 2.05) to C-1 (δC 124.0), C-2 (δC 144.1), and C-3 (δC 133.0) established the location of a methyl at C-2. HMBC correlations between the hydroxy proton (δH 12.20) and C-6 (δC 161.3), C-7 (δC 109.1), and C-5a (δC 113.7), as well as those between the other hydroxy proton (δH 11.83) and C-7 (δC 109.1), C-8 (δC 164.0), and C-9 (δC 111.0), led to the assignment of the phenolic hydroxy groups at C-6 and C-8. Additionally, H-2′ (δH 6.52) showed correlation with the carbon signal of C-3 (δC 133.0) clearly indicated that the hydroxymethyl chromone ring should be located between C-3 and C-4. On the basis of the above evidence, the structure of 1 was established as shown.

Compounds 2 was also obtained as yellow powder, a molecular formula of C19H12O6 was deduced from the HRESIMS data m/z 335.0562 [M-H]- (calcd m/z 335.0556). The 1H and 13C NMR spectra of 2 were very similar to those of 1. The obvious chemical shift differences resulted from the disappearance of a hydroxymethyl signal δC (62.0 t) and δH (4.43 s, 2H), and appearance of a methyl group signal δC (20.0 q) and δH (2.25 s, 3H) in 2. This indicated that the hydroxymethyl group in 1 was substituted by a methyl group in 2. The HMBC correlations of the methyl proton signal (δH 2.25) with C-2′ (δC 108.8) and C-3′ (δC 165.4) also indicated the position of methyl group at C-3′. Thus, the structure of 2 was established as 9,11-dihydroxy-2,5-dimethyl-4H-naphtho[2,3-h]chromene-4,7,12-trione.
Since certain of the anthraquinones exhibit potential antiviral activities,17,18 compounds 1-7 were tested for their anti-TMV activities. The inhibitory activities of compounds 1-7 against TMV replication were tested using the half-leaf method.19 Ningnanmycin, a commercial product for plant disease in China, was used as a positive control. The antiviral inhibition rates of compounds 1-7 at the concentration of 20 μM were listed Table 2. The results showed that compound 7 exhibited high anti-TMV activity with inhibition rate of 35.2%. The inhibition rate is higher than that of positive control. The other compounds also showed potential anti-TMV activities with inhibition rates in the range of 15.2–24.8%, respectively.

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-, 13C, and 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). Second separation was performed by 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 Cassia fistula L., (Leguminosae) were collected in Xishuangbanna Prefecture, Yunnan Province, People’s Republic of China, in September 2011. The identification of the plant material was verified by Prof. Yuan. N (Xishuangbanna Botanical Garden). A voucher specimen (YNNI-2010-9-28) has been deposited in our laboratory.
Extraction and Isolation. The air-dried and powdered leaves and stems of C. fistula (4.8 kg) were extracted four times with 70% acetone (4 × 5 L) at room temperature and filtered. The crude extract (258 g) was applied to silica gel (200–300 mesh) column chromatography, eluting with a MeOH-CHCl3 gradient system (9:1, 8:2, 7:3, 6:4, 5:5), to give five fractions A–E. The further separation of fraction A (9:1, 22.6 g) by silica gel column chromatography, eluted with acetone-CHCl3 (9:1, 8:2, 7:3, 6:4, 1:1), yielded the mixtures A1–A5. The subfraction A1 (9:1, 5.6 g) was subjected to preparative HPLC (65% MeOH, flow rate 12 mL/min) to give 2 (8.57 mg), 3 (12.4 mg), and 4 (22.6 mg). The further separation of subfraction A2 (8:2, 4.8 g) by silica gel column chromatography, and preparative HPLC (60% MeOH, flow rate 12 mL/min) to give 1 (8.8 mg), 5 (16.4 mg), and 6 (15.8 mg). The further separation of subfraction A3 (7:3, 3.4 g) by silica gel column chromatography, and preparative HPLC (50% MeOH, flow rate 12 mL/min) to give 7 (14.9 mg).
Anti-TMV Assays. The Anti TMV activities were tested using the half-leaf method, and ningnanmycin,19 a commercial product for plant disease in China, was used as a positive control.
9,11-Dihydroxy-2-(hydroxymethyl)-5-methyl-4H-naphtho[2,3-h]chromene-4,7,12-trione (1): C19H12O7, obtained as yellow powder; UV (MeOH), λmax (log ε) 367 (3.68), 278 (4.20), 255 (3.87), 210 (4.42) nm; IR (KBr) νmax 3392, 2928, 2876, 1693, 1650, 1605, 1564, 1483, 1418, 1362, 1276, 1158, 1134, 1065, 872, 768 cm-1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), Table 1; ESI-MS (negative ion mode) m/z 351[M-H]-; HR-ESI-MS (negative ion mode) m/z 351.0501 [M-H]- (calcd 351.0505 for C19H11O7).
9,11-Dihydroxy-2,5-dimethyl-4H-naphtho[2,3-h]chromene-4,7,12-trione (2): C19H12O6, obtained as yellow powder; UV (MeOH), λmax (log ε) 364 (3.71), 276 (4.15), 254 (3.85), 210 (4.38) nm; IR (KBr) νmax 3395, 2926, 2874, 1695, 1652, 1608, 1559, 1487, 1438, 1369, 1274, 1152, 1128, 1057, 883, 764 cm-1; 1H and 13C NMR data (CDCl3, 500 and 125 MHz), Table 1; ESI-MS (negative ion mode) m/z 335 [M-H]-; HR-ESI-MS (negative ion mode) m/z 335.0562 [M-H]- (calcd 335.0556 for C19H11O6).

ACKNOWLEDGMENT
This research was supported by the National Natural Science Foundation of China (No. 21302164), 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).

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