HETEROCYCLES

Short Paper | Regular issue | Vol. 87, No. 12, 2013, pp. 2625-2631
Received, 15th September, 2013, Accepted, 15th October, 2013, Published online, 23rd October, 2013.
DOI: 10.3987/COM-13-12842

■ Cytotoxic Xanthones from Hypericum chinense

Yinke Li, Yong Xu, Jia Wu, Sujuan Wang, 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 xanthones, 1-hydroxy-4-(2-hydroxyethyl)-3,8-dimethoxy-9H-xanthen-9-one (1) and (S)-1-hydroxy-4-(1-hydroxy-3-oxobutyl)-3,8-dimethoxy-9H-xanthen-9-one (2), together with three known xanthones (3-5) were isolated from the leaves and stems of Hypericum chinense. Their structures were elucidated by spectroscopic methods, including extensive 1D- and 2D NMR techniques. Compounds 1-5 were tested for their cytotoxicity against five human tumor cell lines (NB4, A549, SHSY5Y, PC3, and MCF7). The results revealed that compound 1 showed high cytotoxicity against A549 and PC3 cell with IC50 values of 2.8 and 3.4 μM, and 2 showed high cytotoxicity with IC50 valves of 2.8 μM, respectively.

The family Clusiaceae is a rich source of xanthones.1,2 Xanthones are typically polysubstituted and occur as either fully aromatized, dihydro-, tetrahydro-, or, more rarely, hexahydro- derivatives.2 This family of compounds appeals to medicinal chemists because of their pronounced biological activity within a notably broad spectrum of disease states, including anti-hepatitis B virus,3 anti-tobacco mosaic virus,4 antibacterial,5,6 antioxidant,7,8 anti-inflammatory,9 tumor-promoting inhibition,10 cytotoxicity,11,12 and the like, as a result of their interaction with a correspondingly diverse range of target biomolecules.
The genus Hypericum belonging to Clusiaceae is distributed widely in temperate regions, and has been used for traditional medicines in various parts of the world. In China, Hypericum. chinese is used as a folk medicine for treatment of female disorders.13 Previous phytochemical investigations on H. chinese resulted in the isolation of xanthones,12 acylphloroglucinols,14 lactones,15 and norlignans.16 With the aim of multipurpose utilization of herb plants and identify bioactive natural products from this genus, the phytochemical investigation on H. chinese was carried out. As a result, two new xanthones (1 and 2), together with three known xanthones (3-5), were isolated. Compound 2 is the first naturally occurring xanthone possessing a 1-hydroxy-3-oxobutyl moiety. The structures of new compounds were elucidated by comprehensive analysis of their NMR data. In addition, the cytotoxicity of 1-5 were evaluated. The details of the isolation, structure elucidation, and cytotoxicity of the isolates are reported in this article.

A 70% aq. acetone extract prepared from the leaves and stems of H. chinense was subjected repeatedly to column chromatography on Silica gel, Sephadex LH-20, RP-18 and Preparative HPLC to afford compounds 1-5, including two new xanthones, named 1-hydroxy-4-(2-hydroxyethyl)-3,8-dimethoxy-9H-xanthen-9-one (1) and (S)-1-hydroxy-4-(1-hydroxy-3-oxobutyl)-3,8-dimethoxy-9H-xanthen-9-one (2), together with three known xanthones, 1,3,7-trihydroxy-2-(2-hydroxy-3-methyl-3-butenyl)xanthone (3),17 1,7-dihydroxy-2,3-[2′′-(1-hydroxy-1-methylethyl)dihydrofurano]xanthone (4),17 cratoxylumxanthone D (5).18 The structures of the compounds 1-5 were shown in Figure 1 and the 1H and 13C NMR data of 1 and 2 were listed in Table 1.
Compound 1 was isolated as a yellow gum. The HRESIMS of 1 gave the pseudomolecular [M+Na]+ ion at m/z 339.0840, corresponding to a molecular formula of C17H16O6. Its UV spectrum showed the maximum absorption at 302, 245, and 210 nm. Strong absorption bands accounting for hydroxy (3432 cm-1), carbonyl (1658 cm-1), and aromatic groups (1602, 1546, 1468 cm-1) could also be observed in its IR spectrum. The 1H- and 13C NMR spectrum (Table 1) displayed signals for all 17 carbons and 16 protons, including a xanthones skeleton19 (C-1 ~ C-9, C-4a, C-8a ~ C-10a; H-2, H-5 ~ H-7), two methoxy groups (δC 56.0 q, 56.2 q; δH 3.85 s, 3.81 s), a hydroxyethyl unit 11 [δC 34.0 t, 63.8 t; δH 2.54 t (7.2), 3.67 t (7.2)], and a phenolic hydroxy group (δH 13.43 s). The typical proton signals of ring A [δH 6.85 d (8.3), 7.42 t (8.3), 6.70 d (8.3)] and ring B (δH 6.59 s) suggested that 1 should be a 1,3,4,8-tetrasubstituted xanthone.19

The HMBC correlation (Figure 2) of one methoxy proton signal (δH 3.85) with C-3 (δC 161.0) showed this methoxy group was located at C-3. The correlation between the proton signal (δH 3.81) and C-8 (δC 165.4) indicated the other methoxy group located at C-8. The long-range correlations of H2-1′ (δH 2.54) to C-3 (δC 161.0), C-4 (δC 108.8) and C-4a (δC 155.3), of H2-2′ (δH 3.67) to C-4 (δC 108.8) were observed in 1. This led us to conclude that the hydroxyethyl unit was located at C-4. Finally, the HMBC correlations between the phenolic hydroxy proton (δH 13.43) and C-1 (δC 162.8), C-2 (δC 98.1), and C-9a (δC 104.1) led to the assignment of the phenolic hydroxy group at C-1. Therefore, compound 1 was assigned as 1-hydroxy-4- (2-hydroxyethyl)-3,8-dimethoxy-9H-xanthen-9-one.

Compounds (2) was also isolated as a yellow gum, and its molecular formula was determined as C19H18O7 through HRESI-MS analysis (pseudomolecular ion [M+Na]+ at m/z 381.0956). The 1H- and 13C spectra data of 2 was very similar to those of 1 (see Table 1), except for the hydroxyethyl unit in 1 was replaced by a 1-hydroxy-3-oxobutyl moiety 20 [δC 63.1 d, 51.6 t, 205.0 s, 31.5 q; δH 5.13 dd (8.8, 3.2), 2.84 dd (15.4, 3.2), 2.43 dd (15.4, 8.8), 2.14 s] in compound 2. Two methoxy groups located at C-3 and C-8, a phenolic hydroxy group located at C-1, and the 1-hydroxy-3-oxobutyl moiety at C-4 were also be concluded by the analysis of its HMBC spectrum. To determine the absolute configuration of 2, the circular dichroism (CD) analysis was employed. The experimental CD spectrum of 2 exhibited a positive Cotton effect (CE) at 219 nm and a negative CE near 246 nm. The CEs, optical rotation, and coupling constant valves of 2 were in excellent agreement with these of known compound,20 (1′S)-7-hydroxy-3-(1′-hydroxy-3′-butanoyl)-chromone-5-carboxylic acid. Thus, compound 2 was determined as (S)-1-hydroxy-4-(1-hydroxy-3-oxobutyl)-3,8-dimethoxy-9H-xanthen-9-one.
Since xanthones are known to exhibit potent cytotoxicity,2,11,12 the cytotoxicity of compounds 1-5 were tested using a previously reported procedure.21 All treatments were performed in triplicate. In the MTT assay, the IC50 was defined as the concentration of the test compound resulting in a 50% reduction of absorbance compared with untreated cells. The cytotoxic abilities against NB4, A549, SHSY5Y, PC3, and MCF7 tumor cell lines by MTT-assay (with taxol as the positive control) were shown in Table 2. The results revealed that compound 1 showed high cytotoxicity against A549 and PC3 cell with IC50 values of 2.8 and 3.4 μM, and 2 showed high cytotoxicity against PC3 cell with IC50 valves of 2.8 μM, respectively. The other compounds also showed moderate cytotoxicity for some tested cell lines with IC50 values below 10.

EXPERIMENTAL
General. Optical rotation was measured in Horiba SEPA-300 high sensitive polarimeter. 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 leaves and stems of Hypericum chinense L. were collected in Xishuangbanna Prefecture, Yunnan Province, People’s Republic of China, in September 2010. The identification of the plant material was verified by Prof. Ren P. Y (Xishuangbanna Botanical Garden). A voucher specimen (YNNI-2010-9-22) has been deposited in our laboratory.
Extraction and Isolation. The air-dried and powdered leaves and stems of H. chinense (4.0 kg) were extracted four times with 70% acetone (4 ×6 L) at room temperature and filtered. The crude extract (256 g) was applied to silica gel (200–300 mesh) column chromatography, eluting with a CHCl3-acetone 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, 18.5 g) by silica gel column chromatography, eluted with petroleum ether-EtOAc (9:1, 8:2, 7:3, 6:4, 1:1), yielded the mixtures A1–A5. The subfraction A2 (8:2, 4.2 g) was subjected to preparative HPLC (68% MeOH, flow rate 12 mL/min) to give 5 (14.8 mg). The further separation of subfraction A3 (7:3, 3.8 g) by silica gel column chromatography, and preparative HPLC (55∼65% MeOH, flow rate 12 mL/min) to give 1 (8.5 mg), 2 (10.3 mg), 3 (15.7 mg), and 4 (16.0 mg).
Cytotoxicity Assay. The IC50 values of compounds were measured using the MTT assay. The MTT assay is a colorimetric assay for measuring the activity of cellular enzymes that reduce the tetrazolium dye, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a yellow tetrazole), to its insoluble formazan, giving a purple color. Firstly, 2500 cells suspended in 100 μL MEM medium were seeded, respectively, in a 96-well plate. After 24 h incubation, fresh medium containing various concentrations of each compound were added into the 96-well plate to replace the old medium. The concentrations were ranged from 100 μM to 1.5625 μM, which was achieved by doing twofold dilutions six times. The OD595 values of the control groups at 0 h and 72 h together with the compound treated groups at 72 h from the MTT assay were measured using a plate reader. IC50 is the concentration of a compound inhibiting 50% of the cell growth.
1-Hydroxy-4-(2-hydroxyethyl)-3,8-dimethoxy-9H-xanthen-9-one (1): Obtained as a yellow gum; UV (MeOH) λmax (log ε) 210 (4.15), 245 (3.38), 302 (3.92) nm; IR (KBr) νmax 3432, 3078, 2930, 2885, 1658, 1602, 1546, 1468, 1375, 1186, 1065, 885, 764 cm-1; ESIMS m/z (positive ion mode) 339 [M+Na]+; HRESIMS (positive ion mode) m/z 339.0840 [M+Na]+ (calcd C17H16O6Na for 339.0845).
(S)-1-Hydroxy-4-(1-hydroxy-3-oxobutyl)-3,8-dimethoxy-9H-xanthen-9-one (2): Obtained as a yellow gum; [α]22.5 D-45.6 (c 0.20, MeOH); UV (MeOH) λmax (log ε) 210 (4.15), 246 (3.78), 305 (3.78) nm; CD (MeOH, c 0.25) Δε219 +0.94, Δε237 -5.56, Δε278 +0.28, Δε324 -0.92; IR (KBr) νmax 3425, 3062, 2872, 2806, 1705, 1649, 1600, 1568, 1472, 1349, 1167, 1059, 875, 764 cm-1; ESIMS m/z (positive ion mode) 381 [M+Na]+; HRESIMS (positive ion mode) m/z 381.0956 [M+Na]+ (calcd C19H18NaO7 for 381.0950).

ACKNOWLEDGMENT
This research was supported by the National Natural Science Foundation of China (No. 21002085 and 21362044), 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), the National Undergraduates Innovating Experimentation Project (2011HX18), and start-up funds of Yunnan University of Nationalities.

References

1. M. M. Pinto, M. E. Sousa, and M. S. Nascimento, Curr. Med. Chem., 2005, 12, 2517. CrossRef
2. K. S. Masters and S. Bräse, Chem. Rev., 2012, 112, 3717. CrossRef
3. T. W. Cao, C. A. Geng, Y. B. Ma, K. He, H. L. Wang, N. J. Zhou, X. M. Zhang, Y. D. Tao, and J. J. Chen, Planta Med., 2013, 79, 697. CrossRef
4. Y. P. Wu, W. Zhao, Z. Y. Xia, G. H. Kong, X. P. Lu, Q. F. Hu, and X. M. Gao, Molecules, 2013, 18, 9663. CrossRef
5. S. Klaiklay, Y. Sukpondma, V. Rukachaisirikul, and S. Phongpaichit, Phytochemistry, 2013, 85, 161. CrossRef
6. H. R. Dharmaratne, Y. Sakagami, K. G. Piyasena, and V. Thevanesam, Nat. Prod. Res., 2013, 27, 938. CrossRef
7. S. Udomchotphruet, P. Phuwapraisirisan, J. Sichaem, and S. Tip-Pyang, Phytochemistry, 2012 73, 148. CrossRef
8. C. Uvarani, K. Chandraprakash, M. Sankaran, A. Ata, and P. S. Mohan, Nat. Prod. Res., 2012, 26, 1265. CrossRef
9. M. Ali, M. Arfan, M. Ahmad, K. Singh, I. Anis, H. Ahmad, M. I. Choudhary, and M. R. Shah, Planta Med., 2011, 77, 2013. CrossRef
10. Q. B. Han, N. Y. Yang, H. L. Tian, C. F. Qiao, J. Z. Song, D. C. Chang, S. L. Chen, K. Q. Luo, and H. X. Xu, Phytochemistry, 2008, 69, 2187. CrossRef
11. Q. F. Hu, D.Y. Niu, X. L. Li, Y. H. Qin, Z. Y. Yang, G. L. Zhao, Z. X. Yang, X. M. Gao, and Z. Y. Chen, Heterocycles, 2013, 87, 1127. CrossRef
12. N. Tanaka, Y. Kashiwada, S. Y. Kim, M. Sekiya, Y. Ikeshiro, and Y. Takaishi, Phytochemistry, 2009, 70, 1456. CrossRef
13. Z. Y. Xiao and Q. Mu, Nat. Prod. Res. Dev., 2007, 19, 344.
14. S. Abe, N. Tanaka, and J. Kobayashi, J. Nat. Prod., 2012, 75, 484. CrossRef
15. N. Tanaka, S. Abe, K. Hasegawa, M. Shiro, and J. Kobayashi, Org. Lett., 2011, 13, 5488. CrossRef
16. W. Wang, Y. H. Zeng, K. Osman, K. Shinde, M. Rahman, S. Gibbons, and Q. Mu, J. Nat. Prod., 2010, 73, 1815. CrossRef
17. N. Tanaka and Y. Takaishi, Phytochemistry, 2006, 67, 2146. CrossRef
18. S. Udomchotphruet, P. Phuwapraisirisan, J. Sichaem, and S. Tip-pyang, Phytochemistry, 2012, 73, 148. CrossRef
19. Y. P. Wu, W. Zhao, Z. Y. Xia, G. H. Kong, X. P. Lu, Q. F. Hu, and X. M. Gao, Phytochem. Lett., 2013, 6, 629. CrossRef
20. M. Gan, Y. Liu, Y. Bai, Y. Guan, L. Li, R. Gao, W. He, X. You, Y. Li, L. Yu, and C. Xiao, J. Nat. Prod., 2013, 76, 1535. CrossRef
21. X. M. Gao, R. R. Wang, D. Y. Niu, C. Y. Meng, L. M. Yang, Y. T. Zheng, G. Y. Yang, Q. F. Hu, H. D. Sun, and W. L. Xiao, J. Nat. Prod., 2013, 76, 1052. CrossRef