Xanthones from the stem bark of Garcinia bracteata with growth inhibitory effects against HL-60 cells

Phytochemistry 77 (2012) 280–286

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Xanthones from the stem bark of Garcinia bracteata with growth inhibitory effects against HL-60 cells Sheng-Li Niu a,b, Zhan-Lin Li a,b, Feng Ji a,b, Gu-Yue Liu c, Nan Zhao a,b, Xiao-Qiu Liu b, Yong-Kui Jing d, Hui-Ming Hua a,b,⇑ a

Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China School of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, Shenyang 110016, PR China School of Life Science and Biopharmaceutics, Shenyang Pharmaceutical University, Shenyang 110016, PR China d Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029, USA b c

a r t i c l e

i n f o

Article history: Received 6 February 2011 Received in revised form 10 November 2011 Available online 9 February 2012 Keywords: Garcinia bracteata Clusiaceae Xanthone Cell growth inhibition HL-60 cell line Bracteaxanthone

a b s t r a c t Five xanthones, 1,4,5,6-tetrahydroxyxanthone (1) and bracteaxanthones III–VI (2–5) together with twenty-six known compounds (6–31), were isolated from the ethanol extract of the stem bark of Garcinia bracteata. Their structures were elucidated via spectroscopic analyses. Growth inhibitory activities of these compounds against the human leukaemic HL-60 cell line were measured in vitro. Compounds 7, 11, and 29 exhibited moderate activities with GI50 values of 2.8, 3.4, and 3.1 lM, respectively, and a preliminary structure–activity relationship is discussed. Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction Plants of the genus Garcinia, which belongs to the Clusiaceae family, are widespread in the lowland tropical rainforests of Southeast Asia, West and East Africa, and Central and South America. They have been extensively investigated from both phytochemical and biological points of view, and they are well known as rich natural sources of xanthones, benzophenones, and biflavonoids (Sordat-Diserens et al., 1989). These phenolic constituents have been reported to possess a wide range of biological and pharmacological properties, such as antibacterial (Rukachaisirikul et al., 2003a), antimalarial (Likhitwitayawuid et al., 1998a), antioxidant (Minami et al., 1994), anti-inflammatory (Mahabusarakam et al., 1987), and cytotoxic (Asano et al., 1996; Han et al., 2006, 2008) activities. In China, Garcinia bracteata C. Y. Wu is distributed in the southern regions of Yunnan and Guangxi provinces. Previous phytochemical investigations of G. bracteata resulted in the isolation of caged prenylxanthones and benzophenones (Thoison et al., 2000, 2005; Na et al., 2010). In the course of our search for anticancer agents from natural sources, a systematic study was launched to investigate the

⇑ Corresponding author at: Key Laboratory of Structure-Based Drug Design & Discovery, Ministry of Education, Shenyang Pharmaceutical University, Shenyang 110016, PR China. Tel./fax: +86 24 23986465. E-mail address: [email protected] (H.-M. Hua). 0031-9422/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2012.01.010

chemical constituents in the ethanol extract of the stem bark of a Chinese G. bracteata. In the present study, thirty-one xanthones, including five new and twenty-six known compounds, have been isolated. Herein, the isolation and structural elucidation of five new xanthones are reported, as well as the inhibitory activities of the isolated compounds against the human leukaemic HL-60 cell line. 2. Results and discussion The 95% ethanol extract of the stem bark of G. bracteata was suspended in water and successively partitioned with CHCl3 and nbutanol. The CHCl3 fraction was subjected to repeated separation by silica gel column chromatography as well as reversed-phase (ODS) column chromatography, Sephadex LH-20 and semi-preparative HPLC, to afford five new xanthones (1–5) (Fig. 1) and twenty-six known xanthones (6–31) (Fig. 3). Compounds 6–31 were identified as 1,4,6-trihydroxy-5-methoxy-7-prenylxanthone (6) (Han et al., 2007), 1,4,5,6-tetrahydroxy-7,8-di(3-methylbut-2-enyl)xanthone (7) (Chanmahasathien et al., 2003), 1,4,5,6-tetrahydroxy-7-prenylxanthone (8) (Han et al., 2007), 1,4,5-trihydroxyxanthone (9) (Iinuma et al., 1995), 1,4-dihydroxy-5,6-dimethoxyxanthone (10) (Govindachari et al., 1967), globuxanthone (11) (Locksel et al., 1966), garciniaxanthone H (12) (Minami et al., 1996a), symphoxanthone (13) (Likhitwitayawuid et al., 1998b), 1-O-methylsymphoxanthone

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Β

Α

3

3

3

Fig. 1. The structures of compounds 1–5 from Garcinia bracteata.

Fig. 2. Key HMBC correlations of 1–5.

(14) (Minami et al., 1996b), morusignin I (15) (Hano et al., 1993), garcinexanthone B (16) (Chen et al., 2008), 6-deoxyjacareubin (17) (Rocha et al., 1994), 1,3,5,6-tetrahydroxyxanthone (18) (Sia et al., 1995), 1,3,6,7-tetrahydroxyxanthone (19) (Frahm and Chaudhuri, 1979), 1,5-dihydroxy-3-methoxyxanthone (20) (Iinuma et al., 1995), 1,5-dihydroxy-3,8-dimethoxyxanthone (21) (Asthana et al., 1991), 1,7-dihydroxyxanthone (22) (Gunatilaka et al., 1982), 1,2,5-trihydroxyxanthone (23) (Minami et al., 1994), 2,6-dihydroxy-1,5-dimethoxyxanthone (24) (Minami et al., 1994), 2,5-dihydroxy-1-methoxyxanthone (25) (Minami et al., 1996b), 1,2, 5-trihydroxy-6-methoxyxanthone (26) (Zhong et al., 2008), 12b-hydroxy-des-D-garcigerrin A (27) (Isabelle et al., 1989), 3-hydroxy-1, 5-dimethoxyxanthone (28) (Ghosal et al., 1976), garciniaxanthone E (29) (Minami et al., 1996b), 6-deoxyisojacareubin (30) (Rukachaisirikul et al., 2003b), and garciduol A (31) (Iinuma et al., 1996a) by comparison of their spectroscopic data with those reported in the literature. Compound 1 was isolated as a yellow powder. Its HRESIMS exhibited a quasi-molecular ion peak at m/z = 261.0394 [M+H]+, which corresponds to a molecular formula of C13H8O6. Its UV spectrum with absorption maxima at 252, 284 and 317 nm was suggestive of a xanthone derivative (Tanaka et al., 2004). The 1H NMR spectrum of 1 (Table 1) exhibited peaks due to a hydrogen-bonded hydroxy group at dH 12.06 (1H, brs, OH-1) along with two sets of ortho-coupled aromatic protons at d 7.53 (1H, d, J = 8.8 Hz, H-8), 6.94 (1H, d, J = 8.8 Hz, H-7), 7.22 (1H, d, J = 8.8 Hz, H-3) and 6.62 (1H, d, J = 8.8 Hz, H-2). Analysis of the 13C NMR spectrum (Table 1) included thirteen carbon signals, including one carbonyl at dC 181.7. The structure of compound 1 was deduced from its HMBC spectrum (Fig. 2). In this HMBC spectrum, a hydroxy group at dH 12.06 showed long-range correlations with the carbon signals at dC 153.0 (C-1), 108.2 (C-9a), and 109.5 (C-2). The carbon resonance at dC 109.5 (C-2) corresponded to one of the ortho-coupled aromatic protons at dH 6.62 (H-2) in the HSQC spectrum. The other ortho-coupled aromatic protons at dH 6.94 and 7.53 were then assigned to H-7 and H-8, respectively, based on the HMBC correlations between H-8 and C-9 (dC 181.7). Consequently, compound 1 was identified as 1,4,5,6-tetrahydroxyxanthone. Compound 2 was obtained as a yellow powder, and its molecular formula was determined to be C19H18O7 based on its HRESIMS data (m/z = 381.0922 [M+Na]+). The UV spectrum of 2 with absorption maxima at 237, 248, 283 and 313 nm was suggestive of a xanthone derivative (Tanaka et al., 2004). A comparison of the 1H NMR

and 13C NMR spectroscopic data of 2 (Table 1) with those of 1,4,6trihydroxy-5-methoxy-7-prenylxanthone (6) (Han et al., 2007) indicated that 2 possesses a 2-hydroxy-3-methylbut-3-enyl unit instead of the prenyl moiety found in 6. Indeed, the 1H NMR spectrum of 2 exhibited peaks due to a hydrogen-bonded hydroxy group at dH 12.21 (1H, s, OH-1), a singlet aromatic proton at dH 7.69 (1H, s, H-8), a pair of ortho-coupled aromatic protons at d 7.25 (1H, d, J = 8.8 Hz, H-3) and 6.61 (1H, d, J = 8.8 Hz, H-2), one methoxy group at dH 3.96 (3H, s, 5-OCH3) and a 2-hydroxy-3methylbut-3-enyl group (Orger et al., 2003), which was deduced from the characteristic proton signals at dH 4.83, 4.72 (each 1H, brs, H2-40 ), 4.21 (1H, dd, J = 8.1, 4.5 Hz, H-20 ), 2.89 (1H, dd, J = 13.8, 4.5 Hz, H-10 ), 2.72 (1H, dd, J = 13.8, 8.1 Hz, H-10 ), and 1.75 (3H, s, H3-50 ). Analyses of the 13C NMR spectrum (Table 1) indicated nineteen carbon resonances (including one set of overlapping carbon signals at dC 108.8) that were classified as two methyl carbons, two methylene carbons, four methine carbons, and eleven quaternary carbons based on the analysis of the HSQC spectrum. The typical methoxy carbon resonance appeared at dC 61.1, which is shifted significantly downfield (ca.+5 ppm) due to steric effects. This result suggested that both of the ortho-positions of this methoxy group are substituted (Iinuma et al., 1996b). The detailed HMBC correlations that are summarised in Fig. 2 were used to confirm the structure of 2. Thus, compound 2 was identified as 1,4,6-trihydroxy-7-(2-hydroxy-3-methylbut-3-enyl)-5methoxyxanthone and was named bracteaxanthone III. Compound 3 was obtained as a yellow powder, and its molecular formula was determined to be C19H18O7 based on its HRESIMS results ([M+Na]+ at m/z = 381.0917). The UV spectrum with absorption maxima at 239, 249, 285 and 317 nm was suggestive of a xanthone derivative (Tanaka et al., 2004). The 1H NMR and 13 C NMR spectra of 3 (Table 1) were very similar to those of 2 except for the signals associated with the side-chain. Indeed, the substitutions at C-6 and C-7 appeared to be consistent with the presence of a 2-(2-hydroxypropan-2-yl)-2,3-dihydrofuran ring (Hashida et al., 2007) based on the following analysis of its characteristic 1H NMR and 13C NMR data: dH 4.81 (1H, t-like, J = 8.7 Hz, H20 ), 4.74 (1H, s, OH-30 ), 3.16 (2H, d-like, J = 8.7 Hz, H2-10 ), 1.19 and 1.16 (each 3H, s, H3-40 ,50 ); dC 91.7 (C-20 ), 73.7 (C-30 ), 31.2 (C-10 ), 26.0 (C-50 ), 25.2 (C-40 ). These results suggest that the five-carbon side-chain at the C-7 position in the structure of 2 was cyclised into a dihydrofuran ring as observed previously in related xanthone compounds (Tanaka and Takaishi, 2006). The location of the

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Fig. 3. The structures of compounds 6–31 from Garcinia bracteata.

dihydrofuran ring was determined following consideration of the HMBC results. The correlations of H-10 with C-6 (dC 148.3), C-7 (dC 125.5) and C-8 (dC 121.9) and of H-20 with C-6 and C-7, as well as that of H-8 with C-10 and C-6 indicated that the dihydrofuran ring was fused to C-6 and C-7 of the xanthone nucleus in a linear arrangement with an ether linkage at C-6. The assigned structure was further verified by a detailed HMBC analysis (Fig. 2). Accordingly, compound 3 was identified as 6,9-dihydroxy-11-methoxy2-(2-hydroxypropan-2-yl)-2,3-dihydrofuro[3.2-b] xanthen-5-one and named bracteaxanthone IV. Compound 4 was obtained as a yellow powder. Its molecular formula was determined to be C19H18O8 from the observed quasi-molecular ion peak at m/z = 373.0920 [MH] in the HRESIMS. The UV spectrum with absorption maxima at 246, 286, and

320 nm was suggestive of a xanthone derivative (Tanaka et al., 2004). A comparison of the 1H and 13C NMR spectroscopic data of 4 (Table 1) with those of 3 showed that the two compounds are closely related analogues in which the only notable difference is the chemical shift of C-10 [dC 70.9 (4); dC 31.2 (3)], suggesting that C-10 is substituted with a hydroxy group. Indeed, the substitution at C-6 and C-7 appeared to be consistent with the presence of a 2-(2-hydroxypropan-2-yl)-3-hydroxy-2,3-dihydrofuran ring (Chin et al., 2008) based on the following analysis of its characteristic 1H NMR and 13C NMR data: dH 5.97 (1H, d, J = 6.1 Hz, OH-10 ), 5.32 (1H, dd, J = 4.9, 4.1 Hz, H-10 ), 4.75 (1H, s, OH-30 ), 4.37 (1H, d, J = 4.1 Hz, H-20 ), 1.21 (3H, s, H3-40 ) and 1.18 (3H, s, H3-50 ); dC 99.5 (C-20 ), 70.9 (C-10 ), 70.2 (C-30 ), 26.1 (C-50 ), 25.9 (C-40 ). The location of this moiety was further confirmed by the results of an HMBC

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S.-L. Niu et al. / Phytochemistry 77 (2012) 280–286 Table 1 1 H and 13C NMR spectroscopic data of compounds 1–5 (in DMSO-d6, d in ppm). Position

1 dC

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 10

2 a

153.0 109.5 123.2 137.7 143.4 133.1 152.5 114.3 116.6 113.4 181.7 108.2 146.1

dHb

(J in Hz)

6.62, d (8.8) 7.22, d (8.8)

6.94, d (8.8) 7.53, d (8.8)

0

b c

152.7 108.8 123.1 137.5 143.9 134.3 156.0 125.3 121.7 108.4 180.9 108.8 149.2 36.6 73.5 148.1 110.1 18.0

2 30 40 50 1-OH 4-OH 5-OCH3 10 -OH 30 -OH a

dC

3 a

12.06, brs 61.1

dHc

(J in Hz)

6.61, d (8.8) 7.25, d (8.8)

7.69, s

2.89, dd (13.8, 4.5) 2.72, dd (13.8, 8.1) 4.21, dd (8.1, 4.5) 4.83, brs, 4.72, brs 1.75, s 12.21, s 3.96, s

dC

4 a

152.9 109.0 123.3 137.7 144.1 134.5 148.3 125.5 121.9 110.1 180.9 108.8 149.4 31.2 91.7 73.7 25.2 26.0

61.4

c

dH (J in Hz) 6.60, d (8.8) 7.23, d (8.8)

7.68, s

3.16, d-like (8.7) 4.81, t-like (8.7) 1.19, s 1.16, s 12.20, s 9.56, brs 4.01, s 4.74, s

dC

5 a

153.0 109.4 123.8 137.9 144.2 131.4 157.3 130.9 116.3 115.2 181.5 108.6 150.7 70.9 99.5 70.2 25.9 26.1

61.0

c

dH (J in Hz) 6.63, d (8.8) 7.26, d (8.8)

7.85, s

5.32, dd (4.9, 4.1) 4.37, d (4.1) 1.21, s 1.18, s 12.06, s 9.63, brs 3.96, s 5.97,d (6.1) 4.75, s

dCa 160.0 101.4 164.4 94.0 155.5 134.8 157.5 114.4 121.1 113.2 179.3 110.1 150.6 20.9 123.8 135.5 60.0 21.6

61.0

dHb(J in Hz)

6.39, s

6.92, d (8.8) 7.69, d (8.8)

3.26, d (7.2) 5.21, t (7.2) 4.10, s 1.65, s 13.32, s 3.85, s

Recorded at 150 MHz. Recorded at 600 MHz. Recorded at 300 MHz.

Fig. 4. Selected NOESY correlations of compound 4.

experiment (Fig. 2). Further analysis of the HMBC correlations allowed for the unambiguous assignment of the various resonances. The NOESY experiments indicated the correlations of H-10 with H340 and H3-50 and of H-20 with OH-10 , as shown in Fig. 4. Consequently, H-10 and H-20 have a trans relationship to each other (Minami et al., 1995). Hence, compound 4 was assigned as 3,6,9trihydroxy-11-methoxy-2-(2-hydroxypropan-2-yl)-2,3-dihydrofuro[3.2-b] xanthen-5-one and named bracteaxanthone V. Compounds 1–4 are biosynthetically closely related. A hypothetical biosynthetic pathway linking 1–4 is proposed as shown in Scheme 1 based on the related references (Helesbeux et al., 2003a,b). Compound 5 was obtained as a yellow powder, and its HRESIMS exhibited the [MH] ion peak at m/z = 357.0972, which supports the molecular formula of C19H18O7. The UV spectrum with absorption maxima at 216, 246, and 319 nm was suggestive of a xanthone derivative (Tanaka et al., 2004) (Chen et al., 2008). The 1H NMR spectrum of 5 (Table 1) exhibited peaks due to a chelated hydroxy group at dH 13.32 (1H, s, OH-1), a singlet aromatic proton at dH 6.39 (1H, s, H-4), a pair of ortho-coupled aromatic protons at dH 7.69 (1H, d, J = 8.8 Hz, H-8) and 6.92 (1H, d, J = 8.8 Hz, H-7), one methoxy group at dH 3.85 (3H, s, 5-OCH3), and a 4-hydroxy-3-methylbut-2-enyl group (Na Pattalung et al., 1994), which was deduced

from a series of characteristic 1H NMR signals at dH 5.21 (1H, t, J = 7.2 Hz, H-20 ), 4.10 (2H, s, H2-40 ), 3.26 (2H, d, J = 7.2 Hz, H2-10 ) and 1.65 (3H, s, H3-50 ). The positions of the substituents were determined from an analysis of the HMBC spectra (Fig. 2). The ortho-coupled aromatic protons at dH 6.92 and 7.69 were assigned to H-7 and H-8, respectively, due to the HMBC correlations between H-8 and C-9 (dC 179.3) and the downfield chemical shift of H-8 at dH 7.69. The linkage of the side-chain was verified by the HMBC correlations of H-10 (dH 3.26) with C-1 (dC 160.0), C-2 (dC 101.4) and C-3 (dC 164.4). The long-range correlation between a methoxy proton at dH 3.85 and C-5 (dC 134.8) determined the location of the methoxy group at the C-5 position. The geometry of the prenyl side-chain double bond was determined to be Z by the chemical shift of the methyl carbon (dC 21.6). The methyl signal of the E form would appear more high-field because of the increasing c-effect (Asano et al., 1996). Thus, compound 5 was identified as 1,3,6-trihydroxy-2-(4-hydroxy-3-methylbut-2-enyl)-5-methoxyxanthone and was named bracteaxanthone VI. All of the isolated compounds except 10, 21, 24, 28 and 31 were evaluated for their cell growth inhibitory effects against the human leukaemic HL-60 cell line (GI50 values are listed in Table 2). The results demonstrated that almost all of the tested compounds exhibited moderate inhibitory activities against the HL-60 cell line, with GI50 values ranging from 2.8 to 47.9 lM. Of these compounds, the prenylated xanthones (6–8, 13–14, 27 and 29) exhibited more potent effects, of which 7, 11, and 29 are the most effective compounds at inhibiting HL-60 cell growth with GI50 values of 2.8, 3.4, and 3.1 lM, respectively. The inhibitory activities of compounds 8 and 27, which possess prenyl groups, were more potent than those of the corresponding simple xanthones 1 and 9, which indicated that the prenyl group plays an important role in the HL-60 growth inhibition. The presence of an extra prenyl group on the xanthone skeleton in compound 7 increased the activity compared to that of 8, indicating that more prenyl groups results in a more potent effect. This finding is in agreement with the results reported in the literature using the HeLa cell line (Han

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Scheme 1. Hypothetical biosynthetic pathway of 1–4.

Table 2 GI50 values of compounds 1–31 that inhibit HL-60 cell growth.a GI50 ± SD (lM)

GI50 ± SD (lM)

Compound

HL-60 cell line

Compound

HL-60 cell line

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

24.8 ± 1.4 21.0 ± 0.5 18.0 ± 0.7 22.2 ± 0.6 21.0 ± 4.8 9.9 ± 0.8 2.8 ± 1.1 10.1 ± 3.1 28.0 ± 6.8 –b 3.4 ± 0.9 21.9 ± 0.1 9.12 ± 2.2 4.8 ± 1.1 37.7 ± 5.1 22.8 ± 0.4

17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 5-Fuc

18.5 ± 2.2 16.2 ± 5.3 18.0 ± 6.1 17.7 ± 7.1 –b 21.9 ± 0.5 23.1 ± 0.9 –b 47.9 ± 2.0 16.7 ± 2.9 5.4 ± 0.2 –b 3.1 ± 1.0 27.3 ± 1.4 –b 3.8 ± 1.9

a GI50 is the concentration that inhibited 50% of cell growth. The data shown are means ± SD of three independent experiments. b Not soluble in DMSO. c Positive control.

et al., 2007, 2008). The additional prenyl substitution at the prenyl side-chain in compound 29 did not significantly influence the activity of this compound compared to that of 7. The hydroxylation (2 and 5) of the prenyl group or the cyclisation of this group into either a furan or a pyran ring (3, 4, 15, 16, 17 and 30) lowered the activity of the corresponding compounds, suggesting that the hydrophobic unsaturated acyclic prenyl group may be essential for the growth inhibition. 3. Conclusions The prenyl xanthones that were isolated significantly inhibited HL-60 cell growth. The present results offer more information about the SAR of prenyl xanthones in cancer cell growth inhibition. Although xanthones are structurally related to anthraquinones, which are used as anticancer drugs, such as mitoxantrone, there has been no record of xanthone derivatives being used as chemotherapeutic agents for the treatment of cancer. The Garcinia species are rich in prenylated xanthones. Of these compounds, the most notable lead compound is gambogic acid with a caged polyprenylated skeleton, which has been studied in phase II clinical trials in China as a prominent anticancer drug candidate (Zhou and Wang, 2007). Thus, concluded from this study and a literature research is that some xanthones could be drug candidates and may have the

potential to be effective anticancer drugs (Ho et al., 2002). Additionally, compounds with more prenyl moieties should be synthesised to obtain more active cytotoxic compounds. 4. Experimental 4.1. General experimental procedures UV spectra were obtained using a Shimadzu UV-2201 spectrometer, whereas, FT-IR spectra were acquired on a Bruker IFS55 spectrometer (using a KBr disk method). The ESI-MS spectra were obtained on an Agilent 1100 ion-trap spectrometer. The HRESIMS data were recorded on Varian QFT-ESI and Bruker microTOFQ-Q mass spectrometers. The NMR spectra were recorded on Bruker ARX-300 and AV-600 NMR spectrometers using TMS as an internal standard. The optical rotations were measured with a Perkin-Elmer 241 polarimeter. Silica gel (200–300 mesh) for column chromatography (CC) was purchased from Qingdao Ocean Chemical Factory (Qingdao, China), and ODS (50 lm) was purchased from YMC Co. Ltd., Kyoto, Japan. The Sephadex LH-20 was purchased from GE Healthcare. The RP-HPLC analysis and semi-preparation were conducted using a HITACHI L2130 series pumping system equipped with a HITACHI L2400 UV detector and a C-18 column (10 mm  250 mm, 10 lm; YMC Co. Ltd.). 4.2. Plant material Plant material was collected from Yunnan province, China, in October 2008 and was identified as stem bark of Garcinia bracteata C. Y. Wu by Mr. Jingyun Cui. (Xishuangbanna Tropical Botanic Garden of the Chinese Academy of Sciences, People’s Republic of China). A voucher sample (DBTH-20081010) was deposited in the Department of Natural Products Chemistry, Shenyang Pharmaceutical University, Shenyang, China. 4.3. Extraction and isolation Dried stem bark of G. bracteata (1.5 kg) was extracted three times (2 h, 2 h, 1 h) with EtOH–H2O (3  3 L 95:5, v/v) under condition of reflux. The combined EtOH extracts were concentrated in vacuo to yield a brown-yellow gum (420 g). The latter was then suspended in H2O (2 L) and partitioned successively with CHCl3 (3  2 L) and n-BuOH (3  2 L). The CHCl3-soluble extract exhibited a significant growth inhibitory effect against the human leukaemia HL-60 cell line (GI50 < 6.25 lg/mL). The CHCl3 extract (85 g) was separated by a silica gel CC using a gradient system of increasing

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polarity of petroleum ether (P. E., 60  90 °C)-acetone (100:0– 0:100, v/v). The collected fractions were combined based on their TLC characteristics to yield ten fractions (Fr. A-Fr. J). Fr. A (2.5 g) was subjected to Sephadex LH-20 CC eluted with CHCl3–MeOH (1: 1) to yield four fractions (Fr. A1-Fr. A4). Fr. A4 was separated by reversed-phase ODS CC and eluted with a MeOH–H2O system (80:20) to afford 17 (3.1 mg). Fr. B (4.8 g) was submitted to Sephadex LH-20 CC and eluted with CHCl3–MeOH (1:1) to yield three fractions (Fr. B1-Fr. B3). Fr. B2 was repeatedly recrystallised in P. E.-acetone (5:1) to yield 30 (3.7 mg). Fr. B3 was fractionated in a similar way to afford 22 (3.4 mg) and 27 (5.8 mg) by recrystallisation in P. E.-acetone (5:1). Fr. C (3.2 g) was purified by Sephadex LH-20 CC and eluted with CHCl3–MeOH (1:1) to yield three fractions (Fr. C1-Fr. C3), and 20 (5.4 mg). Fr. C2 was further purified by ODS CC and eluted with a MeOH–H2O gradient system (from 60:40 to 90:10) to yield three major subfractions (Fr. C2A- Fr. C2C). Fr. C2B was separated by preparative TLC with a developing solvent system of P. E.-acetone (2:1) to provide 9 (6.1 mg) and 10 (3.2 mg), which were further purified by Sephadex LH-20 CC in MeOH. Compound 16 (4.8 mg) was isolated from Fr. C2C by repeated silica gel CC with P. E.-acetone (20:1). Fr. D (3.5 g) was further separated by HPLC on a semi-preparative YMC C-18 column using MeOH–H2O (75:25) as the mobile phase to provide 11 (4.2 mg), 12 (3.2 mg), and 15 (8.5 mg). Fr. E (5.2 g) was loaded onto a Sephadex LH-20 column and eluted with CHCl3–MeOH (1:1) to yield five fractions (Fr. E1-Fr. E5). Compounds 2 (3.6 mg), 6 (20 mg), 7 (10.7 mg), 13 (9.2 mg) and 25 (7.9 mg) were obtained from Fr. E2 by ODS CC, eluted with a MeOH–H2O gradient system (from 30:70 to 90:10), and were further purified by Sephadex LH20 CC in MeOH. Fr. F (5.5 g) was applied to a Sephadex LH-20 column and eluted with CHCl3–MeOH (1:1) to yield three fractions (Fr. F1-Fr. F3). Fr. F2 was repeatedly recrystallised in P. E.-acetone (5:1) to produce 21 (3.9 mg) and 24 (8.3 mg). Fr. F3 was further purified by ODS CC and eluted with a MeOH–H2O gradient (from 50:50 to 90:10) to yield 8 (3.3 mg), 23 (7.3 mg), and 29 (3.1 mg). Fr. G (4.2 g) was divided into two fractions (Fr. G1- Fr. G2) by Sephadex LH-20 CC and eluted with CHCl3–MeOH (1:1). Fr. G1 was further separated by ODS CC and eluted with a MeOH–H2O gradient (from 20:80 to 90:10) to afford four subfractions (Fr. G1A- Fr. G1D). Compounds 1 (2.6 mg), 26 (2.9 mg), and 28 (3.5 mg) were obtained from Fr. G1A via Sephadex LH-20 CC in MeOH. Repeated chromatography of Fr. G1A on a silica gel column and elution with a gradient system of P. E.-acetone (100:1–100:10) afforded 3 (3.2 mg), 5 (3.6 mg), and 14 (5.1 mg). Fr. H (5.3 g) was loaded onto a Sephadex LH-20 column and eluted with CHCl3– MeOH (1:1) to yield three fractions (Fr. H1-Fr. H3). Compound 31 (3.3 mg) was isolated from Fr. H1 by repeated silica gel open CC and eluted with P. E.-acetone (100:50). Fr. H2 was purified by HPLC on a semi-preparative YMC C-18 column using MeOH–H2O (60:40) as the mobile phase to yield 18 (3.4 mg) and 19 (7.6 mg). Fr. J (2.1 g) was divided into three fractions (Fr. J1- Fr. J3) by Sephadex LH-20 CC and eluted with CHCl3–MeOH (1:1). Fr. J1 was separated by ODS CC, eluted with MeOH–H2O (50:50) and was further purified by silica gel open CC (eluted with P. E.-acetone, 100:50) to afford 4 (3.8 mg). 4.3.1. 1,4,5,6-Tetrahydroxyxanthone (1) Yellow amorphous powder, UV (MeOH) kmax: 252, 284, 317 nm; IR (KBr) mmax: 3428, 1648, 1598, 1498, 1466, 1385, 1283, 1025, 774 cm1; for 1H and 13C NMR (DMSO-d6) spectroscopic data, see Table 1; (-) ESI-MS: m/z = 258.8 [MH]; HRESIMS: m/ z = 261.0394 [M+H]+ (calcd. for C13H9O6, 261.0399).

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1604, 1486, 1384, 1234, 1120, 799 cm1; for 1H and 13C NMR (DMSO-d6) spectroscopic data, see Table 1; HRESIMS: m/z 381.0922 [M+Na]+ (calcd. for C19H18O7Na, 381.0950). 4.3.3. Bracteaxanthone IV (3) Yellow amorphous powder, ½a25 D :-5.2 (c 0.02, MeOH); UV (MeOH) kmax: 239, 249, 285, 317 nm; IR (KBr) mmax: 3431, 2921, 2851, 1650, 1596, 1466, 1382, 1282, 1224, 1087, 802 cm1; for 1 H and 13C NMR (DMSO-d6) spectroscopic data, see Tables 1; HRESIMS: m/z 381.0917 [M+Na]+ (calcd. for C19H18O7Na, 381.0950). 4.3.4. Bracteaxanthone V (4) Yellow amorphous powder, ½a25 D :+9.4 (c 0.02, MeOH); UV (MeOH) kmax: 246, 286, 320 nm; IR (KBr) mmax: 3418, 2926, 1686, 1633, 1598, 1487, 1440, 1285, 1222, 1167, 1105, 799, 620, 526 cm1; for 1H and 13C NMR (DMSO-d6) spectroscopic data, see Table 1; (+) ESI-MS: m/z 397.3 [M+Na]+; HRESIMS: m/z 373.0920 [MH] (calcd. for C19H17O8, 373.0923). 4.3.5. Bracteaxanthone VI (5) Yellow amorphous powder, UV (MeOH) kmax:216, 246, 319 nm; IR (KBr) mmax: 3439, 1649, 1612, 1453, 1401, 1322, 1122, 620 cm1; for 1H and 13C NMR (DMSO-d6) spectroscopic data, see Table 1; () ESI MS: m/z 356.9 [MH]; HRESIMS: m/z 357.0972 [MH] (calcd. for C19H17O7, 357.0974). 4.4. Cell culture and growth inhibition assay Cell growth inhibitory activities of isolated compounds were assayed by the trypan blue method using the human leukaemia HL-60 cell line. The cell line was purchased from American Type Culture Collection, ATCC (Rockville, MD, USA) and cultured in RPMI-1640 medium (Gibco, New York, NY, USA) supplemented with 100 U/mL penicillin, 100 lg/mL streptomycin, 1 mM glutamine and 10% heat-inactivated foetal bovine serum (Gibco). The cell growth inhibition assay was performed as reported previously (Wang et al., 2006). Briefly, cells in logarithmic growth were seeded at a density of 1  105 cells/mL and incubated with various concentrations of the test compounds for 3 days. The compounds were dissolved in DMSO and then diluted to the proper concentrations such that the final concentrations of DMSO were less than 0.1% in the culture medium. Cell viability was determined after staining the cells with trypan blue. The trypan blue-stained (nonviable) cells and the total cell number were determined using a hematocytometer. The growth inhibition in cells after the treatment with different concentrations of the compounds was calculated by comparing these data with those of the control cells, and a half-growth inhibitory concentration (GI50) was obtained from a regression analysis of the concentration response data. 5Fluorouracil (5-Fu), an anticancer agent, was used as a positive control, and 0.1% DMSO was used as a negative control. Three independent experiments were duplicated, and the data are presented as the mean ± SD. Acknowledgements The authors wish to thank Mr. Yi Sha and Mrs. Wen Li (Analytical Testing Center, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China) for their measurements of the NMR data. Appendix A. Supplementary data

4.3.2. Bracteaxanthone III (2) Yellow amorphous powder, ½a25 D :+0.5 (c 0.02, MeOH); UV (MeOH) kmax: 237, 248, 283, 313 nm; IR (KBr) mmax: 3445, 1658,

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2012.01.010.

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