Nitric oxide inhibitory xanthones from the pericarps of Garcinia mangostana

Phytochemistry 131 (2016) 115e123

Contents lists available at ScienceDirect

Phytochemistry journal homepage: www.elsevier.com/locate/phytochem

Nitric oxide inhibitory xanthones from the pericarps of Garcinia mangostana Qianyu Liu a, 1, Dan Li a, 1, Anqi Wang a, Zhen Dong b, Sheng Yin b, Qingwen Zhang a, Yang Ye c, Liangchun Li d, **, Ligen Lin a, * a

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou, Guangdong, 510006, China Department of Natural Products Chemistry, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai, 201203, China d School of Life Science and Engineering, Southwest University of Science and Technology, Mianyang, Sichuan, 621010, China b c

a r t i c l e i n f o

a b s t r a c t

Article history: Received 18 January 2016 Received in revised form 21 July 2016 Accepted 15 August 2016 Available online 22 August 2016

Mangosteen (Garcinia mangostana, Clusiaceae) is called “queen of fruit” in Southeast Asia. In the current study, three dimeric xanthones, garcinoxanthones AeC, and four monomeric xanthones, garcinoxanthones DeG, together with 18 known xanthones, were isolated from the pericarps of G. mangostana, collected in Thailand. The structures of garcinoxanthones AeG were elucidated by analysis of their 1D and 2D NMR and other spectroscopic data, and their absolute configurations were determined by the CD spectra. All seven compounds were tested for nitric oxide (NO) inhibitory activity on lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Garcinoxanthones B and C significantly inhibited NO production with IC50 values of 11.3 ± 1.7 and 18.0 ± 1.8 mM, respectively, which were comparable with the positive control indomethacin (IC50 3.9 ± 0.3 mM). Moreover, garcinoxanthone B suppressed inducible NO synthase expression in a dose-dependent manner. These results reveal the presence of rare dimeric xanthones in G. mangostana and their NO inhibitory effect on LPS-stimulated murine macrophage cells. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Garcinia mangostana Clusiaceae Xanthones Dimeric xanthones NO inhibition iNOS

1. Introduction Nitric oxide (NO) is a key mediator and regulator of inflammatory responses, which is involved in the pathogenesis of a diverse range of disorders (Strowig et al., 2012). Overproduction of NO can damage cells and tissues, resulting in inflammatory diseases, such as septic shock, bacterial sepsis and diabetes (Brownlee, 2005; Forstermann and Sessa, 2012). NO production is mainly mediated by inducible NO synthase (iNOS) in inflammatory cells, in response to cytokines, lipopolysaccharide and other agents (Wei et al., 1995). Therefore, inhibition of NO production by suppressing iNOS expression has been considered as a valuable therapeutic pathway for the treatment of inflammatory diseases. Garcinia mangostana L. (Clusiaceae) is a kind of tropical evergreen tree. Its fruit, mangosteen, is known as the “queen of fruit” in

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (L. Li), [email protected] (L. Lin). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.phytochem.2016.08.007 0031-9422/© 2016 Elsevier Ltd. All rights reserved.

Southeast Asia, due to it being tasty and juicy. In Thailand, the pericarps of G. mangostana have been used as medicine to treat catarrh, cystitis, diarrhea, fever, skin infections and wounds (Gutierrez-Orozco and Failla, 2013; Mahabusarakam et al., 1987). Due to its medicinal potential, many phytochemical investigations have been carried out on different parts of G. mangostana, resulting in the identification of xanthones, benzophenones and biflavonoids (Obolskiy et al., 2009). Xanthones are found to be the major constituents responsible for the various bioactivities of G. mangostana, including anti-oxidation (Jung et al., 2006), cytotoxic activity (Han et al., 2009) and anti-inflammation (Chen et al., 2008; GutierrezOrozco et al., 2013; Syam et al., 2014). To date, about 74 xanthones have been isolated and identified from the pericarps, barks, roots, leaves and other parts of G. mangostana (Liu et al., 2015). As part of our ongoing research into the anti-inflammatory constituents of tropical fruit waste components, the pericarps of G. mangostana were chemically investigated in the current study, which led to the isolation of 25 xanthones, including three new dimeric xanthones (1e3) and four new monomeric xanthones (4e7). All the new compounds were evaluated for their ability to inhibit LPS-induced NO production in RAW264.7 cells. The

116

Q. Liu et al. / Phytochemistry 131 (2016) 115e123

suppressive effect of compound 2 on iNOS expression was also investigated. 2. Results and discussion The chloroform-soluble fraction of the 95% ethanol extract of G. mangostana was purified by column chromatography over silica gel, MCI gel, ODS gel, Sephadex LH-20 and preparative HPLC to afford 25 xanthones, including 7 new compounds (1e7) (Fig. 1) and 18 known ones. The structures of the known xanthones were identified as a-mangostin (Sen et al., 1982), b-mangostin (Likhitwitayawuid et al., 1998), g-mangostin (Sakai et al., 1993), garcinone E (Sakai et al., 1993), gartanin (Bennett et al., 1990), mangosharin (Ee et al., 2006), tovophyllin A (Bennett et al., 1993), 8-deoxygartanin (Nguyen et al., 2003), cudraxanthone G (Ito et al., 1996), 8-hydroxycudraxanthone G (Jung et al., 2006), 7-O-methylgarcinone E (Likhitwitayawuid et al., 1997), 9hydroxycalabaxanthone (Sen et al., 1980), trapezifolixanthone (Seo et al., 1999), garcinone D (Bennett et al., 1993), 11-hydroxy-1isomangostin (Sia et al., 1995), garcinone C (Sen et al., 1982), 1,3,6,7tetrahydroxy-8-prenylxanthone (Zhou et al., 2013) and cratoxyxanthone (Sia et al., 1995) by comparison of their observed and reported spectroscopic and physical data. Among the known compounds 7-O-methylgarcinone E and trapezifolixanthone were isolated from G. mangostana for the first time. Garcinoxanthone A (1) was obtained as a yellow amorphous powder. Its HRESIMS spectrum inferred the molecular formula as C48H50O13 (m/z 835.3301 [MþH]þ), with 24 degrees of unsaturation. Its UV absorptions at 210, 245 and 318 nm, together with its IR absorptions at 3424 and 1610 cm1, indicated the presence of hydroxy and conjugated carbonyl groups. The 1H NMR spectrum of compound 1 indicated the presence of two methoxy groups [dH 3.76 (s) and 3.68 (s)], three aromatic protons [dH 6.05 (s), 6.50 (s) and 6.69 (s)] and three prenyl groups. The 13C NMR indicated the existence of 48 carbons, including two carbonyl carbons, four benzene rings, four C5 moieties and two methoxy carbons. Thus, compound 1 was inferred to be a dimeric xanthone. The 1H and 13C

NMR data of compound 1 (Table 1) were quite similar to those of cratoxyxanthone (Sia et al., 1995). The main difference was the chemical shifts of three aromatic protons. Detailed analysis of HMBC and HSQC data established that compound 1 possesses the same two subunits as cratoxyxanthone. The HMBC cross-peaks of H-10,11/C-2 (dC 111.5) suggested that a prenyl group [dH 3.18 (2H, d, J ¼ 6.8 Hz, H-10), 5.12 (1H, m, H-11), 1.71 (3H, s, H-10) and 1.57 (3H, s, H-11)] was attached at C-2. Similarly, the other two prenyl groups, [dH 4.04 (2H, m, H-15), 5.24 (1H, m, H-16), 1.80 (3H, s, H-18) and 1.65 (3H, s, H-19)] and [dH 3.96 (2H, m, H-150 ), 5.12 (1H, m, H160 ), 1.71 (3H, s, H-180 ) and 1.55 (3H, s, H-190 )], were determined at C-8 and C-80 by the HMBC correlations of H-15,16/C-8 (dC 136.4) and H-150 ,160 /C-80 (dC 138.4), respectively. The HMBC cross-peaks of OCH3 (dH 3.76)/C-7 (dC 138.4) and OCH3 (dH 3.68)/C-70 (dC 138.4) suggested that two methoxy groups were located at C-7 and C-70, respectively. The aromatic proton dH 6.05 (1H, s) at position 4 was confirmed by the HMBC correlations from the proton signal to C-2 (dC 111.5), C-3 (dC 163.5), C-4a (dC 158.7) and C-9a (dC 103.6). The HMBC cross-peaks from the aromatic proton (6.50, 1H, s) to C-30 (dC 169.0), C-4a0 (dC 158.7), C-20 (dC 111.9) and C-9a0 (dC 104.8) suggested it to be at position 40 . The HMBC correlations from the aromatic proton at dH 6.69 to C-70 (dC 144.9) and C-8a0 (dC 112.1) indicated it as H-50 . In addition, compound 1 displayed NMR signals at dH/dC 5.46 (1H, d, J ¼ 5.6 Hz)/37.4, dH/dC 4.82 (1H, d, J ¼ 5.8 Hz)/ 98.7, dH/dC 1.35 (3H, s)/25.8 and dH/dC 1.27 (3H, s)/24.2, which were assigned to a dihydrofuran group as in cratoxyxanthone (Sia et al., 1995). This moiety was placed at C-20 /C-30 based on the 2J and 3J HMBC correlations between the proton (dH 5.46) and C-20 (dC 111.9) and C-30 (dC 169.0), and between the proton (dH 4.82) and C-30 (dC 169.0) (Table 1). Moreover, the HMBC cross-peaks of H-100 ,110 /C-5 (dC 115.6) suggested a xanthoneexanthone linkage between C-5 and C-100 . Thus, the overall structure of compound 1 was determined. The absolute configuration of compound 1 was determined by an electronic circular dichroism (ECD) calculation. The experimental ECD spectrum of compound 1 was in accordance with the calculated ECD spectra of 100 R, 110 S (Fig. 2). Thus, the absolute

Fig. 1. New xanthones isolated from G. mangostana.

Q. Liu et al. / Phytochemistry 131 (2016) 115e123 Table 1 1 H NMR (600 MHz) and

13

C NMR (150 MHz) spectroscopic data for garcinoxanthones A (1, CD3OD), B (2, CD3OD) and C (3, Acetone-d6): d in ppm (multiplicities, J in Hz).

Position Garcinoxanthone A (1)

dC type

dH (J)

1 2 3 4 4a 5

161.3, C 111.5, C 163.5, C 93.0, CH 6.05 s 158.7, C 115.6, C

5a 6 7 8 8a 9 9a 10

155.6, C 158.0, C 144.4, C 136.4, C 112.6, C 183.3, C 103.6, C 22.2, CH2 123.8, CH 131.6, C 17.9, CH3 25.8, CH3 27.1, CH2 125.3, CH 131.8, C 18.4, CH3 25.9, CH3 155.0, C 111.9, C 169.0, C 88.8, CH 158.7, C 102.7, CH 156.8, C 153.9, C 144.9, C 138.4, C 112.1, C 183.2, C 104.8, C 37.4, CH

11 12 13 14 15 16 17 18 19 10 20 30 40 4a0 50 5a0 60 70 80 8a0 90 9a0 100

117

HMBC

J2:3, 4a; J3:2, 9a

3.18 d (6.8) 5.12 m

J2:2, 11; J3: 1, 3, 12

1.71 s

J3:11, 14

1.57 s

J3:11, 13

4.04 m

J2:8, 16; J3:7, 8a, 17

5.24 m

J3:8, 18, 19

1.80 s

J3:16, 19

1.65 s

J3:16, 18

6.50 s

J2:30 , 4a0 ; J3:20 , 9a0

6.69 s

J2:5a0 ; J3:70 , 8a0

5.46 d (5.6) 110 98.7, CH 4.82 d (5.8) 120 73.4, C 0 13 25.8, 1.35 s CH3 140 24.2, 1.27 s CH3 150 27.0, 3.96 m CH2 160 125.0, 5.12 m CH 0 17 131.8, C 180 18.3, 1.71 s CH3 190 25.9, 1.55 s CH3 7-OCH3 61.9, 3.76 s CH3 0 61.3, 3.68 s 7OCH3 CH3 1-OH 10 -OH

J3:2, 13

Garcinoxanthone B (2)

dC type 161.8, C 106.6, C 161.6, C 106.5, C 157.7, C 102.6, CH 156.8, C 150.6, C 144.7, C 138.4, C 112.0, C 183.5, C 104.0, C 22.7, CH2 123.6, CH 132.0, C 18.3, CH3 26.1, CH3 27.1, CH2 125.2, CH 131.7, C 18.4, CH3 26.0, CH3 164.7, C 111.2, C 167.3, C 94.0, CH 158.3, C 102.9, CH 156.4, C 157.1, C 145.1, C 138.6, C 112.2, C 183.2, C 103.4, C 37.0, CH

dH (J)

6.28 s

J2:5a; J3:7, 8a

3.33 d (6.9)

J2:2, 11; J3: 1, 3, 12

5.23 m

J3:2, 13

1.80 s

J3:11, 14

1.68 s

J3:11, 13

4.01 d (6.4)

J2: 8, 16; J3: 7, 8a, 17

5.29 m

J3:8, 18, 19

1.77 s

J3:16, 19

1.62 s

J3:16, 18

6.19 s

J2:30 , 4a0 ; J3:20 , 9a0

6.72 s

J2:60 ; J3:70 , 8a0

J2: 5, 20 , 110 ; J3: 10 , 30 , 5.41 d (6.5) 120 J2:100 ; J3: 30 , 5, 120 , 140 97.1, CH 4.90 d (6.5) 73.3, C 26.0, CH3 J2:110 ; J3:120 , 130 24.6, CH3 J2: 80 , 160 ; J3: 70 , 8a0 , 170 27.2, CH2 J3:80 , 190 125.0, CH 132.0, C J3:160 , 190 18.0, CH3 J3:160 , 180 25.7, CH3 J3:7 61.3, CH3 J3:70 61.2, CH3 J2:110 ; J3:120 , 140

Garcinoxanthone C (3) HMBC

1.33 s 1.26 s 4.19 dd (12.9, 6.1); 4.08 dd (12.9, 6.7) 5.15 m

1.85 s 1.70 s 3.76 s 3.65 s

dC type 160.1, C 111.0, C 159.8, C 106.6, C 158.1, C 100.8, CH 153.1, C 152.4, C 141.5, C 128.8, C 111.6, C 183.3, C 104.3, C 22.4, CH2 123.5, CH 132.0, C 18.0, CH3 26.0, CH3 26.4, CH2 124.4, CH 131.3, C 18.2, CH3 25.8, CH3 158.8, C 111.7, C 168.5, C 88.3, CH 157.4, C 102.6, CH 156.2, C 153.8, C 144.6, C 138.1, C 111.9, C 183.0, C 104.3, C 37.0, CH

dH (J)

HMBC

6.38 s

J2:5a; J3:7, 8a

3.47 d (6.9) 5.28 m

J2:2, 11; J3: 1, 3, 12 J3:2, 13

1.81 s

J3:11, 14

1.68 s

J3:11, 13

4.11 d (6.8) 5.24 m

J2: 8, 16; J3: 7, 8a, 17 J3:8, 18, 19

1.58 s

J3:16, 19

1.78 s

J3:16, 18

6.46 s

J2:30 , 4a0 ; J3:20 , 9a0

6.84 s

J2:60 ; J3:70 ,8a0

J2:20 ,5, 110 ; J3: 10 , 30 , 5.31 d 120 (5.7) J2:100 ; J3: 30 , 5, 120 , 140 98.5, CH 4.82 d (5.7) 72.7, C 0 0 0 J2:11 ; J3:12 , 14 26.8, 1.38 s CH3 J2:110 ; J3:120 , 130 24.3, 1.35 s CH3 J2: 80 , 160 ; J3: 70 , 8a0 , 26.2, 4.02 d 170 CH2 (6.3) J3:80 , 190 124.6, 5.15 m CH 131.4, C J3:160 , 190 18.3, 1.56 s CH3 J3:160 , 180 26.0, 1.75 s CH3 J3:7 61.3, 3.76 s CH3 0 J3:7 13.91 s 13.47 s

J2:20 ,5, 110 ; J3: 10 , 30 , 120 J2:100 ; J3: 30 , 5, 120 , 140

J2:110 ; J3:120 , 140 J2:110 ; J3:120 , 130 J2: 80 , 160 ; J3: 70 , 8a0 , 170 J3:80 , 190

J3:160 , 190 J3:160 , 180 J3:7

J2:1 J2:10

118

Q. Liu et al. / Phytochemistry 131 (2016) 115e123

configuration of garcinoxanthone A (1) was determined and the detailed assignments of the 1H, 13C NMR and key HMBC correlations are shown in Table 1. Garcinoxanthone B (2), a yellow amorphous powder, was inferred with a molecular formula C48H50O13 by its HRESIMS (m/z 833.3170 [M-H]-). The IR absorptions indicated the presence of hydroxy (3400 cm1) and conjugated carbonyl (1605 cm1) groups in compound 2. The 1H and 13C NMR spectra of compound 2 (Table 1) showed signals for two methoxy groups (dH 3.76 and 3.65), three aromatic protons (dH 6.19, 6.28 and 6.72) and three prenyl groups, which were very similar to those of cratoxyxanthone (Sia et al., 1995). The main difference was the proton signals at dH 5.41 (1H, d, J ¼ 6.5 Hz, H-100 ) and 4.90 (1H, d, J ¼ 6.5 Hz, H-110 ), which suggested that the configurations of C-100 and C-110 in compound 2 might be different from those in cratoxyxanthone. The absolute configuration of compound 2 was determined by experimental and theoretically calculated ECD methods. As shown in Fig. 2, the absolute configurations of positions 100 and 110 in compound 2 were determined as S and R, respectively. The structure of garcinoxanthone B (2) was determined, and the detailed assignments of the 1 H, 13C NMR data, and key HMBC correlations are shown in Table 1. The molecular formula of garcinoxanthone C (3) was established as C47H48O13 based on the ion peak at m/z 819.3038 in its HRESIMS. The 1H NMR spectrum of compound 3 showed signals for a methoxy group (dH 3.76, s), two hydroxy groups (dH 13.91, s and 13.47, s), three aromatic protons [dH 6.38 (1H, s), 6.47 (1H, s) and 6.84 (1H, s)] and three prenyl groups. The 1H and 13C NMR data of compound 3 were very similar to those of cratoxyxanthone (Sia et al., 1995) except for the lack of one methoxy group. On the basis of HSQC and HMBC data, it was deduced that the methoxy group at C-7 of cratoxyxanthone was replaced by a hydroxy group in compound 3. Next, the experimental and theoretically calculated ECD method indicated the configurations of compound 3 as 100 R, 110 S (Fig. 2). The detailed assignments of the 1H, 13C NMR data and key HMBC correlations are shown in Table 1. Garcinoxanthone D (4) was obtained as a yellow amorphous powder. The ion peak at m/z 441.1552 [M-H]- in the HRESIMS of compound 4 suggested its molecular formula as C24H26O8. Its UV spectrum showed absorptions at lmax 209, 246, 305 and 330 nm. The strong IR absorption at 3432 cm1 indicated the presence of hydroxy group. The 1H NMR spectrum of compound 4 showed signals for a methoxy group (dH 3.72, 3H, s), two oxygen-bearing methine protons (dH 4.97, 1H, d, J ¼ 4.7 Hz and 3.71, 1H, d, J ¼ 4.7 Hz), two aromatic protons (dH 6.32, 1H, s and 6.66, 1H, s), and one prenyl group (Table 2). The 13C NMR and DEPT spectra of compound 4 disclosed a total of 24 carbon signals, corresponding to four methyl carbons, one methoxy carbon, one methylene carbon, two oxygen-bearing methine carbons, three sp2 methine carbons, eleven sp2 quaternary carbons, one sp3 quaternary carbon and one carbonyl carbon (Table 3). The above NMR data quite resembled those of laterixanthone (Ren et al., 2010). The structure of compound 4 was further determined by the HMBC and HSQC experiments. The HMBC cross-peaks from the methylene protons (dH 4.02, 2H, m) to C-8 (dC 138.3), C-8a (dC 114.8) and C-7 (dC 144.9), as well as from the olefinic proton (dH 5.25, 1H, m) to C-8, suggested that a prenyl group was attached at C-8 (Supplemental Fig. S1). The methoxy group was assigned at C-7 based on the HMBC cross-peak of OCH3 (dH 3.72)/C-7 (dC 144.9). The HMBC cross-peaks from the aromatic proton (dH 6.32) to C-2 (dC 107.4), C-3 (dC 164.1) and C-4a (dC 159.6) suggested it as H-4. Similarly, another aromatic proton (dH 6.66) was located on C-5, based on the HMBC corrections of H-5 to C-7, C-8 and C-8a. Two oxygen-bearing methine protons were assigned as H-10 (dH 4.97) and H-11 (dH 3.71) based on the HMBC correlations of H-10, 11/C-2 (dC 107.4), H-10/C-1 (dC 156.4) and H10/C-3 (dC 164.1). Combined with the above information, another

prenyl group was connected through C-2 and C-3-O to construct a tetrahydropyran ring. Thus, the structure of compound 4 was determined, which is the same as that of laterixanthone. The absolute configurations of compound 4 was assigned by experimental and theoretical ECD methods (Fig. 2). The CD spectrum indicated the absolute configurations of compound 4 were 10R, 11S, this being different from those in laterixanthone (Ren et al., 2010). Garcinoxanthone E (5) was isolated as a yellow amorphous powder. The molecular formula, C24H26O8, was deduced by its HRESIMS. The 1H NMR spectrum of compound 5 showed signals for a methoxy group (dH 3.65, 3H, s), two oxygen-bearing methine protons (dH 4.64, 1H, d, J ¼ 6.5 Hz and 3.60, 1H, d, J ¼ 6.5 Hz), two aromatic protons (dH 6.27, 1H, s; 6.59, 1H, s), and one prenyl group (Table 2), which were very similar to those of compound 4. The major difference was the oxygen-bearing methine protons of dH 4.64 and 3.60 in compound 5, instead of dH 4.97 and 3.71 in compound 4. This suggested that the configurations of C-10 and C-11 might be different between compounds 5 and 4. Based on the experimental and theoretical ECD results, the configurations of C10 and C-11 in compound 5 were determined as 10R, 11R (Fig. 2). The detailed assignments of the 1H and 13C NMR data are shown in Tables 2 and 3. The molecular formula of garcinoxanthone F (6) was determined as C25H28O8 by its HRESIMS. The NMR data of compound 6 (Tables 2 and 3) were nearly identical to those of compound 4, with one extra methoxy signal [dH 3.50 (3H, s), dC 57.5]. This additional methoxy group was assigned at C-10 on the basis of the correlations from the methoxy protons (dH 3.50) to C-10 (dC 76.4), from H-10 (dH 4.28) to C-10 and C-11 (dC 71.0), and from H-11 (dH 3.81) to C-10 in the HMBC spectrum. The configuration of compound 6 was indicated by ECD experiments. According to the observed CD spectrum, the absolute configurations of compound 6 was deduced to be the same as that of compound 4 (Fig. 2). Thus, the structure of compound 6 was determined. Garcinoxanthone G (7), a yellow amorphous powder, showed a deprotonated molecular ion peak at m/z 409.1294 [M-H]- in the HRESIMS, corresponding to the formula C23H22O7. The 1H and 13C NMR spectra of 7 (Tables 2 and 3) showed the presence of a carbonyl carbon (dC 179.2), two isolated aromatic protons [dH/dC 6.32 (1H, s)/94.5 and 6.65 (1H, s)/103.1], two coupled aromatic protons [dH/dC 7.92 (1H, d, J ¼ 10.2 Hz)/122.5 and 5.80 (1H, d, J ¼ 10.2 Hz)/132.9], an oxygenated methine [dH/dC 3.76 (1H, m)/ 69.5], and ten substituted aromatic carbons, six of which were oxygenated. The positions of all substituents were determined by detailed analysis of the HMBC and HSQC cross-peaks (Supplemental Fig. S1). Confirmed by the HMBC cross-peaks from the aromatic proton (dH 6.65) to C-8a and C-7, it was assigned on C5. Another aromatic proton (dH 6.32) was suggested on C-4 based on the HMBC correlations between H-4 and C-3 and C-4a. The HMBC correlations from the coupled aromatic proton (dH 7.92) to C8 (dC 135.7), C-8a (dC 111.4) and C-7 (dC 139.2) suggested that the 2,2-dimethylchromene ring was located at C-7 and C-8. The lowfield shift of the carbonyl carbon in compound 7 compared with 4e6 (D0.5e1.9) indicated no hydroxy group on C-1 to form a hydrogen bond. Furthermore, the location of the dihydropyran ring was assigned at C-1 and C-2 by the HMBC correlations from H-10 (dH 2.88, dd, J ¼ 16.8 and 5.0 Hz; 2.52, dd, J ¼ 16.8 and 5.0 Hz) to C-2 (dC 106.4) and C-3 (dC 156.2), along with H-11 (dH 3.76, m) to C-2. Therefore, the structure of compound 7 was determined. Two possible isomers, 11S and 11R, were considered, and the ECD spectra were calculated (Fig. 2). The experimental ECD spectrum of 7 was in accordance with the calculated ECD spectrum for 11S, thus establishing the absolute configuration of compound 7. Cratoxyxanthone has been isolated previously (Han et al., 2009; Sia et al., 1995). However, the absolute configuration of

Q. Liu et al. / Phytochemistry 131 (2016) 115e123

119

Fig. 2. (a) Experimental ECD for compound 1 and predicted ECD for 100 R, 110 S-1; (b) experimental ECD for compound 2 and predicted ECD for 100 R, 110 S-2; (c) experimental ECD for compound 3 and predicted ECD for 100 R, 110 S-3; (d) experimental ECD for compound 4 and predicted ECD for 10R, 11S-4; (e) experimental ECD for compound 5 and predicted ECD for 10R, 11R-5; (f) experimental ECD for compound 6 and predicted ECD for 10R, 11S-6; (g) experimental ECD for compound 7 and predicted ECD for S-7.

Table 2 1 H NMR spectroscopic data (600 MHz, CD3OD) for garcinoxanthones DeG (4e7) with J values (in Hz) in parentheses. Position

Garcinoxanthone D (4)

Garcinoxanthone E (5)

Garcinoxanthone F (6)

Garcinoxanthone G (7)

6.32 6.65 2.88 2.52 3.76 1.30 1.25 7.92 5.80 1.48 1.48

dH (J in Hz) 4 5 10

6.32 s 6.66 s 4.97 d (4.7)

6.27 s 6.59 s 4.64 d (6.5)

6.32 s 6.64 s 4.28 d (3.4)

11 13 14 15 16 18 19 7-OCH3 10-OCH3

3.71 1.49 1.43 4.02 5.25 1.80 1.65 3.72

3.60 1.46 1.23 3.94 5.17 1.72 1.57 3.65

3.81 1.43 1.41 3.98 5.20 1.76 1.61 3.69 3.50

d (4.7) s s m m s s s

d (6.5) s s m m s s s

cratoxyxanthone was not determined. Herein, using experimental and theoretically calculated ECD methods, the absolute configurations of cratoxyxanthone was determined as 100 R, 110 S (Supplemental Fig. S2). To the best of our knowledge, cratoxyxanthone is the only dimeric xanthone identified from the pericarps of G. mangostana (Han et al., 2009). In the current work, three dimeric xanthones were identified from the pericarps of G. mangostana linked through

d (3.4) s s d (5.5) m s s s s

s s dd (16.8, 5.0) dd (16.8, 7.2) m s s d (10.2) d (10.2) s s

a sidechain of the two subunits. Henry and Townsend (Henry and Townsend, 2005) described that the tricyclic xanthone core of sterigmatocystin was constructed through a complex sequence of epoxidation, rearrangement, deoxygenation, Baeyer-Villiger oxidation, and further deoxygenation. Wezeman et al. (Wezeman et al., 2015). had speculated on a biaryl linkage involving coupling of two mangostin-derived radicals. Compounds 1e3 might share the same biosynthetic pathway with cratoxyxanthone. They were

120

Q. Liu et al. / Phytochemistry 131 (2016) 115e123

Table 3 13 C NMR spectroscopic data (150 MHz, CD3OD) for garcinoxanthones DeG (4e7). Position

Garcinoxanthone D (4)

Garcinoxanthone E (5)

Garcinoxanthone F (6)

Garcinoxanthone G (7)

154.7, C 106.7, C 162.3, C 100.0, CH 155.6, C 101.0, CH 158.1, C 154.2, C 143.5, C 136.9, C 113.5, C 177.3, C 106.1, 67.2, CH 74.2, CH 79.6, C 19.1, CH3 24.6, CH3 24.6, CH2 124.2, CH 130.1, C 16.9, CH3 24.5, CH3 59.9, CH3

156.9, C 105.9, C 164.0, C 95.1, CH 159.7, C 102.3, CH 155.6, C 156.9, C 144.8, C 138.3, C 114.9, C 178.7, C 107.4, C 76.4, CH 71.0, CH 80.0, C 23.7, CH3 24.3, CH3 27.0, CH2 125.5, CH 131.5, C 18.3, CH3 26.0, CH3 61.3, CH3 57.5, CH3

162.3, C 106.4, C 156.2, C 94.5, CH 156.2, C 103.1, CH 153.0, C 153.3, C 139.2, C 135.7, C 111.4, C 179.2, C 104.5, C 25.6, CH2 69.5, CH 79.6, C 20.7, CH3 15.4, CH3 122.5, CH 132.9, CH 76.6, C 27.1, CH3 27.0, CH3

dC type 1 2 3 4 4a 5 5a 6 7 8 8a 9 9a 10 11 12 13 14 15 16 17 18 19 7-OCH3 10-OCH3

156.4, C 107.4, C 164.1, C 95.5, CH 159.6, C 102.3, CH 155.6, C 157.0, C 144.9, C 138.3, C 114.8, C 178.7, C 102.3, C 64.2, CH 72.2, CH 80.5, C 22.6, CH3 25.7, CH3 27.1, CH2 125.6, CH 131.4, C 18.3, CH3 26.0, CH3 61.2, CH3

bisxanthones with a xanthoneexanthone linkage between an aromatic carbon and a C5 sidechain. All the new compounds were further evaluated for their inhibitory effect on LPS-induced NO production in RAW264.7 cells (Table 4). The results showed garcinoxanthones B (2) and C (3) significantly reduced NO production in LPS-stimulated RAW264.7 cells, with IC50 values of 11.3 ± 1.7 and 18.0 ± 1.8 mM, respectively, which were comparable with that of the positive control indomethacin (IC50 3.9 ± 0.3 mM). The other compounds did not show any inhibitory effect up to 20 mM. Additionally, compound 2 and indomethacin inhibited the iNOS protein expression in a concentration-dependent manner (Fig. 3). On the other hand, compounds 1e7 did not show obvious cytotoxicity at 20 mM against RAW264.7 cells (Table 4). The xanthones from G. mangostana have been reported to possess an anti-inflammatory effect (Chen et al., 2008; GutierrezOrozco et al., 2013; Syam et al., 2014). Two major compounds, amangostin and g-mangostin have been found to significantly inhibit NO production in LPS-stimulated RAW264.7 cells by suppressing iNOS expression (Chen et al., 2008). In this study, two dimeric xanthones from the pericarps of G. mangostana were found to inhibit LPS-stimulated NO production and one of them suppressed iNOS expression dose-dependently in RAW264.7 cells. These findings might further support claims of the antiinflammatory activity of G. mangostana.

Table 4 NO production inhibitory effect and cytotoxicity of compounds 1e7 on RAW264.7 cells.

3. Conclusions

Phosphate-buffered saline (PBS) powder, Dulbecco's modified Eagle's medium (DMEM), penicillin-streptomycin (P/S), fetal bovine serum (FBS) were purchased from Life Technologies (Grand Island, NY, USA). Lipopolysaccharide (LPS) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The primary antibodies, a-tubulin and iNOS, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and Cell Signaling Technology (Boston, MA, USA), respectively. Optical rotations were taken on a Perkin-Elmer 341 polarimeter. IR spectra were recorded on a Bruker Tensor 37 infrared spectrophotometer using KBr disks. The UV spectra were recorded on a JASCO V-650

In summary, the present study describes the isolation and characterization of seven new xanthones from the pericarps of G. mangostana, including three rare dimeric xanthones. The absolute configurations of all new compounds were determined by CD method. Two new dimeric xanthones showed significant inhibitory activity on LPS-induced NO production in RAW264.7 cells. These findings indicate that dimeric xanthones from G. mangostana have potential to be a new class of anti-inflammatory agents. More in vitro and in vivo studies should be carried out to demonstrate

Compounds

NO production (IC50, mM)

Cell viability (%)a

1 2 3 4 5 6 7 DMSOb Indomethacinc Paclitaxel (0.5 mM)d

>20 11.3 ± 1.7 18.0 ± 1.8 >20 >20 >20 >20 e 3.9 ± 0.3 e

96.8 ± 1.7 99.5 ± 3.6 90.7 ± 2.9 93.8 ± 0.9 91.3 ± 2.1 96.1 ± 2.7 90.8 ± 2.7 100.0 ± 0.6 e 33.6 ± 0.7

a b c d

Cell viability at 20 mM. Blank control. Positive control for NO production assay. Positive control for cytotoxicity against RAW264.7 cells.

their anti-inflammatory efficacy, the underlying molecular mechanisms, and the utilization in the pharmaceutical and healthy food industries. 4. Experimental 4.1. General experimental procedures

Q. Liu et al. / Phytochemistry 131 (2016) 115e123

Fig. 3. Compound 2 suppressed LPS-stimulated iNOS elevation in a concentrationedependent manner. Indomethacin was used as the positive control. Represented Western blots of at least three independent experiments.

spectrophotometer. The CD spectra were recorded on a JASCO J-815 spectropolarimeter. The NMR spectra were recorded on Bruker AM400 and Bruker 600 NMR spectrometers. The chemical shift (d) values were given in ppm with TMS as the internal standard, and coupling constants (J) were in Hz. ESIMS and HRESIMS spectra were recorded on a Waters Micromass Q-TOF spectrometer. All solvents were analytical grade (Tianjin Chemical Plant, Tianjin, People's Republic of China). Silica gel used for flash chromatography and precoated silica gel GF254 plates used for TLC were produced by Qingdao Haiyang Chemical Co., Ltd. The TLC spots were viewed at 254 nm and visualized by spraying with 5% H2SO4 in EtOH. Sephadex LH-20 gel (Amersham Biosciences), ODS gel (12 nm, S50 mm, YMC Co., Ltd.) and MCI gel (CHP20P, 75e150 mm, Mitsubishi Chemical Industries Ltd.) were used for column chromatography (CC). Preparative HPLC was performed on a Shimadzu LC-20AP instrument with an SPD-M20A PDA detector. Chromatographic separations were carried out on a C18 column (250  19 mm, 5 mm, Waters, SunFire™), using a gradient solvent system comprising H2O (A) and CH3CN (B) at a flow rate of 10 mL/min. 4.2. Plant material The pericarps of G. mangostana (10.5 kg) were collected in Chiang Mai, Thailand, and identified by Professor Jingui Shen from Shanghai Institute of Materia Medica, Chinese Academy of Sciences. A voucher is deposited at the herbarium of Institute of Chinese Medical Sciences, University of Macau (LL-20130901). 4.3. Extraction and isolation Air-dried pericarps of G. mangostana were ground into powder and extracted with EtOHeH2O (95%, 3 days  3 times, room temperature, each 30 L). After evaporation of the collected percolate, the crude extract (1155.4 g) was suspended in H2O (3.2 L) and extracted with petroleum ether (2 L  3), CH3Cl (3 L  3), EtOAc (3 L  3) and n-BuOH (1 L  3), successively. The CH3Cl extract (200.0 g) was subjected to silica gel CC and eluted with petroleum ethereacetone (20:1 to 2:1, v/v), to yield ten major fractions (A to L). Fractions C and E were applied to silica gel column eluted with petroleum ethereEtOAc (15:1 to 10:1, v/v) to yield eight (C1 to C8) and seven (E1 to E7) subfractions, respectively. C3 was purified with Sephadex LH-20, eluting with CHCl3eCH3OH (1:1, v/v), to yield three fractions (C3A to C3C). C3C was then subjected to silica gel CC eluted with petroleum ethereacetone (20:1, v/v) to yield seven subfractions (C3C1 to C3C7). C3C4 was further separated by

121

preparative HPLC, eluting with H2OeCH3OH (17:3, v/v), to obtain tovophyllin A (8.1 mg), trapezifolixanthone (1.3 mg), cudraxanthone G (10.3 mg) and 8-hydroxycudraxanthone G (6.8 mg). C6 and C9 were subjected to MCI gel CC eluted with H2OeCH3OH (1:1 to 0:1, v/v), respectively, to yield a series of subfractions (C6A to C6F; C9A to C9C). Gartanin (26.0 mg) and 8-deoxygartanin (160.7 mg) were crystallized from C6C, and 9hydroxycalabaxanthone (583.6 mg) was crystallized from C6D. C9C was purified with Sephadex LH-20, eluting with CHCl3eCH3OH (1:1, v/v), to yield b-mangostin (20.1 mg) and 7-O-methylgarcinone E (6.0 mg). E3 was subjected to silica gel CC eluted with petroleum ethereacetone (9:1 to 4:1, v/v) to yield mangosharin (17.6 mg) and garcinone E (23.5 mg). a-mangostin (30.7 g) was crystallized from subfraction E6. Fractions G and L were subjected to MCI gel CC eluted with H2OeCH3OH (7:3 to 0:1, v/v), respectively, to yield a series of subfractions (G1 to G5; L1 to L9). G2 was subjected to silica gel CC eluted with petroleum ethereacetone (7:1 to 3:1, v/v) to yield G2A to G2C. G2B was purified with Sephadex LH-20, eluting with CHCl3eCH3OH (1:1, v/v), to yield garcinone D (90.7 mg), gmangostin (135.5 mg), 1,3,6,7-tetrahydroxy-8-prenylxanthone (71.3 mg) and garcinone C (2.1 mg). G4 was subjected to silica gel CC eluted with petroleum ethereEtOAc (8:1 to 2:1, v/v) to yield six subfractions (G4A to G4F). G4F was subjected to ODS CC eluted with H2OeCH3OH (1:1 to 0:1, v/v) to yield four subfractions (G4F1 to G4F4). G4F3 and G4F4 were further separated by preparative HPLC with H2OeCH3CN (55:45 to 3:7, v/v), to yield garcinoxanthones A (1, 17.2 mg), B (2, 20.6 mg), C (3, 10.8 mg) and cratoxyxanthone (16.1 mg). L5 was purified with Sephadex LH-20, eluting with CHCl3eCH3OH (1:1, v/v), to yield L5A to L5F. L5D was separated by preparative HPLC with H2OeCH3CN (3:2 to 3:7, v/v), to yield garcinoxanthones D (4, 4.7 mg) and E (5, 29.5 mg). Garcinoxanthone F (6, 4.3 mg) was crystallized from L5B. L5C was separated by pre-TLC to yield garcinoxanthone G (7, 5.7 mg) and 11-hydroxy-1isomangostin (32.7 mg). 4.3.1. Garcinoxanthone A (1) Yellow amorphous powder; ½a20 D þ0.7 (c 3.4, CH3OH); UV (CH3OH) lmax (log ε) 319.5 (3.57), 248 (3.74), 210.5 (3.70) nm; CD (CH3OH, nm) lmax (Dε) 229 (0.55), 252 (þ0.72), 275 (þ0.19), 321 (0.38), 338 (þ0.37); IR nmax (KBr) 3411 (strong, broad), 2915, 1646, 1613, 1579, 1455, 1281, 1169, 1087, 980, 816 cm1; For 1H and 13C NMR spectroscopic data, see Table 1; HRESIMS m/z 835.3301 [MþH]þ (calcd for C48H51O13, 835.3330). 4.3.2. Garcinoxanthone B (2) Yellow amorphous powder; ½a20 D þ3.2 (c 0.09, CH3OH); UV (CH3OH) lmax (log ε) 318 (3.48), 259.5 (3.59), 244.5 (3.71), 207.5 (3.69) nm; CD (CH3OH, nm) lmax (Dε) 227 (0.48), 246 (þ0.62), 289 (0.19), 322 (0.71); IR nmax (KBr) 3400 (strong, broad), 2925, 1605, 1464, 1284, 1158, 1093, 983, 842 cm1; For 1H and 13C NMR spectroscopic data, see Table 1; HRSEIMS m/z 833.3170 [MH] (calcd for C48H49O13, 833.3173). 4.3.3. Garcinoxanthone C (3) Yellow amorphous powder; ½a20 D þ3.3 (c 0.22, CH3OH); UV (CH3OH) lmax (log ε) 319.5 (3.51), 245.5 (3.67), 209 (3.69) nm; CD (CH3OH, nm) lmax (Dε) 231 (3.22), 269 (þ1.22), 324 (þ0.97), 357 (0.84), 383 (0.83); IR nmax (KBr) 3164, 2915, 1612, 1462, 1280, 1160, 1091, 1056, 837 cm1; For 1H and 13C NMR spectroscopic data, see Table 1; HRSEIMS m/z 819.3038 [MH] (calcd for C47H47O13, 819.3017). 4.3.4. Garcinoxanthone D (4) Yellow amorphous powder; ½a20 D þ4.4 (c 0.08, CH3OH); UV (CH3OH) lmax (log ε) 330 (2.98), 305 (3.23), 246 (3.53), 209 (3.45)

122

Q. Liu et al. / Phytochemistry 131 (2016) 115e123

nm; CD (CH3OH, nm) lmax (Dε) 210 (0.64), 226 (0.68), 247 (þ0.61), 283 (þ0.14), 295 (0.18), 310 (þ0.14), 340 (0.21), 356 (0.14); IR nmax (KBr) 3432 (strong, broad), 2923, 2853, 1611, 1461, 1373, 1270, 1089, 996 cm1; For 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HREIMS m/z 441.1552 [MH] (calcd for C24H25O8, 441.1549). 4.3.5. Garcinoxanthone E (5) Yellow amorphous powder; ½a20 D þ3.7 (c 0.18, CH3OH); UV (CH3OH) lmax (log ε) 334.5 (2.96), 304.5 (3.20), 245.5 (3.50), 210.5 (3.40) nm; CD (CH3OH, nm) lmax (Dε) 208 (0.56), 231 (0.44), 247 (þ0.08), 255 (0.08), 273 (þ0.22), 290 (0.12), 320 (0.24), 358 (0.22); IR nmax (KBr) 3409 (strong, broad), 2978, 2932, 1612, 1461, 1372, 1271, 1195, 1090, 998 cm1; For 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HREIMS m/z 441.1550 [MH] (calcd for C24H25O8, 441.1549). 4.3.6. Garcinoxanthone F (6) Yellow amorphous powder; ½a20 D þ2.6 (c 0.06, CH3OH); UV (CH3OH) lmax (log ε) 305 (2.82), 245.5 (3.14), 208 (3.11) nm; CD (CH3OH, nm) lmax (Dε) 208 (1.31), 228 (0.81), 248 (þ0.65), 284 (0.32), 303 (þ0.08), 342 (0.21); IR nmax (KBr) 3409 (strong, broad), 2925, 2854, 1609, 1462, 1374, 1271, 1192, 1091 cm1; For 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HREIMS m/z 457.1857 [MþH] (calcd for C25H29O8, 457.1862). 4.3.7. Garcinoxanthone G (7) Yellow amorphous powder; ½a20 D þ6.1 (c 0.08, CH3OH); UV (CH3OH) lmax (log ε) 368.5 (2.25), 320.5 (2.73), 260 (2.85), 243 (2.90), 208 (2.91) nm; CD (CH3OH, nm) lmax (Dε) 226 (0.94), 238 (0.98), 321 (0.30), 350 (0.28); IR nmax (KBr) 3455 (strong, broad), 2923, 2852, 1585, 1445, 1373, 1254, 1133, 1009, 919, 804 cm1; For 1H and 13C NMR spectroscopic data, see Tables 2 and 3; HREIMS m/z 409.1294 [MH] (calcd for C23H21O7, 409.1287). 4.4. Computational methods for ECD spectroscopies The Amber and the MMFF94S force field were used for conformational studies, respectively. The geometries with a relative energy from 0 to 5.5 kcal/mol were selected for calculation at the B3LYP/6-31G(d)//B3LYP/3-21G(d) level to obtain global minima. Conformers within an energy range of 3 kcal/mol from the global minima were subjected to geometrical optimization (DFT/B3LYP/631G(d)) in the gas phase, followed by calculation of vibrational modes to confirm these minima. After the conformational search, 25 conformations with the lowest energy were found for compound 1; 3 conformations for compounds 2 and 3; 7 conformations for compounds 4 and 6; 17 conformations for compound 5, and 4 conformations for compound 7. ECD spectra of the above conformations were calculated at the B3LYP/6-311þG (d,p) level in MeOH (SCRF/IEFPCM). The calculated ECDs were weighted prior to comparison with the experimental results. In this study, all of the reported DFT calculations were performed with the Gaussian 03 package (Frisch et al., 2009). 4.5. Assay for NO production RAW264.7 cells were maintained in DMEM supplemented with 10% FBS at 37  C and 5% CO2. Cells were seeded into a 24-well plate (1  105 cells/well) and allowed to adhere for 24 h. The cells were then treated with the xanthones or vehicle (DMSO) for 1 h, followed by stimulation with 1 mg/mL LPS and incubation for a further 18 h. Indomethacin (Sigma-Aldrich) was used as the positive control (Lee et al., 2016). DMSO was used as vehicle, with the final concentration of DMSO being maintained at 0.2% of all cultures. To

measure the NO content, the culture supernatant was assayed using Griess reagent (Rajasekaran et al., 2012). 4.6. Western blot analysis RAW264.7 cells were treated with xanthones or vehicle for 1 h, followed by LPS (1 mg/mL) stimulation for 18 h. The cells were washed twice with cold PBS and then lysed with cold RIPA buffer containing freshly added phosphatase inhibitor cocktails and phenylmethylsulfonyl fluoride (PMSF) on ice for 30 min. Whole cell lysates were centrifuged at 17,400 g for 20 min and the supernatants were transferred into new tubes. Protein extract (15 mg) was separated by 8% SDS-PAGE. The proteins were then transferred onto PVDF membranes (Bio-Rad Laboratories, Inc.), blocked with 5% nonfat milk in TBST buffer for 1 h at room temperature and incubated with specific primary antibodies overnight at 4  C. After washing with TBST, the membranes were incubated with horseradish peroxidase conjugated secondary antibody for 2 h at room temperature. The proteineantibody complexes were detected by enhanced chemiluminescence (ECL System) and exposed by autoradiography. 4.7. Cell viability assay The effect of compounds 1e7 on RAW264.7 cell viability was assessed by the 2-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, the RAW264.7 cells were seeded at 1  104 cells/well in a 96-well plate and treated with various concentrations of xanthones or vehicles (DMSO). Paclitaxel (0.5 mM) was used as a positive control (Tseng et al., 2014). Each treated or control group contained six parallel wells. After incubation for 12 h, the medium was discarded and 10 mL MTT solution (1 mg/mL in PBS) was added to each well and incubated for 4 h. Subsequently, 100 mL DMSO was added to each well to dissolve the formazan crystals formed by viable cells. Absorbance at 570 nm was recorded on a microplate reader (SpectraMax M5, Molecular Devices). The percentage of cell proliferation was calculated as a ratio of the optical density (OD) value of the sample to the OD value of the vehicle control. All the experiments were performed under the same conditions at least three times. Acknowledgments Financial support by Science and Technology Development Fund, Macao S.A.R (FDCT 120/2013/A3) and the Research Fund of University of Macau (MYRG2014-00020-ICMS-QRCM and MYRG2015-00153-ICMS-QRCM) are gratefully acknowledged. The authors thank Prof. Jingui Shen for identification of herbal material. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2016.08.007. Notes The authors declare no competing financial interest. References Bennett, G.J., Harrison, L.J., Sia, G.L., Sim, K.Y., 1993. Triterpenoids, tocotrienols and xanthones from the bark of Cratoxylum cochinchinense. Phytochemistry 32, 1245e1251. Bennett, G.J., Lee, H.H., Lee, L.P., 1990. Synthesis of minor xanthones from Garcinia mangostana. J. Nat. Prod. 53, 1463e1470. Brownlee, M., 2005. The pathobiology of diabetic complications: a unifying

Q. Liu et al. / Phytochemistry 131 (2016) 115e123 mechanism. Diabetes 54, 1615e1625. Chen, L.G., Yang, L.L., Wang, C.C., 2008. Anti-inflammatory activity of mangostins from Garcinia mangostana. Food Chem. Toxicol. 46, 688e693. Ee, G.C., Daud, S., Taufiq-Yap, Y.H., Ismail, N.H., Rahmani, M., 2006. Xanthones from Garcinia mangostana (Guttiferae). Nat. Prod. Res. 20, 1067e1073. Forstermann, U., Sessa, W.C., 2012. Nitric oxide synthases: regulation and function. Eur. Heart J. 33, 829e837. Frisch, M.J., Trucks, G.W., Schlegel, H.B., Scuseria, G.E., Robb, M.A., Cheeseman, J.R., Scalmani, G., Barone, V., Mennucci, B., Petersson, G.A., Nakatsuji, H., Caricato, M., Li, X., Hratchian, H.P., Izmaylov, A.F., Bloino, J., Zheng, G., Sonnenberg, J.L., Hada, M., Ehara, M., Toyota, K., Fukuda, R., Hasegawa, J., Ishida, M., Nakajima, T., Honda, Y., Kitao, O., Nakai, H., Vreven, T., Montgomery, J.A., Peralta, J., Ogliaro, F., Bearpark, M., Heyd, J.J., Brothers, E., Kudin, K.N., Staroverov, V.N., Kobayashi, R., Normand, J., Raghavachari, K., Rendell, A., Burant, J.C., Iyengar, S.S., Tomasi, J., Cossi, M., Rega, N., Millam, J.M., Klene, M., Knox, J.E., Cross, J.B., Bakken, V., Adamo, C., Jaramillo, J., Gomperts, R., Stratman, R.E., Yazyev, O., Austin, A.J., Cammi, R., Pomelli, C., Ochterski, J.W., Martin, R.L., Morokuma, K., Zakrzewski, V.G., Voth, G.A., Salvador, P., Dannenberg, J.J., Dapprich, S., Daniels, A.D., Farkas, O., Foresman, J.B., Ortiz, J.V., Cioslowski, J., Fox, D.J., 2009. Gaussian 09. Gaussian, Inc., Wallingford CT. Gutierrez-Orozco, F., Chitchumroonchokchai, C., Lesinski, G.B., Suksamrarn, S., Failla, M.L., 2013. alpha-Mangostin: anti-inflammatory activity and metabolism by human cells. J. Agric. Food Chem. 61, 3891e3900. Gutierrez-Orozco, F., Failla, M.L., 2013. Biological activities and bioavailability of mangosteen xanthones: a critical review of the current evidence. Nutrients 5, 3163e3183. Han, A.R., Kim, J.A., Lantvit, D.D., Kardono, L.B., Riswan, S., Chai, H., Carcache de Blanco, E.J., Farnsworth, N.R., Swanson, S.M., Kinghorn, A.D., 2009. Cytotoxic xanthone constituents of the stem bark of Garcinia mangostana (mangosteen). J. Nat. Prod. 72, 2028e2031. Henry, K.M., Townsend, C.A., 2005. Ordering the reductive and cytochrome P450 oxidative steps in demethylsterigmatocystin formation yields general insights into the biosynthesis of aflatoxin and related fungal metabolites. J. Am. Chem. Soc. 127, 3724e3733. Ito, C., Miyamoto, Y., Rao, K.S., Furukawa, H., 1996. A novel dibenzofuran and two new xanthones from Calophyllum panciflorum. Chem. Pharm. Bull. 44, 441e443. Jung, H.A., Su, B.N., Keller, W.J., Mehta, R.G., Kinghorn, A.D., 2006. Antioxidant xanthones from the pericarp of Garcinia mangostana (Mangosteen). J. Agric. Food Chem. 54, 2077e2082. Lee, J.W., Lee, C., Jin, Q.H., Jang, H.R., Lee, D.H., Lee, H.J., Shin, J.W., Han, S.B., Hong, J.T., Kim, Y.S., Lee, M.K., Hwang, B.Y., 2016. Diterpenoids from therof Euphorbia fischeriana with inhibitory effects on nitric oxide production. J. Nat. Prod. 7, 126e131. Likhitwitayawuid, K., Phadungcharoen, T., Krungkrai, J., 1998. Antimalarial xanthones from Garcinia cowa. Planta Med. 64, 70e72. Likhitwitayawuid, K., Phadungcharoen, T., Mahidol, C., Ruchirawatb, S., 1997. 7-Omethylgarcinone from Garcinia cowa. Phytochemistry 45, 1299e1301. Liu, Q.Y., Wang, Y.T., Lin, L.G., 2015. New insights into the anti-obesity activity of

123

xanthones from Garcinia mangostana. Food Func. 6, 383e393. Mahabusarakam, W., Wiriyachtra, P., Taylor, W., 1987. Chemical constituents of Garcinia mangostana. J. Nat. Prod. 50, 474e478. Nguyen, L.H., Vo, H.T., Pham, H.D., Connolly, J.D., Harrison, L.J., 2003. Xanthones from the bark of Garcinia merguensis. Phytochemistry 63, 467e470. Obolskiy, D., Pischel, I., Siriwatanametanon, N., Heinrich, M., 2009. Garcinia mangostana L.: a phytochemical and pharmacological review. Phytother. Res. 23, 1047e1065. Rajasekaran, A., Sivakumar, V., Darlinquine, S., 2012. Role of Blepharis maderaspatensis and Ammannia baccifera plant extracts on in vitro oxygen radical scavenging, secretion of gastric fluid and gastroprotection on ulcer induced rats. Pharm. Bio. 50, 1085e1095. Ren, Y., Lantvit, D.D., Carcache de Blanco, E.J., Kardono, L.B., Riswan, S., Chai, H., Cottrell, C.E., Farnsworth, N.R., Swanson, S.M., Ding, Y., Li, X.C., Marais, J.P., Ferreira, D., Kinghorn, A.D., 2010. Proteasome-inhibitory and cytotoxic constituents of Garcinia lateriflora: absolute configuration of caged xanthones. Tetrahedron 66, 5311e5320. Sakai, S., Katsura, M., Takayama, H., Aimi, N., Chokethaworn, N., Suttajit, M., 1993. The structure of garcinone E. Chem. Pharm. Bull. 41, 958e960. Sen, A.K., Sarkar, K.K., Mazumder, P.C., Banerji, N., Uusvuori, R., Hase, T.A., 1980. A xanthone from Garcinia mangostana. Phytochemistry 19, 2223e2225. Sen, A.K., Sarkar, K.K., Mazumder, P.C., Banerji, N., Uusvuori, R., Hase, T.A., 1982. The structures of garcinones A, B and C-3 new xanthones from Garcinia mangostana. Phytochemistry 21, 1747e1750. Seo, E.K., Wall, M.E., Wani, M.C., Navarro, H., Mukherjee, R., Farnsworth, N.R., Kinghorn, A.D., 1999. Cytotoxic constituents from the roots of Tovomita brevistaminea. Phytochemistry 52, 669e674. Sia, G.L., Bennett, G.J., Harrison, L.J., Sim, K.Y., 1995. Minor xanthones from the bark of Cratoxylum cochinchinense. Phytochemistry 38, 1521e1528. Strowig, T., Henao-Mejia, J., Elinav, E., Flavell, R., 2012. Inflammasomes in health and disease. Nature 481, 278e286. Syam, S., Bustamam, A., Abdullah, R., Sukari, M.A., Hashim, N.M., Mohan, S., Looi, C.Y., Wong, W.F., Yahayu, M.A., Abdelwahab, S.I., 2014. Beta Mangostin suppress LPS-induced inflammatory response in RAW 264.7 macrophages in vitro and carrageenan-induced peritonitis in vivo. J. Ethnopharmacol. 153, 435e445. Tseng, C.K., Lin, C.K., Chang, H.W., Wu, Y.H., Yen, F.L., Chang, F.R., Chen, W.C., Yeh, C.C., Lee, J.C., 2014. Aqueous extract of Gracilaria tenuistipitata suppresses LPS-induced NF-kB and MAPK activation in RAW264.7 and rat peritoneal macrophages and exerts hepatoprotective effects on carbon tetrachloride treated rat. Plos One 9, e86557. Wezeman, T., Brase, S., Masters, K.S., 2015. Xanthone dimers: a compound family which is both common and pricileged. Nat. Prod. Rep. 32, 6e28. Wei, X.Q., Charles, I.G., Smith, A., Ure, J., Feng, G.J., Huang, F.P., Xu, D., Muller, W., Moncada, S., Liew, F.Y., 1995. Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375, 408e411. Zhou, Q., Chen, L., Chen, Q.W., Chen, H.W., Dong, J.X., 2013. Chemical constituents of Cudrania cochinchinensis. Zhongcaoyao 36, 1444e1447.