Microbial metabolism of α-mangostin isolated from Garcinia mangostana L.

Phytochemistry 72 (2011) 730–734

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Microbial metabolism of a-mangostin isolated from Garcinia mangostana L. Panarat Arunrattiyakorn a,⇑, Sunit Suksamrarn a, Nuttika Suwannasai b, Hiroshi Kanzaki c a

Department of Chemistry, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand Department of Biology, Faculty of Science, Srinakharinwirot University, Bangkok 10110, Thailand c The Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan b

a r t i c l e

i n f o

Article history: Received 10 September 2010 Received in revised form 1 December 2010 Available online 4 March 2011 Keywords: Microbial metabolism a-Mangostin Sulfation Anti-mycobacterial activity

a b s t r a c t a-Mangostin (1), a prenylated xanthone isolated from the fruit hull of Garcinia mangostana L., was individually metabolized by two fungi, Colletotrichum gloeosporioides (EYL131) and Neosartorya spathulata (EYR042), repectively. Incubation of 1 with C. gloeosporioides (EYL131) gave four metabolites which were identified as mangostin 3-sulfate (2), mangostanin 6-sulfate (3), 17,18-dihydroxymangostanin 6-sulfate (4)and isomangostanin 3-sulfate (5). Compound 2 was also formed by incubation with N. spathulata (EYR042). The structures of the isolated compounds were elucidated by spectroscopic data analysis. Of the isolated metabolites, 2 exhibited significant anti-mycobacterial activity against Mycobacterium tuberculosis. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Garcinia mangostana L. is commonly known as mangosteen. In Thailand, it is called the queen of fruits. The fruit hull of G. mangostana has been used as a traditional medicine in Southeast Asia, including Thailand, for treatment of diarrhea, inflammation, and ulcers (Mahabusarakam et al., 1987; Peres et al., 2000; Suksamrarn et al., 2006). Recently, mangosteen products have become widely available in United States and are highly popular because of their perceived role in enhancing human health (Garrity et al., 2004). The major secondary metabolites of G. mangostana fruits are prenylated xanthone derivatives, the most abundant of which is a-mangostin (1) (Mahabusarakam et al., 1987). a-Mangostin, a tetraoxygenated diprenylated xanthone, has been reported to exhibit various interesting bioactivities including antibacterial (Sakagami et al., 2005), antifungal (Gopalakrishnan et al., 1997), anti-inflammatory (Nakatani et al., 2004; Deschamps et al., 2007), anti-mycobacterial (Suksamrarn et al., 2003), antioxidant (Jung et al., 2006; Yu et al., 2007), antiplasmodial (Mahabusarakam et al., 2006), and cytotoxic activities (Matsumoto et al., 2005; Suksamrarn et al., 2006). It also has potential as a possible cancer chemopreventive compound by inhibiting alveolar duct formation in a mouse mammary organ culture model and suppression of the carcinogen-induced formation of aberrant crypt foci in a short-term colon carcinogenesis model (Nabandith et al., 2004; Jung et al., 2006). Thus a-mangostin (1) has recently attracted much attention.

⇑ Corresponding author. Tel.: +66 2 649 5000x8461; fax: +66 2 259 2097. E-mail address: [email protected] (P. Arunrattiyakorn). 0031-9422/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phytochem.2011.02.007

In the present study, the microbial metabolism of a-mangostin (1) was investigated using endophytic fungi. This is the first microbial transformation project carried out on 1. Our aims were to obtain various structurally modified derivatives of 1 that might increase the bioactivity by microbial metabolic activation and to attain a better understanding of microbial model metabolism of 1.

2. Results and discussion Preliminary screening of microbial biotransformation of 1 by 27 endophytic fungi from Tiliacora triandra (Colebr.) Diels led to the selection of Colletotrichum gloeosporioides (EYL 131) and Neosartorya spathulata (EYR 042) for further studies. Both strains showed the ability to convert substrate (1) into more higher polar compounds, while C. gloeosporioides (EYL 131) promoted the production of more diverse products. In preparative scale biotransformations with N. spathulata (EYR 042), mangostin 3-sulfate (2) was obtained. Compound 2, mangostanin 6-sulfate (3) 17,18-dihydroxymangostanin 6-sulfate (4), and isomangostanin 3-sulfate (5) were also formed by C. gloeosporioides (EYL 131) (Fig. 1). Metabolite 2 was isolated as a yellow solid. Its negative ion FABMS showed two intense ions at m/z 489 [MH] and m/z 409 [MH80], with the latter ion attributed to the loss of a sulfate group. The distinctive isotope pattern with relative intensity of signals at m/z 489 [MH] and m/z 491 [MH+2], which is caused by a sulfur atom, was also observed. The molecular formula of 2 was also established by its HRESIMS (m/z 489.1218 [MH], for C24H25O9S). A comparison of the 1H (Table 1) and 13C (Table 2) NMR spectroscopic data of 1 and 2 indicated almost identical resonances for both compounds, except for the downfield shift of H-4,

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19

20

16

H3CO HO

O

8

5

8a

9

10a

15

OH 9a

O

11

1

14

O 4a 4

OH

H3CO OH

OR

HO3SO

O

O

3

1: R=H 2: R=SO3H OH

HO

OH

O

OH

O

H3CO

O

H3CO

OH HO3SO

O

O

HO

O

4

OSO3H

5 Fig. 1. Structures of xanthones 1–5.

C-2, C-4, and C-9a (0.9, 4.7, 5.7, and 2.3 ppm, respectively) and the upfield shift of C-3 (6.5 ppm), implying that the sulfate moiety was conjugated with a C-3 hydroxy group in 2. These shifts were consistent with 1-hydroxy-2,3-dimethoxyxanthone-5-O-sulfate, a transformed product of 1-hydroxy-2,3,5-trimethoxyxanthone, because an ipso carbon of the sulfate substituted C-5 experiences an upfield shift while ortho and para carbons are shifted downfield (Yuan et al., 2006). The sulfate position at C-3 was confirmed by HMBC correlation (H-4/C-3, C-2). Compound 2 was thus concluded to be a C-3 sulfated derivative of 1 and assigned as 3,8-dihy droxy-2-methoxy-1,7-bis(3-methylbut-2-enyl)-9-oxo-9H-xanthen6-yl hydrogen sulfate or mangostin 3-sulfate. Metabolite 3 was isolated as a brown solid. Its negative ion FABMS of 3 showed a strong quasi-molecular ion peak at m/z 505 [MH] and a fragment ion peak at m/z 425 [MH80]. The HRESIMS data of m/z 505.1148 [MH] was also consistent with the molecular formula C24H25O10S, suggesting the presence of a sulfate conjugation, which was further supported by the ratio of isotope peaks at m/z 505 [MH] and m/z 507 [MH+2]. The 1H (Table 1) and 13C (Table 2) NMR spectroscopic chemical shifts of 3 was similar to that of 1, with significant differences being the downfield shift of H-5, C-5, C-7, and C-8a (0.9, 5.4, 2.3, and 3.2 ppm, respectively) and the upfield shift of C-6 (5.2 ppm), supporting sulfation at the C-6 hydroxy moiety (Yuan et al., 2006). Furthermore, the C-12/C-13 olefinic resonances of the C-2 prenyl group in 1 [dH 5.21 (1H, m, H-12); dC 124.0 (C-12); dC 132.0 (C-13)] were replaced by oxymethine [dH 4.73 (1H, t-like, J = 8.4 Hz, H-12); dC 93.2 (C-12)] and oxygenated quaternary carbon [dC 72.6 (C-13)] resonances in 3. In this respect, the presence of two methyls [dH 1.27 (3H, s, H15), dC 25.4 (C-15) and dH 1.22 (3H, s, H-14), dC 26.4 (C-14)], a quaternary oxygenated carbon (dC 72.6, C-13), an oxygenated methine [dH 4.73 (1H, t-like, J = 8.4 Hz), H-12), dC 93.2 (C-12)] and a methylene signal [dH 3.07 (2H, d, J = 8.4 Hz, H-11), dC 27.3 (C-11)] were assignable to a 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuran-3ol moiety. A series of HMBC (H-11/C-1, C-2, C-3, C-12; H-12/C-3, C-13) and COSY (H2-11/H-12) correlations confirmed the assignment of the 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuran-3-ol moiety to positions C-2 and C-3. The chemical shifts of the xanthone unit in 3 were in good agreement with that published for mangostanin (Nilar and Harrison, 2002), except that the hydroxyl group at C-6 in mangostanin was replaced by a sulfate group in

3. The HMBC correlations summarized in Fig. 2 were used to confirm the structure of 3. On the basis of the above analysis, 3 was established to be 3,5-dihydro-4-hydroxy-2-(2-hydroxypropan-2yl)-7-methoxy-6-(3-methylbut-2-enyl)-5-oxo-2H-furo[3,2-b]xanthen-8-yl hydrogen sulfate or mangostanin 6-sulfate. Metabolite 4 was isolated as a yellow solid. Its molecular formula was determined to be C24H27O12S on the basis of HRESIMS data (m/z 539.1224 [MH]). The difference in molecular weight of 34 amu between 4 and 3 suggested that the former was a dihydroxyl derivative of 3. A comparison of the 1H (Table 1) and 13 C NMR (Table 2) spectroscopic data of 4 and 3 indicated almost identical resonances for both compounds, except for replacement of C-17/C-18 olefinic resonances of the prenyl group at C-8 in 3 [dH 5.20 (1H, m, H-17); dC 124.9 (C-17); dC 132.2 (C-18)] by an oxygenated methine [dH 3.67 (1H, m, H-17), dC 80.4 (C-17)], and oxygenated quaternary carbon (dC 74.5, C-18) resonances in 4 suggested hydroxylation of the C-17,18 double bond. A series of

O

OH

H3CO OH HO3SO

O

O

3

O

OH O

H3CO HO

O

OSO3H

5 Fig. 2. Selective HMBC correlations of 3 and 5 (arrows denote HMBC correlations from H to C).

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Table 1 1 H NMR spectroscopic data (dH) for 1–5 in CD3OD (J in Hz). Position

1

4 5 11 12 14 15 16 17 19 20 7-OCH3

6.24 6.70 3.27 5.21 1.66 1.78 4.06 5.21 1.83 1.67 3.75

2 (s) (s) (d, 7.3) (m) (s) (s) (d, 5.4) (m) (s) (s) (s)

3

7.14 6.77 3.41 5.23 1.82 1.90 4.08 5.23 1.92 1.84 3.77

(s) (s) (d, 7.2) (m) (s) (s) (d, 5.4) (m) (s) (s) (s)

6.28 7.60 3.07 4.73 1.22 1.27 4.10 5.20 1.82 1.67 3.85

4 (s) (s) (d, 8.4) (t-like, 8.4) (s) (s) (d, 5.1) (m) (s) (s) (s)

HMBC (H2-16/C-8, C-8a, C-7, C-17; H-17/C-16, C-18) and COSY (H216/H-17) correlations confirmed the placement of hydroxyl groups at C-17, and C-18. The chemical shifts of C-16, C-17 and C-18 in 4 were also inconsistent with those reported for mangostenone E (Suksamrarn et al., 2006). The presence of a 2-(1-hydroxy-1-methylethyl)-2,3-dihydrofuran-3-ol moiety was deduced from the signals of two methyls [dH 1.29 (3H, s, H-15), dC 25.4 (C-15) and dH 1.24 (3H, s, H-14), dC 25.4 (C-14)], a quaternary oxygenated carbon (dC 72.6, C-13), an oxygenated methine [dH 4.78 (1H, H-12), dC 93.3 (C-12)], and a methylene resonance [dH 3.13 (2H, d, J = 8.4 Hz, H11), dC 27.5 (C-11)], in addition to HMBC cross-peaks of (H-11/C1, C-2, C-3, C-12; H-12/C-3, C-13). Therefore, 4 was concluded to be 3,5-dihydro-4-hydroxy-6-(2,3-dihydroxy-3-methylbutyl)-2-(2hydroxypropan-2-yl)-7-methoxy-5-oxo-2H-furo[3,2-b]xanthen-8yl hydrogen sulfate or 17,18-dihydroxymangostanin 6-sulfate. Metabolite 5 was isolated as a dark brown solid. Its HRESIMS (m/z 505.1155 [MH]) established the molecular formula to be C24H25O10S with a molecular weight identical to that of 3. The 1H (Table 1) and 13C NMR (Table 2) spectroscopic data of 5 had a closed resemblance to those of 3. These findings suggested that 5 was also a prenyl-cyclization and sulfation product of 1 similar to 3. A 2-(1-hydroxy-1-methylethyl)-dihydrofuran moiety was deduced by the analysis of its 1H (Table 1) and 13C NMR (Table 2) spectroscopic data: two methyls [dH 1.28 (3H, s, H-15), dC 25.4 (C-15) and dH 1.21 (3H, s, H-14), dC 22.9 (C-14)], a quaternary oxyTable 2 13 C NMR spectroscopic data (dC) for 1–5 in CD3OD.

a

Position

1

2

3

4

5

1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 7-OCH3

161.7 111.6 163.8 93.3 156.3 102.9 158.0 144.9 138.6 112.3 183.3 103.9 156.8 22.4 124.0 132.0 26.1 18.1 27.3 125.2 131.9 18.5 27.3 61.5

161.3 116.3 157.3 99.0 155.3 103.0 158.8 145.2 138.8 112.3 183.9 106.2 158.0 23.1 123.5 132.2 26.1 18.3 27.3 125.0 132.4 18.5 26.1 61.5

159.0a 109.2 168.6 89.4 158.8a 108.3 152.8 147.2 138.7 115.5 183.7 105.1 155.5 27.3 93.2 72.6 26.4 25.4 27.5 124.9 132.2 18.5 26.4 62.1

158.9a 109.5 169.1 89.6 159.1a 108.7 153.0 148.2 136.8 116.1 184.5 105.1 155.6 27.5 93.3 72.6 25.4 25.4 29.8 80.4 74.5 25.6 26.1 61.9

154.5 116.1 161.6 101.7 157.1 103.0 158.9 145.4 138.7 113.6 178.7 107.4 157.7 29.2 94.5 73.2 22.9 25.4 27.4 125.4 131.8 18.4 26.1 61.4

Interchangeable within a column.

6.38 7.68 3.13 4.78 1.24 1.29 3.62 3.67 1.34 1.34 3.94

5 (s) (s) (d, 8.4) (overlapped with solvent) (s) (s) (m) (m) (s) (s) (s)

7.05 6.73 3.06 4.93 1.21 1.28 4.06 5.20 1.77 1.66 3.75

(H, s) (1H, s) (dd, 8.8, 16.0) 3.29 (dd, 9.9, 16.0) (overlapped with solvent) (s) (s) (d, 5.4) (m) (s) (s) (s)

genated carbon (dC 73.2, C-13), an oxygenated methine [dH 4.93 (1H, overlapped with solvent, H-12), dC 94.5 (C-12)], and a methylene signal [dH 3.04 (1H, dd, J = 8.8, 16.0 Hz, Ha-11), dH 3.29 (1H, dd, J = 9.9, 16.0 Hz, Hb-11), dC 29.2 (C-11)]. The HMBC correlations of: H2-11/C-2, C-1, C-12, C-13; H-12/C-3; H-4/C-4a, C-3, C-9a, C-2 established that the dihydrofuran ring was fused to C-1 and C-2 with an ether linkage at C-1. In addition, the resonance of C-3 in 5 (dC 161.6) was different from those in 3 (dC 168.6) and 4 (dC 169.1), as well as showing an upfield shift comparable to those of 1 (2.2 ppm), thus excluding conjugation of the dihydrofuran ring at C-2 and C-3. This situation was corroborated by data obtained from reexamination of the 1H NMR spectra of 1 and 5 recorded in DMSO-d6, in which the chelated hydroxyl group at position 1, occurring at 13.71 ppm in 1, disappeared in 5. According to the HMQC and HMBC (Fig. 2) correlations as well as shifts of C-3 and C-4 (+2.2 and 8.4 ppm, respectively) compared to those of 1, C3 should be sulfated substitution. Moreover, it could be deduced that H-4 was significantly downfield shifted (0.81 ppm) because of the ortho effect of the sulfate moiety. Structure 5 was therefore assigned as 3,11-dihydro-8-hydroxy-2-(2-hydroxypropan-2-yl)-9methoxy-10-(3-methylbut-2-enyl)-11-oxo-2H-furo[2,3-a]xanthen4-yl hydrogen sulfate or isomangostanin 3-sulfate. All four isolated metabolites including substrate 1 were evaluated for anti-mycobacterial activity. Compounds 1 and 2 were found to exhibit significant activity (MIC = 15.24 and 6.75 lM for 1 and 2, respectively). By contrast, 3–5 showed no activity (MIC > 50 lg/mL). 3. Concluding remarks In conclusion, metabolism of substrate 1 by Colletotrichum gloeosporioides (EYL 131) and Neosartorya spathulata (EYR 042) led to isolation of four new xanthone derivatives. All of these compounds were sulfated mangostin as well as a combination of sulfation and prenyl-cyclization product of 1. The proposed biogenesis of prenyl cyclization to form a dihydrofuran moiety in the structures of 3, 4, and 5 can be rationalized by an initial epoxidation of the prenyl group by an oxidative enzyme reaction, followed by a spontaneous intramolecular attack of the neighboring hydroxyl. In this case, ring closure can lead to a five-membered ring. In addition, a dihydroxylated prenyl group in 4 probably proceeds by the same enzymatic reaction to form an epoxide at the C-17,18 double bond; however, because it does not possess an OH group ortho to the prenyl side-chain, the epoxy intermediate was hydrolyzed to give a glycol derivative. Although the expected epoxy intermediate was not obtained, this metabolic pathway was proposed based on the prenyl group metabolism in other prenylated compounds (Tahara et al., 1997; Tanaka and Tahara, 1997; Yilmazer et al., 2001; Herath et al., 2003). Furthermore, all prenyl-cyclization products (3–5) exhibited optical activity, suggesting that at least a degree of stereoselectivity in the cyclization process to form a

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dihydrofuran ring which leads to the creation of the C-12 stereocenter with an unidentified absolute configuration. The present report indicated that both strains are capable of performing phase II conjugation in the form of sulfation reactions by which N. spathulata (EYR 042) regiospecifically sulfated substrate 1 at C-3. Sulfate conjugation is an important pathway in mammalian metabolism of xenobiotics, and thus all products obtained in this study could potentially be useful in the study of metabolism of 1 in mammals. Compound 2, the major metabolite generated from 1 by N. spathulata (EYR 042), had a potent antimycobacterial activity that was higher than that of substrate 1. By contrast, 3, 4, and 5, the compounds without prenyl substitutions at C-2 or C-8, showed no anti-mycobacterial activity. These results suggested that presence of prenyl groups at C-2 or C-8 might be involved in enhanced activity. Moreover, the free hydroxy groups at either C-3 or C-6 and at C-1 of the mangostin nucleus should also play a crucial role for the high anti-mycobacterial activity. A similar result was also observed in a previous report (Suksamrarn et al., 2003).

was distributed equally among these 17 flasks, and the incubation was continued for another 6 d. Filtrate from the broth was extracted with EtOAc (3 with a half volume). The combined EtOAc extracts were dried (anhyd. Na2SO4) and concentrated under vacuum to yield a 54 mg residue. The latter residue was subjected to Sephadex LH-20 chromatography eluted with MeOH. Similar fractions, monitored by TLC, were combined to afford 2 (12 mg, 18%).

4. Experimental

The organism was grown at 28 °C for 3 d. in 40 flasks (500 mL), each containing liquid medium (150 mL) and shaken at 125 rpm on a rotary shaker. Substrate 1 (280 mg) dissolved in acetone (20 mL) was distributed equally among the 40 flasks and incubation was continued for another 10 d. The same extraction procedure mentioned above was then utilized, to afford a residue (216 mg). This was then separated into two fractions by Sephadex LH-20 column chromatography (cc), eluted with MeOH. Fraction 1 was subjected to additional Sephadex LH-20 cc eluted with EtOH-H2O (1:1) to yield 2 (1 mg, 0.4%) and 3 (12 mg, 4.3%). Fraction 2 was also fractionated in the same manner to give 4 (2 mg, 0.7%) and 5 (6 mg, 2.1%).

4.1. General experimental procedures Optical rotations were measured with a Jasco P-2200 digital polarimeter whereas UV spectra were determined with a Shimadzu UV-2450 PC spectrophotometer. IR spectra were acquried on a PerkinElmer FT-IR Spectrum BX spectrophotometer. 1H NMR (300 MHz), 13C NMR (75 MHz), and 2D NMR spectra were recorded on a Bruker AVANCE 300 FT-NMR spectrometer. With chemical shifts referenced to CD3OD (dH 3.31, dC 49.15). Mass spectra were performed on a JEOL JMS-SX102A (Ionization: FAB) and Bruker mrcrOTOF 10323 mass spectrometers. 4.2. Substrate

a-Mangostin (1) was isolated from an EtOAc extract of fruit hulls of G. mangostana. Its spectroscopic data was consistent with its structure (Tables 1 and 2). The purity of a-mangostin (1) exceeded 98% as determined by HPLC analysis (Chaivishangkura et al., 2009). 4.3. Microorganisms and culture media N. spathulata (EYR042) and C. gloeosporioides (EYL131) were isolated from the root and leaf tissues of Tiliacora triandra (Colebr.) Diels, respectively. Both strains were identified based on morphological and cultural characteristics as described by Barnett and Hunter (1998). Their nucleotide sequences based on the internal transcribed spacers (ITS) 1 and 2 including 5.8S rDNA regions were also compared to other sequences available in the GenBank database using the BLAST search tool (www.ncbi.nlm.nih.gov/BLAST/). The sequences were then submitted to EMBL database as the accession numbers as FN985089 and FN985090 for C. gloeosporiodes (EYL131) and N. spathulata (EYR042), respectively. Stock cultures were maintained on potato dextrose agar. Czapek-Dox broth containing sucrose (30 g), NaNO3 (3 g), K2HPO4 (1 g), MgSO47 H2O (0.5 g), KCl (0.5 g), and FeSO47 H2O (0.01 g) in H2O (1000 mL) at, pH 7.2, was used for fermentation experiments. 4.4. Preparative scale fermentation of 1 with N. spathulata (EYR042) The organism was grown at 28 °C for 3 d. in 17 flasks (500 mL), each containing liquid medium (100 mL) and shaken at 100 rpm on a rotary shaker. Substrate 1 (68 mg) dissolved in acetone (8.5 mL)

4.4.1. 3,8-Dihydroxy-2-methoxy-1,7-bis(3-methylbut-2-enyl)-9-oxo9H-xanthen-6-yl hydrogen sulfate (2) Yellow solid; UV (MeOH) kmax (log e): 237.5 (4.31), 255.5 (4.28), 309.5 (4.15), 356 (3.66) nm; IR (neat) mmax: 3450, 1635, 1606, 1465, 1276, 1181 cm1; for 1H and 13CNMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 489.1218 [MH] (calcd for C24H25O9S, 489.1225). 4.5. Preparative scale fermentation of 1 with C. gloeosporioides (EYL131)

4.5.1. 3,8-Dihydroxy-2-methoxy-1,7-bis(3-methylbut-2-enyl)-9-oxo9H-xanthen-6-yl hydrogen sulfate (2) Same physical data as transformed product of 1 by N. spathulata (EYR042). 4.5.2. 3,5-Dihydro-4-hydroxy-2-(2-hydroxypropan-2-yl)-7-methoxy6-(3-methylbut-2-enyl)-5-oxo-2H-furo[3,2-b]xanthen-8-yl hydrogen sulfate (3) Brown solid; ½a23 D +9.3 (c 0.22, MeOH); UV (MeOH) kmax (log e): 245 (4.59), 315 (4.43) nm; IR (neat) mmax: 3450, 1665, 1618, 1461, 1276, 1177 cm1; for 1H and 13C NMR spectroscopic data see Tables 1 and 2; HRESIMS m/z 505.1148 [MH] (calcd for C24H25O10S, 505.1174). 4.5.3. 3,5-Dihydro-4-hydroxy-6-(2,3-dihydroxy-3-methylbutyl)-2-(2hydroxypropan-2-yl)-7-methoxy-5-oxo-2H-furo[3,2-b]xanthen-8-yl hydrogen sulfate (4) Yellow solid; ½a23 D 4.8 (c 0.08, MeOH); UV (MeOH) kmax (log e): 245 (4.54), 319 (4.36) nm; IR (neat) mmax: 3427, 2928, 2360, 1727, 1603, 1462, 1275, 1123, 1071 cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2; HRESIMS m/z 539.1224 [MH] (calcd for C24H27O12S, 539.1229). 4.5.4. 3,11-Dihydro-8-hydroxy-2-(2-hydroxypropan-2-yl)-9methoxy-10-(3-methylbut-2-enyl)-11-oxo-2H-furo[2,3-a]xanthen-4yl hydrogen sulfate (5) Dark brown solid; ½a23 D 4.6 (c 0.16, MeOH); UV (MeOH) kmax (log e): 251 (4.43), 300 (4.16), 350 (3.80) nm; IR (neat) mmax: 3445, 1622, 1462, 1277, 1189 cm1; for 1H and 13C NMR spectroscopic data, see Tables 1 and 2. HRESIMS m/z 505.1155 [MH] (calcd for C24H25O10S, 505.1174).

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4.6. Anti-mycobacterial activity The anti-mycobacterial activities of isolated metabolites against Mycobacterium tuberculosis H37Ra were evaluated using a green fluorescent protein microplate assay (GFPMA) (Changsen et al., 2003). The maximum concentration of test samples was 50 lg/ mL. Reference compounds showed MIC of 0.36  102– 1.46  102, 0.29–0.54, 0.17–0.34, and 1.08–2.16 lV for rifampicin, streptomycin, isoniazid, and ofloxacin, respectively. Acknowledgements This work was carried out in part under the collaboration of a scientific cooperation program between the National Research Council of Thailand (NRCT) and the Japan Society for the Promotion of Science (JSPS) under the Asian Core Program on Capacity Building and Development of Microbial Potential and Fermentation Technology towards a New Era. Support from The Strategic Research Grant, The Thailand Research Fund was gratefully acknowledged. The authors wish to express their thanks to Associate Prof. Teruhiko Nitoda of the Graduate School of Natural Science and Technology, Okayama University, for the mass analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.phytochem.2011.02.007. References Barnett, H.L., Hunter, B.B., 1998. Illustrated Genera of Imperfect Fungi, fourth ed. American Phytopathological Society Press, St Paul, Minnesota. Chaivishangkura, A., Malaikaew, Y., Chaovanalikit, A., Jaratrungtawee, A., Panseeta, P., Ratananukul, P., Suksamrarn, S., 2009. Prenylated xanthone composition of the Garcinai mangostana (mangosteen) fruit hull. Chromatographia 69, 315– 318. Changsen, C., Franzblau, S.G., Palittapongarnpim, P., 2003. Improved green fluorescent protein reporter gene-based microplate screening for antituberculosis compounds by utilizing an acetamidase promoter. Antimicrob. Agents Chemother. 47, 3682–3687. Deschamps, J.D., Gautschi, J.T., Whitman, S., Johnson, T.A., Gassner, N.C., Crews, P., Holman, T.R., 2007. Discovery of platelet-type 12-human lipoxygenase selective inhibitors by high-throughput screening of structurally diverse libraries. Bioorg. Med. Chem. 15, 6900–6908.

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