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New depsidones and xanthone from the roots of Garcinia schomburgkiana
Fitoterapia 111 (2016) 73–77
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New depsidones and xanthone from the roots of Garcinia schomburgkiana Edwin Risky Sukandar a, Pongpun Siripong b, Suttira Khumkratok c, Santi Tip-pyang a,⁎ a b c
Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Natural Products Research Section, Research Division, National Cancer Institute, Bangkok 10400, Thailand Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand
a r t i c l e
i n f o
Article history: Received 17 March 2016 Received in revised form 15 April 2016 Accepted 17 April 2016 Available online 19 April 2016 Keywords: Garcinia schomburgkiana Depsidone Xanthone Cytotoxicity
a b s t r a c t Two new depsidones, schomburgdepsidones A and B (1 and 2), and one new xanthone, schomburgxanthone A (3), together with eight known compounds (4–11) were isolated from the roots of Garcinia schomburgkiana. Their chemical structures were established on the basis of spectroscopic analysis. The in vitro cytotoxicity of all 11 compounds was evaluated against the KB, HeLa S-3, HT-29, MCF-7 and Hep G2 human cancer cell lines. Compound 7 performed a good cytotoxicity against the KB, Hela S-3 and MCF-7 cell lines with IC50 values in the range of 3.17–6.07 μM. Compound 3 exhibited a good cytotoxicity against the KB cell line only, with an IC50 value of 8.14 μM. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The genus Garcinia (family Clusiaceae) is comprised of about 29 species in Thailand [1]. The species Garcinia schomburgkiana Pierre, locally named “Ma dan” in Thai, is a small evergreen plant and can be found in Thailand, Laos and Vietnam as an indigenous plant [2, 3]. This plant has been used by Thai people in folk medicine as an expectorant, laxative and menstrual treatment [4]. Previous phytochemical studies on G. schomburgkiana have revealed the presence of xanthones [3–6], biflavonoids [3,7], biphenyl derivatives [3–5], benzophenones [3,8] and steroids [3], and some of these exhibited an antimalarial activity [8] or cytotoxicity against cancer cell lines [3–6]. As a part of our ongoing search for bioactive constituents [9–11], we report herein the isolation and structure elucidation of two new depsidones, schomburgdepsidones A and B (1 and 2), and one new xanthone, schomburgxanthone A (3), along with eight known compounds (4–11) from the roots of G. schomburgkiana. Furthermore, all these 11 compounds were evaluated for their in vitro inhibitory activities against five human cancer cell lines (KB, HeLa S-3, HT-29, MCF-7 and Hep G2). 2. Experimental 2.1. General experiment procedures The UV spectra were analyzed using a UV-2550 UV–vis spectrometer (Shimadzu, Kyoto, Japan), while IR data were obtained on Nicolet 6700 FT-IR spectrometer using the KBr disc method. The 1D- and 2D-NMR ⁎ Corresponding author. E-mail address: [email protected] (S. Tip-pyang).
http://dx.doi.org/10.1016/j.fitote.2016.04.012 0367-326X/© 2016 Elsevier B.V. All rights reserved.
spectra were measured on a Bruker 400 AVANCE spectrometer in CDCl3. The HR-ESI-MS were analyzed using a Bruker MICROTOF model mass spectrometer. Column chromatography (CC) was performed using silica gel 60 (Merck) and Sephadex LH-20. For TLC analysis, precoated silica gel plates (Merck silica gel 60 GF254, 0.25 mm) were used. Spots were visualized under UV light and sprayed with anisaldehyde solution followed by heating. 2.2. Plant material The roots of G. schomburgkiana were collected from a riparian zone along the Chi River, Mahasarakham Province, Thailand. The plant was identified and deposited with a voucher specimen (Khumkratok no. 92-08) by Dr. Suttira Khumkratok, a botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, Thailand. 2.3. Extraction and isolation The air-dried roots of G. schomburgkiana (13.0 kg) were ground into powder and extracted twice with 30 L each time of dichloromethane (CH2Cl2) at room temperature for six days. The solvent was removed from the extract in vacuo to yield 104.0 g of crude extract. The crude CH2Cl2 extract was then fractionated by CC on silica gel (1.5 kg) with a step gradient elution of hexane:CH2Cl2 (80:20, 60:40, 40:60, 20:80 and 0:100, v/v, each 2.0 L), CH2Cl2:EtOAc (80:20, 60:40, 40:60, 20:80 and 0:100, v/v, each 2.0 L) and MeOH, respectively, to obtain 13 fractions (A–M). Fraction F (10.1 g) was further subjected to Sephadex LH-20 CC (300.0 g) eluted with CH2Cl2:MeOH (1:1, v/v) to yield three subfractions (F1–F3). Subfraction F3 (5.3 g) was separated by using
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see Table 1; and HR-ESI-MS m/z 495.2390 [M + H]+ (calcd. for C29H35O7, 495.2383).
silica gel CC (200.0 g) with a gradient of hexane:EtOAc (10:1, 8:1, 6:1, 4:1 and 2:1, v/v, each 300.0 mL) to give ten subfractions (F3.1–F.3.10). Subfraction F3.1 (40.2 mg) was subjected to Sephadex LH-20 CC (30.0 g) eluted by CH2Cl2:MeOH (1:1, v/v) to afford 11 (4.1 mg). Compound 2 (10.4 mg) was obtained by separation of subfraction F3.2 (150.0 mg) on Sephadex LH-20 CC (50.0 g) with CH2Cl2:MeOH (1:1, v/v). Subfraction F3.7 (1.0 g) was applied to CC over silica gel (50.0 g) with a hexane:CH2Cl2 gradient solvent system (50:50, 40:60 and 30:70, v/v, each 200.0 mL) to obtain 1 (11.1 mg), 3 (5.9 mg) and 4 (5.8 mg). Compound 6 (4.3 mg) was afforded by purification of subfraction F3.10 (67.0 mg) on Sephadex LH-20 CC (30.0 g) eluted with CH2Cl2:MeOH (1:1, v/v). Subfraction F3.9 (1.4 g) was chromatographed into Sephadex LH-20 CC (100.0 g) with CH2Cl2:MeOH (1:1, v/v) to give three subfractions (F3.9.1–F3.9.3). Compound 8 (1.6 mg) and 10 (1.8 mg) were achieved from subfraction F3.9.2 (106.8 mg) by repeated Sephadex LH-20 CC (50.0 g) with a CH2Cl2:MeOH (1:1, v/v) solvent system. Subfraction F3.9.3 (570.0 mg) was treated by silica gel CC (50.0 g) eluted with a step gradient of hexane:CH2Cl2 (50:50, 40:60 and 30:70, v/v, each 100.0 mL) to yield 5 (7.1 mg), 7 (4.2 mg) and 9 (14.5 mg).
2.3.3. Schomburgxanthone A (3) Yellow powder; UV (MeOH) λmax: 325, 281 and 224 nm; IR νmax (KBr): 3337, 2965, 1636, 1609, 1433 and 1214 cm−1; for 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Table 1; and HR-ESI-MS m/z 411.1818 [M + H]+ (calcd. for C24H27O6, 411.1808). 2.4. Cytotoxicity assay All isolated compounds (1−11) were evaluated for their in vitro cytotoxic activities against the KB (epidermoid carcinoma), HeLa S-3 (cervix adenocarcinoma), HT-29 (colon adenocarcinoma), MCF-7 (breast adenocarcinoma) and Hep G2 (hepatocellular carcinoma) human cancer cell lines using the MTT colorimetric method [12]. Doxorubicin was used as the reference substance. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (Sigma Chemical Co., USA) was dissolved in saline to make a 5 mg/mL stock solution. Cancer cells (3 × 103 cells) suspended in 100 μg/wells of MEM medium containing 10% fetal calf serum (FCS, Gibco BRL, Life Technologies, NY, USA) were seeded onto a 96-well culture plate (Costar, Corning Incorporated, NY 14831, USA). After 24 h of pre-incubation at 37 °C in a humidified atmosphere of 5% CO2/95% air to allow cellular attachment, various concentrations of test solution (10 μL/well) were added and these were then incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The optical density (OD) of each well was detected using a Microplate reader at 550 nm and for correction at 595 nm. Each determination
2.3.1. Schomburgdepsidone A (1) Brownish gum; UV (MeOH) λmax: 320, 272 and 217 nm; IR νmax (KBr): 3415, 2974, 1656, 1613, 1465 and 1177 cm−1; for 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Table 1; and HR-ESI-MS m/z 503.2051 [M + Na]+ (calcd. for C28H32O7Na, 503.2046). 2.3.2. Schomburgdepsidone B (2) Brownish gum; UV (MeOH) λmax: 316, 279 and 212 nm; IR νmax (KBr): 3408, 2970, 1660, 1621, 1467 and 1201 cm−1; for 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data,
Table 1 1 H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of compounds 1–3 in CDCl3. Position
1 δH (J in Hz)
1 2 3 4 4a 5a 6 7 8 9 9a 11 11a 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 1-OH 3-OH 6-OH 7-OH 3-OMe
6.28 (1H, s)
3.64 (2H, d, 6.0) 5.24 (1H, t, 6.0) 1.79 (3H, s) 1.86 (3H, s) 3.35 (2H, d, 6.4) 5.08 (1H, t, 6.4) 1.71 (3H, s) 1.79 (3H, s) 3.44 (2H, d, 6.4) 5.01 (1H, t, 6.4) 1.67 (3H, s) 1.79 (3H, s) 10.99 (1H, s) 6.28 (1H, brs) 5.83 (1H, brs) 5.42 (1H, brs)
2 δC 163.9 100.8 162.1 111.2 158.9 136.5 133.6 141.0 123.7 123.6 136.8 168.6 99.5 22.5 122.2 137.1 25.9 18.2 25.8 121.9 134.3 25.9 18.1 25.6 122.4 132.4 25.9 18.2
HMBC
δH (J in Hz)
1, 3, 4, 11a
6.32 (1H, s)
3, 4, 4a, 13, 14 15, 16
3.49 (2H, d, 6.0) 5.14 (1H, t, 6.0)
14, 16 13, 14, 15 7, 8, 18, 19 20, 21
1.67 (3H, s) 1.73 (3H, s) 3.57 (2H, d, 6.4) 5.20 (1H, t, 6.4)
18, 19, 21 19, 20 8, 9, 9a, 24 25, 26
1.74 (3H, s) 1.81 (3H, s) 3.55 (2H, d, 7.2) 5.27 (1H, t, 7.2)
23, 24, 26 24, 25 1, 2
1.73 (3H, s) 1.84 (3H, s) 11.30 (1H, s)
Position δC 164.9 96.2 165.0 113.7 158.6 141.9 117.4 140.2 140.1 118.1 136.5 169.4 98.7 22.6 122.8 132.3 25.8 18.0 23.9 121.6 135.5 25.9 18.1 23.8 121.2 135.0 25.9 18.1
1, 4, 11a
3, 4, 4a, 13, 14 15, 16 13, 14, 16 13, 14, 15 5a, 6, 18, 19 20, 21 19 18, 19, 20 8, 9, 9a, 23, 24 25, 26 26 24 1, 2, 11a
5a, 6, 7 5.54 (1H, s) 3.85 (3H, s)
56.2
6, 7 3
3 δH (J in Hz)
HMBC 1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 1-OH 5-OH 6-OH 3-OMe
6.35 (1H, s)
6.78 (1H, s)
3.48 (2H, d, 6.8) 5.22 (1H, t, 6.8) 1.73 1.85 3.97 (2H, d, 7.2) 5.35 (1H, t, 7.2) 1.75 1.73 13.44 (1H, s) 5.47 (1H, brs) 5.90 (1H, brs) 3.90 (3H, s)
δC 162.6 94.6 163.4 106.6 153.2 128.9 148.2 113.4 137.8 111.9 182.8 103.7 146.4 21.9 123.5 131.7 25.7 18.0 33.1 122.7 133.3 26.0 18.1
HMBC 1, 4, 9a
5, 6, 8a, 16
3, 4, 4a, 12, 13
12, 13, 15 12, 13, 14 7, 8, 17, 18
20 17, 18, 19 1, 2, 9a
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represented the average mean of six replicates. The 50% inhibition concentration (IC50 value) was determined by curve fitting.
3. Results and discussion Phytochemical investigation of the CH2Cl2 extract from the roots of G. schomburgkiana led to the isolation of two new depsidones, schomburgdepsidones A and B (1 and 2), and one new xanthone, schomburgxanthone A (3), along with eight known compounds; oliveridepsidone A (4) [13], oliveridepsidone D (5) [13], 1,5dihydroxyxanthone (6) [14], nigrolineaxanthone E (7) [15], 6desoxyjacareubin (8) [16], aucuparin (9) [17], 3-hydroxy-5methoxybiphenyl (10) [18] and methyl-2,6-dihydroxy-4-methoxy3(3′-methyl-2′-butenyl)-benzoate (11) [19]. The structures of the three new isolated compounds were elucidated by using 1D- and 2DNMR spectroscopy. The structures of the known compounds (4–11) were determined and confirmed by comparison of their 1H and 13C NMR spectroscopic data with previously published data. Schomburgdepsidone A (1) was obtained as a brownish gum. Its molecular formula was determined as C28H32O7 by HR-ESI-MS measurement through the sodium adduct ion at m/z 503.2051 [M + Na]+ (calcd. for C28H32O7Na, 503.2046). The UV spectrum displayed absorption bands at λmax 320, 272 and 217 nm. The IR spectrum showed characteristic absorption bands for hydroxy and lactone carbonyl stretching bonds at 3415 and 1656 cm−1, indicating the presence of a depsidone chromophore [13]. The 1H NMR spectrum (Table 1) consisted of signals for a hydrogen-bonded hydroxy proton at δH 10.99 (1H, s, 1-OH); an aromatic proton at δH 6.28 (1H, s, H-2); three hydroxy protons at δH 6.28 (1H, brs, 3-OH), 5.83 (1H, brs, 6-OH) and 5.42 (1H, brs, 7-OH); and three 3-methylbut-2-enyl units, as deduced from the resonances of the three olefinic protons at δH 5.24 (1H, t, J = 6.0 Hz, H-13), 5.08 (1H, t, J = 6.4 Hz, H-18) and 5.01 (1H, t, J = 6.4 Hz, H-23); three sets of methylene protons at δH 3.64 (2H, d, J = 6.0 Hz, H-12), 3.35 (2H, d, J = 6.4 Hz, H17) and 3.44 (2H, d, J = 6.4 Hz, H-22); and six methyl protons at δH
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1.79 (3H, s, H-15), 1.86 (3H, s, H-16), 1.71 (3H, s, H-20), 1.79 (3H, s, H-21), 1.67 (3H, s, H-25) and 1.79 (3H, s, H-26). Based on the HMBC spectrum (Table 1), a hydrogen-bonded hydroxy proton at δH 10.99 (1-OH) correlated to C-1 (δC 163.9) and C-2 (δC 100.8), showing that the hydroxy group was attached to C-1. An aromatic proton was placed at C-2 by the observed crosspeaks of H-2 to the oxygenated quaternary aromatic carbons C-1 and C-3 (δC 162.1), and substituted aromatic carbons C-4 (δC 111.2) and C-11a (δC 99.5). Furthermore, one isoprenyl moiety was attached to C-4 from the correlations of its methylene protons at H-12 to C-3, C-4 and C-4a (δC 158.9), supporting the assignment of the A-ring. The 1H and 13 C NMR spectroscopic data of 1 (Table 1) were similar to those of oliveridepsidone A (4) [13]. The major difference was the position of the remaining two prenyl units and two hydroxy groups in the B-ring. The HMBC spectrum of 1 (Fig. 2) assigned the correlations of methylene protons at H-17 to C-7 (δC 141.0) and C-8 (δC 123.7), and H-22 to C-8, C9 (δC 123.6) and C-9a (δC 136.8), suggesting that the two isoprenyl units were connected to C-8 and C-9 in the B-ring. Consequently, the two remaining hydroxy groups were placed at C-6 (δC 133.6) and C-7 (δC 141.0). (See Fig. 1.) The linkage between two phenyl moieties (the A- and B-ring) was determined by the following spectroscopic data. The carbon signal at δC 168.6 (C-11) in the 13C NMR spectrum and the observed strong absorption at νmax 1656 cm−1 in the IR spectrum indicated a characteristic of lactone carbonyl from a depsidone [13,20]. The 13C NMR spectrum also assigned the signals of oxygenated quaternary aromatic carbons C-4a, C-5a (δC 136.5) and C-9a, and substituted aromatic carbon C-11a bonded to the carbonyl carbon of depsidone. Based on the HMBC experiment (Fig. 2), the correlations of H-2 to C-11a and H-12 to C-4a suggested that the A-ring was connected to the C-ring. Furthermore, the correlations of 6-OH (δH 5.83) to C-5a and H-22 to C-9a suggested that the B-ring was also connected to the C-ring. Therefore, both phenyl moieties were linked by a seven-membered ring (the C-ring) containing an ester linkage and an ether bridge. From the above evidences and by comparison with the literature [13], compound 1 was determined as
Fig. 1. The structures of compounds 1–3 isolated from the roots of G. schomburgkiana.
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Fig. 2. The key HMBC (arrow curves) and COSY (bold lines) correlations of compounds 1–3.
1,3,6,7-tetrahydroxy-4,8,9-tris(3-methylbut-2-enyl)-11H-dibenzo[b,e] [1,4]-dioxepin-11-one. Schomburgdepsidone B (2) was isolated as a brownish gum. Its HRESIMS exhibited a molecular ion peak at m/z 495.2390 [M + H]+ (calcd. for C29H35O7, 495.2383), which corresponded to the molecular formula of C29H34O7. The UV spectrum revealed maximum absorption bands at λmax 316, 279 and 212 nm. The IR spectrum showed hydroxy and lactone carbonyl stretching bands at 3408 and 1660 cm−1, suggesting the presence of a depsidone derivative [13]. The 1H and 13C NMR spectroscopic data of 2 (Table 1) were comparable to an analogue, oliveridepsidone A (4) [13]. The difference was the replacement of the 1 H NMR signal for the hydroxy group at C-3 of 4 with a methoxy group at δH 3.85 (3H, s, 3-OCH3) in 2. This structural assignment was further confirmed by the HMBC correlation of the methoxy signal (δH 3.85) to C-3 (δC 165.0) (Fig. 2). From these data, the structure of 2 was established as 1,7,8-trihydroxy-3-methoxy-4,6,9-tris(3-methylbut-2enyl)-11H-dibenzo[b,e] [1,4]dioxepin −11-one. Schomburgxanthone A (3) was obtained as a yellow powder. The molecular formula was measured by HRESIMS as C24H26O6 (m/z 411.1818 [M + H]+, calcd. for C24H27O6 411.1808). The UV spectrum displayed absorption bands at λmax 325, 281 and 224 nm, which were typical of a xanthone chromophore [21]. The IR spectrum showed absorption bands at 3337 and 1636 cm−1 due to hydroxy and carbonyl stretching bonds, respectively. The 1H NMR spectrum (Table 1) displayed a hydrogen-bonded hydroxy proton at δH 13.44 (1H, s, 1OH); two hydroxy protons at δH 5.47 (1H, brs, 5-OH) and 5.90 (1H, brs, 6-OH); two singlet aromatic protons at δH 6.35 (1H, s, H-2) and δH 6.78 (1H, s, H-7); a methoxy signal at δH 3.90 (3H, s, 3-OCH3); and two 3-methylbut-2-enyl units, as determined by the resonances of the two olefinic protons at δH 5.22 (1H, t, J = 6.8 Hz, H-12) and 5.35 (1H, t, J = 7.2 Hz, H-17); two sets of methylene protons at δH 3.48 (2H, d, J = 6.8 Hz, H-11) and 3.97 (2H, d, J = 7.2 Hz, H-16); and four methyl protons at δH 1.73 (3H, s, H-14), 1.85 (3H, s, H-15), 1.75 (3H, s, H-19), and 1.73 (3H, s, H-20). In the HMBC spectrum of 3 (Fig. 2), the two isoprenyl groups were located at C-4 and C-8 by the crosspeaks of H-11 (δH 3.48) to C-3 (δC
163.4), C-4 (δC 106.6) and C-4a (δC 153.2), and H-16 (δH 3.97) to C-7 (δC 113.4) and C-8 (δC 137.8). In addition, the crosspeak of the methoxy signal (δH 3.90) to C-3 indicated that the methoxy group was attributed at C-3 in the A-ring. The 1H and 13C NMR data of 3 (Table 1) were similar to those of dulxanthone C [20], except for the absence of the 1H NMR signal of the methoxy group at C-6 in 3. Furthermore, the substituent at C-6 was assigned as a hydroxy group according to its 13C NMR chemical shift (δC 148.2). Thus, the structure of 3 was identified as 1,5,6trihydroxy-3-methoxy-4.8-bis(3-methylbut-2-enyl)-9H-xanthen-9one. Depsidones are mainly formed in lichens through polyketide pathway which usually contain 3.8-dioxygenation and methyl groups attached to C-1, C-9 and/or C-6 [13,22]. However, a number of prenylated depsidones were reported from the genus Garcinia [13, 22–25], which well known to have a rich source of shikimate and mevalonate-derived aromatic compounds [22], including xanthones as chemotaxonomic marker of this genus [1]. It is possible that the biosynthesis of the prenylated depsidones lies in Baeyer-Villiger rearrangements from hydroperoxylation reactions of the precursor xanthones, with incorporation of the mevalonate-derived prenyl units [22,26]. The in vitro cytotoxicity assay of all isolated compounds (1–11) against five human cancer cell lines (KB, HeLa S-3, HT-29, MCF-7 and Hep G2) was evaluated using a modified MTT method with doxorubicin as the positive control [12]. The results are summarized in Table 2. The tested compounds mostly showed moderate to inactive against the five human cancer cell lines, except for compound 7 showed a good cytotoxicity (IC50 ≤ 10 μM) against the KB, Hela S-3 and MCF-7 cell lines with IC50 values of 3.17, 5.46, and 6.07 μM, respectively. Compound 3 showed a good cytotoxic activity against the KB cell line only, with an IC50 value of 8.14 μM. Among the depsidones (1, 2, 4 and 5), only compound 5 had a slightly higher cytotoxicity with IC50 values in the range of 11.14–30.40 μM. Conflict of interest The authors have declared that there is no conflict of interest.
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Table 2 In vitro cytotoxicity of compounds 1–11 against five human cancer cell lines. Compound
IC50 (μM) ± SD KB
Hela S-3
HT-29
Hep G2
MCF-7
Schomburgdepsidone A (1) Schomburgdepsidone B (2) Schomburgxanthone A (3) Oliveridepsidone A (4) Oliveridepsidone D (5) 1,5-dihydroxyxanthone (6) Nigrolineaxanthone E (7) 6-desoxyjacareubin (8) Aucuparin (9) 3-hydroxy-5-methoxybiphenyl (10) Methyl-2,6-dihydroxy-4-methoxy-3(3′-methyl-2′-butenyl)-benzoate (11) Doxorubicin
43.39 ± 1.95 52.33 ± 1.99 8.14 ± 0.61 55.94 ± 5.63 11.14 ± 0.86 25.50 ± 0.96 3.17 ± 0.08 18.56 ± 0.15 N100 59.78 ± 1.01 N100 0.22 ± 0.05
N100 95.96 ± 6.06 14.03 ± 0.26 N100 19.27 ± 0.27 N100 5.46 ± 0.08 19.69 ± 0.46 N100 N100 N100 0.11 ± 0.03
N100 N100 N100 58.74 ± 0.11 30.40 ± 1.78 N100 34.08 ± 0.73 69.29 ± 0.79 N100 N100 N100 0.44 ± 0.08
47.86 ± 0.58 N100 48.90 ± 0.88 39.27 ± 0.54 27.83 ± 0.92 N100 16.08 ± 0.22 39.35 ± 1.05 N100 N100 N100 2.80 ± 0.09
43.87 ± 6.68 46.97 ± 0.97 21.56 ± 0.19 36.46 ± 0.76 13.42 ± 0.10 N100 6.07 ± 0.11 17.53 ± 0.06 N100 N100 69.45 ± 0.34 0.42 ± 0.03
(IC50 ≤ 10 = good activity, 10 b IC50 ≤ 30 = moderate activity, and IC50 N 100 = inactive).
Acknowledgments The authors are grateful to the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) and The Thailand Research Fund via Directed Basic Research Grant (Grant no. DBG 5980001) for partially financial support. We also thank the Graduate School of Chulalongkorn University for the ASEAN Scholarship to E.R.S. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2016.04.012. References [1] T. Ritthiwigrom, S. Laphookhieo, S.G. Pyne, Chemical constituents and biological activities of Garcinia cowa Roxb, Maejo Int. J. Sci. Technol. 7 (2) (2013) 212–231. [2] T.K. Lim, Edible Medicinal and Non-Medicinal Plants: Volume 2, Fruits, Springer Science+Business Media, Dordrecht, 2012 (1100 pp.). [3] C. Mungmee, S. Sitthigool, A. Buakeaw, R. Suttisri, A new biphenyl and other constituents from the wood of Garcinia schomburgkiana, Nat. Prod. Res. 27 (2013) 1949–1955. [4] C. Mungmee, S. Sitthigool, A. Buakeaw, R. Suttisri, Xanthones and biphenyls from Garcinia schomburgkiana wood and their cytotoxicity, Thai. J. Pharm. Sci. 36 (2012) 6–9. [5] C. Ito, T. Matsui, E. Noda, N. Ruangrungsi, M. Itoigawa, Biphenyl derivatives from Garcinia schomburgkiana and the cytotoxicity of the isolated compounds, Nat. Prod. Commun. 8 (9) (2013) 1265–1267. [6] H.T. Vo, N.T.T. Nguyen, H.T. Nguyen, K.Q. Do, J.D. Connolly, G. Maas, J. Heilmann, U.R. Werz, H.D. Pham, L.-H.D. Nguyen, Cytotoxic tetraoxygenated xanthones from the bark of Garcinia schomburgkiana, Phytochem. Lett. 5 (3) (2012) 553–557. [7] A. Häfner, A.W. Frahm, Biflavonoids from the heartwood of Garcinia schomburgkiana and their structural elucidation as atropisomers, Planta Med. 59 (1993) A604. [8] H.K. Fun, S. Koysomboon, K. Chantrapromma, S. Chantrapromma, A cocrystal of clusiacitran A, clusiacitran B, fluorinated clusiacitran A and fluorinated clusiacitran B (0.45:0.45:0.05:0.05), Acta Cryst E62 (2006) o3228–o3230. [9] S. Kaennakam, P. Siripong, S. Tip-pyang, Kaennacowanols A–C, three new xanthones and their cytotoxicity from the roots of Garcinia cowa, Fitoterapia 102 (2015) 171–176. [10] S. Kaennakam, P. Siripong, S. Tip-pyang, Velucarpins A–C, three new pterocarpans and their cytotoxicity from the roots of Dalbergia velutina, Fitoterapia 105 (2015) 165–168.
[11] E.R. Sukandar, T. Ersam, S. Fatmawati, P. Siripong, T. Aree, S. Tip-pyang, Cylindroxanthones A–C, three new xanthones and their cytotoxicity from the stem bark of Garcinia cylindrocarpa, Fitoterapia 108 (2016) 62–65. [12] N. Kongkathip, B. Kongkathip, P. Siripong, C. Sangma, S. Luangkamin, M. Niyomdecha, S. Pattanapa, S. Piyaviriyagul, P. Kongsaeree, Potent antitumor activity of synthetic 1,2-naphthoquinones and 1,4-naphthoquinones, Bioorg. Med. Chem. 11 (14) (2003) 3179–3191. [13] L.D. Ha, P.E. Hansen, F. Duus, H.D. Pham, L.H.D. Nguyen, Oliveridepsidones A–D, antioxidant depsidones from Garcinia oliveri, Magn. Reson. Chem. 50 (3) (2012) 242–245. [14] L. Rocha, A. Marston, A.C. Kaplan, H. Storckli-Evans, U. Thull, B. Testa, K. Hostettmann, An antifungal γ-pyrone and xanthones with monoamine oxidase inhibitory activity from Hypericum brasiliense, Phytochemistry 36 (6) (1994) 1381–1385. [15] V. Rukachaisirikul, T. Ritthiwigrom, A. Pinsa, P. Sawangchote, W.C. Taylor, Xanthones from the stem bark of Garcinia nigrolineata, Phytochemistry 64 (6) (2003) 1149–1156. [16] F. Kawamura, A. Muhamud, R. Hashim, O. Sulaiman, S. Ohara, Two antifungal xanthones from the heartwood of Calophyllum symingtonianum, Japan Agric. Res. Q. 46 (2) (2012) 181–185. [17] C. Hüttner, T. Beuerle, H. Scharnhop, L. Ernst, L. Beerhues, Differential effect of elicitors on biphenyl and dibenzofuran formation in Sorbus aucuparia cell cultures, J. Agric. Food Chem. 58 (22) (2010) 11977–11984. [18] M.C. Song, F. Nigussie, T.S. Jeong, C.Y. Lee, F. Regassa, T. Markos, N.I. Baek, Phenolic compounds from the roots of Lindera fruticosa, J. Nat. Prod. 69 (5) (2006) 853–855. [19] H. Feld, D.S. Rycroft, J. Zapp, Chromenes and prenylated benzoic acid derivatives from the liverwort Pedinophyllum interruptum, Z. Naturforsch. 59b (7) (2004) 825–828. [20] C. Ito, Y. Miyamoto, M. Nakayama, Y. Kawai, K.S. Rao, H. Furukawa, A novel depsidone and some new xanthones from Garcinia species, Chem. Pharm. Bull. 45 (9) (1997) 1403–1413. [21] S. Klaiklay, Y. Sukpondma, V. Rukachaisirikul, S. Phongpaichit, Friedolanostanes and xanthones from the twigs of Garcinia hombroniana, Phytochemistry 85 (2013) 161–166. [22] Y.J. Xu, P.Y. Chiang, Y.H. Lai, J.J. Vittal, X.H. Wu, B.K.H. Tan, Z. Imiyabir, S.H. Goh, Cytotoxic prenylated depsidones from Garcinia parvifolia, J. Nat. Prod. 63 (10) (2000) 1361–1363. [23] D. Permana, N.H. Lajis, M.M. Mackeen, A.M. Ali, N. Aimi, M. Kitajima, H. Takayama, Isolation and bioactivities of constituents of the roots of Garcinia atroviridis, J. Nat. Prod. 64 (7) (2001) 976–979. [24] J. Ngoupayo, T.K. Tabopda, M.S. Ali, E. Tsamo, A-glucosidase inhibitors from Garcinia brevipedicellata (Clusiaceae), Chem. Pharm. Bull. 56 (10) (2008) 1466–1469. [25] C. Ito, M. Itoigawa, Y. Mishina, H. Tomiyasu, M. Litaudon, J.P. Cosson, T. Mukainaka, H. Tokuda, H. Nishino, H. Furukawa, Cancer chemopreventive agents. New depsidones from Garcinia plants, J. Nat. Prod. 64 (2) (2001) 147–150. [26] S. Kosela, S.G. Cao, X.H. Wu, J.J. Vittal, T. Sukri, Masdianto, S.H. Goh, K.Y. Sim, Lateriflorone, a cytotoxic spiroxalactone with a novel skeleton from Garcinia lateriflora Bl, Tetrahedron Lett. 40 (1) (1999) 157–160.
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Fitoterapia journal homepage: www.elsevier.com/locate/fitote
New depsidones and xanthone from the roots of Garcinia schomburgkiana Edwin Risky Sukandar a, Pongpun Siripong b, Suttira Khumkratok c, Santi Tip-pyang a,⁎ a b c
Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Natural Products Research Section, Research Division, National Cancer Institute, Bangkok 10400, Thailand Walai Rukhavej Botanical Research Institute, Mahasarakham University, Mahasarakham 44000, Thailand
a r t i c l e
i n f o
Article history: Received 17 March 2016 Received in revised form 15 April 2016 Accepted 17 April 2016 Available online 19 April 2016 Keywords: Garcinia schomburgkiana Depsidone Xanthone Cytotoxicity
a b s t r a c t Two new depsidones, schomburgdepsidones A and B (1 and 2), and one new xanthone, schomburgxanthone A (3), together with eight known compounds (4–11) were isolated from the roots of Garcinia schomburgkiana. Their chemical structures were established on the basis of spectroscopic analysis. The in vitro cytotoxicity of all 11 compounds was evaluated against the KB, HeLa S-3, HT-29, MCF-7 and Hep G2 human cancer cell lines. Compound 7 performed a good cytotoxicity against the KB, Hela S-3 and MCF-7 cell lines with IC50 values in the range of 3.17–6.07 μM. Compound 3 exhibited a good cytotoxicity against the KB cell line only, with an IC50 value of 8.14 μM. © 2016 Elsevier B.V. All rights reserved.
1. Introduction The genus Garcinia (family Clusiaceae) is comprised of about 29 species in Thailand [1]. The species Garcinia schomburgkiana Pierre, locally named “Ma dan” in Thai, is a small evergreen plant and can be found in Thailand, Laos and Vietnam as an indigenous plant [2, 3]. This plant has been used by Thai people in folk medicine as an expectorant, laxative and menstrual treatment [4]. Previous phytochemical studies on G. schomburgkiana have revealed the presence of xanthones [3–6], biflavonoids [3,7], biphenyl derivatives [3–5], benzophenones [3,8] and steroids [3], and some of these exhibited an antimalarial activity [8] or cytotoxicity against cancer cell lines [3–6]. As a part of our ongoing search for bioactive constituents [9–11], we report herein the isolation and structure elucidation of two new depsidones, schomburgdepsidones A and B (1 and 2), and one new xanthone, schomburgxanthone A (3), along with eight known compounds (4–11) from the roots of G. schomburgkiana. Furthermore, all these 11 compounds were evaluated for their in vitro inhibitory activities against five human cancer cell lines (KB, HeLa S-3, HT-29, MCF-7 and Hep G2). 2. Experimental 2.1. General experiment procedures The UV spectra were analyzed using a UV-2550 UV–vis spectrometer (Shimadzu, Kyoto, Japan), while IR data were obtained on Nicolet 6700 FT-IR spectrometer using the KBr disc method. The 1D- and 2D-NMR ⁎ Corresponding author. E-mail address: [email protected] (S. Tip-pyang).
http://dx.doi.org/10.1016/j.fitote.2016.04.012 0367-326X/© 2016 Elsevier B.V. All rights reserved.
spectra were measured on a Bruker 400 AVANCE spectrometer in CDCl3. The HR-ESI-MS were analyzed using a Bruker MICROTOF model mass spectrometer. Column chromatography (CC) was performed using silica gel 60 (Merck) and Sephadex LH-20. For TLC analysis, precoated silica gel plates (Merck silica gel 60 GF254, 0.25 mm) were used. Spots were visualized under UV light and sprayed with anisaldehyde solution followed by heating. 2.2. Plant material The roots of G. schomburgkiana were collected from a riparian zone along the Chi River, Mahasarakham Province, Thailand. The plant was identified and deposited with a voucher specimen (Khumkratok no. 92-08) by Dr. Suttira Khumkratok, a botanist at the Walai Rukhavej Botanical Research Institute, Mahasarakham University, Thailand. 2.3. Extraction and isolation The air-dried roots of G. schomburgkiana (13.0 kg) were ground into powder and extracted twice with 30 L each time of dichloromethane (CH2Cl2) at room temperature for six days. The solvent was removed from the extract in vacuo to yield 104.0 g of crude extract. The crude CH2Cl2 extract was then fractionated by CC on silica gel (1.5 kg) with a step gradient elution of hexane:CH2Cl2 (80:20, 60:40, 40:60, 20:80 and 0:100, v/v, each 2.0 L), CH2Cl2:EtOAc (80:20, 60:40, 40:60, 20:80 and 0:100, v/v, each 2.0 L) and MeOH, respectively, to obtain 13 fractions (A–M). Fraction F (10.1 g) was further subjected to Sephadex LH-20 CC (300.0 g) eluted with CH2Cl2:MeOH (1:1, v/v) to yield three subfractions (F1–F3). Subfraction F3 (5.3 g) was separated by using
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see Table 1; and HR-ESI-MS m/z 495.2390 [M + H]+ (calcd. for C29H35O7, 495.2383).
silica gel CC (200.0 g) with a gradient of hexane:EtOAc (10:1, 8:1, 6:1, 4:1 and 2:1, v/v, each 300.0 mL) to give ten subfractions (F3.1–F.3.10). Subfraction F3.1 (40.2 mg) was subjected to Sephadex LH-20 CC (30.0 g) eluted by CH2Cl2:MeOH (1:1, v/v) to afford 11 (4.1 mg). Compound 2 (10.4 mg) was obtained by separation of subfraction F3.2 (150.0 mg) on Sephadex LH-20 CC (50.0 g) with CH2Cl2:MeOH (1:1, v/v). Subfraction F3.7 (1.0 g) was applied to CC over silica gel (50.0 g) with a hexane:CH2Cl2 gradient solvent system (50:50, 40:60 and 30:70, v/v, each 200.0 mL) to obtain 1 (11.1 mg), 3 (5.9 mg) and 4 (5.8 mg). Compound 6 (4.3 mg) was afforded by purification of subfraction F3.10 (67.0 mg) on Sephadex LH-20 CC (30.0 g) eluted with CH2Cl2:MeOH (1:1, v/v). Subfraction F3.9 (1.4 g) was chromatographed into Sephadex LH-20 CC (100.0 g) with CH2Cl2:MeOH (1:1, v/v) to give three subfractions (F3.9.1–F3.9.3). Compound 8 (1.6 mg) and 10 (1.8 mg) were achieved from subfraction F3.9.2 (106.8 mg) by repeated Sephadex LH-20 CC (50.0 g) with a CH2Cl2:MeOH (1:1, v/v) solvent system. Subfraction F3.9.3 (570.0 mg) was treated by silica gel CC (50.0 g) eluted with a step gradient of hexane:CH2Cl2 (50:50, 40:60 and 30:70, v/v, each 100.0 mL) to yield 5 (7.1 mg), 7 (4.2 mg) and 9 (14.5 mg).
2.3.3. Schomburgxanthone A (3) Yellow powder; UV (MeOH) λmax: 325, 281 and 224 nm; IR νmax (KBr): 3337, 2965, 1636, 1609, 1433 and 1214 cm−1; for 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Table 1; and HR-ESI-MS m/z 411.1818 [M + H]+ (calcd. for C24H27O6, 411.1808). 2.4. Cytotoxicity assay All isolated compounds (1−11) were evaluated for their in vitro cytotoxic activities against the KB (epidermoid carcinoma), HeLa S-3 (cervix adenocarcinoma), HT-29 (colon adenocarcinoma), MCF-7 (breast adenocarcinoma) and Hep G2 (hepatocellular carcinoma) human cancer cell lines using the MTT colorimetric method [12]. Doxorubicin was used as the reference substance. The 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl-tetrazolium bromide (Sigma Chemical Co., USA) was dissolved in saline to make a 5 mg/mL stock solution. Cancer cells (3 × 103 cells) suspended in 100 μg/wells of MEM medium containing 10% fetal calf serum (FCS, Gibco BRL, Life Technologies, NY, USA) were seeded onto a 96-well culture plate (Costar, Corning Incorporated, NY 14831, USA). After 24 h of pre-incubation at 37 °C in a humidified atmosphere of 5% CO2/95% air to allow cellular attachment, various concentrations of test solution (10 μL/well) were added and these were then incubated for 48 h under the above conditions. At the end of the incubation, 10 μL of tetrazolium reagent was added into each well followed by further incubation at 37 °C for 4 h. The supernatant was decanted, and DMSO (100 μL/well) was added to allow formosan solubilization. The optical density (OD) of each well was detected using a Microplate reader at 550 nm and for correction at 595 nm. Each determination
2.3.1. Schomburgdepsidone A (1) Brownish gum; UV (MeOH) λmax: 320, 272 and 217 nm; IR νmax (KBr): 3415, 2974, 1656, 1613, 1465 and 1177 cm−1; for 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data, see Table 1; and HR-ESI-MS m/z 503.2051 [M + Na]+ (calcd. for C28H32O7Na, 503.2046). 2.3.2. Schomburgdepsidone B (2) Brownish gum; UV (MeOH) λmax: 316, 279 and 212 nm; IR νmax (KBr): 3408, 2970, 1660, 1621, 1467 and 1201 cm−1; for 1H (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) spectroscopic data,
Table 1 1 H (400 MHz) and 13C (100 MHz) NMR spectroscopic data of compounds 1–3 in CDCl3. Position
1 δH (J in Hz)
1 2 3 4 4a 5a 6 7 8 9 9a 11 11a 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 1-OH 3-OH 6-OH 7-OH 3-OMe
6.28 (1H, s)
3.64 (2H, d, 6.0) 5.24 (1H, t, 6.0) 1.79 (3H, s) 1.86 (3H, s) 3.35 (2H, d, 6.4) 5.08 (1H, t, 6.4) 1.71 (3H, s) 1.79 (3H, s) 3.44 (2H, d, 6.4) 5.01 (1H, t, 6.4) 1.67 (3H, s) 1.79 (3H, s) 10.99 (1H, s) 6.28 (1H, brs) 5.83 (1H, brs) 5.42 (1H, brs)
2 δC 163.9 100.8 162.1 111.2 158.9 136.5 133.6 141.0 123.7 123.6 136.8 168.6 99.5 22.5 122.2 137.1 25.9 18.2 25.8 121.9 134.3 25.9 18.1 25.6 122.4 132.4 25.9 18.2
HMBC
δH (J in Hz)
1, 3, 4, 11a
6.32 (1H, s)
3, 4, 4a, 13, 14 15, 16
3.49 (2H, d, 6.0) 5.14 (1H, t, 6.0)
14, 16 13, 14, 15 7, 8, 18, 19 20, 21
1.67 (3H, s) 1.73 (3H, s) 3.57 (2H, d, 6.4) 5.20 (1H, t, 6.4)
18, 19, 21 19, 20 8, 9, 9a, 24 25, 26
1.74 (3H, s) 1.81 (3H, s) 3.55 (2H, d, 7.2) 5.27 (1H, t, 7.2)
23, 24, 26 24, 25 1, 2
1.73 (3H, s) 1.84 (3H, s) 11.30 (1H, s)
Position δC 164.9 96.2 165.0 113.7 158.6 141.9 117.4 140.2 140.1 118.1 136.5 169.4 98.7 22.6 122.8 132.3 25.8 18.0 23.9 121.6 135.5 25.9 18.1 23.8 121.2 135.0 25.9 18.1
1, 4, 11a
3, 4, 4a, 13, 14 15, 16 13, 14, 16 13, 14, 15 5a, 6, 18, 19 20, 21 19 18, 19, 20 8, 9, 9a, 23, 24 25, 26 26 24 1, 2, 11a
5a, 6, 7 5.54 (1H, s) 3.85 (3H, s)
56.2
6, 7 3
3 δH (J in Hz)
HMBC 1 2 3 4 4a 5 6 7 8 8a 9 9a 10a 11 12 13 14 15 16 17 18 19 20 1-OH 5-OH 6-OH 3-OMe
6.35 (1H, s)
6.78 (1H, s)
3.48 (2H, d, 6.8) 5.22 (1H, t, 6.8) 1.73 1.85 3.97 (2H, d, 7.2) 5.35 (1H, t, 7.2) 1.75 1.73 13.44 (1H, s) 5.47 (1H, brs) 5.90 (1H, brs) 3.90 (3H, s)
δC 162.6 94.6 163.4 106.6 153.2 128.9 148.2 113.4 137.8 111.9 182.8 103.7 146.4 21.9 123.5 131.7 25.7 18.0 33.1 122.7 133.3 26.0 18.1
HMBC 1, 4, 9a
5, 6, 8a, 16
3, 4, 4a, 12, 13
12, 13, 15 12, 13, 14 7, 8, 17, 18
20 17, 18, 19 1, 2, 9a
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represented the average mean of six replicates. The 50% inhibition concentration (IC50 value) was determined by curve fitting.
3. Results and discussion Phytochemical investigation of the CH2Cl2 extract from the roots of G. schomburgkiana led to the isolation of two new depsidones, schomburgdepsidones A and B (1 and 2), and one new xanthone, schomburgxanthone A (3), along with eight known compounds; oliveridepsidone A (4) [13], oliveridepsidone D (5) [13], 1,5dihydroxyxanthone (6) [14], nigrolineaxanthone E (7) [15], 6desoxyjacareubin (8) [16], aucuparin (9) [17], 3-hydroxy-5methoxybiphenyl (10) [18] and methyl-2,6-dihydroxy-4-methoxy3(3′-methyl-2′-butenyl)-benzoate (11) [19]. The structures of the three new isolated compounds were elucidated by using 1D- and 2DNMR spectroscopy. The structures of the known compounds (4–11) were determined and confirmed by comparison of their 1H and 13C NMR spectroscopic data with previously published data. Schomburgdepsidone A (1) was obtained as a brownish gum. Its molecular formula was determined as C28H32O7 by HR-ESI-MS measurement through the sodium adduct ion at m/z 503.2051 [M + Na]+ (calcd. for C28H32O7Na, 503.2046). The UV spectrum displayed absorption bands at λmax 320, 272 and 217 nm. The IR spectrum showed characteristic absorption bands for hydroxy and lactone carbonyl stretching bonds at 3415 and 1656 cm−1, indicating the presence of a depsidone chromophore [13]. The 1H NMR spectrum (Table 1) consisted of signals for a hydrogen-bonded hydroxy proton at δH 10.99 (1H, s, 1-OH); an aromatic proton at δH 6.28 (1H, s, H-2); three hydroxy protons at δH 6.28 (1H, brs, 3-OH), 5.83 (1H, brs, 6-OH) and 5.42 (1H, brs, 7-OH); and three 3-methylbut-2-enyl units, as deduced from the resonances of the three olefinic protons at δH 5.24 (1H, t, J = 6.0 Hz, H-13), 5.08 (1H, t, J = 6.4 Hz, H-18) and 5.01 (1H, t, J = 6.4 Hz, H-23); three sets of methylene protons at δH 3.64 (2H, d, J = 6.0 Hz, H-12), 3.35 (2H, d, J = 6.4 Hz, H17) and 3.44 (2H, d, J = 6.4 Hz, H-22); and six methyl protons at δH
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1.79 (3H, s, H-15), 1.86 (3H, s, H-16), 1.71 (3H, s, H-20), 1.79 (3H, s, H-21), 1.67 (3H, s, H-25) and 1.79 (3H, s, H-26). Based on the HMBC spectrum (Table 1), a hydrogen-bonded hydroxy proton at δH 10.99 (1-OH) correlated to C-1 (δC 163.9) and C-2 (δC 100.8), showing that the hydroxy group was attached to C-1. An aromatic proton was placed at C-2 by the observed crosspeaks of H-2 to the oxygenated quaternary aromatic carbons C-1 and C-3 (δC 162.1), and substituted aromatic carbons C-4 (δC 111.2) and C-11a (δC 99.5). Furthermore, one isoprenyl moiety was attached to C-4 from the correlations of its methylene protons at H-12 to C-3, C-4 and C-4a (δC 158.9), supporting the assignment of the A-ring. The 1H and 13 C NMR spectroscopic data of 1 (Table 1) were similar to those of oliveridepsidone A (4) [13]. The major difference was the position of the remaining two prenyl units and two hydroxy groups in the B-ring. The HMBC spectrum of 1 (Fig. 2) assigned the correlations of methylene protons at H-17 to C-7 (δC 141.0) and C-8 (δC 123.7), and H-22 to C-8, C9 (δC 123.6) and C-9a (δC 136.8), suggesting that the two isoprenyl units were connected to C-8 and C-9 in the B-ring. Consequently, the two remaining hydroxy groups were placed at C-6 (δC 133.6) and C-7 (δC 141.0). (See Fig. 1.) The linkage between two phenyl moieties (the A- and B-ring) was determined by the following spectroscopic data. The carbon signal at δC 168.6 (C-11) in the 13C NMR spectrum and the observed strong absorption at νmax 1656 cm−1 in the IR spectrum indicated a characteristic of lactone carbonyl from a depsidone [13,20]. The 13C NMR spectrum also assigned the signals of oxygenated quaternary aromatic carbons C-4a, C-5a (δC 136.5) and C-9a, and substituted aromatic carbon C-11a bonded to the carbonyl carbon of depsidone. Based on the HMBC experiment (Fig. 2), the correlations of H-2 to C-11a and H-12 to C-4a suggested that the A-ring was connected to the C-ring. Furthermore, the correlations of 6-OH (δH 5.83) to C-5a and H-22 to C-9a suggested that the B-ring was also connected to the C-ring. Therefore, both phenyl moieties were linked by a seven-membered ring (the C-ring) containing an ester linkage and an ether bridge. From the above evidences and by comparison with the literature [13], compound 1 was determined as
Fig. 1. The structures of compounds 1–3 isolated from the roots of G. schomburgkiana.
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Fig. 2. The key HMBC (arrow curves) and COSY (bold lines) correlations of compounds 1–3.
1,3,6,7-tetrahydroxy-4,8,9-tris(3-methylbut-2-enyl)-11H-dibenzo[b,e] [1,4]-dioxepin-11-one. Schomburgdepsidone B (2) was isolated as a brownish gum. Its HRESIMS exhibited a molecular ion peak at m/z 495.2390 [M + H]+ (calcd. for C29H35O7, 495.2383), which corresponded to the molecular formula of C29H34O7. The UV spectrum revealed maximum absorption bands at λmax 316, 279 and 212 nm. The IR spectrum showed hydroxy and lactone carbonyl stretching bands at 3408 and 1660 cm−1, suggesting the presence of a depsidone derivative [13]. The 1H and 13C NMR spectroscopic data of 2 (Table 1) were comparable to an analogue, oliveridepsidone A (4) [13]. The difference was the replacement of the 1 H NMR signal for the hydroxy group at C-3 of 4 with a methoxy group at δH 3.85 (3H, s, 3-OCH3) in 2. This structural assignment was further confirmed by the HMBC correlation of the methoxy signal (δH 3.85) to C-3 (δC 165.0) (Fig. 2). From these data, the structure of 2 was established as 1,7,8-trihydroxy-3-methoxy-4,6,9-tris(3-methylbut-2enyl)-11H-dibenzo[b,e] [1,4]dioxepin −11-one. Schomburgxanthone A (3) was obtained as a yellow powder. The molecular formula was measured by HRESIMS as C24H26O6 (m/z 411.1818 [M + H]+, calcd. for C24H27O6 411.1808). The UV spectrum displayed absorption bands at λmax 325, 281 and 224 nm, which were typical of a xanthone chromophore [21]. The IR spectrum showed absorption bands at 3337 and 1636 cm−1 due to hydroxy and carbonyl stretching bonds, respectively. The 1H NMR spectrum (Table 1) displayed a hydrogen-bonded hydroxy proton at δH 13.44 (1H, s, 1OH); two hydroxy protons at δH 5.47 (1H, brs, 5-OH) and 5.90 (1H, brs, 6-OH); two singlet aromatic protons at δH 6.35 (1H, s, H-2) and δH 6.78 (1H, s, H-7); a methoxy signal at δH 3.90 (3H, s, 3-OCH3); and two 3-methylbut-2-enyl units, as determined by the resonances of the two olefinic protons at δH 5.22 (1H, t, J = 6.8 Hz, H-12) and 5.35 (1H, t, J = 7.2 Hz, H-17); two sets of methylene protons at δH 3.48 (2H, d, J = 6.8 Hz, H-11) and 3.97 (2H, d, J = 7.2 Hz, H-16); and four methyl protons at δH 1.73 (3H, s, H-14), 1.85 (3H, s, H-15), 1.75 (3H, s, H-19), and 1.73 (3H, s, H-20). In the HMBC spectrum of 3 (Fig. 2), the two isoprenyl groups were located at C-4 and C-8 by the crosspeaks of H-11 (δH 3.48) to C-3 (δC
163.4), C-4 (δC 106.6) and C-4a (δC 153.2), and H-16 (δH 3.97) to C-7 (δC 113.4) and C-8 (δC 137.8). In addition, the crosspeak of the methoxy signal (δH 3.90) to C-3 indicated that the methoxy group was attributed at C-3 in the A-ring. The 1H and 13C NMR data of 3 (Table 1) were similar to those of dulxanthone C [20], except for the absence of the 1H NMR signal of the methoxy group at C-6 in 3. Furthermore, the substituent at C-6 was assigned as a hydroxy group according to its 13C NMR chemical shift (δC 148.2). Thus, the structure of 3 was identified as 1,5,6trihydroxy-3-methoxy-4.8-bis(3-methylbut-2-enyl)-9H-xanthen-9one. Depsidones are mainly formed in lichens through polyketide pathway which usually contain 3.8-dioxygenation and methyl groups attached to C-1, C-9 and/or C-6 [13,22]. However, a number of prenylated depsidones were reported from the genus Garcinia [13, 22–25], which well known to have a rich source of shikimate and mevalonate-derived aromatic compounds [22], including xanthones as chemotaxonomic marker of this genus [1]. It is possible that the biosynthesis of the prenylated depsidones lies in Baeyer-Villiger rearrangements from hydroperoxylation reactions of the precursor xanthones, with incorporation of the mevalonate-derived prenyl units [22,26]. The in vitro cytotoxicity assay of all isolated compounds (1–11) against five human cancer cell lines (KB, HeLa S-3, HT-29, MCF-7 and Hep G2) was evaluated using a modified MTT method with doxorubicin as the positive control [12]. The results are summarized in Table 2. The tested compounds mostly showed moderate to inactive against the five human cancer cell lines, except for compound 7 showed a good cytotoxicity (IC50 ≤ 10 μM) against the KB, Hela S-3 and MCF-7 cell lines with IC50 values of 3.17, 5.46, and 6.07 μM, respectively. Compound 3 showed a good cytotoxic activity against the KB cell line only, with an IC50 value of 8.14 μM. Among the depsidones (1, 2, 4 and 5), only compound 5 had a slightly higher cytotoxicity with IC50 values in the range of 11.14–30.40 μM. Conflict of interest The authors have declared that there is no conflict of interest.
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Table 2 In vitro cytotoxicity of compounds 1–11 against five human cancer cell lines. Compound
IC50 (μM) ± SD KB
Hela S-3
HT-29
Hep G2
MCF-7
Schomburgdepsidone A (1) Schomburgdepsidone B (2) Schomburgxanthone A (3) Oliveridepsidone A (4) Oliveridepsidone D (5) 1,5-dihydroxyxanthone (6) Nigrolineaxanthone E (7) 6-desoxyjacareubin (8) Aucuparin (9) 3-hydroxy-5-methoxybiphenyl (10) Methyl-2,6-dihydroxy-4-methoxy-3(3′-methyl-2′-butenyl)-benzoate (11) Doxorubicin
43.39 ± 1.95 52.33 ± 1.99 8.14 ± 0.61 55.94 ± 5.63 11.14 ± 0.86 25.50 ± 0.96 3.17 ± 0.08 18.56 ± 0.15 N100 59.78 ± 1.01 N100 0.22 ± 0.05
N100 95.96 ± 6.06 14.03 ± 0.26 N100 19.27 ± 0.27 N100 5.46 ± 0.08 19.69 ± 0.46 N100 N100 N100 0.11 ± 0.03
N100 N100 N100 58.74 ± 0.11 30.40 ± 1.78 N100 34.08 ± 0.73 69.29 ± 0.79 N100 N100 N100 0.44 ± 0.08
47.86 ± 0.58 N100 48.90 ± 0.88 39.27 ± 0.54 27.83 ± 0.92 N100 16.08 ± 0.22 39.35 ± 1.05 N100 N100 N100 2.80 ± 0.09
43.87 ± 6.68 46.97 ± 0.97 21.56 ± 0.19 36.46 ± 0.76 13.42 ± 0.10 N100 6.07 ± 0.11 17.53 ± 0.06 N100 N100 69.45 ± 0.34 0.42 ± 0.03
(IC50 ≤ 10 = good activity, 10 b IC50 ≤ 30 = moderate activity, and IC50 N 100 = inactive).
Acknowledgments The authors are grateful to the 90th Anniversary of Chulalongkorn University Fund (Ratchadaphiseksomphot Endowment Fund) and The Thailand Research Fund via Directed Basic Research Grant (Grant no. DBG 5980001) for partially financial support. We also thank the Graduate School of Chulalongkorn University for the ASEAN Scholarship to E.R.S. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2016.04.012. References [1] T. Ritthiwigrom, S. Laphookhieo, S.G. Pyne, Chemical constituents and biological activities of Garcinia cowa Roxb, Maejo Int. J. Sci. Technol. 7 (2) (2013) 212–231. [2] T.K. Lim, Edible Medicinal and Non-Medicinal Plants: Volume 2, Fruits, Springer Science+Business Media, Dordrecht, 2012 (1100 pp.). [3] C. Mungmee, S. Sitthigool, A. Buakeaw, R. Suttisri, A new biphenyl and other constituents from the wood of Garcinia schomburgkiana, Nat. Prod. Res. 27 (2013) 1949–1955. [4] C. Mungmee, S. Sitthigool, A. Buakeaw, R. Suttisri, Xanthones and biphenyls from Garcinia schomburgkiana wood and their cytotoxicity, Thai. J. Pharm. Sci. 36 (2012) 6–9. [5] C. Ito, T. Matsui, E. Noda, N. Ruangrungsi, M. Itoigawa, Biphenyl derivatives from Garcinia schomburgkiana and the cytotoxicity of the isolated compounds, Nat. Prod. Commun. 8 (9) (2013) 1265–1267. [6] H.T. Vo, N.T.T. Nguyen, H.T. Nguyen, K.Q. Do, J.D. Connolly, G. Maas, J. Heilmann, U.R. Werz, H.D. Pham, L.-H.D. Nguyen, Cytotoxic tetraoxygenated xanthones from the bark of Garcinia schomburgkiana, Phytochem. Lett. 5 (3) (2012) 553–557. [7] A. Häfner, A.W. Frahm, Biflavonoids from the heartwood of Garcinia schomburgkiana and their structural elucidation as atropisomers, Planta Med. 59 (1993) A604. [8] H.K. Fun, S. Koysomboon, K. Chantrapromma, S. Chantrapromma, A cocrystal of clusiacitran A, clusiacitran B, fluorinated clusiacitran A and fluorinated clusiacitran B (0.45:0.45:0.05:0.05), Acta Cryst E62 (2006) o3228–o3230. [9] S. Kaennakam, P. Siripong, S. Tip-pyang, Kaennacowanols A–C, three new xanthones and their cytotoxicity from the roots of Garcinia cowa, Fitoterapia 102 (2015) 171–176. [10] S. Kaennakam, P. Siripong, S. Tip-pyang, Velucarpins A–C, three new pterocarpans and their cytotoxicity from the roots of Dalbergia velutina, Fitoterapia 105 (2015) 165–168.
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