Fitoterapia 108 (2016) 62–65

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Cylindroxanthones A–C, three new xanthones and their cytotoxicity from the stem bark of Garcinia cylindrocarpa Edwin Risky Sukandar a,b, Taslim Ersam b,⁎, Sri Fatmawati b, Pongpun Siripong c, Thammarat Aree a, Santi Tip-pyang a,⁎ a

Natural Products Research Unit, Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand Natural Products and Synthesis Chemistry Research Laboratory, Department of Chemistry, Faculty of Mathematics and Natural Sciences, Institut Teknologi Sepuluh Nopember, Kampus ITS-Sukolilo, Surabaya 60111, Indonesia c Natural Products Research Section, Research Division, National Cancer Institute, Bangkok 10400, Thailand b

a r t i c l e

i n f o

Article history: Received 7 October 2015 Received in revised form 17 November 2015 Accepted 19 November 2015 Available online 22 November 2015 Keywords: Cylindroxanthones A–C Garcinia cylindrocarpa Xanthone Cytotoxicity

a b s t r a c t Three new xanthones, cylindroxanthones A–C (1–3), were isolated from the stem bark of Garcinia cylindrocarpa. The structures were established on the basis of spectroscopic analysis. The molecular structure of 1 was unequivocally confirmed by single-crystal X-ray diffraction analysis. These three xanthones were evaluated regarding their cytotoxicity against KB, HeLa S-3, HT-29, MCF-7, and Hep G2 cancer cell lines. Compound 1 exhibited good cytotoxicity against KB cell with IC50 value of 2.36 μM. © 2015 Elsevier B.V. All rights reserved.

1. Introduction

2. Experimental

The genus Garcinia belongs to the Clusiaceae family and comprises 77 species in Indonesia [1,2]. In the previous phytochemical investigation, this genus was well known to contain xanthones as chemotaxonomic marker [3–7], flavonoids [8], phloroglucinols [9,10], and triterpenoids [11,12]. Many of these compounds have demonstrated a number of interesting biological activities such as anticancer [4,7,12], antioxidant [3], antidiabetes [6,13], and anti-inflammatory [10]. Garcinia cylindrocarpa Kosterm locally known as “Kogbirat” is the woody plant distributed mainly in Maluku Island, Indonesia [14]. This plant has been used in Indonesian traditional medicine as fever remedy. In continuation on phytochemical investigation for bioactive compounds [4,15–18], we report the isolation and structure elucidation of three new xanthones, cylindroxanthones A–C (1–3), as well as cytotoxicity of these compounds against five cancer cell lines (KB, HeLa S-3, HT-29, MCF-7, and Hep G2).

2.1. General experiment procedures The melting points were determined by micro-melting point apparatus Fischer John. UV spectra were analyzed by UV–vis 1700 Shimadzu spectrophotometer. IR data were obtained on FTIR 8400 Shimadzu spectrophotometer using KBr disk methods. 1D and 2D NMR spectra were measured respectively at 600 MHz for 1H NMR and 150 MHz for 13C NMR on JNM ECA-600 spectrometer in CDCl3, with TMS as internal standard. HR-ESI-MS were analyzed using Bruker MICROTOF model mass spectrometer. Vacuum liquid (VLC) and column chromatography were carried out using Merck silica gel 60 GF254 and silica gel 60 (70–230 mesh ASTM). 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 cerium sulfate (Ce(SO4)2) followed by heating. 2.2. Plant material

⁎ Corresponding authors. E-mail addresses: [email protected] (T. Ersam), [email protected] (S. Tip-pyang).

http://dx.doi.org/10.1016/j.fitote.2015.11.017 0367-326X/© 2015 Elsevier B.V. All rights reserved.

The stem bark of G. cylindrocarpa was collected from Saumlaki Forest, Southeast West Maluku Islands, Indonesia. The plant was identified by Mrs. Ritmita Sari (a botanist at Bogor Botanical Garden, Indonesia). A voucher specimen (No. 630) was deposited at the Herbarium Bogoriense, Bogor Botanical Garden, Indonesia.

E.R. Sukandar et al. / Fitoterapia 108 (2016) 62–65

63

Table 1 NMR spectroscopic data (in CDCl3) for 1, 2, and 3. Position

1 δH (J in Hz)

1 2 3 4 4a 4b 5 6 7 8 8a 8b 9 1′ 2′ 3′ 4′ 5′ 1-OH 6-OH 5-OMe 6-OMe 7-OMe 8-OMe

6.36 (1H, s)

6.73 (1H, d, 9.9) 5.59 (1H, d, 9.9) 1.47 (3H, s) 1.47 (3H, s) 13.56 (1H, s) 3.93 (3H, s) 3.97 (3H, s) 3.97 (3H, s) 4.12 (3H, s)

2 δC 158.0 104.8 160.3 94.6 156.1 152.7 137.3 147.4 143.2 149.4 111.1 103.6 180.4 115.6 127.4 78.2 28.4 28.4

61.7 61.8 62.0 62.2

HMBC

9, 4a, 3, 2

δH (J in Hz)

6.35 (1H, s)

1, 2, 3, 4, 3′, 4′, 5′ 2, 3, 3′, 4′, 5′

6.73 (1H, d, 10.2) 5.58 (1H, d, 10.2)

2′, 3′, 5′ 2′, 3′, 4′ 9, 8b, 1, 2, 1′

1.47 (3H, s) 1.47 (3H, s) 13.63 (1H, s) 6.39 (1H, s) 3.90 (3H, s) 4.08 (3H, s) 4.09 (3H, s)

5 6 7 8

2.3. Extraction and isolation The air-dried stem bark of G. cylindrocarpa (3.0 kg) was extracted with methanol (3 × 15 L) by maceration at room temperature for three days. The extract was concentrated in vacuo to yield 138.0 g of brown crude extract and separated then into vacuum liquid chromatography (VLC) on silica gel (1.5 kg) using hexane, CH2Cl2, EtOAc, and MeOH, respectively, for each 3 × 2 L to get hexane, CH2Cl2, EtOAc, and MeOH extracts. CH2Cl2 extract (48.0 g) was further subjected to VLC on silica gel (500.0 g) with gradient of EtOAc–CH2Cl2 (20:80, 40:60, 60:40, 80:20, 100:0, v/v, each 500 mL) to get three fractions (A–C). Fraction A (8.9 g) was separated by VLC on silica gel (300.0 g) with gradient of CH2Cl2–hexane (5:95, 10:90, 15:85, 20:80, 30:70, 40:60, 50:50, v/v, each 300 mL) to yield compound 1 (50.4 mg). Fraction B (10.3 g) was chromatographed into VLC on silica gel (300.0 g) with EtOAc–hexane (5:95, 10:90, 20:80, 30:70, 40:60, 50:50, v/v, each 300 mL) to give six subfractions (B1–B6). Subfraction B5 (1.5 g) was then applied to the same chromatography technique on silica gel (70.0 g) by eluent CH2Cl2–hexane (10:90, 25:85, 40:60, 55:45, 70:30, 85:15, 100:0, v/v,

3 δC 158.0 104.9 160.2 94.6 156.0 147.1 131.3 148.5 137.7 148.5 108.5 103.5 180.3 115.6 127.4 78.1 28.4 28.4

HMBC

9, 8b, 4a, 3, 2

δH (J in Hz)

6.42 (1H, s)

7.40 (1H, s)

δC

HMBC

157.5 104.7 160.3 95.2 156.8 145.9 134.6 145.1 144.8 99.8 113.0 103.2 179.9 115.5 127.6 78.2 28.4 28.4

3, 2′, 3′, 5′ 3, 2′, 3′, 4′

9, 8b, 4a, 3, 2

9, 8a, 7, 6, 5, 4b

2, 3, 2′, 3′, 4′, 5′ 2, 3, 4, 3′, 4′, 5′

6.72 (1H, d, 10.2) 5.60 (1H, d, 10.2)

1, 2, 3, 4, 2′, 3′, 4′, 5′ 2, 3, 3′, 4′, 5′

62.1

1′, 2′, 3′, 5’ 1′, 2′, 3′, 4′ 9, 8b, 1, 2 5, 6, 7 5

1.46 (3H, s) 1.46 (3H, s) 13.24 (1H, s) 6.29 (1H, s) 4.01 (3H, s)

61.7

5, 6 5

61.8 61.8

7 8

4.08 (3H, s)

56.6

7

each 150 mL) to yield four subfractions (B5.1–B5.4). Subfraction B5.1 (220.0 mg) was subjected to column chromatography over silica gel (15.0 g) using EtOAc–CH2Cl2 (5:95) to afford compound 2 (107.2 mg). Compound 3 (89.0 mg) was obtained by separation of subfraction B5.2 (350.0 mg) using column chromatography over silica gel (10.0 g) eluted with a gradient of EtOAc–CH2Cl2 (40:60, 45:55, 50:50, 55:45, v/v, each 100 mL). 2.3.1. Cylindroxanthone A (1) Yellow crystal; mp: 103–105 °C; UV (MeOH) λmax: 330, 283, and 241 nm; IR νmax (KBr): 3310, 2970, 2939, 1649, 1609, 1448, and 1200 cm−1; for 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) spectroscopic data, see Table 1; and HRESIMS m/z 437.1210 [M + Na]+ (calcd. For C22H22O8Na, 437.1212). 2.3.2. Cylindroxanthone B (2) Yellow powder; mp: 144–145 °C; UV (MeOH) λmax: 332, 280, and 243 nm; IR νmax (KBr): 3163, 2974, 2926, 1656, 1605, 1466, and 1180 cm−1; for 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3)

Fig. 1. The structures of 1–3 isolated from the stem bark of G. cylindrocarpa.

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Fig. 2. The key HMBC correlations of 1–3.

spectroscopic data, see Table 1; and HRESIMS m/z 423.1062 [M + Na]+ (calcd. For C21H20O8Na, 423.1056). 2.3.3. Cylindroxanthone C (3) Yellow needle; mp: 187–188 °C; UV (MeOH) λmax: 335, 289, 248 nm; IR νmax (KBr): 3336, 2985, 2945, 1657, 1603, 1433, and 1240 cm−1; for 1H (600 MHz, CDCl3) and 13C NMR (150 MHz, CDCl3) spectroscopic data, see Table 1; and HRESIMS m/z 371.1131 [M + H]+ (calcd. For C20H19O7, 371.1131). 2.4. Single-crystal X-ray crystallography of cylindroxanthone A (1) Crystallographic data of 1 (CCDC 1437057): X-ray diffractometer: Bruker X8 APEX II KAPPA CCD detector; MoKα radiation (λ = 0.71073 Å); T = 296(2) K; formula C22H22O8, MW = 414.39, light yellow, rod-like crystal: 0.10 × 0.22 × 0.32 mm, monoclinic space group P21/c (no. 14), a = 13.3893(11) Å, b = 10.7394(8) Å, c = 13.7177(12) Å, β = 91.339(2)°, V = 1972.0(3) Å3, Z = 4, Dx = 1.396 g cm− 3, μ = 0.107 mm− 1, F(000) = 872; 1.52° ≤ θ ≤ 30.56°; −19 ≤ h ≤ 19, −15 ≤ k ≤ 15, −19 ≤ l ≤ 19; measured/unique reflections: 29,934/6038 (Rint = 0.0522); S = 1.008. The final R1 = 0.0565, wR2 = 0.1268 for 2955 reflections with F2 N 2σ(F2); R1 = 0.1322, wR2 = 0.1723 for all 6038 reflections. 2.5. Cytotoxicity assay All isolated compounds (1–3) were applied to in vitro cytotoxic evaluation against KB (human epidermoid carcinoma), HeLa S-3 (human servix adenocarcinoma), HT-29 (human colon adenocarcinoma), MCF7 (human breast adenocarcinoma), and Hep G2 (human hepatocellular carcinoma) cancer cell lines by using MTT colorimetric method [19]. Doxorubicin was used as the reference substance. The 3-(4,5dimethylthiazol-2-yl)-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 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 CH2Cl2 extract from the stem bark of G. cylindrocarpa led to the isolation of three new xanthones, cylindroxanthones A–C (1–3). The structure of these isolated xanthones was elucidated using 1D and 2D NMR spectroscopy. Cylindroxanthone A (1) was obtained as yellow crystal. Its molecular formula was determined as C22H22O8 by HRESIMS measurement through the pseudomolecular ion peak at m/z = 437.1210 [M + Na]+ (calcd. For C22H22O8Na, 437.1212). The UV spectrum displayed absorption bands at λmax 330, 283 and 241 nm, which were typical of xanthone chromophore [20]. The IR spectrum showed hydroxyl and carbonyl stretching bands at 3310 and 1649 cm− 1, respectively. The 1H NMR spectrum (Table 1) had signals of chelated hydroxy proton at δH 13.56 (1H, s, 1-OH), a singlet aromatic proton at δH 6.36 (1H, s, H-4), and four methoxy groups at δH 3.93, 3.97, 3.97, and 4.12 (each 3H, s, 5,6,7, and 8-OCH3). Furthermore, the presence of dimethylchromene ring was determined from the resonance of two methyl groups at δH 1.47 (each 3H, s, H-4′ and H-5′) and two cis-olefinic protons at δH 5.59 (1H, d, J = 9.9 Hz, H-2′) and 6.73 (1H, d, J = 9.9 Hz, H-1′). The location of

Fig. 3. ORTEP diagram of cylindroxanthone A (1).

E.R. Sukandar et al. / Fitoterapia 108 (2016) 62–65 Table 2 In vitro cytotoxicity of compound 1–3 against five cancer cell lines. Compound Cylindroxanthone A (1) Cylindroxanthone B (2) Cylindroxanthone C (3) Doxorubicin

IC50 (μM) KB

Hela S-3

HT-29

Hep G2

MCF-7

2.36 57.24 59.05 0.17

75.12 71.38 73.65 0.06

25.51 135.13 71.74 0.47

10.41 59.53 37.90 2.84

98.54 168.53 59.05 0.38

65

School of Chulalongkorn University for ASEAN Scholarship (to E.R.S.). We also thank to Mr. Andarias Batlayar for collection of the plant materials, Mrs. Ritmita Sari for plant identification and deposition, and Ms. Oktavina Widorini and Ms. Prasiska Eviati for kindly sharing information on related experimental details. Single-crystal X-ray diffraction facility at the Faculty of Science, Chulalongkorn University was supported through T.A. by the National Research University Project of Thailand (WCU-58-013-FW).

Appendix A. Supplementary data dimethylchromene ring on C-2 (δC 104.8) and its ether linkage on C-3 (δC 160.3) of ring A was confirmed by HMBC (Fig. 2), in which olefinic proton H-1′ was correlated to C-2 and C-3. In the HMBC experiment of 1, the proton of H-4 showed cross peak with C-9 (δC 180.4), C-4a (δC 156.1), C-2, and C-3. On the other hand, the remaining four methoxy groups were placed into positions C-5, C-6, C-7, and C-8 on ring B and also confirmed by comparison the 1H and 13C chemical shifts with those in ring B of known xanthone, laurentixanthone B [21]. Fortunately, a good quality single crystal of 1 was obtained for X-ray crystallographic analysis, which confirmed the detailed molecular structure of 1 as depicted with ORTEP diagram (Fig. 3). Thus, the NMR spectra of cylindroxanthone A was unambiguously assigned (Fig. 1). Cylindroxanthone B (2) was isolated as yellow powder. Its HRESIMS exhibited a pseudomolecular ion peak at m/z = 423.1062 [M + Na]+ (calcd. For C21H20O8Na 423.1056), which corresponded to a molecular formula of C21H20O8. The UV spectrum revealed maximum absorption bands at λmax 332, 280, and 243 nm suggesting the presence of a xanthone chromophore [20]. The IR spectrum showed characteristic absorption bands for hydroxyl and carbonyl stretching bond at 3163 and 1656 cm− 1, respectively. The 1H and 13C NMR spectral data of 2 (Table 1) were closely related to those of 1. The difference was the replacement of the 1H NMR signal for methoxy group at δH 3.97 (3H, s, 6-OCH3) of 1 with hydroxy group at δH 6.39 (1H, s, 6-OH) in 2. The HMBC correlation of 2 (Fig. 2) assigned the correlation of 6-OH to C-5 (δC 131.3), C-6 (δC 148.5), and C-7 (δC 137.7). From the above evidence, the structure of 2 was established as cylindroxanthone B. Cylindroxanthone C (3) was achieved as yellow needle. The molecular formula was measured by HRESIMS as C20H18O7 (m/z 371.1131 [M + H]+, calcd. For C20H19O7 371.1131). The UV spectrum displayed absorption bands at λmax 335, 289 and 248 nm, which were typical of xanthone chromophore [20]. The IR spectrum showed absorption bands at 3336 and 1657 cm−1 due to a hydroxyl and carbonyl stretching bond, respectively. The 1H and 13C NMR data of 3 (Table 1) were structurally closed to 2. The difference was the replacement of the 1H NMR signal for methoxy group at δH 4.09 (3H, s, 8-OCH3) of 2 with proton at δH 7.40 (1H, s, H-8) in 3. In the HMBC correlation of 3 (Fig. 2), the proton H-8 showed cross peak with C-4b (δC 145.9), C-5 (δC 134.6), C-6 (δC 145.1), and C-7 (δC 144.8). Therefore, this compound was determined to be cylindroxanthone C (3). In the previous reports, xanthones were well known to have potent biological activities, especially for cytotoxicity towards cancer cells [4,7]. Thus, we evaluated the cytotoxicity of compounds 1–3 against five cancer cell lines (KB, HeLa S-3, HT-29, MCF-7, and Hep G2) using modified MTT method [19]. The result of cytotoxicity was recorded on Table 2. All tested compounds mostly showed from moderate to weak activities against cancer cell lines, except compound 1 showed good activity against KB cancer cell line with IC50 value of 2.36 μM and compound 2 showed inactive towards HT-29 and MCF-7 cell lines (IC50 N 100 μM). Moderate activities against HT-29 and Hep G2 cancer cell lines were also performed by 1 with IC50 values of 25.51 and 10.41 μM, respectively, which were better than compounds 2 and 3. Acknowledgments The authors are grateful to University Excellent Research Program-ITS (PUPT-ITS) and BOPTN DIKTI (No. 003246.114), Indonesia for partially supporting this project and the Graduate

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.fitote.2015.11.017.

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