HETEROCYCLES

Short Paper | Regular issue | Vol. 83, No. 5, 2011, pp. 1139-1144
Received, 2nd February, 2011, Accepted, 3rd March, 2011, Published online, 8th March, 2011.
DOI: 10.3987/COM-11-12163

■ A New Depsidone from the Twigs of Garcinia cowa

Sarot Cheenpracha, Wong Phakhodee, Thunwadee Ritthiwigrom, Uma Prawat, and Surat Laphookhieo*

Natural Products Research Laboratory, School of Science, Mae Fah Luang University, Tasud, Muang, Chiang Rai 57100, Thailand

Abstract

A new depsidone, cowadepsidone (7), along with six known xanthones (1-6) were isolated from the twigs of Garcinia cowa. Their structures were determined on the basis of spectroscopic methods. The cytotoxicity against KB, MCF-7 and NCI-H187 cancer cell lines of compounds 2-7 were also reported.

The tropical family Clusiaceae is well known to be a rich source of isoprenylated xanthones, depsidones and biflavonoids.1-4 In particular, the genus Garcinia has also provided many bioactive isoprenylated and rearranged xanthonoids and biflavonoids.3-7 In our continuing phytochemical study of Thai medicinal plants, we have found many polyoxygenated xanthones from the barks and dried fruits of Cratoxylum cochinchinense.8,9 In this paper, we describe the isolation and structural elucidation of a new depsidone together with six known xanthones (Figure 1) as well as their cytotoxic activity.

Compound 7 was isolated as a red gum. Its molecular formula was established as C19H18O7 by ESITOFMS at m/z 359.1133 [M+H]+, suggesting the presence of 11 degrees of unsaturations and supported by NMR data (Table 1). The IR spectrum showed absorption bands for hydroxyl group (3363 cm-1) and a lactone carbonyl group chelated to an ortho-hydroxyl group (1658 cm-1). The presence of the latter functionality was confirmed by resonances at δ 168.0 (C-11) and δ 10.98 (OH-1). The 1H NMR data of 7 displayed signals of two aromatic protons at δ 6.70 (1H, s, H-6) and 6.27 (2H, br s, H-2, H-4). Furthermore, the proton signals at δ 5.17 (1H, br s, H-13), 3.47 (2H, d, J = 6.8 Hz, H2-12), 1.80 (3H, s, H3-15), and 1.67 (3H, s, H3-16) suggested the presence of a prenyl moiety in the structure.9 In addition, a methoxyl signal at δ 3.77 (3H, s) was also observed in the 1H NMR spectrum. Analyzing the 2D NMR spectra using HMQC and HMBC techniques enabled the assignment of 1H and 13C NMR signals. By comparing the NMR data of 7 with those of the known compound, garcinisidone-A,4 the possible structure of 7 was established suggesting the same core structure for both compounds.

The HMBC correlations of the methoxyl protons at δ 3.77 with the oxygenated carbon at δ 142.7 (C-8) and those of the methylene protons of a prenyl unit at δ 3.47 (H2-12) with the carbons at δ 142.7 (C-8), 136.0 (C-9a), and 128.3 (C-9) established the attachment of the methoxyl group and the prenyl side chain at C-8 and C-9, respectively. The aromatic proton at δ 6.70 (H-6) showed HMBC connectivity to three aromatic carbons at δ 146.5 (C-5a), 142.7 (C-8) and 136.0 (C-9a), confirming the location of substituents on the B ring. Furthermore, the correlation of aromatic protons at δ 6.27 (H-2, H-4) with the aromatic carbons at δ 165.5 (C-1), 163.7 (C-3), 161.8 (C-4a), 100.6 (C-2), 100.5 (C-4) and 98.8 (C-11a) indicated the orientation of substituents on the A ring. The quaternary carbon signals of δ 165.5 (C-1), 163.7 (C-3), 147.0 (C-7) and its molecular formula C19H18O7 indicated the presence of three hydroxy groups at C-1, C-3 and C-7, respectively. Thus, compound 7 was determined as cowadepsidone which reported for the first time as a metabolite of G. cowa. The remaining compounds were identified as β-mangostin (1),9 cowanin (2),10 3,6-di-O-methyl-γ-mangostin (3),11 cowanol (4),10 norcowanin (5),12 and cowaxanthone (6)10 by the analysis of 1D and 2D NMR spectra and by comparison with their reported physical and spectroscopic data.

As summarized in Table 2, compounds 2-7 were evaluated for their cytotoxicity against KB (oral human epidermal carcinoma), MCF-7 (breast cancer) and NCI-H187 (human, small cell lung cancer) cancer cell lines and all of them exhibited cytotoxic effect against all three cancer cell lines, except a new depsidone 7 was found to be inactive with KB cancer cell line (Table 2). Xanthones 2, 3 and 5 exhibited strong cytotoxicity against KB cancer cell line with the IC50 value ranging from 6.43-7.36 µg/mL, which were stronger than doxorubicin, a standard drug, (IC50 9.61 µg/mL) while 4 and 6 were found to be weakly activity. Xanthones 2, 3, 5 and 6 also showed strong inhibitory effect against NCI-H187 cancer cell line with the range of IC50 3.87-8.58 µg/mL and xanthone 6 had the highest cytotoxicity (IC50 3.87 µg/mL) whereas 4 and 7 exhibited weak activity. In case of cytotoxicity against MCF-7 cancer cell line, xanthone 3 was the best cytotoxicity with the IC50 value of 10.59 µg/mL. All the rest of compounds were found to be moderately to weakly active with the IC50 values ranging from 15.45-36.03 µg/mL.

It is interesting to note that the structural difference between xanthones 2 and 4 is only at C-4′. Xanthone 4 possesses a methylenehydroxyl moiety while 2 has a methyl group which plays an important role in the cytotoxicity against all three human cancer cell lines. In case of xanthones 2 and 5, (2 possesses a methoxyl group at C-7, while 5 contains a hydroxyl group), both of methoxyl and hydroxyl groups seemed to be no effective in the cytotoxicity.

EXPERIMENTAL
GENERAL
UV spectra were recorded with a Perkin-Elmer UV-Vis spectrophotometer. The IR spectra were recorded with a Perkin-Elmer FTS FT-IR spectrophotometer. The NMR spectra were recorded using 400 MHz Bruker spectrometer. Chemical shifts were recorded in parts per million (δ) in CDCl3 with tetramethylsilane (TMS) as an internal reference. The ESITOFMS was obtained from a Micromass LTC mass spectrometer. Quick column chromatography (QCC) and column chromatography (CC) were carried out on silica gel 60 H (Merck, 5-40 μm) and silica gel 100 (Merck, 63-200 μm), respectively. Precoated plates of silica gel 60 F254 were used for analytical purposes.

PLANT MATERIAL
The twigs of G. cowa were collected from Nong Khai Province in March 2010. The plant specimen (MFU-NPR 0014) has been deposited at Natural Products Chemistry Laboratory, Mae Fah Luang University, Chiang Rai, Thailand.

EXTRACTION AND ISOLATION
The air-dried twigs of G. cowa (3.34 kg) were successively extracted with n-hexane and acetone over a period of 3 days each at room temperature. The n-hexane extract (21.36 g) was subjected to QCC over silica gel eluted with a gradient of n-hexane- EtOAc (100% n-hexane to 100% EtOAc) to provide five fractions (A-E). Fraction B (145.9 mg) was further purified by CC with 10% acetone- n-hexane to give compound 3 (6.8 mg) whereas compounds 1 (17.1 mg) and 2 (6.1 mg) were obtained from fraction D (124.2 mg) by repeated CC with 15% acetone-n-hexane. Fraction E (1.63 g) was subjected to repeated CC with 20% acetone- n-hexane to afford compounds 4 (165.2 mg), 5 (40.1 mg), 6 (30.2 mg) and 7 (10.1 mg).
Cowadepsidone (7): Red gum; UV (CHCl3) λmax (log ε): 204 (4.63), 273 (4.10), 313 (3.51), 433 (2.77); IR (neat) νmax cm-1: 3363 (OH), 1658 (C=O); 1H NMR (400 MHz) and 13C NMR (100 MHz) see Table 1; ESITOFMS (m/z): [M+H]+ m/z 359.1133 (calcd for C19H19O7, 359.1131).

CYTOTOXIC ASSAY
The procedures for cytotoxic assay were performed by resazurin microplate assay (REMA) which was a modified method of fluorescent dye for the mammalian cell cytotoxicity according to Brien et al.13 In this study, three cancer cell lines, KB (oral cavity cancer), MCF7 (breast cancer) and NCI-H187 (small cell lung cancer) were used. Ellipticin and doxorubicin were the reference substances in this study and the IC50 values are summarized in Table 2.

ACKNOWLEDGEMENTS
We are grateful to Mae Fah Luang University and University of Phayao for partial financial support and laboratory facilities. We appreciate to Ms. Nareerat Thongtip, Phuket Rajabhat University for recording some NMR spectra and to Mr. Nitirat Chimnoi, Chulabhorn Research Institute, Bangkok for recording ESITOFMS spectra. The Bioassay Research Facility of BIOTEC (Thailand) is also gratefully acknowledged for cytotoxic activity tests.

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