HETEROCYCLES

Note | Regular issue | Vol. 81, No. 10, 2010, pp. 2377-2384
Received, 2nd August, 2010, Accepted, 26th August, 2010, Published online, 26th August, 2010.
DOI: 10.3987/COM-10-12034

■ New Prenylated Xanthone from the Branch of Garcinia costata

Warraphong Nuangnaowarat, Worrapong Phupong,* Kamolphan Intereya, and Masahiko Isaka

School of Science, Walailak University, Thaiburi, Thasala, Nakhon Si Thammarat 80161, Thailand

Abstract

A new triprenylated xanthone, costatin (1), together with five known xanthones (2-6), was isolated from a branch of Garcinia costata (Guttiferae). The structure was elucidated on the basis of spectroscopic data. All xanthone derivatives were evaluated for antimalarial, anti-tuberculosis (TB), cytotoxicity against human breast adenocarcinoma cell line (MCF-7), anti-human KB-cell line and cytotoxicity against African green monkey kidney (Vero) cells. Costatin (1) exhibited significant antimalarial activity (IC50 1.57 μg/mL); however, it also showed comparable cytotoxic activity (IC50 1.12 μg/mL).

Plants in the genus Garcinia are rich sources of bioactive compounds. A number of new compounds with diverse chemical structures have been isolated from several major species such as G. mangostana,1 G. hanburyi,2 G. dulcis,3 G. lancilimba,4 G. subelliptica5 and G. scortechinii. 6,7 One of the major types of compounds present in Garcinia constituents are polyprenylated xanthones, which have been found to possess a broad range of pharmacological properties: antimycobacterial,8 antioxidant,9 HIV-1 protease inhibitory,10 anti-inflammatory,11 antifungal,12 antimalarial,13, 14 anti-cancer and cytotoxic activities.15
Garcinia costata Hemsley ex King, a small tree with orange-brown twigs and large ovate-oblong leaves, is commonly known in Thailand as Mangkhut Paa (wild mangosteens). So far, there has been no report on the phytochemical studies of this species. We found that a crude dichloromethane extract from the branches of G. costata showed significant biological activities against Plasmodium falciparum K1 and Mycobacterium tuberculosis H37Ra. An investigation of a branch of G. costata led to the isolation of 6 xanthones, numbered (1-6), among which compound 1 was a new prenylated xanthone derivative. We report herein the isolation and structural elucidation of the new compound as well as biological activities of these xanthones.

Garcinia costata was collected at Phukradung National Park, Loei Province, Thailand, in July 2006. The crude dichloromethane extract was subjected to isolation by successive column chromatography on Sephadex LH-20 and silica gel to yield compounds 1-6 (Figure 1). The structural elucidation was carried out by means of the spectroscopic analysis and compared with previous reports of the five known compounds.
Compound 1 was obtained as a yellow solid. The molecular formula was determined to be C28H32O6 by HRMS (ESI-TOF), with an observation of an [M + H]+ ion peak at m/z 465.2278. The UV spectrum showed characteristic absorption bands of the xanthone skeleton at λmax 242, 264, 318, and 376 nm. The IR spectrum showed the functionality of a conjugated carbonyl at νmax 1640 cm-1 (C=O stretching) and a broad hydroxyl absorption band at 3401 cm-1. The 1H NMR spectrum (Table 1) showed a sharp signal at δH 13.70 assignable to a chelated hydroxyl group. A phenolic hydroxyl group and a signal of aromatic proton exhibited at δH 6.31 and 6.83, respectively.

The 13C NMR spectrum (Table 1) and DEPT experiments indicated the presence of a carbonyl carbon (δC 182.5), six methyl carbons (δC 25.3 × 3, 17.5, and 17.4 × 2), three methylene carbons (δC 25.5, 21.2 and 21.1), four sp2 methine carbons (δC 121.5, 121.3, 121.0 and 100.7), and fourteen sp2 quaternary carbons (δC 159.3, 158.0, 153.1, 151.6, 150.2, 139.0, 135.1, 134.5, 133.0, 126.9, 110.8, 108.1, 103.8 and 103.2). The chelated OH (δH 13.70) showed HMBC correlations (Figure 2) to C-1 (δC 158.0), C-2 (δC 108.0) and C-9a (δC 103.2), whereas the aromatic proton (δH 6.83) was located on C-5 based on its 2-bond (C-6 and C-10a) and 3-bond (C-7 and C-8a) HMBC correlations.

The remaining signals, olefinic protons at δH 5.28 (3H, overlapped, H-2′, H-2′′and H-2′′′, methyl groups at δH 1.84 (H-4′), 1.86 (H-4′′), 1.88 (H-4′′′), 1.72 (H-5′), 1.76 (H-5′′), 1.78 (H-5′′′) and allylic methylene protons at δH 3.45 (H-1′), 3.49 (H-1′′) and 4.32 (H-1′′′), constitute three prenyl groups which are all attached to quaternary aromatic carbons (C-2, C-4 and C-8) of the xanthone. These were confirmed by the HMBC correlations: from H-1′ to C-2, C-3, C-2′, and C-3′, from H-1″ to C-3, C-4, C-4a, C-2″, and C-3″, and from H-1′″ to C-7, C-8, C-8a, C-2′″, and C-3′″. Consequently, the structure of 1 was determined to be costatin (1,3,6,7-tetrahydroxy-2,4,8-tri-(3-methyl-2-butenyl)xanthone).
Compounds 2–6 were identified respectively as α-mangostin,16-18 brasilixanthone B,19 garcinone E,20 5,9-dihydroxy-8-methoxy-2,2-dimethyl-7-(3-methylbut-2-enyl)-2H,6H-pyrano[3,2-b]xanthen-6-one21 and γ-mangostin10,20 by spectroscopic methods. The 1H and 13C NMR spectroscopic data were identical to those reported in the literature.
Compounds 1-6 were tested for the following biological activities: antimalaria (Plasmodium falciparum K1) with the use of the microculture radioisotope technique, antituberculosis (Mycobacterium tuberculosis H37Ra) and cytotoxicity against Vero cells (African green monkey kidney fibroblasts) with the use of the green fluorescent protein microplate assay (GFPMA), and cytotoxicity against two cancer cell lines (MCF-7 and KB) and nonmalignant Vero cells (Aftrican green monkey kidney fibroblasts) with the use of the resazurin microplate assay. The biological activity test results are summarized in Table 2.

The phytochemical study of Garcinia costata (Guttiferae) is reported here for the first time. Costatin (1) exhibited a significant antimalarial activity (IC50 1.57 µg/mL); however, it also showed comparable cytotoxicity (IC50 1.12 µg/mL). These data were similar to those of other analogues except for the lack of biological activity of 3. Antimalarial activities of these xanthones were well correlated to their cytotoxic activities. In contrast, the structural relationship of the antituberculosis activities among the 6 compounds greatly varied, suggesting a possibility of designing selective anti-TB xanthone drugs.

EXPERIMENTAL
GENERAL
Melting points were measured with the use of a Bibby Stuart Scientific melting point apparatus SMP3. UV spectra were measured with a UNICAM UV-310 spectrophotometer. ESI-TOF mass spectra were recorded with a Micromass LCT spectrometer. NMR spectra (1H, 13C, DEPT, 1H-1H COSY, NOESY, HMQC and HMBC) were recorded with Bruker AV300 spectrometers. Column chromatography was performed with the use of Merck silica gel 60 and Sephadex LH-20.

PLANT MATERIAL
Garcinia costata was collected at Phukradung National Park, Loei Province, Thailand, in July 2006. The plant identification was confirmed by Assistant Professor Dr. Maruay Mekanawakul, a specialist in the field of botanical study. A voucher specimen (WU 1414) is deposited at the botanic garden, Walailak University, Nakhon Si Thammarat, Thailand.

EXTRACTION AND ISOLATION
Dried branches of G. costata (3.10 kg) were macerated in CH2Cl2 (40 L) at room temperature for 3 days and the extract was concentrated under reduced pressure. The evaporated CH2Cl2 extract (26.89 g) was dissolved in MeOH (250 mL), filtered and concentrated under reduced pressure. The MeOH soluble portion (13.33 g) was subjected to Sephadex LH-20 column chromatography (MeOH) to afford seven fractions, 1–7, upon TLC profile. Fraction 6 (1.79 g) was chromatographed again with the use of a Sephadex LH-20 column, yielding nine fractions (6A–6I). Fraction 6F (0.38 g) was chromatographed with Sephadex LH-20 (MeOH) to give six fractions (6F1–6F6). Fraction 6F4 (0.19 g) was subjected to column chromatography with silica gel (CH2Cl2-MeOH, 99:1) to furnish compounds 1 (25 mg), 2 (10 mg), 3 (1 mg), and 4 (13 mg). Fraction 6E (0.42 g) was purified on a silica gel column by elution with hexane-CH2Cl2 (60:40) giving 5 (2 mg). Compound 6 (5 mg) was isolated from fraction 6H by successive column chromatography on Sephadex LH-20 (MeOH) and silica gel (CH2Cl2-MeOH, 98:2) columns.

Costatin [1,3,6,7-tetrahydroxy-2,4,8-tri-(3-methyl-2-butenyl)xanthone] (1): Yellow solid. mp 145–146 °C. UV λmax (MeOH) (log ε): 264 (4.59), 242 (4.51), 318 (4.42), 376 (4.04) nm. IR (neat) νmax: 3401, 1640, 1616, 1581, 1464, 1238, 1172, 1055 cm-1. 1H-NMR (300 MHz, CDCl3) and 13C-NMR (75 MHz, CDCl3) see Table 1. HRMS (ESI-TOF) [M+H]+ m/z: 465.2278 for C28H33O6 (calcd. 465.2277).

BIOLOGICAL ASSAY
An assay for the activity against Plasmodium falciparum K1 was performed with the use of the microculture radioisotope technique.22 The growth inhibitory activity against Mycobacterium tuberculosis H37Ra and the cytotoxicity against Vero cells (African green monkey kidney fibroblasts) were performed with the use of the green fluorescent protein microplate assay (GFPMA).23 Anticancer activities against KB cells (oral human epidermoid carcinoma) and MCF7 cells (human breast cancer) were evaluated with the use of the resazurin microplate assay.24

ACKNOWLEDGEMENTS
The success of this research was partially supported by the Bioresources Research Network, National Center for Genetic Engineering and Biotechnology (BRN010G-48) and the Utilization of Natural Products Research Unit, Walailak University. Warraphong Nuangnaowarat, an author of this paper, would also like to express his gratitude towards the Thailand Graduate Institute of Science and Technology (TGIST) for his doctoral scholarship award.

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