Two New Chemical Constituents from the Stem Bark of Garcinia mangostana

A detailed chemical study on the ethyl acetate and methanol extracts of the stem bark of Garcinia mangostana resulted in the successful isolation of one new prenylated xanthone, mangaxanthone B (1), one new benzophenone, mangaphenone (2), and two known xanthones, mangostanin (3) and mangostenol (4). The structures of these compounds were elucidated through analysis of their spectroscopic data obtained using 1D and 2D NMR and MS techniques.


Introduction
The Clusiaceae family consists of approximately 40 genera [1], including Garcinia, Mesua and Cratoxylum [2]. Some Clusiaceae plants are used in traditional medicine to treat various illnesses. For example, Garcinia schomburgkiana Pierre is used to treat coughs and menstrual problems [3] while Mesua ferrea is used in the treatment for dyspepsis and renal disease [4]. Clusiaceae plants contain numerous biologically active secondary metabolites, such as benzophenones, xanthones, coumarins OPEN ACCESS and flavonoids. The pharmacological properties of these secondary metabolites include antifungal activity in Calophyllum thwaitesii [5], antioxidant activity in Cratoxylum cochinchinense [6], antimicrobial and antibacterial activities in Garcinia cowa [7] and anticancer activity in Garcinia paucinervis [8]. Garcinia plants are mainly found in tropical countries such as Malaysia, Thailand and Brazil [1]. Garcinia plants are currently being more avidly studied due to their abilities to treat dysentery, pain, tapeworm infestations and many more ailments [9]. Being a member of the Garcinia genus, the mangosteen is also well known to be a rich source of xanthones and benzophenones, especially polyprenylated xanthones and oxygenated xanthones [10]. These secondary metabolites have been reported to possess biological properties against fungi [11], bacteria [12,13] and also cancer [14,15]. Ryu and co-workers also reported that some oxygenated xanthones from the seedcases of this plant possess neuraminidase inhibitory activity [16]. The discoveries of these beneficial secondary metabolites have revitalized our interest to investigate more extensively on the stem bark of Garcinia mangostana. Herein, we describe the isolation as well as the characterization of a new prenylated xanthone, mangaxanthone B (1) and a new benzophenone, mangaphenone (2), along with two other known xanthones mangostanin (3) and mangostenol (4).

Results and Discussion
The stem bark of Garcinia mangostana was extracted with ethyl acetate (EtOAc) and methanol (MeOH) followed by fractionation of these extracts to obtain a new prenylated xanthone, mangaxanthone B (1), a new benzophenone, mangaphenone (2) and two other known compounds mangostanin (3) and mangostenol (4). Structural elucidation of these compounds were performed by analysing their spectroscopic data. The structures of compounds 3 and 4 were confirmed by comparing their spectroscopic data with data available from the literature. The structures of compounds 1-4 are illustrated in Figure 1. (1) (3) Compound 1 was isolated as a yellow solid (m.p. = 194-195 °C) and found to have a molecular formula of C 25 H 30 O 7 through the EIMS spectrum, which showed a molecular ion peak at m/z 442. The FTIR absorption indicated the existence of OH (3447 cm −1 ), alkane side chain CH (2938 cm −1 ), aromatic moiety C=C (1457 cm −1 ), CO (1601 cm −1 ) and alkene moiety CH (826 cm −1 ) bands. Besides, the λ max at 209, 245, 262, 316 and 353 nm in the UV-Visible spectrum are the characteristic absorption bands of an aromatic benzene chromophore, which indicated the presence of a xanthone nucleus.

Plant Material
The stem bark of Garcinia mangostana was collected from Melaka, Malaysia. A herbarium specimen (RG221) was deposited at the Herbarium in the Biology Department of UPM.

General
The 1D ( 1 H, 13 C, DEPT) and 2D (COSY, HMQC and HMBC) NMR spectra were recorded on a Unity INOVA 500 MHz NMR instrument using tetramethylsilane (TMS) as the internal standard. EIMS spectra were obtained using a Shimadzu GCMS model QP2010 Plus spectrophotometer. The ultraviolet spectra were recorded on a Shimadzu UV-160A UV-Visible Recording Spectrophotometer. Infrared spectra were obtained using the universal attenuated total reflection (UATR) technique on a Perkin-Elmer 100 Series FT-IR spectrometer. Melting points were measured through Leica Galen III microscope which was equipped with Testo 720 temperature recorder.

Extraction and Isolation
The air-dried powdered stem bark of Garcinia mangostana (2.0 kg) was first de-fatted using hexane followed by extraction with ethyl acetate (EtOAc, 3 × 5 L) for 72 h at room temperature then with 70% methanol (MeOH, 3 × 5 L) for another 72 h. The three extracts were concentrated to give 14.22 g of dark brown residue of EtOAc extract and 278.96 g of dark brown residue of MeOH extract. The EtOAc extract was subjected to vacuum column chromatography by eluting with a stepwise gradient system of hexane, chloroform (CHCl 3 ), ethyl acetate and methanol to afford 6 fractions. The fourth fraction was further fractionated through column chromatography using hexane-CHCl 3 and CHCl 3 -MeOH to give 8 fractions. The last fraction was subjected to repeated chromatography by eluting with hexane-EtOAc (7:3) and CHCl 3 -MeOH (9.8:0.2) to furnish compounds 1, 2 and 3. Meanwhile, the dry MeOH extract (278.96 g) was suspended in H 2 O and then partitioned with n-butanol (n-BuOH, 400 mL). The n-BuOH soluble portion (2.56 g) was chromatographed in a polarity gradient manner (hexane, hexane-CHCl 3 , CHCl 3 , CHCl 3 -EtOAc, EtOAc-MeOH and MeOH) and afforded eight fractions. Fraction 2 was further purified through column chromatography by using CHCl 3 -MeOH (9:1) and compound 4 was thus obtained. Mangostanin (3). Yellow amorphous powder. Spectral data are in agreement with the literature [18].

Conclusions
A new prenylated xanthone, mangaxanthone B and a new benzophenone, mangaphenone, were isolated along with two known xanthones, mangostanin and mangostenol, from the stem bark of Garcinia mangostana. Biological evaluation of these compounds is under way.