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.

1. 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 schomburgkianaPierre 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 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).

2. 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.

Figure 1

Structures of compounds 1–4.

Compound 1 was isolated as a yellow solid (m.p. = 194–195 °C) and found to have a molecular formula of C25H30O7 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 λmax 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.

In the 1H-NMR spectrum, signals at δH 6.38 (s, 1H, H-4) and 6.90 (s, 1H, H-5) imply the presence of a xanthone nucleus. On the other hand, the prenyl moiety was evident from proton resonances at δH 3.32 (br d, 2H, H-1'), 5.25 (t, 1H, J = 6.9 Hz, H-2'), 1.76 (s, 3H, H-4') and 1.62 (s, 3H, H-5') while the characteristic proton resonances of the 3-hydroxy-3-methylbutyl moiety were seen at δH 3.42 (m, 2H, H-1''), 1.70 (m, 2H, H-2'') and 1.28 (s, 6H, H-4'', H-5''). The 1H-NMR spectrum also showed characteristic resonances of a chelated hydroxyl group at δH 13.74 (s, 1H, 1-OH) and two methoxyl groups at δH 4.00 (s, 3H, 6-OCH3) and 3.79 (s, 3H, 7-OCH3) (see Table 1).

Table 1

1H-NMR (500 MHz) and 13C-NMR (125 MHz) spectroscopic data for compound 1 (in Me2CO-d6) and 2 (in CD3OD).

Position	1		2
	δ H	δ C	δ H	δ C
1		160.8		106.4
2		110.3		161.5
3		162.2	5.96 (s)	91.1
4	6.38 (s)	92.3		163.3
4a		154.9
5	6.90 (s)	98.5	5.96 (s)	95.3
5a		155.4
6		158.6		163.3
7		144.1		198.4
8		138.4
8a		111.4
9		182.0
9a		102.9
1'	3.32 ( br d)	21.2		142.7
2'	5.25 (t, J = 6.3 Hz)	122.6	6.52 (d, J = 2.3Hz)	106.5
3'		130.6		158.0
4''	1.76 (s)	17.1	6.39 (t, J = 2.3 Hz)	105.7
5'	1.62 (s)	25.1		158.0
6'			6.52 (d, J = 2.3 Hz)	106.5
1''	3.42 (m)	22.2
2''	1.70 (m)	44.9
3''		69.7
4'' & 5''	1.28 (s)	28.5
1-OH	13.74 (s)
2-OCH3			3.54 (s)	54.6
6-OCH3	4.00 (s)	55.8
7-OCH3	3.79 (s)	60.4

Meanwhile, the DEPT experiment indicated that this compound is composed of three methine (δC 92.3, 98.5 and 122.6), three methylene (δC 21.2, 22.2 and 44.9), four methyl (δC 17.1, 25.1, 28.5 × 2), two methoxyl (δC 55.8 and 60.4) and 13 quaternary carbons (δC 69.7, 102.9, 110.3, 111.4, 130.6, 138.4, 144.1, 154.9, 155.4, 158.6, 160.8, 162.2 and 182.0). These results are consistent with the 13C-NMR spectrum, which indicated the presence of 25 carbons. The presence of a xanthone skeleton was again obvious in the 13C-NMR spectrum, with the signal at δC 182.0, a characteristic signal for the carbonyl group in the xanthone skeleton. Moreover, six oxygenated aromatic carbons [C-7 (δC 144.1), C-4a (δC 154.9), C-5a (δC 155.4), C-6 (δC 158.6), C-1 (δC 160.8) and C-3 (δC 162.2)] were observed in compound 1 after further examination of the DEPT spectrum.

The HMBC long range 3J correlations between the chelated hydroxyl group with C-9a (δC 102.9) and C-2 (δC 110.3) and a 2J correlation with C-1 (δC 160.8) were observed. This allows the assignment of the hydroxyl group to C-1 [δH 13.74 (s, 1H, 1-OH)]. The two prenyl moiety methyl groups resonating at δH 1.76 and 1.62 are correlated to the neighbouring C-3' (δC 130.6) and C-2' (δC 122.6) in the HMBC experiment. Moreover, the linkages between H-1' [δH 3.32 (br d, 2H)] and C-2' (δC 122.6) as well as C-3' (δC 130.6) are seen. The linkages between H-2' [δH 5.25 (t, 1H, J = 6.3 Hz)] and C-4' (δC 17.1) and C-5' (δC 25.1) are also clearly seen in the experiment (Figure 2). These correlations provide evidence for the presence of the prenyl side chain while the 2J correlation between H-1' [δH 3.32 (br d, 2H)] with C-2 (δC 110.3) and the 3J correlations of H-1' [δH 3.32 (br d, 2H)] with C-3 (δC 162.2) indicated that the prenyl moiety is positioned at C-2. In the COSY analysis, the coupling of H-1' and H-2' was very strong.

Figure 2

The HMBC correlation of 3-hydroxy-3-methylbutyl (I) and prenyl (II) moieties in compound 1.

A pair of overlapping aliphatic methyls [δH 1.28 (s, 6H, H-4'', H'')] gave clear correlations with C-2'' (δC 44.9) and C-3'' (δC 69.7) indicating the methyls to be attached to C-3''. H-2'' [δH 1.70 (m, 2H)] gave cross peaks to C-4''(δC 28.5), C-5'' (δC 28.5), C-3'' (δC 69.7) and C-1'' (δC 22.2). Meanwhile H-1'' [δH 3.42 (m, 2H)] showed a 2J correlation with C-2'' (δC 44.9) and a 3J correlation with C-3'' (δC 69.7). These long range correlation signals allow us to conclude a 3-hydroxy-3-methylbutyl moiety (Figure 2). The coupling of H-1'' and H-2'' was seen in the COSY experiment. Moreover, the cross peaks of H-1'' [δH 3.42 (m, 2H)] to C-7 (δC 144.1), C-8 (δC 138.4), C-8a (δC 111.4) and the cross-peak of H-2'' [δH 1.70 (m, 2H)] to C-8 (δC 138.4) in the HMBC analysis were suggestive of the 3-hydroxy-3-methylbutyl moiety location at C-8. In addition, the two aromatic methoxyl groups [6-OCH3 (δH 4.00) and 7-OCH3 (δH 3.79)] are assigned at C-6 and C-7 because of their 3J correlations with δC 158.6 (C-6) and δC 144.1 (C-7). The two remaining aromatic methine protons of the xanthone skeleton were determined to be at the two remaining available carbons, C-4 and C-5. The long range correlations between H-4 (δH 6.38) and C-2 (δC 110.3), C-3 (δC 162.2), C-4a (δC 154.9), C-9 (δC 182.0) and C-9a (δC102.9) places the δH 6.38 (H-4) singlet at C-4. The signal at δH 6.90 (H-5) was assigned to C-5 based on its HMBC correlations with C-5a (δC 155.4), C-6 (δC 158.6), C-7 (δC 144.1), C-8 (δC 138.4), C-8a (δC 111.4) and C-9 (δC 182.0). Compound 1 differs from a closely related compound, garcinone D [17] in the presence of a methoxyl group at C-6 where garcinone D has a hydroxyl group at that position. The methoxyl group in compound 1 was assigned from the observation of a HMBC correlation between 6-OCH3 (δH 4.00) and C-6 (δC 158.6). The HMBC correlations are illustrated in Figure 3. Therefore, compound 1 was elucidated to be 1,3-dihydroxy-8-(3-hydroxy-3-methylbutyl)-6, 7-dimethoxy-2-(3-methyl-2-buten-1-yl)-xanthone and it was named trivially as mangaxanthone B.

Figure 3

Key HMBC correlations between 1H and 13C in compound 1.

Compound 2 was isolated as yellow-brown crystals (m.p. = 245–246 °C) and was found to have a molecular formula of C14H12O6 via the EIMS (m/z 276 [M+]) analysis. The FTIR spectrum exhibited a strong absorption at 1728 cm−1, which suggested the presence of a carbonyl group, and another strong absorption at 2924 cm−1 representing the aromatic C-H stretching band. An aromatic benzene chromophore which is of a benzophenone skeleton, was confirmed by the maximum absorption peaks of 211, 214 and 309 nm in the UV-Visible spectrum.

The 1H-NMR spectrum of compound 2 showed five aromatic proton signals which resonated at δH 5.96 (s, 2H, H-3 and H-5), δH 6.52 (d, 2H, J = 2.3 Hz, H-2' and H-6') and δH 6.39 (t, 1H, J = 2.3 Hz, H-4'). After a detailed inspection on the 13C NMR and DEPT spectra, it was found that the signals in the 13C-NMR spectrum indicated 14 carbons which consisted of one methoxyl (δC 54.6), five methine (δC 91.1, 95.3, 105.7 and 106.5 × 2) and eight quaternary carbons (δC 106.4, 142.7, 158.0 × 2,161.5, 163.3 × 2 and 198.4). The benzophenone skeleton of compound 2 was demonstrated by signals resonating at δC 198.4 for the carbonyl group, as well as signals at δC 158.0, 161.5 and 163.3 for the hydroxylated aromatic carbons (see Table 1).

The structure of compound 2 was deduced based on the HMBC spectrum (See Figure 4). The aromatic protons at H-2' and H-6' [δH 6.52 (d, 2H, J = 2.3 Hz)] showed three-bond connectivities with, C-4' (δC 105.7), and C-7 (δC 198.4). Protons H-2' and H-6' also gave two-bond connectivities with C-3' and C-5' (δC 158.0 × 2) respectively. Meanwhile H-4' has 2J correlations with C-3' and C-5' (δC 158.0 × 2) and 3J correlations with C-2' and C-6' (δC 106.5 × 2). The aromatic proton of H-3 [δH 5.96 (s, 1H)] exhibited three-bond (3J) and two-bond (2J) connectivities with C-1 (δC 106.4) (3J), C-5 (δC 95.3) (3J) and C-4 (δC 163.3) (2J) respectively. H-5 [δH 5.96 (s, 1H)] also showed correlations with C-1 (δC 106.4) (3J), C-3 (δC 91.1) (3J), C-4 and C-6 (δC 163.3 × 2) (2J) in the HMBC spectrum. Moreover, the methoxyl group was assigned to C-2 because of the 3J HMBC correlation of the methoxyl proton [δH 3.54 (s, 3H, 2-OCH3)] with C-2 (δC 161.5). The observation of a 2J correlation between H-3 and C-4 implies position 3 to be occupied by H-3. Moreover, we also observed a 2J correlation between H-5 and C-6 as well as between H-5 and C-4. Therefore, another proton is situated at C-5. 4-OH and 6-OH carbons have the same chemical shift values, thus justifying that the molecule is symmetrical. Hence, the two OH groups are at C-6 and C-4 and not at C-2. Hence, the structure of this compound was elucidated as (4,6-dihydroxy-2-methoxyphenyl)-(3,5-dihydroxyphenyl)methanone and it was given the trivial name mangaphenone.

Figure 4

2J, 3J and 4J HMBC correlations between protons and carbons in compound 2.

3. Experimental

3.1. 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.

3.2. General

The 1D (1H, 13C, 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.

3.3. 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 (CHCl3), ethyl acetate and methanol to afford 6 fractions. The fourth fraction was further fractionated through column chromatography using hexane–CHCl3 and CHCl3–MeOH to give 8 fractions. The last fraction was subjected to repeated chromatography by eluting with hexane–EtOAc (7:3) and CHCl3–MeOH (9.8:0.2) to furnish compounds 1, 2 and 3. Meanwhile, the dry MeOH extract (278.96 g) was suspended in H2O 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–CHCl3, CHCl3, CHCl3–EtOAc, EtOAc–MeOH and MeOH) and afforded eight fractions. Fraction 2 was further purified through column chromatography by using CHCl3–MeOH (9:1) and compound 4 was thus obtained.

3.4. Spectral Data

Mangaxanthone B (1). Yellow crystals; m.p. 194–195 °C; UV (EtOH) λmax (log ε): 209 (4.40), 245 (4.44), 262 (4.42), 316 (4.29) and 353 (3.79) nm; IR ν (cm−1): 3447, 3249, 2938, 1601, 1457, 1277 and 826; 1H-NMR (500 MHz, Me2CO-d6) and 13C-NMR (125 MHZ, Me2CO-d6), see Table 1; EIMS m/z (rel. int.): 442(32), 424(28), 381(74), 369(43), 368(33), 354(23), 353(100), 327(53) and 325(33).

Mangaphenone (2). Brownish-yellow crystals; m.p. 245–246 °C; UV (EtOH) λmax (log ε): 211 (4.11), 214 (4.12) and 309 (3.77) nm; IR ν (cm−1): 3599, 2924, 1728, 1261, 804 and 730; 1H-NMR (500 MHz, CD3OD) and 13C-NMR (125 MHZ, CD3OD), see Table 1; EIMS m/z (rel. int.): 276(67), 260(50), 168(70), 167(100), 153(22) and 69(44).

Mangostanin (3). Yellow amorphous powder. Spectral data are in agreement with the literature [18].

Mangostenol (4). Yellow solid; m.p. 159–160 °C. Spectral data are in agreement with the literature [19].

4. 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.