Cytotoxic Xanthone Constituents of the Stem Bark of Garcinia mangostana (Mangosteen)

Abstract

Bioassay-guided fractionation of a chloroform-soluble extract of Garcinia mangostana stem bark using the HT-29 human colon cancer cell line and an enzyme-based ELISA NF-κB assay, led to the isolation of a new xanthone, 11-hydroxy-3-O-methyl-1-isomangostin (1). The structure of 1 was elucidated by spectroscopic data analysis. In addition, ten other known compounds, 11-hydroxy-1-isomangostin (2), 11α-mangostanin (3), 3-isomangostin (4), α-mangostin (5), β-mangostin (6), garcinone D (7), 9-hydroxycalabaxanthone (8), 8-deoxygartanin (9), gartanin (10), and cratoxyxanthone (11), were isolated. Compounds 4–8 exhibited cytotoxicity against the HT-29 cell line with ED₅₀ values of 4.9, 1.7, 1.7, 2.3, and 9.1 μM, respectively. In an ELISA NF-κB assay, compounds 5–7, 9, and 10 inhibited p65 activation with IC₅₀ values of 15.9, 12.1, 3.2, 11.3, and 19.0 μM, respectively, and 6 showed p50 inhibitory activity with an IC₅₀ value of 7.5 μM. α-Mangostin (5) was further tested in an in vivo hollow fiber assay, using HT-29, LNCaP, and MCF-7 cells, but it was found to be inactive at the highest dose tested (20 mg/kg).

Garcinia mangostana L. (Clusiaceae) is well-known in southeastern Asia for its pleasant-tasting fruits, commonly known as mangosteen, which is now used widely as a botanical dietary supplement in several countries.¹ Xanthones are the most characteristic secondary metabolite constituents of G. mangostana and over 80 compounds of this type have been isolated and characterized from the various parts of this plant.^1,2 The biological effects of the mangosteen xanthones are diverse, and include antioxidant, antibacterial, antifungal, antimalarial, anti-inflammatory, cytotoxic, and HIV-1 inhibitory activities.^1,2 Recent phytochemical investigations on the fruits of G. mangostana at The Ohio State University have resulted in the isolation of xanthones with antioxidant,³ aromatase inhibitory,⁴ and quinone reductase-inducing activities.⁵

As part of a collaborative project directed towards the discovery of novel natural product anticancer agents,⁶ a CHCl₃-soluble extract of the stem bark of G. mangostana collected in Indonesia showed cytotoxic activity against a “gatekeeper” HT-29 human colon cancer cell line with an ED₅₀ value of 1.6 μg/mL. This extract also inhibited p50 and p65 activation with 57% and 67% inhibition at 50 μg/mL, respectively, in an ELISA NF-κB (nuclear factor-kappaB) assay. Therefore, it was subjected to bioactivity-guided fractionation, leading to the isolation of twelve xanthones, including a new compound (1). The structure elucidation of 1 and the biological evaluation of all compounds isolated are described herein.

Compound 1 was obtained as a yellow amorphous powder and produced a molecular ion peak at m/z 463.1729 [M+Na]⁺ in the HRESITOFMS, corresponding to the sodiated elemental formula, C₂₅H₂₈O₇Na. The IR spectrum showed absorption bands at 3350 cm⁻¹ for one or more hydroxy groups and at 1614 and 1456 cm⁻¹ for aromatic groups.⁷ The UV spectrum of 1 exhibited absorption maxima at 242, 254, and 303 nm, indicating the presence of a xanthone system.⁷ The ¹H and ¹³C NMR spectra of 1 were similar to those of the known compound, 11-hydroxy-1-isomangostin (2),⁸ except for the presence of signals for a second methoxy group at δ_H 3.83 (3H, s, OCH₃-3) and δ_C 56.0 (CH₃, OCH₃-3). The positions of two methoxy groups were assigned C-3 and C-7 by the ¹H-¹³C HMBC correlations between signals at δ_H 3.83 (3H, s, OCH₃) to δ_C 162.0 (C, C-3) and δ_H 3.78 (3H, s, OCH₃) to δ_C 142.9 (C, C-7), respectively. Further detailed analysis of the ¹H-¹H COSY, ¹H-¹³C HSQC, and ¹H-¹³C HMBC NMR data (Figure 1) allowed unambiguous assignments for all of the ¹H and ¹³C NMR signals of 1. The Mosher ester method was utilized in an attempt to determine an absolute configuration of 1.⁹ However, the reaction between the secondary hydroxy group at C-11 and the R- and S-MTPA-Cl reagents yielded a mixture of S- and R-MTPA esters and a mixture of R- and S-MTPA esters of 1, respectively, indicating the presence of a racemic mixture, but not in a 1:1 mixtures due to the slightly positive specific rotation observed. Thus, the structure of the new compound 1 was elucidated as 11-hydroxy-3-O-methyl-1-isomangostin.

Figure 1.

Important ¹H-¹H COSY () and ¹H-¹³C HMBC (→) correlations of compound 1.

Compounds 2 and 3 exhibited NMR spectroscopic data identical to those of 11-hydroxy-1-isomangostin⁸ and mangostanin.¹⁰ To attempt to determine the absolute configuration of 2, the Mosher ester method was performed,⁹ but these reactions again produced evidence for the presence of a racemic mixture. As observed for 1, the slightly positive specific rotation value of 2 could be also caused by an unequal ratio of 11R and 11S isomers present. Compound 3 was isolated previously from the fruits of G. mangostana, but its relative configuration has not been reported thus far. The energy-minimized stereostructure of 3 showed a dihedral angle of 26 between H-11 and H-10β, which corresponded to the coupling constant value with ³J_HH = 8~9 Hz, from the Karplus correlation equation (³J_HH = A + B cos Φ + C cos 2Φ; A = 7, B = −1, C = 5, Φ = dihedral angle).^7,11 The dihedral angle between H-11 and H-10α was calculated as 151° in the energy-minimized stereostructure of 3, with the expected calculated coupling constant value ³J_HH = 9~10 Hz.^7,11 These computational calculations were compared to the actual coupling constants observed in the ¹H NMR spectrum for 3 at δ_H 4.83 (1H, dd, J = 9.5, 8.2 Hz, H-11), 3.18 (1H, dd, J = 15.8, 8.2 Hz, H-10β), and 3.12 (1H, dd, J = 15.8, 9.5 Hz, H-10α). Based on these observations, the hydroxy group at C-11 was established as α (Figure 2). Therefore, compound 3 was determined as 11α-mangostanin.

Figure 2.

Dihedral angles in the energy-minimized stereostructure of compound 3.

Other known compounds were identified in the present investigation as 3-isomangostin (4),⁹ α-mangostin (5),¹² β-mangostin (6),¹³ garcinone D (7),¹⁴ 9-hydroxycalabaxanthone (8),¹⁵ 8-deoxygartanin (9),¹⁶ gartanin (10),¹⁷ cratoxyxanthone (11),⁸ and mangostanol,¹⁸ respectively, by comparison of their physical and spectroscopic data with those reported previously. Compounds 2–4, 9, and 10 were isolated as constituents of the stem bark part of this plant for the first time. In addition, cratoxyxanthone (11) has been isolated only from the bark of Cratoxylum cochinchinense (Lour.) Bl. (Clusiaceae) previously,⁸ so this is the first report of its isolation from a plant of the genus Garcinia.

All compounds isolated in the present investigation were tested in vitro for their cytotoxic activity against the HT-29 human colon cancer cell line (Table 1). The major active compounds, α-mangostin (5) and β-mangostin (6), have been found cytotoxic against various human cancer cells,^19–22 including DLD-1 human colon cancer cells,²³ and compounds 4, 7, and 8 have been also reported for their cytotoxicity against epidermoid carcinoma (KB), breast cancer (BC-1), or small cell lung cancer (NCI-H187).¹⁹ However, this is the first report of the evaluation of xanthones from G. mangostana for their cytotoxicity against the HT-29 colon cancer cell line. In an enzyme-based ELISA NF-κB assay, all compounds except for 3 and 4 and mangostanol were tested for their p50 (NF-κB1) and p65 (RelA) inhibitory activities, with β-mangostin (6) and garcinone D (7), respectively, being the most active substances found (Table 1). The major cytotoxic isolate, α-mangostin (5), was chosen for evaluation in an in vivo hollow fiber assay, which is used as a secondary bioassay in our drug discovery program to prioritize leads for subsequent analysis in traditional xenograft models.²⁴ However, compound 5 was found to be inactive against HT-29 and LNCaP (hormone-dependent human prostate cancer) cells implanted at the intraperitoneal (i.p.) site at doses of 2.5, 5, 10, and 20 mg/kg (Figure S1, Supporting Information). Therefore, on the basis of these results in the hollow fiber assay, α-mangostin (5) does not seem to be promising as a potential anticancer agent.

Table 1.

In Vitro Activity of Compounds 4–10 in Cytotoxicity (HT-29 Cell Line) and ELISA NF-κB (p65 and p50) Assays.

compound	4	5	6	7	8	9	10	camptothecind	rocaglamided
cytotoxicitya	4.9	1.7	1.7	2.3	9.1	>10	>10	0.06	-
NF-κB (p65)b	NTc	15.9	12.1	3.2	>20	11.3	19.0	-	0.08
NF-κB (p50)b	NTc	>20	7.5	>20	>20	>20	>20	-	2.0

^aCompounds 1–3 and 11 were inactive (ED₅₀ >10 μM).

^bCompounds 1, 2, and 11 were inactive (IC₅₀ >20 μM).

^cCompounds 3 and 4 were not tested in the NF-κB assays because of the limited amounts available.

^dPositive control substances.

Experimental Section

General Experimental Procedures

The melting point was measured on a Fisher-Johns 12-144 melting point apparatus with a 12-142T thermometer (Fisher Scientific, Pittsburgh, PA), and is uncorrected. Optical rotations were measured with a Perkin-Elmer 343 automatic polarimeter. UV and IR spectra were obtained with a Shimadzu UV 160U spectrophotometer and Thermo Scientific Nicolet^™ 6700 FT-IR spectrometer, respectively. 1D and 2D NMR experiments were performed on Bruker Avance DPX-300 and DRX-400 spectrometers with tetramethylsilane (TMS) as internal standard. Electrospray ionization (ESI) mass spectrometric analyses were performed with a 3-Tesla Finnigan FTMS-2000 Fourier Transform mass spectrometer. Silica gel (65–250 and 230–400 mesh, Sorbent Technologies, Atlanta, GA) and Sephadex LH-20 (Supelco, Bellefonte, PA) were used for column chromatography. Thin-layer chromatographic (TLC) analysis was performed on Silica G (silica gel, 0.2 mm layer thickness, Sorbent Technologies, Atlanta, GA) and RP-18 F_254s (Merck, Germany) TLC plates, with visualization under UV light (254 and 365 nm) and 10% (v/v) sulfuric acid spray followed by heating (120 °C, 2 min). A Sunfire PrepC₁₈ column (150 mm × 19 mm i.d., Waters, Milford, MA) and a Sunfire guard column (5 μm, 10 mm × 19 mm i.d., Waters, Milford, MA) were used for preparative HPLC, along with a Waters system composed of a 600 controller, a 717 Plus autosampler, and a 2487 dual wavelength absorbance detector.

Plant Material

The stem bark of G. mangostana (400 g) was collected at Pangradin village, Jasinga, West Java, Indonesia, by S. R. in August, 2005, who also identified this plant. A voucher specimen (acquisition number 2285414) has been deposited in the John G. Searle Herbarium of the Field Museum of Natural History, Chicago, Illinois.

Extraction and Isolation

The dried stem bark of G. mangostana (400 g) was extracted with MeOH (3 × 1 L) overnight at room temperature. The solvent was evaporated in vacuo to afford a MeOH extract (75 g), which was then suspended in MeOH-H₂O (9:1, 1 L), and partitioned with hexane (3 × 1 L). To the defatted residue, which was dried in vacuo, was added 10% MeOH in H₂O (1 L), and then this was partitioned with CHCl₃ (3 × 1 L). The CHCl₃-soluble layer was washed with 1% aqueous NaCl (3 × 1 L) to provide a partially detannified CHCl₃ extract (14 g). This CHCl₃ extract was subjected to silica gel column chromatography (CC; φ 3.5 cm; 60–250 mesh, 200 g), using gradient mixtures of MeOH in CHCl₃ (0→1%) as mobile phases, affording nine fractions (FI - FIX). These fractions were evaluated against the HT-29 cell line, and, of these fractions, FI - FV were found to be active (ED₅₀ <10 μg/mL). Compounds 6 (300 mg, 0.075% w/w) and 5 (2.5 g, 0.625% w/w) were isolated from fraction FI and FII, respectively, by precipitation in MeOH. The residual portion of fraction FI (464 mg), eluted with 100% CH₂Cl₂ from the first separation, was subjected to silica gel CC (φ 2.5 cm; 230–400 mesh, 50 g), with hexanes-EtOAc (4:1→1:1) as solvent system, yielding 8 (8.0 mg, 0.002% w/w). Fraction FII (3.3 g, after partial removal of α-mangostin), also eluted with 100% CH₂Cl₂ from the first purification step, was separated by silica gel CC (φ 3 cm; 230–400 mesh, 100 g), using gradient mixtures of hexanes-EtOAc-MeOH (20:10:1→10:10:1) for elution, affording seven sub-fractions. Sub-fraction FII-1 (100 mg), eluted with hexanes-EtOAc-MeOH (20:10:1), was further purified by preparative HPLC, using an isocratic mixture of MeOH-H₂O (8:2, 8 mL/min) as solvent system, to afford 9 (t_R 12.1 min, 3.1 mg, 0.00077% w/w) and 10 (t_R 19.5 min, 2.2 mg, 0.00055% w/w). Fraction FIII (987 mg), eluted with 0.1% MeOH in CH₂Cl₂ from the first separation, was subjected again to silica gel CC (φ 2.5 cm; 230–400 mesh, 50 g), using gradient mixtures of MeOH in CHCl₃ (1→10%) as mobile phases, providing ten sub-fractions. Passage over Sephadex LH-20 of the third fraction (210 mg) from this column, which was eluted with 1% MeOH in CHCl₃, using 100% MeOH, afforded mangostanol (2.2 mg, 0.00055% w/w). Fraction FIV (1.1 g), eluted with 0.15% MeOH in CH₂Cl₂ from the first separation, was separated by silica gel CC (φ 3 cm; 230–400 mesh, 70 g), using a gradient solvent system of CH₂Cl₂-acetone (99:1→9:1), providing 16 sub-fractions. Sub-fraction 10 (100 mg), eluted with CH₂Cl₂-acetone (95:5), was subjected to Sephadex LH-20 chromatography using 100% MeOH as solvent, yielding 11 (15.5 mg, 0.0038% w/w). The fourth fraction (10 mg) from this Sephadex LH-20 column was purified by preparative HPLC, using an isocratic mixture of CH₃CN-H₂O (9:1, 8 mL/min) as solvent system, to afford 3 (t_R 18.5 min, 3.4 mg, 0.00085% w/w). The combined sub-fractions 11 and 12 (97 mg), eluted with CH₂Cl₂-acetone (95:5), were subjected to passage over Sephadex LH-20 (100% MeOH), providing four sub-fractions. The first sub-fraction (20.1 mg) from this separation was purified by preparative HPLC using an isocratic mixture of MeOH-H₂O (85:15, 5 mL/min) as solvent system, to obtain 1 (t_R 11.8 min, 5.1 mg, 0.00127%). Combined sub-fractions 13 and 14 (162 mg), eluted with CH₂Cl₂-acetone (95:5), were also separated by Sephadex LH-20 CC (100% MeOH), affording 7 (28.2 mg, 0.007% w/w) and 4 (3.2 mg, 0.0008% w/w). Fraction FV (242 mg), eluted with 0.2% MeOH in CH₂Cl₂ from the first separation, was subjected to Sephadex LH-20 CC, using 100% MeOH as solvent system, and then purified by preparative HPLC using an isocratic mixture of CH₃CN-H₂O (9:1, 8 mL/min) as solvent system, furnishing 2 (t_R 9.2 min, 3.9 mg, 0.00097% w/w).

11-Hydroxy-3-O-methyl-1-isomangostin (1)

yellow amorphous powder; mp 130–132 °C; [α]_D ²⁵ +9.1 (c 0.1, MeOH); UV (MeOH) λ_max (log ε) 242 (4.43), 252 (4.39), 303 (4.18) nm; IR (film) ν_max 3350, 2931, 1614, 1456, 1374, 1272, 1208, 1181, 1138, 1115 cm⁻¹; ¹H NMR (CDCl₃, 400 MHz) δ 6.76 (1H, s, H-5), 6.29 (1H, s, H-4), 5.33 (1H, t, J = 6.4 Hz, H-16), 4.07 (2H, t, J = 6.4 Hz, H-15), 3.85 (1H, t, J = 4.9 Hz, H-11), 3.83 (3H, s, OCH₃-3), 3.78 (3H, s, OCH₃-7), 2.84 (1H, dd, J = 17.3, 4.9 Hz, H-10β), 2.69 (1H, dd, J = 17.3, 4.9 Hz, H-10α), 1.80 (3H, s, H-19), 1.65 (3H, s, H-18), 1.48 (3H, s, H-14), 1.36 (3H, s, H-13); ¹³C NMR (CDCl₃, 100 MHz) δ 176.9 (C, C-9), 162.0 (C, C-3), 157.4 (C, C-4a), 154.6 (C, C-6), 154.3 (C, C-5a), 153.7 (C, C-1), 142.9 (C, C-7), 137.2 (C, C-8), 131.4 (C, C-17), 124.3 (CH, C-16), 115.3 (C, C-8a), 108.0 (C, C-9a), 104.0 (C, C-2), 101.4 (CH, C-5), 90.7 (CH, C-4), 78.2 (C, C-12), 68.8 (CH, C-11), 61.9 (CH₃, OCH₃-7), 56.0 (CH₃, OCH₃-3), 26.6 (CH₂, C-15), 26.5 (CH₂, C-10), 26.1 (CH₃, C-18), 24.7 (CH₃, C-13), 22.6 (CH₃, C-14), 18.5 (CH₃, C-19); ESIMS (positive mode) m/z 463.17 [M+Na]⁺ (100), 322.80 (10), 172.89 (40); HRESITOFMS (positive mode) m/z 463.1729 [M+Na]⁺ (calcd for C₂₅H₂₈O₇, 463.1733).

11-Hydroxy-1-isomangostin (2)

[α]_D²⁵ +11.4 (c 0.1, MeOH).

11α-Mangostanin (3)

[α]_D²⁵ −1.75 (c 0.1, MeOH).

Cratoxyxanthone (11)

[α]_D²⁵ +0.06 (c 0.13, MeOH).

Cytotoxicity Assay

Cytotoxic potential against HT-29 was determined using a established protocol.²⁵ Camptothecin was employed as the positive control (ED₅₀ = 0.06 μM).

Enzyme-based ELISA NF-κB Assay

The NF-κB p65 and p50 inhibitory activity assay was conducted according to a published protocol.^26,27 Rocaglamide was used as the positive control and exhibited IC₅₀ values of 0.08 and 2.0 μM in this assay.

Hollow Fiber Assay

α-Mangostin (5) was evaluated in the in vivo hollow fiber model, using HT-29, LNCaP, and MCF-7 cells, according to a procedure described in the literature.^28,29 Paclitaxel was used as the positive control for this experiment at a dose of 20 mg/kg.