A New Ellagic Acid From the Leaves and Twigs of Irvingia malayana

Abstract

A phytochemical investigation of the leaves and twigs of Irvingia malayana led to the isolation of a new 3,3′,4′-tri-O-methylellagic acid-6″-acetoxy-4-O-β-glucoside (1), along with 3,3′,4′-tri-O-methylellagic acid (2), 3,3′-di-O-methylellagic acid-4-O-β-xyloside (3), 3,3′,4′-tri-O-methylellagic acid-4-β-O-glucoside (4), friedelin (5), friedelinol (6), methyl-3,4,5-trihydroxybenzoate (7), 5,7,4′-trihydroxyflavone-8-C-β-glucopyranoside (8), 5,7,3′,4′-tetrahydroxyflavone-8-C-β-glucopyranoside (9), and 5,3′,4′-trihydroxyflavone-6-C-β-glucopyranoside (10). Their structures were elucidated by means of spectroscopic techniques and direct comparison with literature data. Compounds 4 and 7 showed weak cytotoxic activity against a panel of mammalian cancerous cell lines. Furthermore, compounds 1, 2, 4, and 9 exhibited significant inhibitory activity in the syncytium inhibition assay, whereas compounds 8 and 9 displayed moderate activity in the HIV 1 reverse transcriptase assay.

Irvingia malayana is among the 5 species in the family Irvingiaceae (Simaroubaceae). It is widely distributed in tropical regions including Southeast Asia and India. ¹ Irvingia malayana is locally called in Thai “Kra Bok.” Its seeds are edible and the oil is widely studied for use as diesel fuel. ^2,3 Oil from the seed of I. malayana has the potential to be used in food and nutrient applications. ⁴ In Indonesia, the stem bark extract of I. malayana was used in folk medicine for curing cancer and malarial symptoms. ⁵ Various parts of I. malayana were reported to exhibit antimalarial and cytotoxic activities. ^4-6 Recently, the oil of this plant was demonstrated, in vitro, to inhibit rumen methanogenesis in Holstein dairy cows. ⁷ Herein, an extract from the leaves and twigs of I. malayana was studied as part of our ongoing search for biologically active substances from natural sources, which led to the isolation of a new ellagic acid derivative 1, together with 9 known compounds 2 to 10 (Figure 1). The chemical structures of the isolated compounds were elucidated on the basis of their spectroscopic data and in comparison with those previously reported in the literature. Cytotoxic activity and anti-HIV activity using anti-HIV1 reverse transcriptase (anti-HIV1-RT) and syncytium inhibition assays of the isolated compounds were also evaluated.

Figure 1

Compounds isolated from I. malayana.

Compound 1 was obtained as a white amorphous solid, m.p. 272.6°C to 273.8°C (MeOH-H₂O), and its UV spectrum showed major absorption bands at λ _max 237, 349, and 366 nm. The Fourier transform infrared spectrum (FT-IR) showed absorptions at ν _max 1743 cm⁻¹ (C=O stretching) and 1606 and 1572 cm⁻¹ (C=C stretching). Its molecular formula C₂₅H₂₄O₁₄ was determined on the basis of the electrospray ionization high-resolution time-of-flight mass spectrometry data (found m/z 549.1255, [M+H⁺], calcd m/z 549.1244 for C₂₅H₂₅O₁₄). Fragment ions at m/z 344 [M+H-glucoside⁺] in its electron ionization-mass spectrometry suggested that 1 was a glycoside derivative. The ¹H NMR spectrum of 1 in pyridine-d ₅ (Table 1) displays 2 singlet aromatic resonances at δ _H 8.44 and 7.82 ppm assignable to H-5 and H-5′, respectively, as deduced from the heteronuclear multiple bond correlations (HMBC; H-5 correlated with C-1, C-3, C-4, C-6, and C-7; and H-5′ correlated with C-1′, C-3′, C-4′, C-6′, and C-7′) as summarized in Figure 2.

Table 1

¹H and ¹³C NMR Data of Compound 1. ^a

Position	In pyridine-d5		In DMSO-d6
	δ H (mult., J in Hz)	δ C	δ H (mult., J in Hz)	δ C
1	-	114.2	-	114.3
2	-	141.7 b	-	141.7 b
3	-	142.5	-	142.3
4	-	152.9	-	152.4
5	8.44 (s)	112.8	7.82 (s)	112.4
6	-	113.2	-	112.8
7	-	158.8	-	158.9
1′	-	113.3	-	113.8
2′	-	141.8 b	-	141.5 b
3′	-	141.9	-	141.7
4′	-	154.9	-	154.7
5′	7.82 (s)	108.0	7.64 (s)	108.2
6′	-	113.2	-	113.2
7′	-	158.8	-	158.97
1″	5.80 (d, 9.0)	102.7	5.16 (d, 7.2)	101.6
2″	4.41 (m)	74.5	3.38 (m)	73.6
3″	4.38 (m)	78.1	3.40 (m)	76.6
4″	4.19 (m)	71.2	3.15 (m)	70.5
5″	4.32 (m)	75.6	3.73 (m)	74.6
6″	5.10 (m),	64.5	4.37 (br d, 10.4),	64.2
	4.66 (dd, 11.7, 7.7)		3.97 (m)
OCH3-3	4.16 (s)	61.4	4.02 (s)	61.8
OCH3-4	3.83 (s)	56.4	3.99 (s)	57.3
OCH3-3′	4.24 (s)	61.7	4.07 (s)	66.2
C(O)CH3	2.21 (s)	170.7, 20.6	2.03 (s)	170.9, 20.9
OH-2	-	-	5.58 (d, 5.2)	-
OH-3	-	-	5.30 (d, 5.5)	-
OH-4	-	-	5.37 (d, 4.2)	-

DMSO, dimethyl sulfoxide.

^aThe spectral data were recorded in ¹H (400 MHz) and ¹³C (100 MHz) NMR spectrometer.

^bMay be interchangeable.

Figure 2

Key ¹H-¹H COSY (bold line), HMBC (single-headed arrow), and NOESY (double-headed arrow) correlations of compound 1.

Three singlet resonances at δ _H 4.24, 4.16, and 3.83 ppm belonged to 3 methoxy groups connected to carbons at δ _C 142.5 (C-3), 141.9 (C-3′), and 154.9 (C-4′) ppm, respectively, as confirmed by HMBC correlations. On the same basis, a singlet resonance at δ _H 2.21 ppm was ascribed to methyl protons of an acetoxy group at C-6″ (Figure 2). Finally, resonances in the range of δ _H 5.10 to 4.19 ppm were assigned to a glucoside moiety as confirmed by the ¹H-¹H COSY and HMBC correlations (Figure 2). Notably, a large coupling constant of the anomeric proton (H-1″) [δ _H 5.80 (d, J = 9.0 Hz, H-1″) ppm in pyridine-d ₅ or δ _H 5.16 (d, J = 7.2 Hz, H-1″) ppm in dimethyl sulfoxide (DMSO)- d ₆] indicated that the glucoside moiety was a β-anomer. ⁸ The NOESY correlation (H-1″ with H-5) and HMBC correlation of H-1″ (δ _H 5.80) with C-4 (δ _C 152.9) were employed to confirm the location of the glucoside moiety to locate at C-4 of the ellagic acid core (Figure 2). Based on the spectroscopic data described earlier and in comparison with those of the known compound 4, ^9,10 compound 1 was identified as 3,3′,4′-tri-O-methylellagic acid-6″-acetoxy-4-O-β-glucoside.

The known compounds were identified as 3,3′,4′-tri-O-methylellagic acid (2), ¹¹ 3,3′-di-O-methylellagic acid-4-O-β-xyloside (3), ¹⁰ 3,3′,4′-tri-O-methylellagic acid-4-β-O-glucoside (4), ^9,10 friedelin (5), ¹² friedelinol (6), ¹³ methyl-3,4,5-trihydroxybenzoate (7), ¹⁴ 5,7,4′-trihydroxyflavone-8-C-β-glucopyranoside (8), ¹⁵ 5,7,3′,4′-tetrahydroxyflavone-8-C-β-glucopyranoside (9), ¹⁵ and 5,3′,4′-trihydroxyflavone-6-C-β-glucopyranoside (10) ¹⁵ by direct comparison of their physical properties and spectroscopic data with those previously reported in the literature.

All isolated compounds were evaluated for their cytotoxic effects against a panel of mammalian cancer cell lines (Table 2) including P-388, murine lymphocytic leukemia; KB, human oral nasopharyngeal carcinoma; HT-29, human colorectal adenocarcinoma; MCF-7, human breast carcinoma; A-549, human lung carcinoma; ASK, rat glioma cell; CL, Chang liver normal cell, together with anti-HIV1 activities (Table 3) both in syncytium inhibition and RT assays. Compounds 4 and 7 exhibited weak cytotoxic activity with ED₅₀ values in the range of 6.92 to 33.92 µM (Table 2). For anti-HIV1 activities, compounds 1, 2, 4, and 9 were found active in the syncytium inhibition assay. In particular, compound 1 showed significant inhibitory activity by significantly reducing syncytium formation with an ED₅₀ value of 7.9 µM (SI = 5.9) (Table 3). In addition, compounds 8 and 9 were found moderately active against the RT enzyme (Table 3).

Table 2

Cytotoxic Activity of Compounds 1 to 10.

Compounds	Cytotoxic assay; cell line (ED50, µM)a,b,c
	P-388	KB	HT-29	MCF-7	A-549	ASK	CL
1	-	-	-	-	-	-	-
2	-	-	-	-	-	-	-
3	-	-	-	-	-	-	-
4	10.70	29.77	-	33.92	6.92	18.62	29.65
5	-	-	-	-	-	-	-
6	-	-	-	-	-	-	-
7	17.54	-	-	-	28.89	-	21.50
8	-	-	-	-	-	-	-
9	-	-	-	-	-	-	-
10	-	-	-	-	-	-	-
Ellipticine d	1.90	2.15	2.36	2.15	1.91	2.07	1.99

^aEach ED₅₀ value presented in micromolar was obtained from 3 independent experiments which were conducted in triplicates.

^bP-388, murine lymphocytic leukemia; KB, human oral nasopharyngeal carcinoma; HT-29, human colorectal adenocarcinoma; MCF-7, human breast carcinoma; A-549, human lung carcinoma; ASK, rat glioma cell; CL, Chang liver normal cell.

^c“-” denotes ED₅₀ >50 µM.

^dEllipticine was used as a positive control for the cytotoxic assay.

Table 3

Anti-HIV1 Activities of Compounds 1 to10.

Compounds	Cytotoxic and syncytium assays				HIV1-RT assay e
	IC50 (μM) a	EC50 (μM) b	SI c	Activity d	% inhibition at 200 µg/mL	Activity f
1	46.3	7.9	5.9	A	3.9	I
2	12.7	5.5	2.3	A	27.9	I
3	226.7	>250	<0.9	I	17.92	I
4	231.7	103.0	2.2	A	14.2	I
5	>250	>250	−	I	−1.0	I
6	>250	>250	−	I	−13.2	I
7	>250	>250	−	I	25.3	I
8	11.9	16.9	0.7	I	56.9	M
9	71.6	19.4	3.7	A	52.0	M
10	>250	>250	−	I	42.9	W

AZT, azidothymidine; RT, reverse transcriptase.

^aCytotoxic assay: IC₅₀ = dose of compound that inhibited 50% metabolic activity of uninfected 1A2 cells. AZT, averaged from 3 experiments, IC₅₀ >10⁻² µM (less than 50% inhibition at this concentration).

^bEC₅₀ = dose of compound that reduced syncytium formation by ^ΔTat/RevMC99 virus in 1A2 cells by 50%. AZT, averaged from 3 experiments, EC₅₀ 3.95 × 10⁻³ µM.

^cSI, selectivity index: IC₅₀/EC₅₀.

^dActivity: A, active (SI >1); I, inactive (SI <1).

^eRT assay: Compounds were prescreened at 200 µg/mL.

^fActivity: I = inactive; M = moderate; W = weak. Nevirapine, positive control, averaged from 2 experiments, 6.8 µM.

Experimental

General

Melting points were recorded in degree Celsius and were measured on an electrothermal melting point apparatus and are uncorrected. Optical rotations were determined on a JASCO DIP-370 digital polarimeter using a 50-mm microcell. Infrared spectra (FT-IR) were recorded using Perkin Elmer System 2000 FT-IR spectrophotometer and major bands (ν _max) were recorded in wave number (cm⁻¹). UV absorption spectra were recorded by using Melton Roy Spectronic 3000 Array and JASCO UNIDE-650 spectrophotometers. Principal bands (λ _max) were recorded as wavelength (nanometer) and log ε in ethanol solution. NMR spectra were recorded on a Bruker Ascend^TM 400 spectrometer (at 400 MHz for ¹H and 100 MHz for ¹³C) in deuterated chloroform (CDCl₃), DMSO- d ₆, and pyridine (pyridine-d ₅) solutions using tetramethylsilane or residual nondeuterated solvent peak as an internal standard. Mass spectra were recorded on a Finnigan Mat INCOS 50 mass spectrometer. Solvents for extraction, chromatography, and crystallization were distilled at their boiling point ranges prior to use. Precoated thin-layer chromatography (TLC) aluminum sheets of silica gel 60 PF₂₅₄ (Merck) were used for analytical purposes and the compounds were visualized under UV light (254 and/or 366 nm) and/or spraying 12% H₂SO₄ in ethanol and/or anisaldehyde spraying reagents. Plates of silica gel F₂₅₄ (Merck, 5-40 μm) were activated at 120°C for 2 hours, prior to use, in the case of preparative TLC technique. Bands were visualized by UV light either at 254 and/or 366 nm. Column chromatography was performed by using silica gel 60 (Merck, 60-200 mm or 70-230 mesh American Standard Test Sieve Series).

Plant Materials

Leaves and twigs of I. malayana were collected from Nakornayok, Thailand, and identified by one of us (N.N.). A voucher specimen (BKF 057918) was deposited at the Herbarium, Royal Forest Department, Bangkok, Thailand.

Extraction and Isolation

The air-dried and finely powdered twigs (2.3 kg) and leaves (8.3 kg) of I. malayana were separately and successively percolated with hexanes, chloroform, and methanol (5 × 8 L for each solvent). After filtration and removal of the solvents from each extract by rotary evaporator under reduced pressure, the crude extracts were obtained; from twigs: hexanes extract (12.7 g), chloroform extract (6.2 g), and methanol extract (325.2 g) and from leaves: hexanes extract (103.4 g), chloroform extract (102.4 g), and methanol extract (1200.1 g).

The hexanes extract from twigs (12.7 g) was purified by preparative TLC, eluted with chloroform-hexanes (1:1 v/v) to give 2 compounds, friedeline (5) (119.5 mg) and friedelinol (6) (213.0 mg), respectively. Because of the paucity of the quantity together with a very complex TLC pattern of the chloroform extract, no further investigation on the material was carried out. The crude methanol extract from the twigs (325.2 g) was suspended with water (2 L) and was sequentially partitioned with EtOAc (10 L) and n-BuOH (6 L) to give EtOAc (75.8 g) and n-BuOH (103.9 g) fractions, respectively, after removal of organic solvents on a rotary evaporator at reduced pressure. Moreover, the two extracts were freeze-dried to remove trace of solvents. The EtOAc fraction was purified by using silica gel column chromatography (SiO₂-CC), eluted with gradient proportions of hexanes-EtOAc (0:1 to 1:0 v/v), then with gradient proportions of MeOH-EtOAc (0:1 to 1:0 v/v). The solvents were evaporated to dryness to afford 29 subfractions (A1-A29). The combined subfractions A12 to A14 (6.01 g), combined on the basis of TLC characteristics, were further separated by SiO₂-CC, eluted with gradient proportions of CHCl₃:MeOH:H₂O (200:3:1 v/v to 20:3:1 v/v). Fractions (1 L each) were collected and combined on the basis of their TLC characteristics to give five subfractions (B1-B5). Subfractions B2 (441.7 mg) and B4 (4.17 g) were combined and were further purified by preparative TLC, eluted with CHCl₃:MeOH:H₂O (40:3:1 v/v) to yield compound 2 (17.1 mg) and compound 7 (1.01 g). Subfraction A23 (3.72 g) was further purified by SiO₂-CC, eluted with CHCl₃:MeOH:H₂O (50:3:1 v/v) to give two ellagic glycosides 1 (32.8 mg) and 3 (6.8 mg) after recrystallization. Furthermore, the n-BuOH fraction (90.0 g) partitioned from methanol extract of twigs part was chromatographed on SiO₂-CC, eluted with gradient proportions of CHCl₃:MeOH:H₂O (50:3:1 v/v to 6:4:1 v/v). Fractions (1 L each) were collected and combined on the basis of their TLC characteristics to give 12 subfractions (C1-C12). Subfractions C2 (34.1 mg) and C6 (322.0 mg) were identified to be compounds 2 and 4, respectively. The ¹H NMR spectra of the hexanes extract from the leaves showed mainly fats, therefore it was not further investigated. The chloroform fraction was deeply green. Its TLC pattern and NMR spectrum were very complex and no tangible signals could be identified; consequently, no further fractionation was carried out. A portion of the sequential methanol extracts from the leaves (400 g) was further separated by using SiO₂-CC, eluted with gradient proportions of EtOAc-hexanes (0:1 v/v to 1:0 v/v), then with gradient proportions of MeOH-EtOAc (0:1 v/v to 1:0 v/v), and finally with MeOH. Fractions (1 L each) were collected and combined on the basis of their TLC characteristics to afford 19 subfractions (D1-D19). Based on similar patterns on their TLC characteristics, subfractions D11, D12, and D13 were combined (25 g) and were further separated by SiO₂-CC, eluted with CHCl₃, followed by gradient proportions of CHCl₃:MeOH:H₂O (100:3:1 v/v to 7:3:1 v/v), and finally with MeOH. Fractions (1 L each) were collected and combined on the basis of their TLC characteristics to yield 14 subfractions (E1-E14). Subfraction E5 was characterized to be compound 1 (34.0 mg), after recrystallization from MeOH:H₂O. Subfraction E7 (75.5 mg) was recrystallized from a CHCl₃:MeOH:H₂O mixture to yield a white powder of compound 3 (49.1 mg). Compound 8 (47.0 mg) and compound 9 (6.7 mg) were obtained from subfractions E10 and E12, respectively, after recrystallization with the mixture of MeOH:H₂O. Subfraction D15 (25.0 g) was further purified by SiO₂-CC, eluted with CHCl₃:MeOH:H₂O (30:3:1 v/v) to give 14 subfractions (F1-F14). Compound 10 (449.9 mg) was obtained from subfractions F7 and F8, after recrystallization from the mixture of CHCl₃:MeOH:H₂O. Subfraction D16 was subjected to SiO₂-CC, eluted with CHCl₃ followed by the gradient proportion of CHCl₃:MeOH:H₂O (100:3:1 v/v to 7:3:1 v/v) and finally with MeOH. Nine subfractions (G1-G9) were obtained after removal of the solvent. Subfractions G4 (158.0 mg) and G8 (1.38 g) were recrystallized to provide compounds 4 (68.4 mg) and 9 (38.9 mg), respectively.

3,3′,4′-Tri-O-Methylellagic Acid-6′′-Acetoxy-4-O-β-Glucoside

M.p.: 272.6°C to 273.8°C (MeOH-H₂O)

[α]²⁶ _D : +77.9 (c 1.00, C₅H₅N)

R _f: 0.27 (CHCl₃:MeOH:H₂O, 30:3:1 v/v)

IR (nujol): 3455, 2901, 1743, 1606, and 1572 cm⁻¹

UV/Vis λ _max (EtOH) nm (log ε): 237 (3.75), 349 (3.11), 366 (2.96)

¹H NMR (400 MHz, pyridine-d ₅ and DMSO- d ₆): Table 1

¹³C NMR (100 MHz, pyridine-d ₅ and DMSO- d ₆): Table 1

MS (EI, 70 eV): m/z (%) =344 ([M+H-glucoside⁺], 17), 329, 302, 255, 189, 145, 105, 83, 79

HRMS-ESI-TOF: m/z [M+H⁺] calcd for C₂₅H₂₅O₁₄: 549.1244; found 549.1255

Cytotoxic Assay

The cytotoxic activities of the crude extracts and the isolated compounds were determined by using the standard in vitro sulforhodamine B assay in 96-well microtiter plates. ^16,17 Ellipticine, a potent cytotoxic plant alkaloid, was used as a positive control. Seven cell lines were employed, including P-388, murine lymphocytic leukemia; KB, human oral nasopharyngeal carcinoma; HT-29, human colorectal adenocarcinoma; MCF-7, human breast carcinoma; A-549, human lung carcinoma; ASK, rat glioma cell; and CL, Chang liver normal cell. The potency for cytotoxic activity was expressed as 50% effective dose (ED₅₀).

The Syncytium Assay

Cell-based assay using ^ΔTat/RevMC99 virus and 1A2 cell line system was used. ¹⁸ The experiment was carried out in triplicate, starting at the final concentration of 3.9 to 125 µg/mL of compound. Virus control and cell control wells contained neither the extract nor the virus; cytotoxicity control wells containing cells with the extract/compound and a positive control, that is, azidothymidine were included. The result was expressed as 50% effective concentration (EC₅₀). Colorimetric cytotoxicity assay using XTT tetrazolium salt and phenazine methosulfate, made as described ¹⁹ , was also performed in parallel. The procedure was similar to the syncytium assay but the virus was replaced by the medium and tested in duplicate. Control wells included medium, drug, and cell controls. After the soluble formazan developed, the optical density at A450 was measured with a reference at A650. The result was expressed as 50% inhibitory concentration (IC₅₀).

Anti-HIV 1 RT Assay

Samples were prepared in DMSO to obtain a final concentration of 0.2 mg/mL. The assay was performed according to the method previously described. ²⁰ In a 96-well plate, a standardized amount of HIV1-RT (Amersham Pharmacia Biotech Asia Pacific Ltd., Hong Kong) and the sample (0.2 mg/mL) were added to the reaction mixture containing radio-labeled thymidine triphosphate ([³H]TTP) and polyadenylic acid (polyA) as an RT substrate and RNA template, respectively. HIV1-RT activity was detected by measuring the incorporation of [³H]TTP to the polyA template. Positive controls included fagaronine chloride and nevirapine, known RT inhibitors, in place of the tested sample, whereas negative controls were absent of tested samples. RT enzyme was standardized with fagaronine chloride. The experiments were done in duplicate. The data were averaged, and percent inhibition compared with the negative control was calculated. For samples with greater than 70% inhibition, their 50% IC₅₀ were further determined by testing RT activities in the presence of various concentrations of the samples. The IC₅₀ values were then estimated from the dose-response curves.