Meliglabrin, A New Flavonol Derivative from the leaves of Melicope glabra (Blume) T.G. Hartley

Ratih Dewi Saputri; Tjitjik Srie Tjahjandarie; Mulyadi Tanjung

doi:10.20307/nps.2018.24.3.155

Abstract

A new flavonol derivative, meliglabrin (1) along with three known flavonols, ternatin (2), meliternatin (3), and 5,4′-dihydroxy-3,7,3′-trimethoxyflavon (4) were isolated from the leaves of Melicope glabra (Blume) T.G. Hartley. Their structures were determined using extensive spectroscopic methods, including UV, IR, HRESIMS, 1D and 2D NMR. Compounds 1 – 4 were evaluated for their cytotoxicity against murine leukemia P-388 cells, compound 4 showed moderate activity.

Introduction

Melicope glabra (Blume) T.G. Hartley locally known as ‘Ki Sampang’ belongs to the Rutaceae family found in all of Indonesia Island. The aqueous decoction of leaves of M. glabra are used in Indonesia as traditional medicine for the treatment of fever, infections, and cough.1 The Melicope genus has been shown to be prolific a number of secondary metabolites, particularly alkaloids,2 3 flavonoids,4 5 coumarins6 7 and showed biological activities such as anticancer, antifungal and antioxidant.

The phytochemical survey from the bark of M. glabra were isolated coumarins and lignan but the leaves until now has not been reported.6 In this paper, we wish to report the isolation and structural elucidation of a new flavonol, meliglabrin (1) along with three known compounds, ternatin (2), meliternatin (3), and 5,4′-dihydroxy-3,7,3′-trimethoxyflavon (4) from the leaves of M. glabra. The cytotoxic activity of compounds 1 – 4 against murine leukemia P-388 cells from this plant are also reported.

Experimental

General experimental procedures

UV spectra were measured with a Shimadzu 1800 spectrometer, FTIR spectrum One Perkin-Elmer instrument, respectively. ¹H and ¹³C NMR spectra were recorded with a JEOL ECA 400 spectrometer operating at 400 (¹H) and 100 (¹³C) MHz in CDCl₃. Mass spectra were measured on an ESI-TOF Waters LCT Premier XE producing pseudo-molecular ions, [M-H]⁻ negative ion mode. Vacuum liquid chromatography (VLC) and planar radial chromatography were carried out using Si gel 60 GF₂₅₄ and Si gel 60 PF₂₅₄, for TLC analysis, pre-coated silica gel plates (Merck Kieselgel 60 GF₂₅₄, 0.25 mm thickness) were used.

Plant materials

The leaves of M. glabra were collected in March 2017 from Gunung Salak, Bogor, West Java, Indonesia. The plant material was identified by Mr. Ismail Rachman from the Herbarium Bogoriense, Bogor. A voucher specimen (PL 60325) was deposited in Herbarium Bogoriense, Center of Biological Research and Development, National Institute of Science, Bogor, Indonesia.

Extraction and isolation

The powdered and dried leaves of M. glabra (1.7 kg) were macerated in methanol at room temperature two times and, after evaporation of the methanol extract, gave a dark residue (210 g). The extract was redissolved in MeOH-water (9:1) and partitioned with n-hexane (95 g) and ethyl acetate (30 g) fractions. The ethylacetate extract (29 g) was further fractionated by vacuum liquid chromatography on silica gel (150 g) eluted with n-hexane-ethyl acetate of increasing polarity (9:1, 4:1; 7:3, 1:1, and 1:4) to give three major fractions A-C. Fraction A (4.68 g) was separated by column chromatography eluted with n-hexane-ethyl acetate (9:1 to 7:3) to produce subfractions A₁–A₃. Subfraction A₁ was purified by planar radial chromatography using n-hexane-CHCl₃ (from 4:1 to 1:4) to yield compound 1 (20 mg), 3 (15 mg), and 4 (23 mg). Fraction B (13 g) was refractionated using column chromatography and eluted n-hexane- ethyl acetate (from 8:2 to 3:7) to produce subfractions B₁–B₂. Subfraction B₁ was purified by planar radial chromatography using n-hexane-acetone (from 9:1 to 1:1) to yield compound 2 (9 mg).

Meliglabrin (1)

Yellow solid, mp. 119 – 121 ℃, UV (MeOH) λ_max nm (log ε) : 245 (4.20), 255 (3.99), 296 (4.01) and 344 (4.20). IR (KBr) νmax cm⁻¹: 3421, 1645, 1560, 1481 and 1132. ¹H and ¹³C NMR see Table 1. HRESIMS: m/z [M-H]⁻ calcd. for C₁₈H₁₃O₈ 357.0610, found 357.0613.

Ternatin (2)

Yellow solid. ¹H NMR (CDCl₃ 400 MHz): δ_H 12.44 (1H, s, 5-OH), 7.79 (1H, dd, J = 9.1, 2.0 Hz, H-6′), 7.78 (1H, d, J = 2.0 Hz, H-2′), 7.06 (1H, d, J = 9.1 Hz, H-5′), 6.42 (1H, s, H-6), 6.01 (1H, s, 4′-OH), 3.98 (3H, s, 3′-OCH₃), 3.94 (3H, s, 7-OCH₃), 3.92 (3H, s, 8-OCH₃), 3.87 (3H, s, 3-OCH₃). ¹³C NMR (CDCl₃ 100 MHz): δ_C 179.1 (C-4), 158.4 (C-7), 157.4 (C-5), 155.8 (C-2), 148.5 (C-8a/4′), 146.4 (C-3′), 139.4 (C-3), 122.9 (C-6′), 128.8 (C-8), 122.7 (C-1′), 114.8 (C-5′), 110.8 (C-2′), 105.4 (C-4a), 95.5 (C-6), 61.7 (8-OCH₃), 60.2 (3-OCH₃), 56.5 (7-OCH₃), 56.1 (3′-OCH₃). HRESIMS: m/z [M-H]⁻ calcd. for C₁₉H₁₇O₈ 373.1916, found 373.1912. The ¹H and ¹³C NMR spectral data are consistent with published data.8

Meliternatin (3)

Pale white solid, mp. 167 – 169 ℃. UV (MeOH) λ_max nm (log ε) : 247 (4.22), 270 (4.08) and 336 (4.34). IR (KBr) ν_max cm⁻¹: 1641, 1524, 1479 and 1114. ¹H NMR (CDCl₃ 400 MHz): δ_H 7.63 (1H, dd, J = 8.4, 1.8 Hz, H-6′), 7.56 (1H, d, J = 1.8 Hz, H-2′), 6.91 (1H, d, J = 8.4 Hz, H-5′), 6.65 (1H, s, H-8), 6.05 (2H, s, 3′,4′-OCH₂-O), 6.04 (2H, s, 6,7-OCH₂-O), 4.12 (3H, s, 5-OCH₃), 3.86 (3H, s, 3-OCH₃). ¹³C NMR (CDCl₃ 100 MHz): δ_C 174.0 (C-4), 153.7 (C-7), 153.7 (C-8a), 153.0 (C-4′), 152.6 (C-2), 149.4 (C-3′), 141.1 (C-5), 140.8 (C-3), 134.8 (C-6), 124.5 (C-1′), 123.1 (C-6′), 113.1 (C-4a), 108.5 (C-5′; 3′,4′-OCH₂-O), 108.4 (C-2′; 6,7-OCH₂-O), 93.0 (C-8), 61.3 (5-OCH₃), 59.9 (3-OCH₃). ). HRESIMS: m/z [M-H]⁻ calcd. for C₁₈H₁₃O₈ 357.0610, found 357.0613. The ¹H and ¹³C NMR spectral data are consistent with published data.9

5,4′-Dihydroxy-3,7,3′-trimethoxyflavon (4)

Yellow solid, ¹H NMR (acetone-d₆, 400 MHz): δ_H 12.72 (1H, s, 5-OH), 8.64 (1H, s, 4′-OH), 7.75 (1H, d, J = 2.0 Hz, H-2′), 7.67 (1H, dd, J = 8.4, 2.0 Hz, H-6′), 6.97 (1H, d, J = 8.4 Hz, H-5′), 6.62 (1H, d, J = 2.4 Hz, H-8), 6.27 (1H, d, J = 2.4 Hz, H-6), 3.91 (3H, s, 3′-OCH₃), 3.87 (3H, s, 7-OCH₃), 3.86 (3H, s, 3-OCH₃). ¹³C NMR (acetone-d₆, 100 MHz): δ_C 179.5 (C-4), 166.5 (C-7), 162.7 (C-5), 157.6 (C-8a), 156.8 (C-2), 150.5 (C-4′), 148.2 (C-3′), 139.3 (C-3), 123.3 (C-6′), 122.6 (C-1′), 116.1 (C-5′), 112.5 (C-2′), 106.4 (C-4a), 98.4 (C-6), 92.8 (C-8), 61.3 (3′-OCH₃), 56.4 (7-OCH₃), 56.5 (3-OCH₃). HRESIMS: m/z [M-H]⁻ calcd. for C₁₈H₁₇O₇ 344.0896, found 344.0900. The ¹H and ¹³C NMR spectral data are consistent with published data.10

Cytotoxic activity

All isolated compounds (1 – 4) were subjected to cytotoxic evaluation against murine leukemia P-388 cells according to the MTT method with artonin E as the positive control.11 12 The P-388 cells were seeded into each 96-well cell culture plate at a density of 3 × 10⁴ cells/well and incubated at 37 ℃ for 48 h. The number of cells that inhibited by each of compounds 1 – 4 were measured using microplate reader spectrometer at λ 540 nm after incubation for 24 hours in CO₂ incubator at 37 ℃. All of isolated compounds by variations in concentration of 1000; 100; 30; 10; 3; 1; 0.3 and 0.1 µg/mL with triplicate treatment tested on cell cultures murine leukemia P-388. The IC₅₀ value₅₀ can be calculated through extrapolation 50% absorption lines to various concentrations of each compound using regression analysis.

Result and Discussion

Compound (1) was isolated as yellow solid, mp. 119 – 121 ℃. The HRESIMS displayed a negative molecular ion peak [M-H]⁻ at m/z 357.0613 (calcd. 357.0610) indicating a molecular formula of C₁₈H₁₄O₈. The UV maximum absorption at λ_max 245 (4.20), 255 (3.99), 296 (4.01) and 344 (4.20) nm typical for a flavonol chromophore.12 The IR spectrum indicated absorptions for hydroxyl (3421 cm⁻¹), conjugated carbonyl (1645 cm⁻¹), aromatic (1560 – 1481 cm⁻¹) and ether (1132 cm⁻¹) groups, respectively13. The ¹H NMR (Table 1) spectrum of 1 showed an ABX system at δ_H 7.68 (1H, dd, J = 8.4; 1.8 Hz, H-6′), 7.59 (1H, d, J = 1.8 Hz, H-2′), 6.95 (1H, d, J = 8.4 Hz, H-5′), and a singlet at δ_H 6.54 (1H, s, H-8) in the aromatic region. The ¹H NMR spectrum of 1 also showed a chelated hydroxyl group at δ_H 12.88 (1H, s, 5-OH), a hydroxyl signals at δ_H 6.50 (1H, s, 7-OH), two methoxyls at δ_H 4.04 (3H, s, 6-OCH₃), 3.85 (3H, s, 3-OCH₃), and a methylenedioxy at δ_H 6.08 (2H, s, 3′,4′-OCH₂-O). Eighteen carbon signals were observed by ¹³C NMR spectrum. Two of them signals at δ_C 138.2 and δ_C 179.3 are characteristic for C-3 and C-4 of a flavonol structure.12 The placement of hydroxyl, methoxyl and methylenedioxy groups in flavonol structure was established by HMQC and HMBC spectra (Fig. 2). The proton signal of a chelated hydroxyl group (δ_H 12.33, 5-OH) correlated with three quaternary carbons [δ_C 151.8 (C-5); 130.1 (C-6); 106.3 (C-4a)]. The proton signal of methoxyl group at δ_H 4.04 correlated to δ_C 130.1 (C-6) showing that a methoxyl group was placed at C-6. A hydroxyl proton signal at δ_H 6.50 (7-OH) correlated with one quaternary carbon signal δ_C 155.1 (C-7), and one methine carbon signal δ_C 93.2 (C-8) indicating that a hydroxyl group was placed at C-7. The aromatic proton signal (δ_H 6.54, H-8) showed long-range correlations with four quaternary carbons [δ_C 155.1 (C-7), 153.9 (C-8a)], 130.1 (C-6); 106.3 (C-4a)]. In the ¹H NMR spectrum, proton signal of an ABX system in the aromatic region at ring B indicated a methylenedioxy group fused at C-3′ and C-4′. Therefore, another methoxyl group was placed at C-3. The proton signal of methoxyl group at δ_H 3.85 correlated to δ_C 138.2 showing that a methoxyl group was placed at C-3. The proton signal of a methylenedioxy group (δ_H 6.08, 3′,4′-OCH₂-O-) showed long-range correlations with two quaternary carbons at δ_C 149.7 (C-3′) and δ_C 150.0 (C-4′). One of aromatic proton signal of ABX system (δ_H 7.59, H-2′) showed correlations with two quaternary carbons δ_C 155.8 (C-2), 150.0 (C-4′), and one methine carbon signal δ_C 123.8 (C-6′). The aromatic proton signal (δ_H 6.95, H-5′) showed correlations with two quaternary carbons δ_C 124.2 (C-1′), and 149.7 (C-3′). Furthermore, the aromatic proton signal (δ_H 7.68, H-6′) showed correlations with two quaternary carbons δ_C 155.8 (C-2), 150.0 (C-4′), and one methine carbon signal δ_C 108.7 (C-2′). From these NMR data analysis, meliglabrin (1) was assigned as 5,7-dihydroxy-3,6-dimethoxy-3′,4′-methylenedioxyflavone. Other HMBC correlations consistent with the structure 1 are shown in Table 1 and Fig. 2.

The isolated compounds 1 – 4 were assessed for their anticancer activity against murine leukemia P-388 cells. The result of anticancer activity are presented in Table 2, showing their IC₅₀ were 48.30, 15.98, 30.04, and 5.02 µg/ mL, respectively (artonin E as a positive control, IC₅₀ 1.33 µg/mL). These anticancer activity data suggested that the compounds 1 – 3 were inactive and compound 4 showed moderate activity. The hydroxy group at C-4′ and methoxy group at C-3′ (compounds 2 and 4) enhances activity than methylenedioxy group at C-3′ and C-4′ (compounds 1 and 3). The same thing, the structureactivity relationship of flavonol from M. triphylla, the presence of hydroxy group at C-4′ and methoxy group at C-3′ showed moderate activity against murine leukemia P-388 cells.14 The presence of methoxy group at C-8 in compound 2 decreases anticancer activity compared to compound 4.