A new havanensin-type limonoid from Chisocheton macrophyllus

Abstract

A new havanensin-type limonoid, 16β-hydroxydysobinin (1), along with four known limonoids (2–5), have been isolated from the seeds of Chisocheton macrophyllus. The chemical structure of the new compound was determined by referencing spectroscopic data, and by comparison to those related spectra previously reported. Each compound was evaluated for their cytotoxic effects against Michigan Cancer Foundation-7 (MCF-7) breast cancer cells and display no significant activity.

Introduction

Limonoids, known as degraded triterpenes, are derived from a precursor with a 4,4,8-trimethyl-17-furanylsteroid four-ring skeleton labelled as A, B, C and D rings [1]. Limonoids are a class of secondary metabolites found in the order Rutales and the Meliaceae and Rutaceae family [2]. Meliaceae is a family of timber trees that are a rich source for limonoids and are widely distributed in tropical and subtropical regions with 50 genera and more than 1400 species [3, 4]. Limonoids isolated from species of the family Meliaceae have been reported to have biologically activity as antifeedant, antimicrobial, antimalarial, and cytotoxins [5,6,7,8]. Genus Chisocheton is the second largest in the family Meliaceae, consisting of more than 50 species distributed across Nepal, India, Bhutan, Myanmar, South China, Thailand, Indonesia, Malaysia, and Papua New Guinea [7, 9].

Chisocheton macrophyllus is a species distributed in the Nicobar Islands, peninsular Thailand, peninsular Malaysia, Singapore, Sumatera, Anambas Islands, Java and Borneo Islands [10]. Its seeds have been reported to yield bioactive limonoids such as dysobinol, 7α-hydroxyneotricilenone, dysobinin and nimonol with cytotoxic activity against P-388 murine leukemia cells [11], whereas the leaves to yield Epstein-Barr virus activation of Triterpenoids [12]. After further investigations for cytototoxic limonoids from the seeds of C. macrophyllus, we found and structural elucidation of a new havenensin-type limonoids (1) and four known limonoids (2–5), along with their cytotoxic activity against MCF-7 breast cancer cells. Herein, the isolation, structural elucidation and cytotoxic activity against MCF-7 breast cancer cells are discussed.

Materials and methods

Plant materials

Seeds of C. macrophyllus were collected from Bogor Botanical Garden, Bogor, West Java Province, Indonesia. The plant was identified by Mr. Harto, the staff of Bogoriense Herbarium, Research Center for Biology, Indonesia Science Institute, Bogor, Indonesia and a voucher specimen (No. Bo-1295453) was deposited at the Herbarium.

Instruments and reagents

Optical rotations were measured on a Perkin Elmer 341 Polarimeter (Waltham, MA, USA). UV spectra was measured using a TECAN Infinite M200 pro with MeOH. Furthermore, the IR spectra and mass spectra were recorded on a One PerkinElmer spectrum-100 FT-IR in KBr and Waters Xevo QTOF MS, respectively. NMR spectra were obtained with Bruker Topspin at 500 MHz for ¹H and 125 MHz for ¹³C (compound 1) and for compounds 2–5 using JEOL JNM-ECZ500R/S1 at 500 MHz for ¹H and 125 MHz for ¹³C, using tetra methylsilane (TMS) as the internal standard. Chromatographic separations were carried out on the silica gel 60 (70–230 and 230–400 mesh, Merck). Thin layer chromatography (TLC) analysis was carried out on 60 GF₂₅₄ (Merck, 0.25 mm) using various solvent systems, and measured by irradiation under ultraviolet–visible light Vilber Lourmat (λ 254 nm dan 365 nm) followed by heating of silica gel plates, sprayed with 10% H₂SO₄ in ethanol and Ehrlich’s reagent (p-Dimethylaminobenzaldehyde in ethanol).

Extraction and isolation of C. macrophyllus

The dried and powdered seeds of C. macrophyllus (2.5 kg) were extracted with methanol at room temperature for 3 days (3 × 5 L). After removal of the solvent under a vacuum, a total of 360 g of methanol extract was obtained and partitioned with n-hexane (3 × 3 L), ethyl acetate (3 × 2 L) and n-butanol (3 × 2 L). Evaporation resulted in crude extracts of n-hexane (146.6 g), ethyl acetate (60.8 g) and n-butanol (14.6 g) respectively. The n-hexane soluble fraction (140 g) was subjected to vacuum-liquid chromatography (VLC) column packed with silica gel 60 using a gradient of n-hexane, ethyl acetate and methanol (10% stepwise) to afford thirteen fractions (A-M). Fraction D (5.4 g) was subjected to silica gel column chromatography using a gradient of n-hexane and ethyl acetate (5% stepwise) as eluting solvent to afford five subfractions (D1-D5). Subfraction D2 (165.7 mg) was chromatographed on a column of silica gel eluted with n-hexane: dichloromethane: ethyl acetate (2:7.5:0.5) to give 1 (15.3 mg). Fraction F (4.4 g) was subjected to a silica gel column chromatography using a gradient of n-hexane and ethyl acetate (5% stepwise) as eluting solvent to afford twelve subfractions (F1-F12). Subfraction F5 (1.2 g) was chromatographed on a column of silica gel eluted with n-hexane: dichloromethane: ethyl acetate (2:7.5:0.5) to give 3 (19.7 mg) and four subfractions (F5A-F5D). Furthermore, subfraction F5D (308.3 mg) was chromatographed on a column of silica gel eluted with n-hexane: dichloromethane: ethyl acetate (1:8.5:0.5) to give 2 (12.8 mg). Fraction H (1.8 g) was subjected to a silica gel column chromatography using a gradient of n-hexane and ethyl acetate (5% stepwise) as eluting solvent to give 5 (12.0 mg). Fraction J (1.5 g) was subjected to a silica gel column chromatography using a gradient of n-hexane and ethyl acetate (5% stepwise) to afford nineteen subfractions (J1-J19). Subfraction J9 (50.3 mg) was chromatographed on a column of silica gel eluted with n-hexane: dichloromethane: ethyl acetate (4:5.5:0.5) to give 4 (3.0 mg).

16β-hydroxydysobinin (1): Colorless needle crystals; mp: 205–207 °C; [$\alpha$]²⁷_D $+1$22.5° (c 0.2, MeOH); UV (MeOH) λ_max 284 nm; IR (KBr) v_max 3509, 2929, 1744, 1670, 1502, 1366, 1386, 1248 cm⁻¹; HR-TOFMS m/z 511.2634 [M + H]⁺, (calcd. for C₃₀H₃₉O₇ m/z 511.2696); ¹H-NMR (CDCl₃, 500 MHz) and ¹³C-NMR (CDCl₃, 125 MHz) see Table 1.

Table 1 NMR spectral data for 1 (500 MHz for ¹H and 125 MHz for ¹³C in CDCl₃)

Cytotoxic activity test

The cytotoxicity of compounds 1–5 was determined with a cell viability test using PrestoBlue® assay. The cells were maintained in a Roswell Park Memorial Institute (RPMI) medium with 10% (v/v) Fetal Bovine Serum (FBS) and 1 $\upmu$L/1 mL antibiotics (1% Penicillin–Streptomycin). Cultures were incubated at 37 °C in a humidified atmosphere of 5% CO₂. MCF-7 cells plated in 96 multiwell culture plates at a density of 1.7 $\times$ 10⁴ cells/well. After twenty-four hours, the medium was discarded and fresh medium containing sample with different concentrations 7.81, 15.63, 31.25, 62.50, 125.00, 250.00, 500.00, 1000.00 μg/mL and control was added. After incubation with the sample for 24 h, PrestoBlue® reagent (resazurin dye) was added. The PrestoBlue® assay results were read using a multimode reader at 570 nm. The IC₅₀ values were determined by linier regression method using Microsoft Excel software. The IC₅₀ value corresponds to the concentration of compounds that decreases by 50% the number of viable cells and the absorbance in control corresponds to 100% viability.

Results and discussion

The n-hexane fraction from the seeds of C. macrophyllus was subjected to vacuum-liquid chromatography (VLC) column packed with silica gel 60 by gradient elution. The VLC fractions were repeatedly subjected to normal phase column chromatography on silica gel to yield compounds 1–5 (Fig. 1).

Fig. 1

Structures of compounds 1–5

Compound 1 was isolated as colorless needle crystals. The molecular formula was determined to be C₃₀H₃₉O₇ based on the high resolution time-of-flight mass spectrometry (HR-TOFMS) spectra (Additional file 1; Fig. S8) at m/z 511.2634 [M + H]⁺ (calcd. for C₃₀H₃₉O₇ m/z 511.2696) and nuclear magnetic resonance (NMR) data (Table 1), indicating the presence of twelve degrees of unsaturation. The ultraviolet (UV) spectrum showed maximum absorption at 284 nm, indicating the presence of an $\alpha ,\beta$-unsaturated ketone [13, 14]. Infrared (IR) absorptions spectra suggested the presence of hydroxyl (3509 cm⁻¹), aliphatic (2929 cm⁻¹), carbonyl ester (1744 cm⁻¹), $\alpha ,\beta$-unsaturated carbonyl (1670 cm⁻¹), olefinic (1502 cm⁻¹), gem-dimethyl (1366 and 1386 cm⁻¹) and ether groups (1248 cm⁻¹).

The ¹H-NMR spectrum (Additional file 1; Fig. S1) showed five tertiary methyls at δ_H 0.95 (3H, s, Me-28), 1.20 (3H, s, Me-29), 1.28 (3H, s, Me-19), and 1.33 (6H, s, Me-18 and Me-30) as well as two acetoxyl groups at δ_H 2.04 (3H, s, Me-1′) and 2.07 (3H, s, Me-1″). In addition, three oxygenated protons δ_H 5.46 (1H, m, H-6), 5.41 (1H, d, J = 2.6 Hz, H-7) and 4.49 (1H, t, J = 7.7 Hz, H-16), a β-furan moiety δ_H 6.30 (1H, d, J = 1.45 Hz, H-22), 7.28 (1H, s, H-21), and 7.40 (1H, d, J = 1.45 Hz, H-23) and three olefinic protons at δ_H 5.48 (1H, d, J = 7.7 Hz, H-15), 5.88 (1H, d, J = 10.5 Hz, H-2) and 8.32 (1H, d, J = 10.5 Hz, H-1) were also observed in the ¹H-NMR spectrum. The ¹³C NMR (Additional file 1; Fig. S2) along with distortions enhancement by polarization transfer (DEPT) (Additional file 1; Fig. S3) and heteronuclear single quantum coherence (HSQC) spectra (Additional file 1; Fig. S4) showed thirty carbons consisting of an $\alpha$,$\beta$-unsaturated carbonyl at δ_C 204.4 (C-3), two acetoxyl groups at δ_C 21.3 (C-1′), 170.1 (C-2′), 22.0 (C-1″) and 170.3 (C-2″) and five methyls at δ_C 20.5 (Me-28), 20.6 (Me-29), 20.9 (Me-30), 28.7 (Me-18) and 31.6 (Me-19). The spectra also showed two methylene carbons at δ_C 34.3 (C-11) and 46.4 (C-12), three sp³ methine carbons at δ_C 45.4 (C-9), 47.5 (C-5) and 51.2 (C-17), four sp² methine carbons at δ_C 110.9 (C-22), 119.3 (C-15), 124.5 (C-2) and 158.1 (C-1), three oxygenated sp³ methine carbons at δ_C 67.2 (C-16), 69.7 (C-6) and 74.0 (C-7), two oxygenated sp² methine carbons at δ_C 139.7 (C-21) and 142.7 (C-23), four sp³ quaternary carbons at δ_C 41.6 (C-8), 42.7 (C-10), 45.0 (C-4), and 46.8 (C-13) and two sp² quaternary carbons at δ_C 124.0 (C-20) and 160.6 (C-14). These functionalities accounted for seven out of the twelve degrees of unsaturation, while the remaining five degrees of unsaturation corresponded to the pentacyclic limonoid structure [6, 11, 15, 16]. The NMR spectra data of 1 resembled those of previously reported dysobinin [16, 17], except for the appearance of oxygenated signals [δ_H 4.49 (1H, t, J = 7.7 Hz), δ_C 67.2], thus suggesting that 1 was a hydroxyl analog of dysobinin. Position of the hydroxyl group at C-16 was determined through the ¹H-¹H correlated spectroscopy (¹H-¹H COSY) and proton multiple bond connectivity (HMBC) experiments (Fig. 2, Additional file 1; Fig. S5 and Fig. S6). Correlations from methyl protons at δ_H 1.33 (CH₃-18) to δ_C 51.2 (C-17), oxygenated sp³ methine at δ_H 4.49 (H-16) to δ_C 124.0 (C-20) and methyne proton δ_H 2.82 (H-17) to δ_C 139.7 (C-21) and δ_C 110.9 (C-22) were used to assign the hydroxyl group and a furan ring attached at C-16 and C-17, respectively.

Fig. 2

Selected HMBC and ¹H-¹H COSY correlations for 1

Based on the ¹H-¹H COSY spectrum of 1, correlation in H₁-H₂, H₅-H₆-H₇, H₉-H₁₁-H₁₂, H₁₆-H₁₇ and H₂₂-H₂₃ supported the presence of a havanensin-type limonoid structure in 1 [15, 17, 18]. The HMBC spectrum showed ³ J correlations between sp² methine proton signal δ_H 8.32 (H-1) to δ_C 47.5 (C-5) and carbonyl at δ_C 204.4 (C-3) and δ_H 5.88 (H-2) to δ_C 42.7 (C-10), indicating the presence of α, β-unsaturation ketone located at C-1, C-2 and C-3, respectively. Correlations from oxygenated sp³ methine protons at δ_H 5.46 (H-6) to δ_C 45.0 (C-4) and δ_C 170.1 (C-2′) and δ_H 5.41 (H-7) to δ_C 45.4 (C-9) as well as δ_H 2.04 (H-1′) to δ_C 170.1 (C-2′) and δ_H 2.07 (H-1″) to δ_C 170.3 (C-2″), indicate that an acetyl group was attached at C-6 and C-7, respectively.

The relative stereochemistry of hydroxyl group at C-16 of 1 was determined by a nuclear overhauser and exchange spectroscopy (NOESY) experiment (Fig. 3 and Additional file 1; Fig. S7). Comparison of oxygenated sp³ methine protons at δ_H 4.49 (H-16) and CH₃-18 (δ_H 1.33) with $\alpha$-oriented, indicated that H-16 was $\alpha$-oriented and hydroxyl group at C-16 is $\beta$-oriented. Correlations between δ_H 5.41 (H-7) and CH₃-30 (δ_H 1.33) with $\beta$-oriented, indicated that H-7 was $\beta$-oriented and acetyl group at C-7 is $\alpha$-oriented. Correlations between δ_H 5.46 (H-6) and CH₃-19 (δ_H 1.28) with $\beta$-oriented, indicated that H-6 was $\beta$-oriented and acetyl group at C-6 is $\alpha$-oriented. Furthermore, the optical rotation of 1, [$\alpha$]²⁷_D $+1$22.5° (c 0.2, MeOH) is the same sign to those of previously reported dysobinin (6) ([α]²⁰_D + 150°) [17, 18]. Therefore, the structure of 1 was elucidated as the new havanensin-type of limonoid derivative and named 16β-hydroxydysobinin.

Fig. 3

Selected NOESY correlations for 1

Four known compounds, 7-deacetylepoxyazadiradione (2), were previously synthesized as a derivative of epoxyazadiradione [19, 20], but isolated from a natural source for the first time. In addition, 6$\mathrm{\alpha }$-acetoxyepoxyazadiradione (3) and 6$\mathrm{\alpha }$-acetoxygedunin (4) [21] as well as dysobinin (5) [11, 17, 18] were identified by comparison of their spectroscopic data with previously reported values.

Cytotoxic activity

All isolated compounds were evaluated for the cytotoxic activity against MCF-7 breast cancer cell line and cisplatin is used as a positive control according to the method previously described [16, 22] and the results are shown in Table 2. Compound 1 showed the strongest activity against MCF-7 breast cancer cell with IC₅₀ (inhibitory concentration, 50%) values of 45.91 $\upmu$M, suggesting that the presence of hydroxyl at C-16 can increase the cytotoxic activity. In addition, the presence of epoxy ring and ketone group, like in compound 2–4, showed weak activity, indicated the presence of epoxy ring and ketone group can decrease activity.

Table 2 Cytotoxicity activity of compounds 1–5 against MCF-7 breast cancer cell line