A bioactive compound isolated from Duku (Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line

Plant material

The fruits of L. domesticum were collected on March 2018 from Bantul, Yogyakarta (GPS : -7.871098, 110.394854) and identified at the Department of Pharmaceutical Biology, Faculty of Pharmacy, Universitas Gadjah Mada.

Chemicals and equipment

Organic solvents (methanol, ethyl acetate, chloroform, n-hexane) used were pro analytical grades obtained from Merck. Silica gel F₂₅₄, Silica gel PF₂₅₄, (Merck), RPMI 1640, Fetal Bovine Serum, Penicillin-Streptomycin, Fungizon, Sodium bicarbonate (Gibco), HEPES (Invitrogen), Phosphate Buffered Saline, MTT (Sigma Aldrich cat. M5655), Doxorubicin (Sigma Aldrich). Infrared (KBr) spectrum was obtained from spectrophotometer (Shimadzu) using a method previously described by Ashokkumar and Ramaswamy⁷. Ultraviolet spectrum (CHCl₃) was obtained from UV spectrophotometer (Hitachi UH 5300). Sample (1 mg) were diluted in 1 mL CHCl₃ and was run between 200–400 nm. Spectra of ¹H- and ¹³C- NMR in CDCl₃ solvent were measured using JEOL JNM-ECZ 500R/S1 at 500 MHz.

Extraction and fractionation

The peel was separated from the fruit, dried in oven 50°C for 24 hours and powdered using a blender. Powdered L. domesticum fruit peel (200g) was macerated with ethyl acetate (EtOAc; 2 L) overnight. This solution was filtrated (0.45μm) and the filtrate was evaporated to dryness with a rotary evaporator set at 50°C, to give dried EtOAc extract (A, 50.13 g). In order to separate the extract into non-polar and polar fractions, 6 g of extract A was diluted 5 times using n-hexane (20 mL) to give soluble n-hexane fraction B (supernatant; 3.03 g) and insoluble n-hexane fraction C (residue; 2.83 g).

Fraction C was the most active among other fractions (see Results), and was therefore further fractionated using vacuum liquid chromatography as described by Mae Sri Hartati et al.⁸. In brief, using silica gel preparation grade (15g) as stationary phase this was eluted with n-hexane and increasing amounts of ethyl acetate. Six subfractions were obtained and subfraction 2 contained a major compound which appeared as white crystals (referred to as compound 1). Compound 1 was obtained as a single compound from subfraction 2, while the other subfractions still contained various compounds. Compound 1 (142.6 mg), had a melting point at 140–150°C (Figure 1). Compound 1 was identified using spectroscopy data such as ultraviolet (UV), infrared (IR), ¹³C-NMR and ¹H-NMR (see section Chemicals and equipment).

Figure 1. Isolation process of cytotoxic compound 1 (subfraction 2) from Lansium domesticum fruit peels.

Cytotoxic assay

The bioassay followed the methodology described by Bahuguna et al.⁹ with modifications. In brief, 100 μl T47D and Vero cell line (in RPMI mediaFaculty of Medicine, Universitas Gadjah Mada) were placed in each well of a 96 well micr oplates, resulting in 1 × 10⁴ cells/well. The cells were incubated for 24 hours at 37°C in a CO₂ incubator.

Extract A, fractions B and C and compound 1 (5mg) were dissolved in DMSO (50 μL). Serial concentrations of extract and fractions (50, 25, 12.5, 6.25, 3.125 μg/mL), compound 1 (25, 12.5, 6.25, 3.125 μg/mL) and doxorubicin (positive control; 0.5, 0.25, 0.125, 0.0625, 0.0312 μg/mL) were obtained. Cells were treated with the dose dependent samples and incubated for 24 hours at 37°C. The culture medium was removed by pipette, and MTT solution (100μL) was added to each well and incubated for 4 hours at 37°C. After incubation, stop solution (10% SDS, 100 μL) was added to each well and let stand at room temperature for 24 hours.

Absorbance was measured by microplate reader (Bio Rad) at 595 nm. positive control The data generated were used to plot a dose-response curve and IC₅₀ of the samples was determined.

Statistical analysis

The IC₅₀ values were analyzed by one-way ANOVA with statistical significance P < 0.05 using IBM SPSS ver.23.

Compound 1 characterization

Identified as Lamesticumin A.

White crystal. IR (KBr) v_max cm^-1: 3074, 2960, 1712; UV (MeOH)λ_max 236,5; ¹H,¹³C-NMR: see Table 1; m/z 502; (Calculated for C₃₁H₅₀O₅)

The infrared spectroscopy (KBr) spectrum of 1 showed a broad band at 3400–2800 cm^-1, which indicated the presence of –OH group, specifically –COOH due to intermolecular bonding. This data is supported by the appearance of –C=O at 1712 cm^-1. Compound 1 displayed UV absorption at 236,5 nm. The ¹³C-NMR spectrum (500 MHz, CDCl₃) of compound 1 showed 30 carbons (Table 1). There were two down field carbon signals (δ, 147.6 and 148.1 ppm) identified as C=O signal carbons. Two characteristic terminals =CH₂ signals (δ, 107.4 and 114.2 ppm) were observed, and this identity was confirmed by 2D (Het-Cor) NMR technique. Based on ¹³C-NMR and ¹H-NMR data, compound 1 (Figure 2) was identified as Lamesticumin A (C₃₁H₅₀O₅, m/z, 502) which was previously isolated from L. domesticum twigs⁵.

Figure 2. Isolated compound 1, Lamesticumin A.

Cytotoxicity of extract A, fractions B and C, and compound 1

The cytotoxicity of extract A, fractions B and C, compound 1 and doxorubicin (positive control) is shown in Table 2. Fraction C was the most cytotoxic (IC₅₀ 25.57 μg/mL) compared with extract A (29.41 μg/mL) and fraction B (43.51 μg/mL). The IC₅₀ of the isolated compound from fraction C, compound 1/Lamesticumin A was 15.68 μg/mL. All samples inhibited T47D cell growth in a dose dependent behavior (Figure 3).

Figure 3. T47D breast cancer cell line viability after treatments with extracts from Lansium domesticum fruit peels.

Error bar shows standar deviation (SD, n=3). symbol (*) indicates statistical significance (p<0.05)

Error bars shows standard deviation.

Underlying data

Zenodo: A bioactive compound isolated from Duku (Lansium domesticum Corr) fruit peels exhibits cytotoxicity against T47D cell line, http://doi.org/10.5281/zenodo.3539670²³.

This project contains the following underlying data:

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).