3-Hydroxy-8,14-secogammacera-7,14-dien-21-one: A New Onoceranoid Triterpenes from Lansium domesticum Corr. cv kokossan

Abstract

A new onoceranoid triterpenes, namely 3-hydroxy-8,14-secogammacera-7,14-dien-21-one (1), has been isolated from the fruit peels of Lansium domesticum Corr. cv kokossan. The structure of 1 was determined on the basis of spectroscopic data including infrared, 1D and 2D-NMR, as well as high resolution mass spectroscopy analysis. Compound 1 showed a weak activity against MCF-7 breast cancer cell lines.

1. Introduction

Lansium domesticum Corr. (Meliaceae) is a popular plant that widely grows in southeastern Asia [1,2]. L. domesticum Corr. cv kokossan (Meliaceae) is a higher tree growing up to 30 m in height, commonly called “kokosan” in Indonesia [3]. Several bioactive triterpenoids have been isolated from L. domesticum [4,5,6,7,8,9], of which some have shown potential anticancer [10], antibacterial [11], insecticides [12], antimalarial [13], antimutagenic [14,15], and antifeedant activities [16]. Previously, we isolated six triterpenes from the seeds and bark of L. domesticum Corr. cv kokossan [2,16]. In this paper, we report the isolation and structural elucidation of the new onoceranoid triterpenes 1 (Figure 1), along with its cytotoxic activity against MCF-7 breast cancer lines.

2. Results

Extraction and Isolation

The dried fruit peel (1.7 kg) was extracted with n-hexane, ethyl acetate, and methanol three times, respectively, for 24 h, at room temperature. After solvent removal under reduced pressure, we obtained the n-hexane (86 g), ethyl acetate (110 g), and methanol (75 g) crude extracts. A portion of n-hexane (60 g) was subjected to vacuum liquid chromatography over silica gel using a 10% gradient of n-hexane/EtOAc (10:0–0:10) to afford eight fractions (A–H). Fraction D (6.7 g) was subjected to vacuum liquid chromatography, eluted by a 1% gradient of n-hexane/EtOAc (100:0–85:15) to afford five combined fractions (D1–D5). Fraction D4 was then subjected to silica gel column chromatography, eluted with n-hexane:acetone:methanol (7:2.5:0.5), resulting in four fractions (D4A–D4D). Compound 1 was obtained from fraction D4B as a white crystal (0.27 g).

3-Hydroxy-8,14-secogammacera-7,14-dien-21-one (1), white crystal, m.p. 153–155 °C, [α]²⁰_D − 3.0° (c 0.1, MeOH), HR-TOF-MS m/z 463.3569 [M + Na]⁺ (calcd. for C₃₀H₄₈O₂Na, m/z 463.3552); IR (KBr) ν_max (cm⁻¹): 3533, 2932, 1700, 1456; ¹H-NMR and ¹³C-NMR showed in the

Table 1. ¹³C and ¹H NMR Spectral Data of Compounds 1 in CDCl₃.

Position	δC ppm	δH ppm (Int, mult., J = Hz)
1	37.5	1.14; 1.86 (2H, m)
2	27.4	1.65 (2H, m)
3	79.1	3.29 (1H, dd, 11.3; 4.3)
4	38.7	-
5	51.5	1.62 (1H, m)
6	29.8	1.35; 1.50 (2H, m)
7	121.7	5.40 (1H, brs)
8	135.4	-
9	55.3	1.66 (1H, m)
10	36.5	-
11	24.1	1.99; 2.10 (2H, m)
12	23.5	1.98; 2.10 (2H, m)
13	56.0	1.59 (1H, m)
14	134.9	-
15	122.3	5.40 (1H, brs)
16	29.9	1.35; 1.50 (2H, m)
17	49.6	1.20 (1H, m)
18	36.5	-
19	38.4	1.51; 2.15 (2H, m)
20	34.7	2.29; 2.77 (2H, m)
21	217.0	-
22	47.5	-
23	27.9	1.00 (3H, s)
24	15.1	0.87 (3H, s)
25	13.3	0.99 (3H, s)
26	22.3	1.75 (3H, s)
27	22.4	1.72 (3H, s)
28	13.6	0.76 (3H, s)
29	22.1	1.12 (3H, s)
30	25.0	1.08 (3H, s)

. Compound 1 was evaluated for its cytotoxic acitivity against MCF-7 breast cancer cell line, and compared to doxorubicin (35.7 µM) as a positive control. Compound 1 exhibited weak activity against MCF-7 with an IC₅₀ value of 717.5 µM.

3. Discussion

The molecular composition of 1 was proposed as C₃₀H₄₈O₂ with seven degrees of unsaturation, based on HR-TOF-MS and NMR spectral data. Mass spectra showed molecular ion peak at m/z 463.3569 [M + Na]⁺, with calculated m/z 463.3552 for C₃₀H₄₈O₂Na. The IR spectrum exhibited bands at ν_max (cm⁻¹) 3533 (hydroxy), 2932 (C-H stretching of aliphatic), 1700 (ketone), and 1456 (gem dimethyl).

The ¹³C NMR data (Table 1) with DEPT and HSQC experiments (Figures S2–S4) revealed the presence of total of 30 carbon signals, which were classified as eight methyls, eight methylenes, seven methines (two olefinic and one oxygenated), and seven quaternary carbons (two olefinic and one carbonyl). The two trisubstituted double bonds and the carbonyl in a system with seven degrees of unsaturation suggested that 1 possess a tetracyclic structure. Previous studies supported that evidence, indicating that compound 1 has an onoceranoid-type triterpenoid [3,14].

The ¹H NMR spectrum of 1 (Figure S1) displayed the presence of eight methyl groups at δ_H (ppm) 1.00 (3H, Me-23), 0.87 (3H, Me-24), 0.99 (3H, Me-25), 1.75 (3H, Me-26), 1.72 (3H, Me-27), 0.76 (3H, Me-28), 1.12 (3H, Me-29), and 1.08 (3H, Me-30). Two olefinic protons (H-7 and H-15) were observed at δ_H (ppm) 5.45 and 5.42 (each 1H, brs), together with two vinyl methyls proton resonating at δ_H (ppm) 1.75 (3H, s, H-26) and δ_H 1.72 (3H, s, H-27), indicating two trisubstituted double bonds of 1. One oxygenated methine signal was also observed at δ_H 3.29 ppm (1H, dd, H-3).

The structure of 1 was further defined by ¹H-¹H COSY (correlated spectroscopy) and HMBC (heteronuclear multiple bond connectivity) spectra (Figure 2, Figures S5 and S6). The ¹H-¹H COSY spectra showed couplings between H-1 (δ_H 1.14)/H-2 (δ_H 1.65), H-2 (δ_H 1.65)/H-3 (δ_H 3.29), H-9 (δ_H 1.66)/H-11 (δ_H 2.10), H-11 (δ_H 2.10)/H-12 (δ_H 1.98), H-12 (δ_H 1.98)/H-13 (δ_H 1.59), and H-19 (δ_H 1.51)/H-20 (δ_H 2.77), supporting the presence of a onoceranoid-type triterpenoid structure. The oxygenated methine bearing a hydroxy group was located at C-3 by the HMBC correlations of H₂-1 (δ_H 1.86), H₃-23 (δ_H 1.00) and H₃-24 (δ_H 0.87) to C-3 (δ_C 79.1). The Δ^7,8 and Δ^14,15 double bonds were assigned by the HMBC correlations from H₃-26 (δ_H 1.75) to C-7 (δ_C 121.7), C-8 (δ_C 135.4), C-9 (δ_C 55.3); and H₃-27(δ_H 1.72) to C-13 (δ_C 56.0), C-14 (δ_C 134.9), C-15 (δ_C 122.3). The ketone group was attached to C-21 by the HMBC correlations of H₂-20 (δ_H 2.77) and H₃-29 (δ_H 1.12) to C-21 (δ_C 217.0). This analysis indicated that compound 1 was similar to 3β-hydroxyonocera-8(26),14-dien-21-one [5], uniquely differing on the double bond position in the B ring. Based on the coupling constants of H-3 (dd, J = 11.4; 4.3 Hz), the configuration of the hydroxyl group at C-3 was indicated in the β-equatorial position [5,17]. From the analyses, compound 1 was determined as 3-hydroxy-8,14-secogammacera-7,14-dien-21-one.

4. Materials and Methods

4.1. General Experimental Procedures

Mass spectrum was recorded on a waters Xevo QTOFMS (Waters, Milford, MA, USA). IR spectrum was measured on a One Perkin Elmer infrared-100 (Waltham, MA, USA) in KBr. NMR data were recorded on a Brucker spectrometer (Billerica, MA, USA) at 400 MHz for ¹H and 120 MHz for ¹³C using TMS as an internal standard. Chromatographic separations were carried out on silica gel G60 (Merck, Darmstadt, Germany) and RP18 (Merck). TLC plates were precoated with silica gel GF254 (Merck, 0.25 mm) and detection was achieved by spraying with 10% (v/v) H₂SO₄ in ethanol, followed by heating.

4.2. Plant Material

The fruit peels of L. domesticum Corr. cv kokossan (Meliaceae) was collected from Cililin, West Java, Indonesia in April 2018. The plant was identified and deposited in The Herbarium of Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Indonesia (Identification Number: 195/HB/08/2018).

4.3. Cytotoxic Bioassay

Cytotoxicity of the compound against MCF-7 human breast cancer cells was measured using MTT (Methyl Thiazoldiphenyl-Tetrazoliumbromide) assay. Stock culture was grown in flasks, containing Roswell Park Memorial Institute (RPMI) medium, respectively supplemented with 10% (v/v) feta bovine serum (FBS) and 1% (v/v) penicillin-streptomicin as an antibiotic. The culture was incubated at 37 °C for 24 h. The medium was changed, and tumor cells were detached and seeded in 96-well microliter plates. After, 24 h, compounds were added to the wells. After 48 h, cell viability was determined by measuring the metabolic conversion of yellow salt or 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide to its insoluble formazan, which has a purple color product resulting from reduction in viable cells. Insoluble formazan was diluted with DMSO. The MTT assay results were read using spectrophotometer UV at 450 nm. Compound 1 was evaluated at eight concentrations (15.9; 34.0; 70.3; 140.7; 283.6; 567.3; 1134.6; 2269.1 µM) in 100% DMSO with the final concentration of DMSO of 2.5% (v/v) in each well. Doxorubicin was used as a positive control. IC₅₀ values were calculated by the linear regression method using Microsoft Excel software.

5. Conclusions

A new onoceranoid triterpene, namely 3-hydroxy-8,14-secogammacera-7,14-dien-21-one (1), was isolated from fruit peels of L. domesticum Corr. cv kokossan (Meliaceae). This compound exhibited weak cytotoxic activity against MCF-7 human breast cancer cell lines with IC₅₀ value of 717.5 µM.