A New Lignan Derivative, Lasiocarpone, from the Stembark of Chisocheton lasiocarpus (Meliaceae)

Introduction

The Chisocheton plant genera as a second largest plant of Meliaceae family, consisting of 50 plants and widely distributed in the tropics. Previous phytochemical studies of this genera have produced some compounds with interesting biological activity, such as anti-plasmodial limonoid,² cytotoxic limonoid,³ and anti-inflammatory limonoids,⁴ NO production inhibitory activity limonoids,⁵ cell growth inhibitory activity limonoids,⁶ anti-inflammatory protolimonoids,⁷ cytotoxic triterpenoid⁸ and cytotoxic tetranortriterpenoid.⁹

Our previous phytochemical studies on Chisocheton plants, we had found new limonoids, pentandrice and dysobinol from C. pentandrus¹⁰ and C. mocrophyllus¹¹ as well as a triterpenoid-type lanostane from C. cumingianus.¹² In the further study, we focus the stembark of C. lasiocarpus that showed significant cytotoxic activity on MCF7 breast cancer cells in vitro.

C. lasiocarpus is up to 20 m high plant and widely distributed in the tropical regions.¹³ Traditionally, the stembark of C. lasiocarpus are used for treatment of fever and skin diseases.^13-15  There is no phytochemical study on C. lasiocarpus previously. In this paper, isolation, structure determination and cytotoxic activity of a new lignan derivative are described.

Material and Method

Instruments

Optical rotation values were measured with a Perkin-Elmer 341 polarimeter. UV spectrum was measured with Shimadzu-1800 spectrophotometer. IR spectrum was measured on Perkin-Elmer 1760X spectrophotometer. Mass spectra was measured with a Qtof HR-MS XEVotm mass spectrometer instruments. NMR data were recorded on a Bruker Topspin spectrometer and used TMS as an internal standard. Column chromatography on SiO₂ (Merck). TLC analysis on SiO₂ GF₂₅₄, stain was observed on UV light and heated on the hotplate after spraying with 10% H₂SO₄ in ethanol.

Plant Material

C. lasiocarpus stembarks were obtained from Bogor Botanical Garden, Indonesia in July 2015. Plant identifications were made from Bogoriense Herbarium, Indonesia. Specimens (No. Bo-1295453) are saved in the Bogoriense Herbarium.

Cytotoxic Assay

Determination of cytotoxic activity is performed according to the procedure described in the previous paper.¹⁰ Harvest suspension of breast cancer cells (MCF-7) by centrifugation. Determine the amount and viability cells (with trypan blue exclusion), and resuspend cells with final 4 × 10⁵ cells / mL supplemented with 10% phosphoric buffer solution (FBS) and 1% Penicillin-Streptomycin. Dispense 50 μL of cell suspension (20,000 cells) into all wells on microplate, then incubated for 24 hours. On different microplate samples were prepared. Samples or standards to be measured, diluted in an EMEM medium containing 10% FBS and 1% Penicillin-Streptomycin, then dilutions. The diluted 50 μL sample was transferred into well on the microplate containing the incubated cell. Then re-incubated for 48 hours. After that, a salt 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (MTS) reagent was introduced to each microplate of 20 μL and incubated for 2-4 hours until the orange formazan was seen. Furthermore, the colored formazan that has been produced measured its absorbance at a wavelength of 570 nm using a multimode reader. The IC₅₀ value was obtained from percentage live cells compared to control (%), versus the tested concentration of compounds (µM).

Results

Extraction and Purification

Dried ground stembarks of C. lasiocarpus (2.1 kg) were soaked with methanol for three days and filtered. Evaporate of the methanol on vacuum produced a concentrated methanol extract (209.4 g) and subsequently partitioned to n-hexane, EtOAc and n-BuOH. A portion of EtOAc (28.3 g) was vacuum liquid chromatographed on SiO₂ with n-hexane-EtOAc-MeOH as a gradient solvent to yield seven fractions (I-VII). Fraction IV (3.20 g) was column chromatographed on SiO₂ with n-hexane-EtOAc (10:0-1:1) as a developing solvent to give seven subfractions (IVa-IVg). Subfraction IVf (420 mg) was further separated by column chromatography on SiO₂ with choroform: methanol (9:1) as a solvent to give five subfractions (IVf.1-5). Subfraction IVf.3 (62.5 mg) was further purified by preparative TLC on SiO₂ GF₂₅₄ with n-hexane: ethyl acetate (8:2) as a solvent to give 1 (5 mg) as a minor compound.

Lasiocarpone (1). Yellowish gum; [α]^D₂₀ + 25.1^o (c 0.1, CH₃OH); UV λ_max 280 (e 2.80); IR (KBr) v_max (cm^-1): 3394, 3060, 1731,  1720, 1560, 1464, 1024; ¹H- (500 MHz) and ¹³C NMR (125 MHz) in DMSO-d₆, Table 1. HR-TOFMS m/z 355.1015 [M+H] ⁺ (calcd for C₂₀H₁₈O₆, m/z 354.1025).

Discussion

An aqueous methanol extract was concentrated and partitioned to n-hexane, ethyl acetate, and n-butanol. The ethyl acetate fraction was column chromatographed on SiO₂ and separated on preparative TLC on SiO₂ GF₂₅₄  produce a new lignan derivative, lasiocarpone (1).

Scheme 1 Click here to View scheme

 

Lasiocarpone (1),  [α]²⁵_D + 25.1^o (c 0.1, CH₃OH), was isolated as a yellowish gum with molecular formula, C₂₀H₁₈O₆ by high resolution time of flight mass spectra (HR-TOFMS), requiring twelve double bond equivalents. UV absorption of 1 in MeOH exhibited at  at 280 nm, indicating the presence on benzene ring. IR spectrum showed peaks at 3394, 1731,1720, 1560 and 1024 cm^-1, due to the presence of hydroxyl, carboxyl ester lactone, benzene and ether groups. ¹H-NMR spectrum of 1 revealed p-disubstituted benzene ring at δ_H 7.52 (2H, d, J=6.8 Hz, H-2′, H-6′) and 7.43 (2H, d, J=6.4 Hz, H-3′, H-5′). In ¹H-NMR spectrum showed also m-disubstituted benzene ring at δ_H 7.26 [(1H, d, J=7.1 Hz, H-4), 7.70 (1H, dd, J=7.1, 8.2 Hz, H-5), 7.77 (1H, d, J=8.2 Hz, H-6) and 7.18 (1H, s, H-2)]. In addition, deshielded methylene protons at δ_H [4.45 (1H, dd, J=7.1, 8.5 Hz), 4.54 (1H, dd, J=8.5, 9.4 Hz)], methine signals at δ_H [4.67 (1H, dd, J=4.7, 7.1 Hz), 4.19 (1H, m)] and four methylene signals at δ_H 2.87 (1H, dd, J=5.5, 13.8 Hz), 3.24 (1H, dd, J=7.1, 13.8 Hz), 3.05 (1H, dd, J=10.4, 13.5 Hz) and 3.20 (1H, dd, J=4.7, 13.5 Hz) also were observed and supporting of a dibenzyl butyrolactone-type lignin.¹⁶ The ¹³C-NMR spectrum together with DEPT spectrum of 1 displayed twenty carbon signals, including a lactone (δ_C 171.9), two carboxyl carbons (δ_C 167.2, 166.7), four sp² quartenary carbons, eight sp² methines, an oxygenated sp³ methylene (δ_C 65.9), two sp³ methylenes and two sp³ methines, featuring a dibenzyl butyrolactone-type lignan.^16,17 A NMR data of 1 are similar those of (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4,5-trimethoxy)benzylbuyrolactone,¹⁷ except the disappear of methoxyl and hydroxyl groups and the appearance of carboxyl group. In order to clarify the position of functional groups, 2D NMR experiments were conducted (Figure 1). Correlations in H2′-H3′, H5′-H6′, H4-H5-H6, H7-H8 and H7′-H8′-H9′, supporting the presence of a 1, 4-disubtituted and a 1,3-disubtituted benzene ring from a dibenzyl butyrolactone-type lignan.

Figure 1: Selected correlations for 1 Click here to View figure

 

All of aromatic protons were correlated to carboxyl and used for assigment of the carboxyl groups were attached in meta and para orientation, respectively. Furthermore, a correlation between an oxygenated methylene at δ_H 4.45 to C-9 (δ_C 171.9) and C-8′ (δ_C 50.3) and a methine proton at δ_H 4.67 to C-9 (δ_C 171.9) were used to assign a lactone ring at C-8, C-9, C-9′ and C-8′, which characteristic for butyrolactone-type of lignan. Correlation from methylene protons at δ_H 3.07 and 2.87 to aromatic carbons at δ_C 137.8 (C-1), 138.2 (C-1′) and to methine carbons at δ_C 54.8 (C-8) and 50.3 (C-8′), supporting the dibenzyl butyrolactone-type of lignan. It was clearly confirmed that compound 1 contains a 3-carboxybenzyl at C-9 and 4′-carboxybenzyl unit C-9′ on a butyrolactone skeleton and the relative configuration of the dibenzyl units is trans. A relative stereochemistry of C-8 and C-8′ was supported also by NOESY spectra. There are no crosspeak between H-8 and H-8′ In the NOE spectra, consequently both protons are trans-orientation.

Table 1: NMR data for 1

Position of C	δC (mult.)	δH (SH., Integral., J=Hz)
1	137.8 (s)	–
2	129.6 (d)	7.18 (1H, s)
3	134.0 (s)	–
4	126.9 (d)	7.26 (1H, d, 7.1)
5	127.6 (d)	7.70 (1H, dd, 7.1, 8.2)
6	128.5 (d)	7.77 (1H, d, 8.2)
7	36.4 (t)	3.05 (1H, dd, 10.4, 13.5)
		3.20 (1H, dd, 4.7, 13.5)
8	54.5 (d)	4.67 (1H, dd, 4.7, 7.1)
9	171.8 (s)	–
1′	138.0 (s)	–
2′	131.5 (d)	7.52 (1H, d, 6.8)
3′	128.6 (d)	7.43 (1H, d, 6.4)
4′	134.8 (s)	–
5′	128.6 (d)	7.40 (1H, d, 6.4)
6′	131.5 (d)	7.60 (1H, d, 6.8)
7′	36.7 (t)	2.87 (1H, dd, 5.5,13.8)
		3.24 (1H, dd, 7.1, 13.8)
8′	50.3 (d)	4.19 (1H, m)
9′	65.9 (t)	4.45 (1H, dd, 7.1, 8.5)
		4.54 (1H, dd, 8.5, 9.4)
4′-CO2H	166.7 (s)	–
5-CO2H	167.2 (s)	–

 

This results was supported also by comparing to those of trans-dibenzylbutyrolactone¹⁸ and the specific optical rotation of 1 ([α]²⁰ _D – 24.1^o, c 0.1, CH₃OH) is same negative sign to that of the previously reported, (2R,3R)-2-(4-hydroxy-3-methoxybenzyl)-3-(3,4,5-trimethoxy)benzylbuyrolactone ([α]¹⁸_D -25.1 (c 0.55, EtOH)].¹⁷ Therefore, compound 1 was elucidated to be a new dibenzyl butyrolactone-type lignan, (2R,3R)-5-carboxybenzyl-4′-carboxybenzylbutyrolactone and was named lasiocarpone.

Lasiocarpone (1) was checked its cytotoxic activity on MCF7 breast cancer cells according to a method described^10,19 and cisplatin (IC₅₀ 27.0 µM) was used as a positive control.²⁰ Lasiocarpone displayed an IC₅₀ value of 42.5 mM against MCF7 breast cancer cell, indicating showed moderate cytotoxic activity.

Conclusions

A new dibenzyl butyrolactone-type lignan derivative, lasiocarpone (1), was isolated from the stembark of Chisocheton lasiocarpus as a minor compound. This investigation confirm that Chisocheton genus is capable to produce a lignan derivative.

Acknowledgments

We express sincerely thank to Universitas Padjadjaran for financial support (Academic Leadership Grant 2016-2018 by US). We are thank also to Dr. Yuni Elsa at Central Laboratory, Universitas Padjadjaran, Bandung, Indonesia for MCF7 assay.

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