A New Lignan Derivative , Lasiocarpone , from The Stembark of Chisocheton iasiocarpus ( Meliaceae )

A new lignan derivative, named lasiocarpone (1), was isolated from the stembark of Chisocheton lasiocarpus. The chemical structure of 1 was determined by extensive NMR and MS spectra analyses as well as by comparing with analogue compound from previous studies. Lasiocarpone showed moderate cytotoxic activity against MCF7 breast cancer cells with an IC50 value of 42.5 mM.


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 1 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 triterpenoid 8 and cytotoxic tetranortriterpenoid 9 .
Our previous phytochemical studies on Chisocheton plants, we had found new limonoids, pentandrice and dysobinol from C. pentandrus 10 and C. mocrophyllus 11 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 MCF-7 breast cancer cells in vitro.
Chisocheton 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][14][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.

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 SiO 2 (Merck & Co.).TLC analysis on SiO 2 GF 254 , stain was observed on UV light and heated on the hotplate after spraying with 10% H 2 SO 4 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 × 10 5 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-carboxy -methoxyphenyl)-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 IC 50 value was obtained from percentage live cells compared to control (%), versus the tested concentration of compounds (μM).

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 par titioned to n-hexane, EtOAc and n-BuOH.A portion of EtOAc (28.3 g) was vacuum liquid chromatographed on SiO 2 with n-hexane-EtOAc-MeOH as a gradient solvent to yield seven fractions (I-VII).Fraction IV (3.20 g) was column chromatographed on SiO 2 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 SiO 2 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 SiO 2 GF 254 with n-hexane: ethyl acetate (8:2) as a solvent to give 1 (5 mg) as a minor compound.
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 d  Lasiocarpone (1) was checked its cytotoxic activity on MCF-7 breast cancer cells according to a method described 10,19 and cisplatin (IC 50 27.0μM) was used as a positive control 20

CONCLUSION
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.
H 4.45 to C-9 (d C 171.9) and C-8′(d C 50.3) and a methine proton at d H 4.67 to C-9 (d 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 d H 3.07 and 2.87 to aromatic carbons at d C 137.8 (C-1), 138.2 (C-1′) and to methine carbons at d 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.