Chemical constituents and cytotoxic effect from the barks of Goniothalamus chinensis Merr . & Chun . growing in Vietnam

Article history: Received on: 29/01/2016 Revised on: 17/02/2016 Accepted on: 09/03/2016 Available online: 30/04/2016 Goniothalamus chinensis Merr. & Chun is a medicinal plant which has cytotoxicity, antitumor, antifungal, antimalaria and antituberculosis and antioxidant activity. The styryl-lactone, alkaloid and acetogenin are the main group compounds in species of Goniothalamus. In this study, we aimed to investigate phytochemical and cytotoxic effect of compounds from bark of Goniothalamus chinensis Merr. & Chun. The bark of Goniothalamus chinensis Merr. & Chun grown in Vietnam was extracted by methanol. Three compounds were isolated using on Sephadex LH-20 and preparative glass-backed TLC plates. The compound's structure were characterized on the basis of spectroscopic data and by comparing their physicochemical and spectral data with those published in literatures. The cytotoxicity of three compounds was evaluated against four cancer cell lines: epithelial cancer (KB), liver (Hep G2); breast cancer (MCF-7) and lung (LU-1). We have been isolated three compounds. Their structures were identified as Goniothalamin (1), Aristolactam BII (2), 3-Methyl-1Hbenz[f]indole-4,9-dione (3). Isolated compounds were tested cytotoxic activity against different cancer cell lines. The compound 1 demonstrated the highest potency against the epithelial cancer cell line with an IC50 of 4.18 ± 0.60μg/mL. Other compounds have weak cytotoxic activity.


INTRODUCTION
With the tropical climate, Vietnam has diversity of plant and animals species.In total, Vietnam has more than 12000 plant species; of which nearly 4000 species can be used in traditional medicine.The genus of Goniothalamus contains about 160 species in the world, mostly in Southeast Asia, among which about 19 are distributed in Vietnam (Wiart, 2007).Some species have been used as folk medicines for healing, for digestion and as a tonic plant.Main compounds in Goniothalamus are styryllactone, alkaloid and acetogenin which have cytotoxic, antitumor, insecticides, antifungal, antimalaria, antituberculosis and antioxidant activities (Blázquez et al., 1999).Orlikova et al., have showed goniothalamin, a styryl-lactone isolated from Goniothalamus macrophyllus, have cytotoxic and antiinflammatory effects.Thí compound also inhibits the tumor necrosis factor-α-induced NF-κB activation and have antileukemic potential (Orlikova et al., 2013).Other study demonstrated that goniolactone C, a styryl lactone, was extracted from Goniothalamus cheliensis, strongly inhibited platelet-derived growth factor-BB-induced vascular smooth muscle cell migration and proliferation (Sun et al., 2014).Goniothalamin, an active compound extracted from Goniothalamus griffithii, also inhibits human leukemic cells undergo apoptosis via intrinsic and extrinsic pathways (Petsophonsakul et al., 2013) and cervical cancer cells (HeLa) (Alabsi et al., 2012).Furthermore, goniothalamin also showed potent gastroprotective activity on gastric ulcers models in rats.The mechanism is goniothalamin may induce the production of sulfhydryl compounds and prostaglandins (Vendramini-Costa et al., 2014).For now, species of Goniothalamus continue being interested by many researches.However, there has been no report about the studies on the chemical constituents and bioactivity of this plant growing in Vietnam.

General experimental procedures
Melting points were measured on Mikroskopheiztisch PHMK-50 (VEB WaegetechnikRapido, Germany).The FT-IR spectra were recorded on an IMPACT-410FT-IR spectrometer (CARL ZEISS JENA).The NMR [ 1 H (500 MHz), 13 C (125 MHz), and DEPT-90 and 135 MHz)] spectra were recorded on an AVANCE spectrometer AV 500 (Brucker, Germany) in the Institute of Chemistry, Vietnam Academy of Science and Technology (VAST).Chemical shifts were reported in ppm downfield from TMS with J in Hz.Electrospray Ionization Mass Spectra (ESI-MS) were recorded on a Varian Agilent 1100 LC-MSD mass spectrometer.Analytical TLC was performed on Kieselgel 60 F 254 (Merck) plates (silica gel, 0.25 mm layer thickness) and RP-18 F 254 (Merck) plates (0.25 mm layer thickness).Spots were visualized using ultraviolet radiation (at 254 and 365 nm) and by spraying with 10% H 2 SO 4 , followed by heating with a heat gun.Column chromatography was performed on silica gel (70-230 and 230-400 mesh, Merck).Organic solvents were of analytical grade.

Cytotoxicity Assay
Cytotoxicity of these compounds was evaluated against four types of cancer cell lines: epithelial cancer (KB), liver (Hep G2); breast cancer (MCF-7) and lung (LU-1) by 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.Cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM) with 10% foetal bovine serum, at 37 o C, 5% CO2 and 95% humidity.Cells were plated in 96-well flat bottom tissue culture plates and allowed to density of approximately 3x10 4 /mL then begin experiment.The cells were incubated with 100 μL of compound at a range of concentration (from 1 μg/mL to 1000 μg/mL) for 72 hours.Then, 0.2 mg/mL MTT reagent was added to each well, and the plate was incubated for 4 hours at 37°C.Next, DMSO (100 uL) was added and the plates shaken for 5 min.The absorbance for each well was measured at 540 nm in a microtiter plate reader and the percentage cell viability (CV) was calculated manually using the formula:

CV verage abs of duplicate drug wells verage abs of control wells 100
The effects of compounds were expressed by IC 50 values (the drug concentration reducing the absorbance of treated cells by 50% with respect to untreated cells) (Monks et al., 1991).

Bioactivity
The result of cytotoxicity tests of isolated compounds against epithelial cancer (KB), liver (Hep G2), breast cancer (MCF-7) and lung (LU-1) cell lines are shown in Table 1.Only compound 1 exhibited strong cytotoxic activity against epithelial cancer (KB), liver (Hep G2), breast cancer (MCF-7) and lung (LU-1) cell lines.Previous study have demonstrated Goniothalamin possess anticancer potential activity through mechanism of induces apoptosis and inhibits TNF-α-induced NF-κB activation (Orlikova et al., 2013;Seyed et al., 2014).For the compounds 2, 3, we have found the IC 50 against fours tested cancer cell line were greater over than ten times as compared with compound 1.
are in trans position.The coupling constants J (9.5 Hz) between H-3 and H-4 indicated that two protons are in cis position.Based on the above evidence and the literature data C 163.8), one CH 2 group at δ C 29.9, one sp 3 methine o ygenated carbons at δ C 77.9; nine methine sp 2 group at δ C 121.7-144.5 and one quaternary carbon sp 3 at δ C 135.8.The signal in COSY spectra allowed correlating from C-3 to C-8.The coupling constants J(16.0 Hz) between H-7 and H-8 indicated that two protons

Table 1 :
Cytotoxicity of isolated compounds