NAPHTHALENE DERIVATES FROM BAWANG TIWAI BULB Eleutherine bulbosa IN BORNEO

Naphthalene derivative compounds named Eleutherol ( 1 ) and Eleutherol C ( 2 ) have been isolated from Bawang Tiwai Bulb Eleutherine bulbosa with moderate cytotoxic activity against T47D breast cancer cells. This research was conducted to evaluate cytotoxic activity against T47D breast cancer cells, the compounds Eleutherol ( 1 ) and Eleutherol C ( 2 ) are naphthalene derivatives from Eleutherine bulbosa , where previously it has been reported that naphthalene derivative compounds have activity against breast cancer cells, especially T47D. After isolating the compounds Eleutherol ( 1 ) and Eleutherol C ( 2 ) from the EtOH extract of Bawang Tiwai Bulb Eleutherine bulbosa using conventional chromatography methods, their cytotoxic activity was tested against T47D breast cancer cells in vitro. Their chemical structures were elucidated based on spectroscopic analysis including IR, HR-TOFMS, 1D, and 2D NMR, and by comparison to those related spectra previously reported. Two naphthalene derivatives named Eleutherol ( 1 ) and Eleutherol C ( 2 ) have been isolated from the Bawang Tiwai Bulb of Eleutherine bulbosa . Compounds 1 and 2 were tested for their cytotoxic effects against T47D breast cancer cells and showed moderate cytotoxicity against T47D breast cancer cells with IC 50 values 117.15 and 80.21 µM, respectively, compared with cisplatin 24.07 µM. This research shows moderate cytotoxic activity of the compounds Eleutherol ( 1 ) and Eleutherol C ( 2 ) from the EtOH extract of Bawang Tiwai Bulb Eleutherine bulbosa. Testing of the cytotoxic effects on T47D breast cancer cells of compounds 1 and 2 of naphthalene derivatives was first reported.


INTRODUCTION
E. bulbosa is an herbaceous plant belonging to the Iridaceae family that is distributed mainly in South Africa, South America, and Southeast Asia and grows mostly in sulfur areas, 600-2000 m above sea level [1].In Indonesia, E. tubers are widely consumed by local tribal communities in Kalimantan, namely the Dayak tribe, and are traditionally used as a medicinal plant to increase breast milk production and treat diabetes, breast cancer, stroke, hypertension, and sexual disorders [2].Research has been conducted to identify the compounds responsible for the various properties of E. bulbosa tubers.Naphthalene, anthraquinone, and naphthoquinone are the main constituents of E. bulbosa [3,4] which exhibits various pharmacological properties such as anti-diabetic [2], anti-inflammatory [5], anti-microbial [6], antimelanogenesis [7] and anti-cancer [8].As an anticancer, much research has been carried out on isolated compounds from E. bulbosa.For example, the compounds Eleutherinoside C and Isoeleutherin showed selective cytotoxic activity and inhibited TCF/β-catecin transcription in SW480 cancer cells in a dose-dependent manner [8], the compound isoeleutherol showed potent activity in both cancer cell lines against HeLa and MCF-7 cells with LC50 values of 35.4 and 23.8 ppm respectively [9].This study is a report on naphthalene derivative compounds that were isolated from the ethanolic extract of E. bulbosa.Eleutherol (1) and Eleutherol C (2) which are derivatives of naphthalene compounds have been isolated from E. bulbosa, along with their cytotoxic activity against T47D breast cancer cells in vitro.Activity tests were carried out on T47D because this naphthalene group has very good activity against cytotoxic, especially breast cancer cells.

General Experimental Procedures
The UV spectrum was measured using a TECAN Infinite M200 pro (Mannedorf, Switzerland) with MeOH.The IR spectra were recorded on SHIMADZU IR Prestige-21 in KBr (Kyoto, Japan).The high-resolution of time-offlight mass spectrometry (HR-TOFMS) data was recorded with Water Xevo QTOF MS (Milford, Massachusetts, USA).
NMR spectrum was evaluated using JEOL ECZ-500 (Tokyo, Japan) at 500 MHz for 1 H and 125 MHz for 13 C using tetramethylsilane (TMS) as the internal standard.In addition, the chemical shifts are expressed in ppm, concerning the CDCl 3 (δ H 7.26/δ C 77.2) signals.

Plant Material
The bulbs of E. bulbosa were collected from Batu Cermin, Samarinda, East Kalimantan, Indonesia.The plant was identified at the Laboratory of Ecology of Tropical Forest Biodiversity, Faculty of Forestry, Mulawarman University, Samarinda, East Kalimantan.Indonesia.The plant was shade-dried (<45°C), coarsely powdered, and stored in an airtight container.

Extraction And Isolation
The dried E. bulbosa (200 g) was extracted with distilled ethanol at room temperature for 4 days (4×2.5 L) and concentrated using a vacuum rotary evaporator, yielding a concentrated extract (24 g).About 20 g of ethanol was chromatographed using silica gel column CC (70-230 mesh) with n-hexane: ethyl acetate as eluent (5% stepwise) and obtained 5 combined fractions (Fraction A-E).

Bioassays for Cytotoxic Activity
The T47D cells were seeded into 96-well plates at a density of 3 x 10 4 cells/well and incubated in a humidified CO2 incubator for 24 h.Varying concentrations of the test compound were dissolved in DMSO, followed by six desirable concentrations prepared using PBS (phosphoric buffer solution, pH 7.30-7.65).each concentration of the compound was added to the wells in triplicate and incubated in a humidified CO2 incubator for 48 h, the negative control wells received only DMSO, and cisplatin was used as the positive control.After an incubation period, MTT reagent [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] was added to all test and control well and the incubation was continued for another 4 h.The MTT-stop solution containing SDS (sodium dodecyl sulphate) was added to wells and incubated again for another 24 h.The absorbance values were read using a microplate reader at a wavelength of 550 nm.IC50 values were determined by the linear regression method using Microsoft Excel software.The IC50 is the concentration required for 50% growth inhibition.

CONCLUSION
Naphthalene derivatives, namely Eleutherol (1) and Eleutherol C (2) were isolated from the bulbs of E. bulbosa.The cytotoxic activity was evaluated against the T47D breast cancer cell line in vitro, Compounds 1 and 2 showed weak and no cytotoxic activity with IC50 values of 117.15 and 80.21 µM, respectively compared with cisplatin 24.07 µM.Suggesting that the location of the hydroxyl group can increase cytotoxic activity.