RESORCINOL COMPOUNDS ISOLATED FORM THE BARK OF MYRISTICA FATUA HOUTT

Myristica fatua Houtt. is one of the endemic plants growth in Wallace region. Isolation of the secondary metabolite compounds from the ethyl acetate fraction of the bark of Myristica fatua Houtt. has been done using some chromatography techniques afforded two resorcinol compounds, malabaricone C ( 1 ), and malabaricone B ( 2 ). The structures of 1 - 2 were determined using spectroscopic techniques, including mass spectrometry, 1D-NMR and 2D-NMR. Both compounds showed in vitro cytotoxic activity against the breast carcinoma cancer cell lines MCF-7 using alamar blue assay method, with IC 50 of 2.38 and 0.71 μg/mL, respectively.


Myristica
(Myristicaceae) widely distributed from India and South East Asia to North Australia and the Pacific Islands.Myristica comprising of 72 species, and one of them is Myristica fatua Houtt (Zachariah, 2008).M. fatua is endemic to the Kalimantan, Sulawesi, Moluccas in Indonesia, beside the other region such as India, Philippines, New Guinea, Solomon Island, Pacific Islands, and New Caledonia.Mekongga forest is a conservation forest in Kolaka District, Northeast Sulawesi, Indonesia, which have so many uniques and endemic bioresources (Lim, 2012).
There is no reported study on M. fatua, except a study for antioxidant and antibacterial activities (Viveka, 2016) and its potency as αglucosidase inhibitor (Sivasothy, 2016).It is encourages us to do a phytochemical study about secondary metabolite compounds contained in Myristica fatua Houtt.In this paper we will describe the isolation of the acylphenols from M. fatua, the structure of malabaricone B (1) and C (2), and their cytotoxic activity against breast carcinoma MCF-7 cell lines.

General.
The 1D-and 2D-NMR spectra were recorded by JEOL JNM-ECA 500 spectrometer with CD3OD as a solvent and TMS as an internal standard.LC-ESI-MS were measured by the Mariner Biospectrometry-Finnigan instrument with methanol p.a (Merck) as a solvent.

Plant material.
The bark of Myristica fatua were collected from the Mekongga Forest, District of Kolaka, Southeast Sulawesi, Indonesia.The plant was determined and deposite as a specimen in Herbarium Bogoriense, Research Centre for Biology, Indonesian Institute of Sciences, Cibinong-Bogor, Indonesia.

Cancer cells line preparations
In this study, we used breast carcinoma (MCF-7) cells line as cancer cells.Cancer cell was cultured at 37ºC of temperature with 95% moisture content and 5% CO2 in DME medium contained 10% of FBS for 3 days (until the cell cultures undergo confluent 60-70%).Removed and replace the medium with a new medium and continued the incubation for next 24h.Wash Cultured cells with PBS 1-2 times and suspended using trypsin-EDTA solution.

Cytotoxic assay
Cytotoxic assay carried out using Alamar Blue method(Alamar Blue Assay, U.S. Patent No.5,501,959).100µL of cells line was supplemented with 10L of samples, incubated at temperature 37°C for 24h.The coloring process was done through the addition of Alamar blue solution for 4h.The color intensity of the cells line was measured by using ELISA plate reader at 560nm (excitation) and 590nm (emission).Percent viability is calculated using the formula as follows: OD (cell+sample)-OD (negative control) x 100 = % viability OD (cells) -OD (negative control) While IC50 of the active samples was calculated using linear regression analysis between percent of survival against the concentration of the sample
The HMQC ( 1 J(C-H)) and HMBC ( 2 J(C-H) to 4 J(C-H)) correlations of compound 1 (Figure 2).The HMQC correlation between a downfield proton atom at δ 3.11 with a downfield methylene carbon atom at δ 45.7 indicated that both atom were located near the electronegatif  Based on the NMR data, supported by ESI-MS data and reference (Table I), compound 1 was identified as malabaricone C (Figure 2) (Pham, 2000).
Compound 2 was isolated as yellowish brown, its has positive ESI-MS [M+H]+at m/z 343. 1 H-NMR data showed that compound 2 has 7 aromatic proton at δ 6.33 (2H), 7.19 in 1 st aromatic ring, 6.96 (2H), 6.67 (2H) in 2 nd aromatic ring and 8 pair of methylene proton groups at δ 3.10, 1.66, 1.33 (4-CH2), 1.55, and 2.49.Two proton peaks at δ 6.67 (2H,d,J 8.43 Hz) and 6.96 (2H,d,J 8.43 Hz)   of aromatic ring proton system, its mean that in 1st aromatic ring has four symmetric proton peaks with one type of ortho aromatic proton position at δ 6.96 and 6.67 (Figure 4a).In addition, two proton peaks at δ 6.33 (2H, d, J 8.43 Hz) and 7.19 (1H, t, J 8.43 Hz) in 2nd ring showed the AB2 pattern of aromatic ring proton system(Figure 4b), indicated that 2nd aromatic ring has two simetris proton peak at δ 6.33, which has correlation with proton peak at δ 7.19 in ortho position.Both AA'XX' and AB2 patterns were supported by HMQC and HMBC correlation data (Figure 5).The HMQC ( 1 J(C-H)) and HMBC ( 2 J(C-H) to 4 J(C-H)) correlations of compound 2 illustrated in Figure 5.The HMQC correlation between a downfield proton atom at δ 3.10 with a downfield methylene carbon atom at δ 45.8 indicated that both atom were located near the electronegatif atom, were supported by the HMBC correlation between proton atom at δ 3.10 with carbonyl group at δ 209.8, the same things happen to methylene group at δH 1.66/δC 25.8.Other HMBC correlation between a downfield proton atom at δ 2.49 with carbon quaternary sp2 (1st aromatic ring) at δ 135.0 showed the influence of the aromatic ring to the chemical shift value of the carbon atom at δ 36.1.The ESI-MS spectra (Figure 6) showed a molecular ion [M+H]+ 343.45 or [M+] 342.45 indicates the molecular weight of the compound corresponds to molecular formula C21H26O4.
Based on the NMR data, supported by ESI-MS data and reference (Table II), compound 2 was identified as malabaricone B (Figure 5) (Pham, 2000).

Cytotoxic assay
Compound 1-2 were examined for their cytotoxicity against the breast carcinoma (MCF-7) cells line (Figure 7) and showed that both compounds were active as anticancer compounds against breast carcinoma (MCF-7) cells line with the IC50 values 2.68 and 0.71µg/mL, respectively.

CONCLUSION
Isolation and purification of ethyl acetate fraction 5 (F-5) of the bark of M. fatua using column chromatography method, and structure elucidation based on spectroscopic data analysis result have been done.Can be concluded that compound 1 and 2 are malabaricone C and malabaricone B, respectively.Both compounds have high cytotoxicity activity against breast carcinoma (MCF-7) cells line with IC50 values are 2.68 and 0.71µg/mL, respectively.
Tabel I.13C-and 1 H-NMR date for malabaricone C (1) ( 1 H:500 MHz, 13 C: 125 MHz, in CD3OD; carbon numbering does not follow the official rule and is used here for simplicity) in 1st ring indicated the presence of AA'XX' pattern Tabel II. 13 C-and 1 H-NMR data for malabariconesB (2) ( 1 H:500 MHz, 13 C: 125 MHz, in CD3OD; carbon numbering does not followthe official rule and is used here for simplicity)