Antiproliferative activity of natural coumarins from Calophyllum incrasaptum M . R Henderson-Wyatt Smith against human breast cancer cells MCF

Objective: to evaluated the antiproliferative activity of natural coumarin from Calophyllum incrasaptum M.R Henderson-Wytt Smith against human breast cancer cells MCF-7. Methode : Coumarin from ethyl acetate fraction of C. incrasaptum M.R HendersonWyatt Smith was isolated by coloumn chromatographyic and structure elucidated by using spectroscopic methods and isolate compound was evaluated for their antiproliferative activities in the alamar blue assay. Result: Coumarin have antiproliferative activity against MCF-7 cancer cell lines through alamar blue assay for 4 h after treatment. Conclusions: coumarin showed good activity against cancer cell lines with IC50 value of 2.23 μg/mL.


Introduction
Cancer is a fatal disease standing next to the cardiovascular disease.Cancer, a diverse group of diseases characterized by uncontrolled growth and spread of abnormal cells, if the spread is not controlled, it can result in death or mortality, estimate 1,660,299 new cancer cases are expected to be diagnosed in 2013, and die about 580,350 in 2013 and almost 1,600 people die per day.New cases of breast cancer in the US during 2013 is 232,340 expected in women and 2,240 in men.Deaths : an estimated 40,030 breast cancer death (39,620 women, 410 men).Risk factors breast cancer for women is bigger than men, potentially risk factor after age 18, being overweight or obese (for postmenopausal breast cancer), used of menopausal hormone therapy (combined estrogen and progestin), risk is also increased by a family history of breast cancer [1]) and the number of deaths from cancer will continue to rise, with an estimated 13.1 million people dying in 2030 [2].
Emergency and rapid spread of cancer, represent a major problem for prophylaxis and treatment cancer which becomes more difficult, and the medicines used as treatment cancer have clear limitation and unfortunately cancer projected as the primary cause of death in the future [3].Therefore, it is necessary to discovery anticancer drugs from traditional medicine plant for curing cancer.
Some experiments have been done to develop compound which it's having anticancer activity, such as: Cao et al 1998 have isolated some of the phenyl coumarins., which it's showed cytotoxic activity against mouse leukemia P388, mouse fibrosarcoma (WEH 1640), human monocytic leukemia (HTP-1) and human lymphoblastic leukemia (MOLT4) [4].: Guilet et al., Preventing of cancer by administrating a 6-substituted coumarin derivative was patented, The substituent at the 6-position may have five or more carbon atoms such as ten or preferable fifteen carbon atom [(C-5(6prenyloxycoumarins), C-10(6-prenyloxycoumarins), C-15(6-prenyloxycoumarins)], [10].Preventing of cancer by using coumarin/calcone derivative was described by (Carrico-Moniz).The structure of coumarin/calcone as anticancer was saw in figure 1, 2,3,4 [9] The structure of coumarin derivate with substituted C-5, C10 and C15 at position C-6 Since coumarin showed interesting antiproliferative or anticancer activity, we decided to investigated Calophyllum species led to the isolation of one chemically distinct group of coumarin.In these study we are described the antiproliferative activity of the phenylcoumarin to MCF-7 cell lines from C. incrasaptum.

Materials
Stem bark of C. incrasaptum M.R Henderson-Wyatt Smith was collected from Kalimantan Island in Indonesia.The sample was identified at the Herbarium Unit, Department of Botany Indonesian Institute of Sciences (LIPI).Solvents and chemicals used in this study are analytical grade and were purchased from Merck (Darmstadt, Germany).

General aspects
1 H and 13 C NMR spectra were recorded on a JEOL NMR spectrometer at 500 and 125 MHz, respectively.The abbreviations s, d, and br used to describe 1 H spectra refer to singlet, doublet, and broad, respectively.NMR spectra were recorded in CDCl3 solution on a Jeol JNM ECA 500 MHz instrument, using TMS as the internal standard.Silica gel (60-230 mesh and 230-400 mesh) were used for column chromatography and preparative TLC plates coated with silica gel (2000 micron thickness) was purchased from Merck (Darmstadt, Germany).Precoated Si gel plates SIL G/UV254, 0.25 mm were used for TLC.The compounds were detected under UV light at 254 and 366 nm.Melting points were determined on an Electrothermal Fisher melting point apparatus (Scientific serial 903N0056) and are uncorrected.IR spectra were recorded on a FTIR Prestige 21 Shimadzu spectrophotometer (KBr dish) and UV spectra was taken on a Hitachi U-2000 spectrophotometer.LC-MS were recorded on Mariner Bio Spectrometric equipment with pneumatically assisted electronspray ionization (EIS) source.

Extraction procedure
The air-dried stems bark of C. incrasaptum (2.1 kg) were minced finely and shredded in an electric mill, macerated three time with MeOH (6L x72h weight of extract after solvent was removed : 400 gr), then was diluted in H2O and partitioned with between n-hexane and H2O: EtOAc and H2O : and butanol -H20 .The n-hexane, EtOAc and buthanol was removed under reduced pressure to give 65.8 g, 138.8 g and 609.4 g n-hexane, EtOAc and BuOH extract respectively.

Preparation of Cancer Cells Line
Cancer cells line used in this study is breast carcinoma (MFC-7), and the cell line was cultured in DME Medium with FBS 10%.These cell was cultured at 37 o C with moisture content of 95% and 5% CO2 for 3 days until the cell cultures undergo confluence 60-70%.After that the old media removed, replaced with new medium and incubated again for 24 hours.Culture cells then washed with PBS 1-2 times as much and was suspended using trypsin-EDTA solution.Cells that have been suspended added with new.

Fig 8. HMBC spectrum of isolate compound
All NMR 1D and 2D data was reported in Figure 9 and Table 1.Hormon oestrogen has the crucial role in development of the breast cancer, the most frequent malignant disease in women, therefore many therapies are designed to block this activity.Cinnamoyl-coumarin derivatives were especially effective in eostrogen-dependent cancer, such as breast (MCF7) cancer cell line.The phenylcoumarin in these paper have anticancer activity too, becauce coumarin are proven to posses a wide range of biological activitied , e. i as anticancer.

Results of cell viability
Inhibition of cell viability caused by prenyl coumarins including phenylcoumarin from C. incrasaptum was examined using Alamar Blue assay.The cytotoxic effect of pure test compound (phenyl coumarin) against MCF-7 breast cancer cells was determined with alamar blue assay at range of 0-1.5 ppm after 24 h of treatment period.The cell viability can be qualitatively assessed by the color intensity of alamar blue.The intensity of the alamar blue dye increased when the concentration of pure test compound decreased.The results are graphically represented in Figure 11`.Cell viability (%) was observed to be conversely proportioned to the concentration of the pure test compound, these results showed that these phenylcoumarin compound indicated decreased cell viability (that treatments showed growth inhibition) cel MCF7 responded in a dose-dependent.The pure test compound showed effective cytotoxic effect with the IC50 value of 2,6 ppm in MCF-7 breast cancer cells

Discussion
According to the results, we treated the MCF7 cell line with different concentrations of phenylcoumarin for 24 h.The results indicated phenylcoumarin induced cell death in concentration -dependent manner.
Phenyl coumarin is a group of compounds which have shown to psses preventive and therapeutic effects on breast cancer.

Conclusions
Using a combination of NMR techniques ( 1 H and 13 C NMR, HMQC, HMBC and COSY) and IR spectroscopy combined with DEPT calculations, it has been possible to determine the structure of phenyl coumarin (Figure 10) Phenylcoumarin exerts antiproliferative effect in breast carcinoma cell line, and can be considered for furher mechanistic evaluation in human its biosafety and anticancer effect.
The results demonstrated that treatmen of MCF7 cells with phenylcoumarin results in a significant decrease in cell viabilty, IC50 values against MCF7 is 2.23 µg/mL.

Fig 1 .Fig 2 .Fig 3 .Fig 4 .
Fig 1. Structure of C-5(6-prenyloxycoumarins) Colourring process was done by adding Alamar blue solution for 4 hours.The colour intensity of the cell line was measured by using ELISA plate reader at 560 nm (excitation) and 590 nm (emission) wavelength.Percent viability is calculated as in equation 1.While IC50 of the active extract was calculated by linear regression analysis between percent survival showed in equation 2. Alamar Blue was used to color the sample, and the IC50 of the pure compound (Alamar Blue Assay, U.S. Patent No.5,501,959).[12]OD (cell+sample)-OD (negative control % Viability = - 100 OD (cells) -OD (negative control) ) as an yellow amourphous from an EtOAC extract of steam bark of C incrasaptum M.R Henderson-Wytt Smith, mp 184-185 o C. The UV absorbtion of phenylcoumarin characterized a 5,7,10 threeoxygenated coumarin, and give a positive test with a vaniline/H2SO4 reagent, an intense deep blue spot develops over 24 hour period.LC-MS revealed the molecular formula to be C21H18O5 (m/z 351,40 [M+1] + ), by ESIMS analysis, i,e., the molecule had threeteen degrees of unsaturation.The UV spectrum exhibited two maxima at λ max 256 and 319 nm were indicative of a dimethylchromanone derivative, whilst the FTIR spectra showed important peaks explaining the bending, stretching, and double-bond absorptions of the coumarin.

Fig 5 .
Fig 5. IR spectrum of isolate compound Determination of structure was performed usinga NMR device.The 1-NMR spectrum of isolaed compound

Fig 6 .
Fig 6. 1 H-NMR spectrum of isolate compound The 13 C-NMR data shows 21 peaks of carbon atoms, nine C quartener atoms (including two carbonyl downfield signals at δ 159.43 and 191.65), seven C-H aromatic, two aliphatic protons (δH 1.52, m, H-8 & δH 2.55, m, H-9), the presence of two methyl groups (CH3) is supported by 13 C-NMR data with a carbon chemical shift of 19.65 and 10.49 ppm and one methoxy group at 62.77 ppm (Figure 7).

Fig 9 .
Fig 9. HMBC correlation of of isolate compoundThis paper report the crystal structure of one isolated compound from C. incrasaptum as phenyl coumarin (Figure10).

Table 1 .
Data NMR 1D and 2D and COSY chemical shift (ppm) of phenyl coumarin