Mai Mohammed Farid，Sameh Reda Hussein*，Lamiaa Fawzy Ibrahim，Mohammed Ali El Desouky，Amr Mohammed Elsayed，Ahmad Ali El Oqlah，Mahmoud Mohammed SakerDepartment of Phytochemistry and Plant Systematic，National Research Center， Cairo，Egypt

2Department of Biochemistry，Cairo University，Cairo，Egypt

3Department of Biological Sciences，Yarmouk University，Jordan

4Department of Plant Biotechnology，National Research Center，12311 Cairo，Egypt

Cytotoxic activity and phytochemical analysis of Arum palaestinum Boiss.

Mai Mohammed Farid1，Sameh Reda Hussein1*，Lamiaa Fawzy Ibrahim1，Mohammed Ali El Desouky2，Amr Mohammed Elsayed2，Ahmad Ali El Oqlah3，Mahmoud Mohammed Saker4
1Department of Phytochemistry and Plant Systematic，National Research Center，12311 Cairo，Egypt

2Department of Biochemistry，Cairo University，Cairo，Egypt

3Department of Biological Sciences，Yarmouk University，Jordan

4Department of Plant Biotechnology，National Research Center，12311 Cairo，Egypt

ARTICLE INFO

Article history：

Accepted 15 Jul 2015

Available online 18 Aug 2015

Black calla lily

Araceae

Flavonoids

Cytotoxic activity

Gas chromatography-mass spectrometry

Objective：To evaluate the in vitro cytotoxic activity of the fractionated extract as well as isolated compounds of Arum palaestinum Boiss.（A.palaestinum）（black calla lily），and to identify the volatile components which may be responsible for the potential antitumor activity.

Methods：A.palaestinum was collected from its natural habitats and subjected to phytochemical analysis for separation of pure compounds.In vitro cytotoxic activity was investigated against four human carcinoma cell lines Hep2，HeLa，HepG2 and MCF7 for the fractionated extract and isolated compounds.While，the diethyl ether fraction was subjected to GC-MS analysis as it exhibited the most potent cytotoxic effect to evaluate the active constituents responsible for the cytotoxic activities.

Results：Four flavonoid compounds were isolated（luteolin，chrysoeriol，isoorientin，isovitexin）from the diethyl ether and ethyl acetate.The extracts and the pure isolated compounds showed a significant high antiproliferative activity against all investigated cell lines.The GC-MS analysis revealed the separation and identification of 15 compounds representing 95.01%of the extract and belonging to different groups of chemical compounds.

Conclusions：The present study is considered to be the first report on the cytotoxic activitiescarriedoutondifferentselectedfractionsandpurecompoundsof A.palaestinum to provide evidences for its strong antitumor activities.In addition，chrysoeriol and isovitexin compounds were isolated for the first time from the studied taxa.

Original articlehttp://dx.doi.org/10.1016/j.apjtb.2015.07.019

1.Introduction

Arum L.is a genus of flowering plants belonging to family Araceae，represented by 26 species and distributed in Northern Africa，Mediterranean Region，Western Asia，and Europe［1］. Arum palaestinum Boiss.（A.palaestinum），known as black calla lily，it is edible and considered as ornamental plant，and used in folk medicine to cure several chronic diseases such asstomach acidity，atherosclerosis，cancer and diabetes［2，3］.The preservation and conservation of A.palaestinum were carried out by Shibli et al.and Farid et al.［4，5］.

A few publications are about the phytochemical investigation and biological activity of A.palaestinum.It is characterized by presence of alkaloids，proanthocyanidins，flavones and their C-glycosides and flavonols［6，7］.A comprehensive metabolite profilingofA.palaestinumleavesbyusingliquid chromatography tandem mass spectrometry was studied by Abu-Reidah et al.［8］.It is worth to mention that in all published reports of its phytochemical studies，leaves and flowers of A.palaestinum were used and evidences for strong antitumor activities of its extracts and isolated compounds were provided［1，9］.

The present study aims to evaluate the in vitro cytotoxic activity of the fractionated extract as well as isolated compounds of A.palaestinum against four human carcinoma cell lines Hep2，HeLa，HepG2 and MCF7，and to identify the volatile components which may be responsible for the potential antitumor activity by using gas chromatography-mass spectrometry（GCMS）through preliminary analysis.

2.Materials and methods

2.1.Plant collection

A.palaestinum Boiss.was collected from their growing habitats in Bergesh protected area，Irbid，Jordan（latitude: 32°25′43.1682′′Nandlongitude:35°46′47.0094′′E）in February 2012 by Dr.Ahmed El-Oqlah，Department of Biological Sciences，Yarmouk University，Jordan.

2.2.Extraction and isolation

The aerial parts of A.palaestinum were air dried and ground in the laboratory，and the obtained powder（1 450 g）was extracted three times at room temperature with 70%methanol for an interval of six days.The aqueous-methanol extract was evaporated under reduced pressure and temperature to obtain a residue of 302 g.The residue was dissolved in distilled water（500 mL）and fractionated by using diethyl ether（44 g），dichloromethane（26 g），ethyl acetate（40 g），butanol（75 g），methanol（56 g）and water（58 g）according to their polarity，then the fractions were subjected to biological analysis.Diethyl ether and ethyl acetate fractions were subjected to 3 mm paper chromatography by using bulk acoustic wave as eluent to yield compound 1（7.0 mg），compound 2（3.8 mg），and compound 3（3.5 mg）from the diethyl ether fraction；and compound 4（4.3 mg）was isolated from the ethyl acetate fraction.Each individual compound was subjected to a Sephadex LH-20 column for the final purification.

2.3.Structure elucidation of the isolated compounds

The identification of compounds was determined by Rfvalues，color reactions，ultraviolet spectrophotometry，1D-NMR spectroscopy，andelectronimpactionsource/massspectrometery.Tracecompoundswereidentifiedbycochromatography with reference samples which were obtained from Department of Phytochemistry and Plant Systematics，National Research Center，Egypt.

2.4.Cell culture and in vitro cytotoxic activity

Four human tumor cell lines，epidermal carcinoma of larynx（Hep2），cervix（HeLa），liver（HepG2）and breast（MCF7）were used in the study and obtained from the American Type Culture Collection，Minisota，USA.The cell lines were maintained at the National Cancer Institute，Cairo，Egypt，by serial subculturing. The samples were prepared by dissolving 1:1 stock solution in dimethylsulfoxide at 100 mmol/L and stored at-20°C.

The cytotoxic activity of the fractionated extract and pure compounds were carried out by using sulforhodamine-B（SRB）assay following the method reported by Vichai and Kirtikara［10］. SRB is a bright pink aminoxanthrene dye with two sulphonic groups.It is a protein stain that binds to the amino groups of intracellular proteins under mildly acidic conditions to provide a sensitive index of cellular protein content.Cells were seeded in 96-well microtiter plates at initial concentration of 3×103cell/ well in a 150μL fresh medium for 24 h before treatment with the extract to allow attachment of cells to the wall of the plate.The extract as well as pure compounds at different concentrations（0.0，5.0，12.5，25.0 and 50.0μg/mL）were added to the cell monolayer in triplicate.Monolayer cells were incubated with the compounds for 48 h at 37°C in an atmosphere of 5%CO2.After 48 h，cells were fixed，washed and stained with SRB.Excess stain was washed with acetic acid and attached stain was recoveredwithethylenediaminetetraaceticacid-Trisbuffer. Color intensity was measured at 570 nm with an ELISA reader. The relation between surviving fraction and drug concentration was plotted to obtain the survival curve of each tumor cell line as compared with doxorubicin，the control anticancer drug.

2.5.GC-MS

GC-MS analysis was performed on a thermo scientific TRACE GC ultra gas chromatograph directly coupled to ISQ single quadrupole mass spectrometer，by using non-polar 5% phenylmethylpolysiloxanecapillarycolumn（30.00m× 0.25 mm×0.25μm）type TG-5MS under the following conditions:oven temperature program from 60°C（3 min）to 280°C at 5°C/min，then isothermal at 280°C for 5 min.The carrier gas was helium at flow rate of 1 mL/min.The volume of injected sample was 1μL of sample in diethyl ether using splitless injection technique，ionization energy 70 eV，in the electronic ionization mode.The identification of the compounds were carried out by matching their fragmentation patterns in mass spectra with those in Wiley Registry 9th and NIST11 GC-MS libraries.

3.Results

3.1.Structure elucidation of the isolated compounds

The present work dealed with the isolation and characterization of four compounds:luteolin（Compound 1），chrysoeriol（Compound 2），isoorientin（Compound 3），isovitexin（Compound 4）.Compounds 2 and 4 were isolated for the first time from the studied taxa.

Table 1 IC50of the antitumor activity of fractionated extract and pure compounds of A.palaestinum against the four carcinoma cell lines.μg/mL.

3.2.In vitro cytotoxic activity

The in vitro cytotoxicity of the fractionated extract and the isolated compounds of A.palaestinum showed its significant antitumor activity against the four cell lines Hep2，HeLa，HepG2 and MCF7（Table 1，F(xiàn)igures 1 and 2）.

The results suggested that diethyl ether fraction exhibited obviously higher antiproliferative activity compared with the regular regimen for cancer treatment in which doxorubicin was used as a protocol even in a relatively low dose of IC50of 3.83，4.13，3.68，and 3.53 against Hep2，HeLa，HepG2 and MCF7 cell lines，respectively.Also，dichloromethane fraction revealed strong activity against all tested cell lines with IC50of 10.30，11.30，8.63，and 6.23，respectively.

The four isolated flavonoid compounds（luteolin，chrysoeriol，isoorientin，isovitexin）were tested against the four cell lines and showed moderate antitumor activity in comparison with isolated fractions（Table 1，F(xiàn)igure 2）.

3.3.GC-MS analysis

The diethyl ether fraction of A.palaestinum was subjected to GC-MS analysis to identify the volatile constituents and compounds of which are biologically active.A total of 15 compounds were identified and represented 95.01%of the extract（Table 2）.The most important compounds were phytol，phytol acetate，linolenic，linoleic and hexadecanoic acid which represented by 25.29%，5.05%，22.37%，6.54%and 21.89%respectively，of the total extract.These compounds may be responsible for the higher anticancer activity of diethyl ether fraction.

4.Discussion

The cytotoxicity results suggested that the diethyl ether fraction exhibited an obviously higher antiproliferative activity compared with the regular regimen for cancer treatment in which doxorubicin was used as a protocol even in a low dose.The obtained results are in agreement with previous studies which showed that luteolin and isoorientin have antimutagenic activity on different cell lines［11］.In addition，isovitexin has an effect on reducinginflammationandcarcinogenesis［12］.Toour knowledge，there are no previous data recorded on the antitumor activity of chrysoeriol.From GC-MS data，phytol is a major compound with a cytotoxic activity against some cell lines［13］.It was reported that phytol acetate had antimicrobial，anti-inflammatory，anticancer and antidiuretic properties［14］.Linolenic acid and linoleic acid are essential fatty acids which belong to theω3 andω6 fatty acids respectively，while hexadecanoic acid is saturated fatty acid.These fatty acids have recently drawn attention and are recently marketed as health supplements；their methyl ester derivatives are of much biological importance，and they have antioxidant，anticancer and antihistaminic properties［15］.

Table 2 GC-MS analysis of diethyl ether fraction of A.palaestinum.

The present study is considered to be a preliminary investigation for future development of naturally occurring antitumor drugs，though some synergistic or antagonistic effects needed to beclarified.Theunderlyingantitumormechanismof A.palaestinum fractionated extract and the isolated compounds as well as preparation in a large scale also need to be further studied.

Conflict of interest statement

We declare that we have no conflict of interest.

Acknowledgments

Financial support from Science and Technology Development Fund（Egypt），with Grant No.4402 is highly appreciated. The authors are thankful to Prof.Ahmed El Oqlah，Department of Biological Sciences，Yarmouk University，Jordan，for his cooperation in collecting the plant.

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28 May 2015

Sameh Reda Hussein，Department of Phytochemistry and Plant Systematic，National Research Center，12311 Cairo，Egypt.

Tel:+20 1110238700

E-mail:sameh_reda@hotmail.com

Peer review under responsibility of Hainan Medical University.

Foundation Project:Support by Science and Technology Development Fund，with Grant No.4402.

in revised form 25 Jun，2nd revised form 26 Jun 2015