Use of GC / MS to identify chemical constituents and cytotoxic activity of the leaves of Phoradendron mucronatum and Phoradendron microphyllum ( Viscaceae )

Phoradendron mucronatum and P. microphyllum are plants that found in tropical and subtropical areas, used in traditional medicine and popularly known as mistle-thrush. The aim of this study was to identify the chemical constituents of different leaf extracts from P. mucronatum and P. microphyllum and assess cytotoxic activity against strains from a human tumour cells. Extracts obtained with hexane, dichloromethane, chloroform and ethyl acetate from the leaves were analysed by gas chromatography coupled with mass spectrometry (GC-MS) and the cytotoxicity was assessed by the MTT method (bromide (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)). The tested human tumour cells were NCI-H292 (human pulmonar mucoepidermoid carcinoma), MCF-7 (human breast adenocarcinoma) and HEp-2 (epidermoid carcinoma of the larynx). Analysis by GC/MS of the extracts from leaves of P. microphyllum and P. mucronatum detected 51 different compounds, such as alkaloids, diterpenes, triterpenes, sterols, alcohols, aldehydes, fatty acids and hydrocarbons. In the cytotoxic evaluation, hexane and ethyl acetate extracts from the leaves P. microphyllum inhibited cell growth of NCI-H292 strains (72.97%) and HEp-2 (87.53%), respectively. The extracts of P. mucronatum species showed an inhibitory effect towards NCI-H292 (83.19%/hexane), MCF-7 (88.69%/dichloromethane) and HEp-2 (93.40%/ hexane). The extracts showed cytotoxic activity against the tested strains, especially the P. mucronatum, which presented the highest percentages of inhibition of cell growth.


INTRODUCTION
Most current drugs are derived, directly or indirectly from the chemical constituents of higher plants.About 60% of the drugs used for the treatment of cancer have been isolated from vegetable products (Gordaliza 2007).
The analysis of anticancer activity is based on assays that use established cell lines where the toxic effect of plant extracts can be measured.Cytotoxic screening models provide important preliminary data for selecting plant extracts or compounds with antineoplasic properties (Alonso-Castro et al. 2011).
Phoradendron is a genus of the Viscaceae family with approximately 234 species.Mistletoes are plants that occur in tropical and subtropical areas of both hemispheres (Cronquist 1981).Several species have wide use in traditional medicine.For example, P. carneum Urb., P. galeottii Trel., P. reichenbachianum (Seem.)Oliver and P. serotinum (Raf.)M.C.Johnst are used to treat dermatological disorders and skin tumors (Alonso-Castro et al. 2011).
Phoratoxinas isolated from P. tomentosum manifests cytotoxic activity in different cell lines (Johansson et al. 2003).The methanol extract of the leaves of P. vernicosum Greenm.presented activity against nasopharyngeal carcinoma (Caamal-Fuentes et al. 2011).The aqueous extract the leaves of P. serotinum (Raf.)MC Johnst.caused toxic effects against breast carcinoma (Jacobo-Salcedo et al. 2011) and ethanol extract shows cytotoxic activity against lung epithelial cells (Alonso- Castro et al. 2012).
The Phordendron microphyllum (Pohl ex DC.) Trel and Phordendron mucronatum (DC.)Krug & Urb species are popularly known as "herbal" or "bird dung" and can be found as parasite species in the northeastern semiarid region of Brazil (Ferreira et al. 2007).
Scientific reports concerning this species are scarce.The present study then was mounted to identify the chemical constituents of hexane, dichlomethane, chloroform and ethyl acetate extracts from the leaves of P. mucronatum and P. microphyllum and to evaluate their cytotoxic activity against human tumour cell lines.

PREPARATION OF THE EXTRACT
A dry power of the P. microphyllum and P. mucronatum (30 g) leaves was extracted at room temperature for 30 minutes three times consecutively by maceration in an ultrasonic bath (Uniqu USC -1400) using solvents with different polarities: hexane (Hx), dichloromethane (DCM), chloroform (CF) and ethyl acetate (EtOAc).Then it was filtered and the crude extracts were obtained after evaporation of the solvents under reduced pressure at 40 °C.Ten mg of the extracts was dissolved in 2 ml of ethyl acetate for analysis by Gas Chromatography coupled to Mass Spectrometry (GC/MS).The initial column temperature was 100 °C for 4 min, followed by an increase of 3 °C min -1 up to 300 °C and kept constant for 90 min.The split ratio was 5:1.The mass spectrometer was programmed to perform readings in a range of 50 to 500 Da at intervals of 0.30 s, with ionization energy of 70 eV.
One μl of the different extracts was injected (10 mg dissolved in 2 ml ethyl acetate).A mixture of linear hydrocarbons (C9-C20, C21-C40) was injected under the same conditions in order to identify the chemical constituents.The identification of the constituents was performed by analyzing and comparing the mass spectra based on data libraries (FFNSC1.3.lib,WILEy7.LIB, NIST08s.LIB, My LIBRARy.lib) using a GC/MS instrument, whose índices showed a similarity of greater than or equal to 90%.Relative quantification of the components of each sample was obtained from the relative area of the peaks in the chromatograms.

CyTOTOXIC ACTIVITy
The cytotoxic activity was performed using the MTT method, which consists of a dosage based on the colorimetric conversion of the salt 3-(4,5-dimethyl-2-thiazole)-2,5 diphenyl-2-H bromide tetrazolium (MTT) which moved from a yellow color when insoluble [formazan crystals], moving to purple when precipitated due to the action of succinyl-dehydrogenase enzyme present only in metabolically active cells of mitochondria (Alley et al. 1988, Mosmann 1983).The human tumour cell lines used were NCI-H292 (lung mucoepidermoid carcinoma), MCF-7 (breast adenocarcinoma) and HEp-2 (epidermoid carcinoma of the larynx) obtained from the section of cell cultures of Bank Cell in Rio de Janeiro and maintained in accordance with the protocol established by the Cell Culture Laboratory, Department of Antibiotics of the Federal University of Pernambuco.Cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% antibiotic solution (penicillin and streptomycin) and L-glutamine, grown in culture plates with 96 wells at a concentration of 2 x 10 5 cells/well, incubated at 37 °C in a humid atmosphere (5% CO 2 ) for 24 hours (Butler and Dawson 1992).Then the extracts Hx, DCM, CF and EtOAc leaves dissolved in dimethylsulfoxide (DMSO) (stock concentration 10 mg/ml) were added to the wells in a single concentration (50 μL/ml) and doxorubicin (5 mg/ml) was used as a positive control.This procedure was performed in triplicate.After 72 hours of plate reincubation, 25 μL of MTT (5 mg/ml) was added to each well.The culture medium with MTT was aspirated, after 3 hours incubation, 100 μL of DMSO was added to each well to dissolve the formazan crystals.The absorbance was measured in a spectrophotometer at a wavelength of 540 nm.Cell growth inhibition percentage (CI%) was determined considering the mean of negative control as 100% proliferation.
An intensity scale was used to assess the cytotoxic potential of the extracts which were as follows: samples without activity, with little activity (cell growth inhibition ranging from 1 to 50%), with moderate activity (cell growth inhibition ranging ISLA V.G.A. BASTOS et al. from 50 to 75%) and high activity (growth inhibition ranging 75 to 100%) (Fouche et al. 2008).

RESULTS AND DISCUSSION
Analysis by GC/MS of the Hx, DCM, CF and EtOAc extracts from leaves of P. microphyllum and P. mucronatum detected 51 constituents (Table I and Table II).These extracts contained a mixture of different compounds such as alkaloids, diterpenes, triterpenes, sterols, alcohols, aldehydes, fatty acids and hydrocarbons.
Consistent with the diversity of compounds detected, extracts from the leaves of P. microphyllum and P. mucronatum showed cytotoxic activity against strains analyzed (Table III).The percentages of inhibition of cell growth of extracts from P. microphyllum front line HEp-2 were 86.94% for DCM, 82.08% and 87.53% for EtOAc.The latter extract showed moderate activity (72.97%) against NCI-H292.
The cytotoxic activity of the Phoradendron genus has been reported.The ethanol and aqueous extracts of P. crassifolium (Polh) Eichier showed low toxicity in cultured HeLa cells (human cervix carcinoma) (Abad et al. 1999); the methanol extract of the leaves of P. vernicosum Greenm showed cytotoxic activity against cell line nasopharyngeal carcinoma (KB) (Caamal-Fuentes et al. 2011); the aqueous extract of the leaves of P. serotinum (Raf.)The percentage of inhibition of cell growth in different extracts may be related to the presence of certain groups of compounds such as alkaloids and triterpenes, known for their anticancer properties (Alonso-Castro et al. 2012).
The lupeol has been extensively studied, particularly in research involving the discovery of antitumor compounds (Saleem 2009).Some investigations have been carried out to evaluate the cytotoxic effects of lupeol on tumour cell lines such as Vero (African Green Monkey kidney), B16F10 (low wall melanoma) and HEp-2 (Badami et al. 2003).
The extracts (Hx, DCM, CF and EtOAc) of P. microphyllum and P. mucronatum showed cell percentages inhibition above 70% in the HEp-2 strain.It was observed that the lupeol was present in all extracts, but in higher percentages in the extracts of P. microphyllum.
Indole alkaloids were found in the two species under study.In the DCM, CF and EtOAc extracts of P. microphyllum have 2-(1H-indol-3-yl)-N,Ndimethylethanamine, also called Dimetiltriptamine (DMT).In Hx, DCM, CF and EtOAc extracts from P. mucronatum Aspidospermine was found and Aspidospermidine in the latter two extracts.
Alkaloids with amine groups and indole rings have been studied and various activities have been found, among them: antitumor, antiviral, antifungal and anti-inflammatory (Mcnulty et al. 2007, Griffin et al. 2007, Bao et al. 2004, Dassonneville et al. 2000, Xu et al. 2006).
Other research has been carried out with the purpose of showing that the alkaloids have a significant antiproliferative activity for solid tumours.These compounds have also selective cytotoxicity, a fact that stimulates a better investigation of their anticarcinogenic activity (Zhang et al. 2007, Wang et al. 2005).
Any of the extracts as well as the positive control (doxorubicin) presented an inhibition percentage above 75% (considered with high activity) against the MCF-7 line, except the DCM extract of the leaves of P. mucronatum.It is known that the breast cancer MCF-7 cells are resistant to chemotherapy, has low susceptibility to conventional drugs such as doxorubicin and cisplatin.This effect can be attributed to aberrant apoptotic pathway (Del Bufalo et al. 2002, Wesierska-Gadek et al. 2003).

CONCLUSIONS
Analysis by GC/MS allowed detection of 51 constituents in extracts of leaves of P. microphyllum and P. mucronatum.The constituents with higher percentages were lupenone (23.07%), lupeol (18.53%) and dimethyltryptamine (18.36%) for P. microphyllum and pentacosane (11.37%), hexacosane (10.72%) and lupenone (10.21%) for P. mucronatum.The different extracts of P. microphyllum showed cytotoxic activity against cell lines NCI-H292 and Hep-2, while the extracts of the species P. mucronatum showed inhibitory action towards the cell lines of NCI-H292, MCF-7, HEp-2.The major cell growth inhibition percentages were observed in the species P. mucronatum possibly due to the wide variety of constituents detected.

TABLE I Constituents identified in extracts of leaves of P. mucronatum by GC/MS with the highest similarity indices or equal to 90%.
An Acad BrasCienc (2017) 89 (2)