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 main plant-derived compounds with anticancer properties are alkaloids and terpenoids (^{Gupta et al. 2005}). Examples are derivatives of etoposide and teniposide lignans (^{Allen et al. 2003}, ^{Choi et al. 2008}), vincristine and vinblastine from Catharanthus roseus (L.) G. Don (^{Johnson et al. 1963}, ^{Carvalhaes et al. 2002}), semi-synthetic derivatives of podophyllotoxin isolated from species of the genus Podophyllum (Berberidaceae) (Jacobo-Herrera et al. 2016), taxol, and docetaxel from Taxus brevifolia Nutt. (^{Wani et al. 1971}) and semi-synthetic derivatives from camptothecin, irinotecan and topotecan isolated from Camptotheca acuminata (Nyssaceae) (^{Cragg et al. 1993}, 1994, ^{Wang 1998}).

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.

MATERIALS AND METHODS

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.

The constituents that showed relative larger areas (%), from the extracts of P. microphyllum leaves, were Lup-20(29)-en-3-one (lupenone, 23.07%) and (3β)-Lup-20(29)-en-3-ol (lupeol, 16.95%) in the Hx. In the DCM and CF extracts were lupeol (18.53% and 16.19%), lupenone (14.47% and 12.99%) and (3β)-Estigmast-5-en-3-ol (β-sitosterol, 9.74% and 9.45%), and N,N-dimethyl-1H-indole-3-ethanamine (dimetiltriptamine 18.36%) in CF. In the EtOAc extract, the compounds that had higher percentages were lupeol (20.86%) and lupenone (18.69%).

Regarding the extracts of the leaves of P. mucronatum, the constituents with the highest percentages were: lupeol (8.33%), triacontane (8.71%), pentacosane (11.37%) in Hx; in DCM, (Z)-11-octadecenoic acid (cis-vaccenic acid, 7.83%), pentacosane (8.35%); the CF, cis-vaccenic acid (8:35%), tetracosane (7.85%) and EtOAc, hexacosane (10.72%), tetratetracontane (8.2%), lupenone (10.21%) and lupeol (22.8%).

Studies with ethanol extract of the aerial parts of P. greggii identified and isolated oleanoic acid (^{Dominguez et al. 1971}). ^{Rios et al. (2001}) isolated a tetracyclic triterpene (3,4-seco-olean-18-ene-3,28-dioic acid) from the aerial parts of P. reichenhachianum. López-Martínez et al. (2013) identified and isolated from the acetone extract of the leaves of P. brachystachyum morolic acid as the major component, and β-sitosterol, stigmasterol, triacontanol, squalene, α- and β-amyrin, lupeol, lupenone, betulin, oleanolic aldehyde, betulinic acid, betulonic acid, moronic acid, morolic acid, oleanolic acid.

Some of these compounds, such as β-sitosterol, squalene, lupeol and lupenone were detected in this study, indicating that various constituents are common among the different species of the genus Phoradendron. In addition, common constituents in different extracts of P. microphyllum and P. mucronatum were observed. These are: 7,11,15-trimethyl-3-methylene-1-hexadecene; (2E)-3,7,11,15-tetramethyl-2-hexadecen-1-ol; (2R)-2,5,7,8 tetramethyl-2-[(4R,8R)-4,8,12 trimethyltridecyl]-6-chromanol; 4,14-dimethyl-9,19-cycloergost-24(28)-en-3-yl acetate and stigmast-4-en-3-one.

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 extract Hx of P. mucronatum inhibited cell growth of NCI-H292 and Hep-2 at 83.19% and 93.40% respectively. The DCM extracts showed inhibitory effect on HEp-2 (85.47%) and MCF-7 (88.69%). The CF showed inhibition of 77.75% for NCI-H292, and 91.46% for HEp-2 and EtOAc to extract inhibited Hep-2 line (72.17%).

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.) MC Johnst exerted toxic effects against cancer cells MCF-7 (adenocarcinoma human breast) (Jacobo-Salcedo et al. 2011); and the ethanol extract showed cytotoxic activity against TC-1 cells (derived from lung epithelium) (Alonso-Castro et al. 2012).

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,N-dimethylethanamine, 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.