14 , 15-epoxygeranylgeraniol and extracts isolated from Pterodon emarginatus Vog . fruits : antitumor activity on glioblastoma cells

Article history: Received on: 03/09/2012 Revised on: 14/09/2012 Accepted on: 20/09/2012 Available online: 28/09/2012 Plant-derived substances have traditionally played important roles in the treatment of human diseases, including of great significance to cancer therapy. Plants of the genus Pterodon (Fabaceae, Leguminosae), commonly known as ‘sucupira’, are disseminated throughout the central region of Brazil and have been used frequently in popular medicine. In recent years, interest in these plants has increased considerably. The biological effects of their extracts and pure metabolites have been investigated in several experimental models in vivo and in vitro. Until the present day, the antitumor effect of Pterodon plants on brain tumors is unknown. Therefore, the aim of this work was to investigate the action of P. emarginatus Vogel extracts and its fractions on glioblastoma cells. The hexane (HE), dichloromethane (DE) and ethanol (EE) extracts were obtained from seeds powder in each solvent. The diterpene 14,15-epoxygeranylgeraniol was obtained from HE fractionation. For tumorigenic assays, the extracts and fractions were added to U87MG, a human glioblastoma cells line. The cell viability assay showed that the proliferation of U87MG was inhibited by both extracts and the 14,15epoxygeranylgeraniol. Further trials in vivo will help to confirm these results, and may contribute to generate natural compounds for the treatment of this type of cancer.


INTRODUCTION
Human beings have used some plant constituents for centuries, e.g., to prepare poisonous spearheads for warfare and hunting.The plants make use sophisticated signaling mechanisms and an elaborate chemical arsenal of deadly weapons such as terpenes to poison the soil to inhibit competitors, and alkaloids which make them unpalatable to insects and predators.Plantderived substances have traditionally played important roles in the treatment of human diseases, including of great significance to cancer therapy (Mans et al., 2000).. Plant of the genus Pterodon (Fabaceae, Leguminosae), commonly known as 'sucupira' or 'faveira', comprises four native species: Pterodon abruptus Benth., Pterodon apparicioi Pedersoli., Pterodon polygalaeflorus Benth.and Pterodon emarginatus Vogel.synonym Pterodon pubescens Benth.(Carvalho, 2004).These genus are disseminated throughout the central region of Brazil and have been used frequently in popular medicine for its anti-rheumatic (Sabino et al., 1999;Coelho et al., 2004), analgesic (Spindola et al., 2011;Galceran et al., 2011) andanti-inflammatory (Dutra et al., 2009;Moraes et al., 2011) properties.In recent years, interest in these plants has increased considerably and, the biological effects of different phytoextracts and pure metabolites have been investigated in several experimental models in vivo and in vitro (Hansen et al., 2010).
The investigations on the pharmacological properties of 'sucupira' surpass those of its anti-inflammatory and antirheumatic activities.The literature describes the presences of flavonoids, diterpenes, steroids, besides proteins from this genus (Hansen et al., 2010).The interest in the Pterodon genus began when Mors et al. (1967) isolated the geranylgeraniol and 14,15epoxygeranylgeraniol by hexane extraction of the essential oil from fruits of P. pubescens fruit essencial oil with hexane, showing the chemoprophylactic effect on schistosomiasis (Mors et al., 1966;1967).The geranylgeraniol obtained of this plant presented antiplatelet (Calixto et al., 2007) and anti-Trypanosoma cruzi action with effect on proliferation of epimastogotes and trypomastigotes (Menna-Barreto et al., 2008).Spindola et al. (2010) evaluated the contribution of geranylgeraniol in the antinociceptive activity of the crude extracts from seeds of P. pubescens.
Related to antitumor activity of Pterodon genus, until now, little has been investigated.Some subfractions from crude ethanolic extract and the diterpene vouacapan-6α, 7β, 14β, 19tetraol of seeds from P. pubescens have presented effect on human melanoma cells (Vieira et al., 2008) and furanoditerpenes have presented action in prostate cells (Spindola et al., 2009).In recent study, a terpenic subfraction presenting a furane diterpene, induced apoptosis of K562 leukemic cells (Pereira et al., 2011).In relation to P. polygalaeflorus species, the antiproliferative effect on human cancer cells was studied by Euzébio et al., (2009).
Until the present day, the effects of the Pterodon extract or metabolites on glioblastoma cells obtained from brain tumors are unknown.The highest-grade malignant astrocytoma, glioblastoma (GBM), is the most common and the major lethal type of tumor in the central nervous system, leading to a mean survival time of approximately 16 months after removal of tumor and radiotherapy (Valente et al., 2009).This discouraging prognosis is due to both the infiltrative nature of the tumor and the resistance of tumor cells to cytotoxic treatments (Omuro and Delattre, 2007;Dent et al., 2009;Agarwal et al., 2011).
The results reported here shows the action of extracts and of the diterpene 14,15-epoxygeranylgeraniol obtained from P. emarginatus fruits on proliferation of U87MG human glioblastoma cells line.

Plant Material
The dry fruits were collected in the Cerrado region of the municipality of Urutaí, Goiás State, Brazil (17 o 39'3.3''S/ 48 o 14'5.6''W), on August 2009.The plant was identified for botanic at the Botanic Institute Herbarium of São Paulo and a voucher specimen was deposited at the Biological Institute of São Paulo, under number DH2009/001.
The thin layer chromatography (TLC) and the UV spectroscopy were employed to characterize chemical skeletons, presenting compounds of terpene nature.The extracts were analyzed according its R f (retention factor) on TLC, using silica gel 60 F 254 Merck as stationary phase, hexane/ethyl acetate solution (2:1, v/v) as moving phase and was revealed with vanillin/H 2 SO 4 at 100 o C.

Hexanic extract fractionation (C1)
The HE (30 g) was fractionated using column chromatography packed with silica gel 60 Merck KGaA (Darmstadt, Germany) and eluted with hexane and ethyl acetate, increasing order of polarity (fractions C1 1 to C1 40 ).The fractions were monitored by TLC employing a plastic plate impregnated with silica gel 60G F 254 Merck KGaA (Darmstadt, Germany), developed with hexane/ethyl acetate solution (2:1, v/v) and revealed with vanillin/H 2 SO 4 at 100 o C.

Isolation and characterization of compound
Based on TLC dates, the fraction C1 13 (290 mg) resultant of chromatography C1, was fractionated in a new column chromatography (C2) with the same stationary phase as C1 and was eluted with hexane and gradients of hexane/ethyl acetate solution (95:5 v/v) up to 35% of ethyl acetate.The fractions were analyzed according its R f on TLC, using the same stationary and moving phases as used in C1.
The identification of the compounds was carried out using proton ( 1 H-NMR) and carbon nuclear magnetic resonance ( 13 C-NMR).All spectra were recorded on a Bruker DPX300 spectrometer, operating at 300 MHz.

Cell culture
The U87MG human glioblastoma cancer cell line was kindly provided by Dr. Suely K. N. Marie from the Laboratory of Medical Investigation (LIM15) at the University of São Paulo.Cells were maintained in Dulbecco's-modified Eagle's Medium-Low Glucose (DMEM-LG, Invitrogen), supplemented with 2 mM L-glutamine, 10% bovine fetal serum, 100 U/mL penicillin, and 100 µg/mL streptomycin, in a humidified atmosphere at 37 o C with 5% CO 2 .

Cell viability assay
The effect of the extracts HE and its fractions, DE and EE on the viability of U87MG glioblastoma cells line was determined using a MTT-based assay (Carimichael et al., 1987).Briefly, exponential-phase cells were collected and transferred to a microtiter plate (10 3 cells/0.1 ml).The cells were then incubated for 24, 48 and 72 hs with various concentrations of the extracts and HE fractions.After incubation, 0.1 mg MTT (Sigma, St. Louis, MO, USA) was added to each well, and the cells were incubated at 37 o C for 2 h.Then, the medium was carefully removed and added isopropanol to each well to dissolve formazan crystals.After 30 minutes of incubation at 37 o C, the plates were read immediately at 620 nm on a Packard SpectraCount microplate reader.The percentage of cell viability was calculated based on the following formula: mean value of (control group -treated group/control group) x 100%.All results were assessed in triplicate for each concentration.

Calculations and statistical analysis
Cell viability experiments results were expressed as the percentage cell viability of control (untreated cells).All results were submitted to one way analysis of variance (ANOVA) with repeated measurements.The inhibitory concentration value (IC 50 ) was derived from a nonlinear regression model (curve fit) based on sigmoidal dose response curve and computed using GraphPad Prism, version 5.00 for Windows, GraphPad Software, San Diego, CA.All assays were performed in triplicate.

RESULTS AND DISCUSSION
The preliminary results achieved by cell viability assay indicated that the crude hexane (HE), dichloromethane (DE) and ethanol extracts (EE) from P. emarginatus Vogel fruits inhibited at least 50% of the proliferation of U87MG human glioblastoma cells after 48 and 72 hours of incubation in the concentration of 10 mg/ml (Fig. 1a and 1c).After 24 hours of incubation, the inhibition wasn´t significant, as shown in Fig. 1a.Given these initial results, we decided investigated the HE in a first moment.Thus, the phytochemical study of the HE presented compounds of terpene nature; the TLC shows an intense spot with R f 0.35, which was also predominant in C1 13 , C2 120 and neighboring fractions (Fig. 2).The NMR spectra through its carbon and hydrogen chemical shifts indicate that the molecule corresponding to C2 120 fraction is the diterpene 14,15-epoxygeranylgeraniol (Fig. 3), previously isolated for Mors et al. (1967).
To perform the cell viability assay to the fractions obtained from HE, U87MG cells were treated with 10 -3 to 10 3 µg/ml of the C1 13 and C2 120 samples and incubated for 24, 48 and 72 hours.The calculated IC 50 values of HE, C1 13 and C2 120 fractions after 72 hours of incubation were 0.031, 4.622 and 9.706 µg/ml, and were obtained from the dose-response curve shown in Fig. 4a, 4b and 4c, respectively.
Natural product derived drugs constitute a vast majority of the chemotherapeutic agents currently in use for all types of cancers (Baker et al., 2007).Preliminary testing of antitumoral activity of P. emarginatus extracts and 14,15epoxygeranylgeraniol on glioblastoma cells presented promising results and the continuation of these studies, in additional trials in vivo, could contribute to generate new bioactive molecules or chemotherapeutic agents for this type of cancer.

Fig. 4 :
Fig. 4: Cell viability test of P. emarginatus HE and its fractions.Dose-response curve of U87MG human glioblastoma cells following 72 hours exposure to (a) HE, (b) C113 and (c) C2120.Cell viability was achieved by the MTT assay and expressed as percentage of untreated cells.

Fig. 2 :
Fig. 2: Chromatogram on TLC of the hexane extract and its fractions.The TLC shows an intense spot with Rf 0.35, which remained predominant after fractionation.HE: hexanic extract; C113: fraction 13 from chromatography 1 and C2120: fraction 120 from chromatography 2.

Fig. 3 :
Fig. 3: Chemical structure of 14,15-epoxygeranylgeraniol.The NMR spectra through its carbon and hydrogen chemical shifts indicate that the molecule corresponding to C2120 is the diterpene previously isolated for Mors et al. (1967).