Prenylated flavonoids from roots of Dahlstedtia glaziovii (Fabaceae)

ARTICLE

Prenylated flavonoids from roots of Dahlstedtia glaziovii (Fabaceae)

Edione F. Canzi^I; Francisco A. Marques^I; Sirlei D. Teixeira^II; Ana Maria G. A. Tozzi^III; Marcos J. Silva^IV; Renata Maria T. Duarte^V; Marta Cristina T. Duarte^V; Ana Lúcia T. G. Ruiz^VI; Paula A. Monteiro^VI; João E. de Carvalho^VI; Beatriz Helena L. N. Sales Maia^{I, *}

^IDepartamento de Química, Universidade Federal do Paraná (UFPR), Centro Politécnico, Jardim das Américas, P.O. Box 19081, 81531-990 Curitiba-PR, Brazil

^IIDepartamento de Química, Universidade Tecnológica Federal do Paraná (UTFPR), P.O. Box 571, 85503-390 Pato Branco-PR, Brazil

^IIIDepartamento de Biologia Vegetal, Instituto de Biologia, Universidade Estadual de Campinas (Unicamp), P.O. Box 6109, 13083-970 Campinas-SP, Brazil

^IVInstituto de Ciências Biológicas, Universidade Federal de Goiás (UFG), P.O. Box 131, 74001-970 Goiânia-GO, Brazil

^VDivisão de Microbiologia , CPQBA, Universidade Estadual de Campinas (Unicamp), P.O. Box 6171, 13083-970 Campinas-SP, Brazil

^VIDivisão de Farmacologia e Toxicologia, CPQBA, Universidade Estadual de Campinas (Unicamp), P.O. Box 6171, 13083-970 Campinas-SP, Brazil

ABSTRACT

A phytochemical study of roots of Dahlstedtia glaziovii (Fabaceae) furnished a new dibenzoylmethane (glaziovione), along with eighteen known compounds. Their structures were determined through 1D and 2D nuclear magnetic resonance (NMR) (heteronuclear single quantum coherence, HSQC, and heteronuclear multiple bond correlation, HMBC) and high-resolution mass spectrometry (HRMS) spectral analyses. The antiproliferative activity was investigated for the crude extracts, the dibenzoylmethanes 2'-methoxy-8-(α-α-dimethylallyl)-furano-[4",5":3',4']-dibenzoylmethane, 3,4-methylenedioxy-2'-methoxy-8-(α-α-dimethylallyl)-furano-[4",5":3',4']-dibenzoylmethane and pongamol, and the flavones lanceolatin B, karanjin, pongapin and 3',4'-methylenedioxy-2",2"-dimethylpyrano-[5",6":8,7]-flavone. The dibenzoylmethanes were more active than the flavones.The extracts were evaluated for their antimicrobial effects, but none was shown to be active.

Keywords: Lonchocarpus, prenylated flavonoids, dibenzoylmethane, Dahlstedtia glaziovii, antiproliferative activity

RESUMO

O estudo fitoquímico das raízes de Dahlstedtia glaziovii (Fabaceae) forneceu um novo dibenzoilmetano (glaziovione), juntamente com dezoito compostos conhecidos. Suas estruturas foram determinadas através de métodos espectroscópicos de ressonância magnética nuclear (NMR) 1D e 2D (correlação heteronuclear única quântica, HSQC, e correlação heteronuclear de múltipla ligação, HMBC) e espectrometria de massas de alta resolução (HRMS). A atividade antiproliferativa foi investigada para os extratos, para os dibenzoilmetanos 2'-metóxi-8-(α-α-dimetilalil)-furano-[4",5":3',4']-dibenzoilmetano, 3,4-metilenodioxi-2'-metóxi-8-(α-α-dimetilalil)-furano-[4",5":3',4']-dibenzoilmetano e pongamol, e para as flavonas lanceolatina B, karanjina, pongapina e 3',4'-metilenodioxi-2",2"-dimetilpirano-[5",6":8,7]-flavona. Os dibenzoilmetanos foram mais ativos do que as flavonas. A atividade antimicrobiana foi investigada para os extratos, sendo que estes não se apresentaram ativos.

Introduction

The genera Dahlstedtia Malme and Lonchocarpus Kunth (Fabaceae) are very similar in terms of taxonomy as well as in the biosynthesis of secondary metabolites. A recent study investigated the relationships of Lonchocarpus and allied genera based on nuclear and plastid DNA markers.¹ This genus is native to the southern portion of South America, including southern and southeastern Brazil. Dahlstedtia glaziovii (Taub.) M.J. Silva & A.M.G. Azevedo (previously Lonchocarpus glaziovii Taub.) is restricted to Rio de Janeiro state, municipalities of Nova Friburgo and Alto Macaé, where it is popularly known as "Guaraná-timbó".¹

Dahlstedtia has been characterized by producing prenylated flavonoids, usually containing furan and dimethylpyran moieties.²

Flavonoids are one of the largest groups of secondary metabolites, with a wide range of biological activities including antioxidant,³ photoprotective,^4,5 antimicrobial,⁶ anti-inflammatory,⁷ anticancer,^8,9 memory effects and reduction of cardiovascular diseases,¹⁰ antifungal,^9,11 and antiviral,⁹ among others.¹²

In this context and continuing our research on native Fabaceae species, we conducted a phytochemical investigation of the roots of D. glaziovii, which led to the isolation and structural determination of a new dibenzoylmethane (1), in addition to eighteen known flavonoids (2-19) (Figure 1). Antibacterial and anti-Candida activities were assessed for the crude extracts. The antiproliferative in vitro activity of the extracts and some flavonoids was evaluated.

Experimental

General experimental procedures

Fourier transform infrared (FTIR) spectrum was acquired using a BIORAD FTS-3500 FTIR spectrometer, in CHCl₃ and as KBr discs. The UV-Vis spectra were obtained in CHCl₃ on a UV-2401PC (Shimadzu) spectrophotometer system. Optical rotations were measured in CHCl₃ solutions at room temperature on a Jasco polarimeter model P-2000. High-resolution mass spectra were run on a quadrupole LC-MS MicroQTOF II (Bruker) spectrometer equipped with an electrospray ionization source. Nuclear magnetic resonance (NMR) data were recorded at ambient temperature in CDCl₃ on a Bruker Avance 400, operating at 9.4 T, observing ¹H and ¹³C at 400.1 and 100.6 MHz, respectively, and on a Bruker Avance III 600 MHz, operating at 14.1 T (¹H at 600 MHz; ¹³C at 150 MHz) equipped with 5 mm cryo probe with automatic tuning matching (ATMA) and field gradient in z. The chemical shifts (δ) are given in ppm relative to tetramethylsilane (TMS, δ 0.00 ppm) as internal standard and coupling constants (J) in Hz. High-performance liquid chromatography (HPLC) analyses were performed on a Waters chromatograph consisting of quaternary pump, auto injector, 2998 PDA detector and coupled to an Empower software data acquisition system. The analyses were carried out using an analytic reversed phase column X-Terra C18, 5 µm, 250 × 4.6 mm and guard column at 20 ºC (Waters). The mobile phase consisted of an isocratic elution from acetonitrile:water (55:45, v/v), flow rate of 1 mL min^–1. The acetonitrile (ACN) was HPLC grade, filtered under vacuum through nylon membrane (0.45 µm) of Millipore^® and degassed for 30 min in ultrasound bath. Ultrapure water was obtained by Milli-Q system. The samples were filtered through Millex^® HV PVDF (0.45 µm) of Millipore^® (Cork, Ireland). The separations of fractions by HPLC were carried out using a semi-preparative column X-Terra C18, 10 µm, 300 × 7.8 mm (Waters). For open column chromatography silica gel 60 (70-230 mesh) was used whereas in flash column chromatography silica gel (230-400 mesh, Merck) was used. Radial chromatography was performed on Chromatotron 7924T (Harrison Research Chromatotron, San Bruno, California, USA) using silica gel (60 GF₂₅₄ Merck, 1 mm). Aluminum pre-coated silica-gel plates (60 F₂₅₄ Merck, 0.25 mm) were used for thin layer chromatography (TLC) analyses and glass pre-coated silica-gel plates (60 PF₂₅₄ Merck, 1 mm) were used for preparative TLC. The spots were detected by spraying with p-anisaldehyde reagent, followed by heating.

Plant material

The roots of Dahlstedtia glaziovii were collected in August 2009, in Nova Friburgo (Rio de Janeiro state, Brazil) at 22º20'2.2" S, 42º41'42.3" W and 1.221 m high, and identified by the taxonomists Prof Ana Maria Tozzi from Unicamp and Prof Marcos Silva from UFG. A voucher specimen (Marcos J. Silva 1077) was deposited at the Herbarium UEC, Institute of Biology, Unicamp.

Extraction and isolation

Dried root bark (315 g) of D. glaziovii was powdered and successively extracted at room temperature with petroleum ether (PE), dichloromethane (CH₂Cl₂) and methanol (MeOH). The solvents were removed under reduced pressure to afford the PE (1.1 g), CH₂Cl₂ (2.3 g) and MeOH (21.5 g) extracts. The dichloromethane extract (2.3 g) was subjected to silica gel flash column chromatography (diameter 3.5 cm), with gradient elution of PE/CH₂Cl₂ from 100:0 to 25:75, CH₂Cl₂/EtOAc from 100:0 to 25:75 and EtOAc/MeOH from 100:0 to 0:100, affording a total of 41 fractions (F1-F41) of 150 mL each. Fraction 16 (349 mg) was purified by radial chromatography with gradient elution of PE/CH₂Cl₂ from 70:30 to 0:100 yielding 12 (42 mg). Fraction 17 (500 mg) was subjected to a silica gel column chromatography eluted with PE/CH₂Cl₂ from 30:70 to 10:90, CH₂Cl₂/EtOAc from 100:0 to 20:80, EtOAc/MeOH from 100:0 to 50:50 and MeOH affording 31 fractions (F17-1 to F17-31) of 50 mL each. Fraction F17-6 (74 mg) was purified by preparative TLC, eluted with PE/acetone (75:25) (four times) yielding 13 (2.7 mg), 12 (3.0 mg) and 16 (1.0 mg). Fraction F17-9 (60 mg) was subjected to HPLC (H₂O/ACN (45:55), flow rate 1 mL min^-1) to give 19 (2.1 mg), 11 (2.5 mg), 13 (1.6 mg), 15 (3.0 mg) and 17 (2.1 mg). Fraction F17-14 was subjected to preparative TLC eluted with PE/acetone (80:20) (four times) yielding a mixture of 18 and 14 (8.2 mg). Fraction 14 (60.0 mg) was subjected to preparative TLC eluted with PE/EtOAc (80:20) yielding 2 (32.9 mg), 3 (12.5 mg), 5 (1.0 mg), 10 (2.6 mg) and 6 (2.9 mg). Fraction 12 (30.0 mg) was purified by preparative TLC eluted with PE/ethyl ether (7:3) (four times) yielding 6 fractions (F12-1 to F12-6). Fractions F12-2 (9.8 mg) and F12-4 (6.6 mg) were subjected to preparative TLC eluted with PE/ethyl ether (8:2) (twice) yielding 2 (2.2 mg) and 9 (1.7 mg), respectively. Fraction 8 (12.3 mg) was purified by preparative TLC with PE/ethyl ether (9.8:0.2) (three times) yielding 7 (1.4 mg), 4 (3.7 mg) and 1 (1.4 mg). Fraction 6 (5.8 mg) was subjected to preparative TLC with PE/ethyl ether (9:1) (twice) yielding 8 (2.1 mg).

Glaziovione (1): yellow oil; [α]_D²⁰ –2.31 (c 0.0014, CHCl₃); λ_max/nm 249, 281, 336; IR (film) ν_max/cm^-1 3450, 2921, 2850, 1640, 1637, 1580, 1292; ¹H NMR (CDCl₃, 400.1 MHz) and ¹³C NMR (CDCl₃, 100.6 MHz) data see Table 1; HRMS (pESI) calcd. for C₂₂H₂₀O₄ [M+Na]⁺: 371.1255; found: 371.1255.

In vitro antimicrobial activity

The antimicrobial activity of the crude extracts was tested against eight microorganisms (Escherichia coli ATCC 11775, Pseudomonas aeruginosa ATCC 13388, Salmonella choleraesuis ATCC 10708, Staphylococcus aureus ATCC 6538, Streptococcus pneumonia ATCC 11733, Candida albicans ATCC 10231, Candida dubliniensis CBS 7987 and Candida krusei CBS 573).

The microorganisms were subcultured overnight at 36 ºC using nutrient agar (Merck) for bacteria, and Sabouraud dextrose agar for Candida spp. Inocula for the assays were prepared by diluting a scraped cell mass in 0.85% NaCl solution, adjusted to McFarland scale 0.5 and confirmed by spectrophotometer reading at 625 nm (bacteria) or 530 nm (yeasts). Cell suspensions were finally diluted to 10⁴ colony forming units (CFU) mL^-1 or 10³ for use in the activity assays, respectively for bacteria and yeasts. Minimal inhibitory concentration (MIC) tests were carried out according to the Clinical and Laboratory Standards Institute (CLSI),^13,14 using Müller-Hinton broth on a tissue-culture test plate (96 wells) or Roswell Park Memorial Institute (RPMI)-1640 broth. The stock solution of crude extracts were diluted and transferred into the first well, and serial dilutions were made so that concentrations in the range of 1.0-0.015 mg mL^-1 were obtained. Chloramphenicol and nistatin (Merck) were used as the reference antibiotic control in the range of 0.25-0.002 mg mL^-1. The inoculum was added to every well, and the plates were incubated at 36 ºC for 48 h. Each concentration was screened in triplicate. Antibacterial activity was detected by adding 20 µL of 0.5% triphenyltetrazolium chloride (TTC, Merck) aqueous solution. MIC was defined as the lowest concentration of the sample that inhibited visible growth, as indicated by TTC staining (dead cells are not stained by TTC). In the case of Candida spp, after the incubation period the RPMI-1640 medium color changed from pink (original color) to yellow. The change indicates an acidification of the medium by the microorganism growth.

In vitro antiproliferative activity assay

Human tumor cell lines U251 (glioma), MCF-7 (breast), 786-0 (renal), and HT-29 (colon) were kindly provided by the Frederick Cancer Research & Development Center, National Cancer Institute, Frederick, MA, USA. Human keratinocyte (HaCaT) cell line was donated by Dr Ricardo Della Coletta (Piracicaba Dental School, University of Campinas). Stock cultures were grown in medium containing 5 mL RPMI-1640 (GIBCOR BRL) supplemented with 5% fetal bovine serum. Penicillin/streptomycin (1000 µg mL^-1:1000 UI mL^-1, 1 mL L^-1) was added to experimental cultures. Cells in 96 well plates (100.0 µL cells well^-1) were exposed to sample concentrations in dimethylsulfoxide (DMSO)/RPMI (0.25, 2.5, 25 and 250 µg mL^-1) at 37 ºC, 5% of CO₂ in air for 48 h. Final DMSO concentration did not affect cell viability. Afterwards, cells were fixed with 50% trichloroacetic acid and cell proliferation determined by spectrophotometric quantification (540 nm) of cellular protein content using sulforhodamine B assay, by measuring absorbance at the beginning of incubation (time zero, T0) and 48 hours post-incubation for compound-free (T1) and tested (T) cells.¹⁵ Cell proliferation was determined according to the equation 100 × [(T – T0) / T1 – T0], for T0 < T < T1, and 100 × [(T – T0) / T0], for T < T0. Using the concentration-response curve for each cell line, the concentration that elicits 50% growth inhibition (GI₅₀) was determined through non-linear regression analysis (Table 2) using ORIGIN 8.0 (OriginLab Corporation) software.

Results and Discussion

The dichloromethane extract afforded nineteen flavonoids, including one new (1) and five known dibenzoylmethanes (2-6), two chalcones (7 and 8), two flavanones (9 and 10), eight flavones (11-18) and one rotenoid (19) (Figure 1).

Glaziovione (1) was obtained as a yellow oil with the –2.31 (c 0.0014, CHCl₃). Its molecular formula C₂₂H₂₀O₄ was calculated by HRESIMS in positive mode, which revealed a peak at m/z 371.1255, relative to the pseudo-molecular ion [M+Na]⁺. The ¹H NMR spectrum (Table 1) showed a singlet at δ 5.57 (s, 1H, H-8), which showed correlations with two carbonyl carbons at δ 193.9 (C-7) and δ 200.0 (C-9) in the heteronuclear multiple bond correlation (HMBC) spectrum, suggesting a dibenzoylmethane diketone skeleton.¹⁶ Additionally, the ¹H NMR spectrum exhibited characteristic signals of a dimethylallyl group at δ 6.20 (dd, 1H, J 17.5, 10.7 Hz, H-2"'), 5.02 (dd, 1H, J 17.5, 0.9 Hz, H-3a"') and 4.98 (dd, 1H, J 10.7, 0.9 Hz, H-3b"'), as well as signals referring to methyl groups at δ 1.33 and 1.34 (s, 3H each). The location of the dimethylallyl group at C-8 (Figure 2) was determined from the HMBC spectrum. The presence of a monosubstituted aromatic ring was also corroborated by signals at δ 7.95 (m, 2H, H-2 and H-6), 7.42 (m, 2H, H-3 and H-5) and 7.53 (m, 1H, H-4). Two doublets were observed at δ 7.57 (d, 1H, J 2.2 Hz, H-2") coupling with δ 6.99 (d, 1H, J 2.2, 0.9 Hz, H-3") that were assigned to a furan ring; its angular position in the B ring was determined by the aromatic hydrogens at δ 7.07 (dd, 1H, J 9.0, 0.9 Hz, H-5') and δ 7.83 (d, 1H, J 9.0 Hz, H-6'), featuring ortho coupling between H-5' and H-6' (J 9.0 Hz), together with a long distance coupling (J 0.9 Hz) between H-5' and H-3" from the furan ring.

When comparing the ¹H NMR spectrum of 1 with that of 2'-methoxy-8-(α-α-dimethylallyl)-furano-[4",5":3',4']-dibenzoylmethane (2)¹⁶ (Table 1), it was observed that 1 differed by the presence of a hydroxyl group characterized by the singlet at δ 13.30 (s, 1H, 2'-OH). This could be attributed to an internal hydrogen bond to a 4-oxo group, correlating to a carbonyl in C-9 (Table 1), in place of the methoxyl group observed for compound 2.

The complete structure elucidation and unambiguous ¹H and ¹³C NMR chemical shift assignments were supported by the heteronuclear single quantum coherence (HSQC) and HMBC experiments (Table 1, Figure 2) and by comparison with the spectral data of 2.¹⁶ Therefore, 1 was determined as 2'-hydroxy-8-(α,α-dimethylallyl)-furano-[4",5":3',4']-dibenzoylmethane and named glaziovione.

Compounds 2-19 (Figure 1) were identified by comparison of their spectral data with those described in the literature, as 2'-methoxy-8-(α-α-dimethylallyl)-furano-[4",5":3',4']-dibenzoylmethane (2),¹⁶ 3,4-methylene­dioxy-2'-methoxy-8-(α-α-dimethylallyl)-furano-[4",5":3',4']-dibenzoylmethane (3),¹⁶ pongamol (4),¹⁷ ovalitenone (5),¹⁷ 2'-methoxy-8-(α,α-dimethylallyl)-2",2"-dimethylpyrano-(5",6":3',4')-dibenzoylmethane (6),¹⁸ glabrachromene-II (7),¹⁹ pongachalcone (8),²⁰ (–)-isolonchocarpin (9),²¹ ovalichromene B (10),²² lanceolatin B (11),²³ karanjin (12),²³ pongapin (13),²⁴ pongaglabrone (14),²⁵ 2",2"-dimethylpyrano-[5",6":8,7]-flavone (15),² karanjachromene (16),² pongachromene (17),²⁶ 3',4'-methylenedioxy-2",2"-dimethylpyrano-[5",6":8,7]-flavone (18),² and tephrosin (19).²⁷

The crude extracts were evaluated for antibacterial and anti-Candida activity. None of the extracts showed significant activity against any of the microorganisms tested.

In vitro antiproliferative activity was evaluated for the three extracts, dibenzoylmethanes 2-4, and flavones 11-13 and 18 (Table 2).

Considering the mean value of GI₅₀, the PE and CH₂Cl₂ extracts showed better antiproliferative activity, with mean GI₅₀ of 5.1 and 7.1 µg mL^-1, respectively. The MeOH extract showed weak activity, with a mean GI₅₀ of 57.4 µg mL^-1. Both PE and CH₂Cl₂ extracts showed a similar activity against the cell lines U251 (glioma), MCF7 (breast) and 786-0 (renal); the PE extract inhibited the HT29 (colon) cell line more strongly than the CH₂Cl₂ extract. Although they showed similar results for antiproliferative activity, the CH₂Cl₂ extract was obtained in a larger amount than the PE extract. Therefore, the CH₂Cl₂ extract was fractionated and some of the isolated flavonoids (2-4, 11-13 and 18) were isolated in sufficient quantity to assay against tumor cell lines.

Considering all the flavonoids assayed and their mean GI₅₀, the members of the dibenzoylmethane series were more active than the flavones. Dibenzoylmethanes 2-4 exhibited the best antiproliferative activity, with mean GI₅₀ of 25.1, 32.0 and 33.7 µmol L^-1, respectively. Flavones 11 and 18 showed similar activity (mean GI₅₀ = 145.8 and 130.7 µmol L^-1, respectively). Flavone 13 was the least active of the series, with mean GI₅₀ > 215.2 µmol L^-1. Flavone 12 showed no activity (mean GI₅₀ > 856.2 µmol L^-1).

These results suggest that the cyclization to form the C ring decreased the antiproliferative activity, since the flavonoids with an open chain (the central part of the molecule) were more active (dibenzoylmethanes 2-4) than those with a C ring (flavones 11-13 and 18).

Comparison of dibenzoylmethanes 2 and 3 indicated that the presence of the methylenedioxy group in 3 decreased the mean GI₅₀ activity (25.1 and 32.0 µmol L^-1, respectively). Comparing compounds 2 and 4, the structural difference is the presence of the α,α-dimethylallyl group at C-8 in 2, which leads to an increase of the mean GI₅₀ activity (25.1 and 33.7 µmol L^-1, respectively).

Evaluating the antiproliferative activity on the non-tumor cell line HaCat (keratinocyte), the extracts and the prenylated flavonoids affected the proliferation of these cells within the same order of magnitude as their effect on tumor cells. The same trend was observed for the chemotherapeutic agent doxorubicin, while the prenylated flavonoids assayed showed less toxicity than doxorubicin. Thus, in vivo investigation is needed to determine the safety of using these flavonoids as potential chemotherapeutic agents.

Conclusions

The isolation of the prenylated flavonoids (1-19) from D. glaziovii roots concurs with the reclassification suggested by da Silva et al..¹ Flavonoids with prenyl cyclized groups such as a furan ring and 2",2"-dimethylpyran, which have been used as indicators of the evolution of the genus Dahlstedtia, are found at the angular position in the main skeleton.²

Many compounds isolated from D. glaziovii have been found in other species, including D. pinnata² (Benth.) Malme and D. penthaphylla² (Taub.) Burkart. This indicates the chemical similarity between the species, in concordance with the morphological affinity cited by da Silva et al..¹ In addition, other species reclassified as Dahlstedtia,¹ previously studied, have some chemical similarity to D. glaziovii, including D. floribunda (Vogel) M.L. Silva & A.M.G. Azevedo (Lonchocarpus subglaucescens Benth)²⁸ and D. muelbergiana (Hassl.) M.J. Silva & A.M.G. Azevedo (L. muehbergianus Hassl.).²⁹

Dibenzoylmethane 1 is a new metabolite, and flavonoids 6, 7, 13 and 19 are reported for the first time in the genus Dahlstedtia. This is the second report of dibenzoylmethane 6, which was isolated for the first time in Muellera montana M.J. Silva & A.M.G. Azevedo (Lonchocarpus montanus).²⁰

The antiproliferative assays showed that the members of the dibenzoylmethane series were more active than the flavones, and also showed that these flavonoids have promising activity.

Supplementary Information

Supplementary information (Figures S1-S26) is available free of charge at http://jbcs.sbq.org.br as a PDF file.

Acknowledgements

The authors are grateful to Prof Massuo J. Kato, Universidade de São Paulo, USP, SP, Brazil for the HRESIMS analyses, to CAPES-PROCAD and to Fundação Araucária-Paraná, for financial support. E. F. C. is grateful to CAPES for the award of a scholarship. B. H. L. N. S. M. is also grateful to Fundação Araucária for a Productivity Fellowship.

Submitted: February 25, 2014

Published online: April 8, 2014

FAPESP has sponsored the publication of this article.

Supplementary Data

The supplementary data is available in pdf: [Supplementary material]

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Publication Dates

• Publication in this collection
  01 July 2014
• Date of issue
  June 2014

History

• Accepted
  04 Aug 2014
• Received
  25 Feb 2014