Antiproliferative activity of methanol extracts of four species of Croton on different human cell lines

Antiproliferative activity of methanol extracts of four species of Croton on different human cell lines

Jóice P. Savietto^I; Cláudia M. Furlan^I; Lucimar B. Motta^I; Maria Luiza F. Salatino^I; João E. Carvalho^II; Ana Lucia T. G. Ruiz^II; Antonio Salatino^I; Déborah Yara A. C. Santos^I,*

^IDepartamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Brazil

^IIDivisão de Farmacologia e Toxicologia, CPQBA, Universidade Estadual de Campinas, Brazil

ABSTRACT

Several species of Croton have been described with biological activities, mainly due to diterpenes, alkaloids and/or other secondary metabolites. These activities account for the traditional use of Croton species to treat certain diseases in South America, Asia and Western Africa. The crude methanol extracts obtained from leaves and steam bark of Croton dichrous Müll. Arg., C. erythroxyloides Baill., C. myrianthus Müll. Arg. and C. splendidus Mart. ex Colla were tested for antiproliferative activity against ten human cancer cell lines. Chemical analyses of all extracts were carried out by GC/MS and HPLC/MS/MS. The leaf extract obtained from C. erythroxyloides showed potent activity against PC-3 (prostate) and OVCAR-3 (ovary) cell lines. Lupeol is suggested to be involved in such activity. Tiliroside, an acyl-glycosilated flavonoid ubiquitous in all tested extracts, seems to play an important role in the observed moderate activity of most extracts against the leukemia K562 cell lineage.

Keywords: antiproliferative activity, Croton. Euphorbiaceae, human cell lines, lupeol, methanol extract

Introduction

Several species of Euphorbiaceae are known in many parts of the world for their toxic and/or medicinal properties (Salatino et al., 2007). Such diversity of effects is a reflection of the variety of secondary metabolites which have already been described for this family.

Croton L. is one of the giant genera of angiosperms, comprising over 1200 species of herbs, shrubs, trees and occasionally vines. Recent studies have estimated that approximately two-thirds of the Croton species occur in the New World, the other third being distributed across the Old World (van Ee et al., 2011). It is the second largest genus in Euphorbiaceae, with various species used in folk medicine in many regions of the world.

Hypoglycemic, hypolipidemic, anti-estrogenic, anti-tumor and trypanocidal effects have been ascribed to the trans-terpenoid dehydrocrotonin obtained from< C.cajucara(Maciel et al., 2002; Campos et al., 2010). Antiulcerogenic effect in gastric lesion of the hydroalcoholic extract ofC. campestrisleaves has been demonstrated in laboratory assays with mice (Júnior et al. 2013). Both latex and the alkaloid taspine, isolated fromC. lechleri, show cytotoxic activity (Montopoli et al., 2012), and plaunotol, an open-chain diterpene found in Croton species, acts in synergism with common drugs against Helicobacter pylori (peptic ulcer-causing bacteria) (Sasaki et al., 2007) and as antiangiogenic (Kawai et al., 2005).

The biological activities of these compounds account for the traditional use of Croton species to treat certain diseases in South America, Asia and Western Africa (Sampson et al., 2000). Cancer is a disease recognized by several hallmarks, one of the main ones being the proliferation of atypical cells (Mantovani, 2009). According to the World Health Organization, cancer is a leading cause of death worldwide. The organization estimates that 84 million people will die from cancer between 2005 and 2015 (WHO, 2007).

The potential use of natural products against cancer has been recognized since 1950 by the National Cancer Institute (USA) (Cragg & Newman, 2005). Some important drugs currently available were discovered by the investigation of secondary metabolites of plants, some examples being taxol, extracted from the stem of Taxus brevifolia, Taxaceae (Wani et al., 1971) and vincristine, an alkaloid from the roots of Catharanthus roseus, Apocynaceae (Johnson et al., 1963).

The antiproliferative activity of crude extracts of some species of Croton have already been described. Stem bark essential oil obtained from C. lechleri showed mutagen-protective efficacy (Rossi et al., 2013) and crude extracts of stems of C. cajucara (Maciel et al., 2007) exert antitumor activity against the K562 leukemic cell line. The dicloromethane extract of C. macrobothrys leaves exhibited antiproliferative activity against several cell lines, in particular on NCI-H460 (lung) and K562 (leukemia) (Motta et al., 2011).

In this study, we investigated the antiproliferative activity of methanol extracts from four Brazilian native species of Croton, and suggest compounds involved in the observed effects. For this, ten human tumor cell lines commonly investigated were used: 786-0 (kidney), HT-29 (colon), K562 (leukemia), NCI-ADR/RES (drug resistant ovary), NCI-H460 (lung), MCF-7 (mammary), PC-3 (prostate), OVCAR-3 (ovary), U251 (glioma), UACC-62 (melanoma).

Materials and Methods

Plant material

Samples of Croton dichrous Müll. Arg. (Cordeiro 3066, Campos do Jordão-SP), C. erythroxyloides Baill. (Cordeiro 3067, Campos do Jordão-SP), C. myrianthus Müll. Arg. (Lima 296, Cambará do Sul-RS) and C. splendidus Mart. ex Colla (Cordeiro 3041, Santa Barbara-MG) were collected and voucher specimens were deposited in the Herbarium of the Institute of Botany -São Paulo (SP), and in the Herbarium of the Institute of Biosciences, São Paulo (SPF).

Crude extract preparation and analysis

Dried leaves and stems (2 g) were powdered separately, and extracted with methanol under reflux for 1 h (three times). The extracts were pooled and dried under reduced pressure. An aliquot of 10 mg of the dried extract was used for the antiproliferative tests.The samples were submmited to HPLC, HPLC/MS/MS and GC/MS analysis.

HPLC and HPLC/MS/MS

The methanol extracts were analysed by HPLC-DAD (Agilent - HP 1090 II) using a reverse phase column (Zorbax C18 - 4.6 x 250 mm, 5 µm) and a gradient of 0.1% acetic acid (solvent A) and acetonitrile (solvent B) with the following program: 0-5 min 12% of B; 5-8 min 12% - 20% of B; 8-28 min 20% of B; 28-38 min 20% - 50% of B; 38-48 min 50% - 65% of B. The solvent flux was 0.5 mL.min^-1, the column temperature was 40 ºC and the detection at λ=352 nm (Motta et al., 2009). Samples were also analysed by HPLC/MS/MS (Shimadzu M10AVP/Esquire 3000 Plus, Bruker Daltonics). The analysis was developed under the same conditions, but the solvent flux of 90 µL.min^-1. The mass spectrometer parameters involved skimmer voltage at 4000 V, nebulizer at 27 psi, dry gas at 320 ºC with flux of 7,0 L/min. Major compounds identification used MS information and the available private library constructed in the Phytochemistry Lab, Department of Botany, University of São Paulo.

GC/MS analysis

The methanol extracts were submitted to GC/ MS (Agilent GC/MS 6859/5975B) employing a DB-5 HT column (30 m x 0,25 mm x 0,25 µm) and He as carrier gas at 1.2 mL.min^-1. The injector temperature was 250 ºC, and the heating column program was: 150 ºC, 1 min, heating ramp of 6 ºC.min^-1 until 310 ºC, keeping this temperature for 3 min. The samples were analysed in the splitless mode. The temperatures of the MS source and the quadrupole were 230 and 150 ºC, respectively. The ions were detected in the positive mode, with 2.7 scan.seg^-1, in a mass range of 40-450 a.m.u. The electron multiplier voltage was adjusted to 70 eV. The compounds identification followed comparison with NIST 05 MS Library Bundle (Agilent).

Cell cultures

Ten human tumor cell lines were used [786-0 (kidney), HT-29 (colon), K562 (leukemia), NCI-ADR/ RES (drug resistant ovary), NCI-H460 (lung), MCF-7 (mammary), PC-3 (prostate), OVCAR-3 (ovary), U251 (glioma), UACC-62 (melanoma)] and were kindly provided by National Cancer Institute at Frederick MA-USA (NCI). Also a non-tumor cell line (VERO, renal, green monkey) was used. Stock cell cultures were grown in medium containing RPMI 1640, supplemented with 5% of fetal bovine serum. Experimental cultures were supplemented also with peniciline:streptomicine (10 µg/mL:10 UI/mL).

Antiproliferative assay

Cells (100 µL cells/well, inoculation density from 3-6 x10⁴ cell/mL) in 96-well plates were exposed to various sample concentrations (0.25 to 250 µg/mL, 100 µL/well) in DMSO/RPMI 1640 at 37 ºC, 5% of CO₂ in air for 48 h. Final DMSO concentration (0.2%) did not affect cell viability. Cells, before (T₀) and after 48 h exposition (T₁), were then fixed with 50% trichloroacetic acid and cell proliferation was determined by spectrophotometric quantification of cellular protein content at 540 nm, using the sulforhodamine B assay. Doxorubicin (DOX; 0.025, 25 µg/mL) was used as positive control. Three measurements were obtained at the beginning of incubation (time zero, T₀) and 48 h post-incubation for compound-free (T₁) and tested (T) cells. Cell proliferation was determined according to the equation 100 x [(T-T₀) / (T1-T₀)], for T₀< T< T₁, and 100 x [(T-T₀)/T₀], for T < T₀ and a concentration-response curve for each cell line was plotted using software Origin 7.5 (OriginLab Corporation) (Monks et al., 1991).

Data analysis

Using the concentration-response curve for each cell line, the GI50 (concentration causing 50% growth inhibition) (Shoemaker, 2006) of each cell line was determined by non-linear regression analysis, using software Origin 7.5. The average activity (mean of log GI50) of the analysed extracts was also determined using MS Excel software (Fouche et al., 2008). The average activity is a parameter used by the National Cancer Institute to classify the test-extract as: I: inactive (mean of log GI50 >1.5); W: week activity (mean of log GI50>1.1-1.5); M: moderate activity (mean of log GI50>0-1.1); and P: potent activity (mean of log GI50<0).

Results and Discussion

Table 1 presents the results of antiproliferative activity obtained with the methanol extracts of Croton dichrous Müll. Arg., C. erythroxyloides Baill., C. myrianthus Müll. Arg. and C. splendidus Mart. ex Colla against human tumor cell lines. Using the National Cancer Institute criteria, which means looking through the mean value of the logarithm of GI50 for all the tumor cell lines used, except the non tumor VERO, only the extract obtained from leaves of C. erythroxyloides showed moderate activity (mean of log GI50=1.00) against cancer cells. This extract also presented a high selectivity for PC-3 (prostate, GI50=0.80 µg.mL^-1) and OVCAR-3 (ovarian, GI50=0.81 µg.mL^-1) cell lines. Based on the NCI criteria, these individual GI50 values indicate potent activity (log GI50<0), similar to doxorubicin, an effective drug used in cancer control.

Although an excellent growth inhibiter of cancer cells, doxorubicin also affects the growth of non-tumor cells, for example, the ones from the VERO cell line (Table 1). In this regard, all assayed Croton extracts seem to be safer than doxorubicin, since toxicity to healthy cells had been reached only with high concentrations. For example, the extract of C. erythroxyloides leaves, the most active against cancer cell lines, reaches an GI50 higher than 250 µg.mL^-1 for the VERO cell line.

On the other hand, extract of stems of C. splendidus had the lowest inhibition potential (mean of log GI50=1.64), while all other six extracts presented at least a weak antiproliferative activity (mean of log GI50 from 1.20 to 1.48) (Table 1). Among leaf extracts, C. dichrous extract showed a slightly selectivity for MCF-7 (breast, GI50=16.07 µg.mL^-1) cell line, C. splendidus extract presented some selectivity against H460 (lung, GI50=6.08 µg.mL^-1) and K562 (leukemia, GI50=8.00 µg.mL^-1) cell lines, while C. myrianthus was even more selective for MCF-7 (breast, GI50=1.29 µg.mL^-1) and U251 (glioma, GI50=1.34 µg.mL^-1) cell lines. Looking at the stem extracts, C. dichrous extract showed selectivity for K562 (leukemia, GI50=2.11 µg.mL^-1) and OVCAR-3 (ovarian, GI50=9.00 µg.mL^-1) cell line, C. myrianthus was more selective for U251 (glioma, GI50=3.34 µg.mL^-1), K562 (leukemia, GI50=4.05 µg.mL^-1), MCF-7 (breast, GI50=4.19 µg.mL^-1) and OVCAR-3 (ovarian, GI50=6.60 µg.mL^-1) cell lines, and C. erythroxyloides extract presented some selectivity against K562 (leukemia, GI50=7.48 µg.mL^-1), U251 (glioma, GI50=8.51 µg.mL^-1) and PC-3 (prostate, GI50=9.28 µg.mL^-1) cell lines.

Manthey & Guthrie (2002) proposed that the activity of extracts with GI50 values lower than 10 µg.mL^-1 should be regarded as strong. Considering this new perspective, only two extracts, naming the leaf extract of C.dichrous and the stem extract of C. splendidus, showed no significant activity against the cell strains analyzed (Table 1). The stem extract of C. myrianthus also provided promising results toward four cell lines (U251, glioma; MCF-7, breast; OVCAR-3, ovary; K562, leukemia). Five of the eight extracts tested showed antiproliferative activity against K562 (leukemia). Notwithstanding, no extract presented considerable activity against strains UACC-62 (glioma), ADR/RES (resistant ovarian), 786-0 (kidney) and HT-29 (lung) (Table 1).

All methanol extracts were submitted to HPLC and GC/MS analysis. Table 2 shows the major compounds identified. Major compounds detected by HPLC analysis were flavonoids. Apigenin dihexoside was detected in leaf extracts of C. dichrous, C. myrianthus and C. erithroxyloides and tiliroside (kaempferol-pcoumaroylglucoside) was ubiquitous. These results suggest that these flavonoids are not key compounds accounting for the better antiproliferative effects of C. erythroxyloides, since they are present also in extracts with weak or no growth inhibition activity, sometimes in higher contents. In addition, C. dichrous, a species with no promising antiproliferative activity (Table 1), contains significant amounts of these flavonoids.

Apigenin glycosides from chamomile (Matricaria chamomilla L.) have been reported as having antiproliferative activity, but much lower in comparison with the corresponding aglycone (Srivastava & Gupta, 2007).

A study conducted with flavonoids from Citrus demonstrated loss of antiproliferative activity by different aglycones when they were glycosylated (Manthey & Guthrie, 2002). However, Rao et al. (2007) demonstrated a potent activity of tiliroside against Jurkat (lymphocytic leukemia) and HepG2 (hepatoma) cancer cell lines. Interestingly, for the leukemia K562 lineage, used in our study, there seems to be a correlation between tiliroside content and antiproliferative activity (Table 1). Most stem extracts have over 50% of tiliroside and moderate activity against K562 cell line (Table 2). Even C. splendidus, with lower tiliroside content, inhibited the growth of this cell line at a moderate rate. The leaf extract of C. erythroxyloides had the highest activity toward leukemia line (GI50=1.86 µg.mL^-1; Table 1) and the highest concentration of tiliroside (Table 2). A test of the activity of tiliroside against K562 cells would be interesting.

Vitexin appears only in the leaves of C. splendidus, which has moderate activity against K562 (log GI50=0.90) and H460 (log GI50=0.78). This compound was isolated from a medicinal plant from cerrado, Luehea candicans, Tiliaceae, which showed antiproliferative activity against various cell lines, including K562 (Silva et al., 2012).

The GC/MS analysis unveiled the triterpene lupeol in extracts of C. erythroxyloides. As mentioned before, only these extracts showed moderate antiproliferative activity based on NCI criteria (Table 1). In fact, antitumor activity of lupeol has been demonstrated against several cancer lines, including ovarian (Chaturvedula et al., 2004) and prostate (Prasad et al., 2005). The stem extract of the same species also contains lupeol, although in smaller amounts than in leaves. Nevertheless, its activity against PC-3 is moderate (log GI50=0.97) and higher than the activities of stem extracts from other species.

On the other hand, β-sitosterol was detected in five extracts and, besides its known effect at treatment of benign prostate hyperplasia (Scholtysek et al., 2009), no clear correlation was noted between the presence of β-sitosterol in analyzed Croton species and antiproliferative activity against PC-3.

Among the leaf and stem extracts of the four studied species of Croton, the leaf extract of Croton erythroxyloides seems to be a promising source of substances against at least two different cancer cell lines with the additional benefit of exhibiting low activity against normal cells. This finding turns C. erythroxyloides a strong candidate for further in-depth studies about antiproliferative activity.

Acknowledgements

The authors thank FAPESP for provision of funds (Processo no 07/02518-6), and to CNPq for the fellowship of JPS. We also thank the Frederick Cancer Research & Development Center of the National Cancer (Frederick, MA, USA) for provision of the cell lines, Dra Giusepina Negri for assistance in the MS analyses, and Drs. Inês Cordeiro, Ricarda Riina and Beatriz Caruzo for assistance in plant collection and identification. AS, MLFS and JEC are fellow researchers of CNPq.

Authors' contributions

JPS (MS student) contributed in collecting plant sample, running the laboratory work, analysis of the data and drafted the paper. CMF contributed in intelectual discussion during all development of the project and critical reading of the manuscript. LBM contributed in collecting plant sample, in intelectual discussion during all development of the project and critical reading of the manuscript. MLFS contributed in intelectual discussion during all development of the project. JEC contributed with discussion of the results related to antiproliferative assays. ALTGR contributed running the laboratory work of antiproliferative assays, discussion of the results, and critical reading of the manuscript. AS was the main researcher of the project, applied for provision of funds, contributed in intelectual discussion during all development of the project and critical reading of the manuscript. DYACS contributed with JPS orientation at MS thesis, supervised the laboratory work, with intelectual discussion during all development of the project, critical reading of the manuscript and final submittion.

Received 10 Jun 2013

Accepted 2 Aug 2013

• Campos MCO, Salomão K, Castro-Pinto DB, Leon LL, Barbosa HS, Maciel MAM, Castro SL 2010. Croton cajucara crude extract and isolated terpenes: activity on Trypanosoma cruzi. Parasitol Res 107:1193-1204.
• Chaturvedula VSP, Schilling JK, Miller JS, Andriantsiferana R, Rasamison VE, Kingston DGI 2004. New cytotoxic terpenoids from the wood of Vepris punctata from the Madagascar rainforest. J Nat Prod 67:895-898.
• Cragg GM, Newman DJ 2005. Plants as a source of anti-cancer agents. J Ethnopharmacol 100:72-79.
• Fouche G, Cragg GM, Pillay P, Kolesnikova N, Maharaj VJ, Senabe J 2008. In vitro anticancer screening of South African plants. J Ethnopharmacol 119:455-461.
• Johnson IS, Armstrong JG, Gorman M, Burnett JP 1963. The vinca alkaloids: a new class of oncolytic agents. Cancer Res 23:1390-1427.
• Júnior FEB, Oliveira DR, Bento EB, Leite LHI, Souza DO, Siebra ALA, Sampaio RS, Martins AOPB, Ramos AGB, Tintino SR, Lacerda-Neto LJ, Figueiredo PRL, Oliveira LR, Rodrigues CKS, Sales VS, Figueiredo FRSDN, Nascimento EP, Monteiro AB, Amaro EN, Costa JGM, Coutinho HDM, Menezes IRA, Kerntopf MR 2013. Antiulcerogenic Activity of the Hydroalcoholic Extract of Leaves of Croton campestris A. St.-Hill in Rodents. Evid-Based Compl Alt, Article ID 579346, 10 pages.
• Kawai K, Tsuno NH, Kitayama J, Okaji Y, Yazawa K, Asakage M, Yamashita H, Watanabe T, Takahashi K, Nagawa H 2005. Anti-angiogenic properties of plaunotol. Anti-Cancer Drug 16:401-407.
• Maciel MAM, Pinto AC, Veiga VF, Grynberg NF, Echevarria A 2002. Medicinal plants: the need for multidisciplinary scientific studies. Química Nova 25:429-438.
• Maciel MAM, Martins JR, Pinto AC, Kaiser CR, Esteves-Souza A, Echevarria A 2007. Natural and semi-synthetic clerodanes of Croton cajucara and their cytotoxic effects against ehrlich carcinoma and human K562 leukemia cells. J Braz Chem Soc 18:391-396.
• Manthey JA, Guthrie N 2002. Antiproliferative activities of Citrus flavonoids against six human cancer cell lines. J Agric Food Chem 50:5837-5843.
• Mantovani A 2009. Inflaming metastasis. Nature 457:36-37.
• Monks A, Scudeiro D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolff A, Gray-Goodrich M, Campbell H, Mayo J, Boyd M 1991. Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83:757-766.
• Montopoli M, Bertin R, Chen Z, Bolcato J, Caparrotta L, Froldi G 2012. Croton lechleri sap and isolated alkaloid taspine exhibit inhibition against human melanoma SK23 and colon cancer HT29 cell lines. J Ethnopharmacol 144:747-753.
• Motta LB, Furlan CM, Salatino A, Salatino MLF 2009. Flavonoids and the taxonomy of Camarea (Malpighiaceae). Biochem Syst Ecol 37:201-205.
• Motta L.B, Furlan CM, Santos DYAC, Salatino MLF, Duarte-Almeida JM, Negri G, Carvalho JE, Ruiz ALTG, Cordeiro I, Salatino A 2011. Constituents and antiproliferative activity of extracts from leaves of Croton macrobothrys (Euphorbiaceae). Rev Bras Farmacogn 21:972-977.
• Prasad S, Nigam N, Kalra N, Shukla Y 2008. Regulation of signaling pathways involved in lupeol induced inhibition of proliferation and induction of apoptosis in human prostate cancer cells. Mol Carcinogen 47:916-924.
• Rao YK, Geethangili M, Fang S, Tzeng Y 2007. Antioxidant and cytotoxic activities of naturally occurring phenolic and related compounds: a comparative study. Food Chem Toxicol 45:1770-1776.
• Rossi D, Guerrini A, Paganetto G, Bernacchia G, Conforti F, Statti G, Maietti S, Poppi I, Tacchini M, Sacchett G 2013. Croton lechleri Müll. Arg. (Euphorbiaceae) stem bark essential oil as possible mutagen-protective food ingredient against heterocyclic amines from cooked food. Food Chem 139:439-447.
• Salatino A, Salatino MLF, Negri G 2007. Traditional uses, chemistry and pharmacology of Croton species (Euphorbiaceae). J Braz Chem Soc 18:11-33.
• Sampson JH, Phillipson JD, Bowery NG, O'Neill MJ, Houston JG, Lewis JA 2000. Ethnomedicinally selected plants as sources of potential analgesic compounds: indication of in vitro biological activity in receptor binding assays. Phytother Res 14:24-29.
• Sasaki M, Mizoshita T, Mizushima T, Inoue H, Kamiya T, Kataoka H, Ogaswara N, Wada T, Kubota E, Mori Y, Shimura T, Hirata H, Ando K, Okamoto Y, Ohara H, Nakao H, Joh T 2007. Effect of plaunotol in combination with clarithromycin against clarithromycin-resistant Helicobacter pylori in vitro and in vivo. J Antimicrob Chemother 60:1060-1063.
• Scholtysek C, Krukiewicz AA, Alonso JL, Sharma KP, Sharma PC, Goldmann WH 2009. Characterizing components of the Saw Palmetto Berry Extract (SPBE) on prostate cancer cell growth and traction. Biochem Biophys Res Commun 379:795-798.
• Shoemaker RH 2006. The NCI60 human tumour cell line anticancer drug screen. Nat Rev Cancer 6:813-826.
• Silva DA, Alves VG, Franco DMM, Ribeiro LC, Souza MC, Kato L, Carvalho JE, Kohn LK, Oliveira CMA, Silva CC 2012. Antiproliferative activity of Luehea candicans Mart. et Zucc. (Tiliaceae). Natl Prod Res 26:364-369.
• Srivastava JK, Gupta S 2007. Antiproliferative and apoptotic effects of chamomile extract in various human cancer cells. J Agric Food Chem 55:9470-9478.
• van Ee BW, Riina R, Berry P 2011. A revised infrageneric classification and molecular phylogeny of New World Croton (Euphorbiaceae). Taxon 60:1-33.
• Wani MC, Taylor HL, Wall ME, Coggon P, Mcphail AT 1971. Plant antitumor agents. VI. The isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus brevifolia. J Am Chem Soc 93:2325-2327.
• World Health Organization 2007. Cancer. Retrieved from http://www.who.int/cancer/en/ April 2011.

Publication Dates

• Publication in this collection
  20 Aug 2013
• Date of issue
  Aug 2013

History

• Received
  10 June 2013
• Accepted
  02 Aug 2013