Abstracts
Purification of the root extract of Colubrina greggii var. yucatanensis resulted in the isolation and identification of 3-O-acetyl ceanothic acid as a new natural ceanothane triterpene, together with the known metabolites ceanothic acid, cenothenic acid, betulinic acid, discarine B and chrysophanein. The natural products and the semisynthetic esters acetyl dimethyl ceanothate, dimethyl ceanothate and chrysophanein peracetate showed moderate to low leishmanicidal and trypanocidal activities. None of the metabolites showed cytotoxic or antiproliferative effects. The results also suggested that betulinic acid contributes to the antiplasmodial activity originally detected in the crude root extract of C. greggii var. yucatanensis.
Colubrina greggii var. yucatanensis; Rhamnaceae; antiprotozoan; cytotoxic ceanothane
A purificação do extrato da raiz de Colubrina greggii var. yucatanensis levou ao isolamento e identificação do ácido 3-O-acetil ceanótico, um novo triterpeno natural, juntamente com os metabólitos já descritos: ácido ceanótico, ácido cenoténico, ácido betulínico, discarina B e crisofaneína. Os produtos naturais e os derivados semi-sintéticos éster de acetil dimetil ceanotato, dimetil ceanotato e peracetato de crisofaneína mostraram moderada a baixa atividade leishmanicida e tripanocida. Nenhum dos metabólitos mostrou ser citotóxicos ou ter atividade antiproliferativa. Os resultados também sugerem que o ácido betulínico contribui para a atividade antiplasmódica inicialmente detectada na raiz do extrato bruto de C. greggii var. yucatanensis.
ARTICLE
Metabolites from roots of Colubrina greggii var. yucatanensis and evaluation of their antiprotozoan, cytotoxic and antiproliferative activities
Dafne B. Domínguez-CarmonaI; Fabiola Escalante-ErosaI; Karlina García-SosaI; Grace Ruiz-PinellII; David Gutierrez-YapuII; Manuel J. Chan-BacabIII; Rosa E. Moo-PucIV; Nigel C. VeitchV; Alberto Giménez-TurbaII; Luis M. Peña-RodríguezI,** e-mail: lmanuel@cicy.mx
IUnidad de Biotecnología, Centro de Investigación Científica de Yucatán. Calle 43, N. 130, Col. Chuburná, Mérida, Yucatán, 97200 México
IIInstituto de Investigaciones Fármaco-Bioquímicas, Universidad Mayor de San Andrés, Av. Saavedra 2224, La Paz, Bolivia
IIIDepartamento de Microbiología Ambiental y Biotecnología, Universidad Autónoma de Campeche, Agustín Melgar s/n, Campeche, Campeche, México
IVUnidad de Investigación Médica Yucatán, Unidad Médica de Alta Especialidad, Centro Médico Ignacio García Téllez IMSS, Calle 41, N. 439, Col. Industrial, Mérida, Yucatán, 97150 México
VJodrell Laboratory, Royal Botanic Gardens Kew, Richmond, Surrey, TW9 3AB UK
ABSTRACT
Purification of the root extract of Colubrina greggii var. yucatanensis resulted in the isolation and identification of 3-O-acetyl ceanothic acid as a new natural ceanothane triterpene, together with the known metabolites ceanothic acid, cenothenic acid, betulinic acid, discarine B and chrysophanein. The natural products and the semisynthetic esters acetyl dimethyl ceanothate, dimethyl ceanothate and chrysophanein peracetate showed moderate to low leishmanicidal and trypanocidal activities. None of the metabolites showed cytotoxic or antiproliferative effects. The results also suggested that betulinic acid contributes to the antiplasmodial activity originally detected in the crude root extract of C. greggii var. yucatanensis.
Keywords:Colubrina greggii var. yucatanensis, Rhamnaceae, antiprotozoan, cytotoxic ceanothane
RESUMO
A purificação do extrato da raiz de Colubrina greggii var. yucatanensis levou ao isolamento e identificação do ácido 3-O-acetil ceanótico, um novo triterpeno natural, juntamente com os metabólitos já descritos: ácido ceanótico, ácido cenoténico, ácido betulínico, discarina B e crisofaneína. Os produtos naturais e os derivados semi-sintéticos éster de acetil dimetil ceanotato, dimetil ceanotato e peracetato de crisofaneína mostraram moderada a baixa atividade leishmanicida e tripanocida. Nenhum dos metabólitos mostrou ser citotóxicos ou ter atividade antiproliferativa. Os resultados também sugerem que o ácido betulínico contribui para a atividade antiplasmódica inicialmente detectada na raiz do extrato bruto de C. greggii var. yucatanensis.
Introduction
Leishmaniosis, trypanosomiosis and malaria are a group of protozoan diseases considered of significant importance due to their incidence and rate of mortality in developing countries.1 The low effectiveness, limited availability and high toxicity of existing treatments for these illnesses emphasize the importance of continuing the search for new antiprotozoan pharmaceuticals.2 Plants are considered an important source of biologically active natural products,3 including a number of them with antiprotozoan activity.4-6 During the screening of extracts from native Yucatecan medicinal plants as potential sources of bioactive metabolites, the root extract of Colubrina greggii S. Watson var. yucatanensis M. C. Johnst., a shrub used for the treatment of liver diseases, ulcerations, abscesses, asthma and tuberculosis,7 showed trypanocidal, antimalarial, leishmanicidal and cytostatic activity.8,9 Previous phytochemical studies of the genus Colubrina (Rhamnaceae), which include 31 species,10 reported the presence of a wide structural diversity of metabolites, including ansa macrolides, saponins, aporphinic alkaloids, phenolic acids, flavones and triterpenoid acids.11-18 To date, only chrysophanol, an anthraquinone with antimicrobial activity, has been reported as a metabolite from C. greggii.19 In the present study we report the leishmanicidal, trypanocidal, antiplasmodial, cytotoxic and antiproliferative activity of the crude extract, semi-purified fractions and isolated metabolites from the root extract of C. greggii var. yucatanensis.
Results and Discussion
Fractionation of the bioactive root extract of C. greggii var. yucatanensis yielded a low polarity fraction with leishmanicidal, trypanocidal and antiplasmodial activities and a medium-polarity fraction with leishmanicidal activity. Successive purification of the medium-polarity fraction resulted in the isolation of 3-O-acetyl-ceanoth-20(30)-en-1,28-dioic acid (3-O-acetyl ceanothic acid) (1) as a new natural ceanothane triterpene, together with the known metabolites ceanothic acid (2), discarine B (5) and chrysophanein (6). Similarly, purification of the low-polarity fraction led to the isolation and identification of ceanothenic acid (3) and betulinic acid (4) (Figure 1).
The FTIR spectrum of 1 showed absorption bands at 3073 (CH=C), 1731 (ester) and 1681 cm-1 (carboxyl). The pseudo molecular ion peak [M + Na]+ at m/z 551.3524 in HRMS (high resolution mass spectrometry) indicated a molecular formula C32H48O6, implying nine degrees of unsaturation. Carbon multiplicity, deduced from HMQC (heteronuclear multiple quantum coherence) and DEPT (distortionless enhancement by polarization transfer) experiments, indicated the presence of seven methyl groups, nine methylene, seven methine, and nine quaternary carbons. The 1H NMR spectrum of 1 (Table S1, Figure S1) displayed seven three-proton singlets at δ 0.88, 0.98, 0.99, 1.06, 1.16, 1.68 and 2.03 consistent with methyl groups attached to quaternary carbons. The presence of an acetyl group was confirmed by the HMBC (heteronuclear multiple bond coherence) correlation of the methyl at δ 2.03 with the carbonyl at δ 172.5, whereas an isopropenyl group was assigned from NMR signals corresponding to a methyl group (δH 1.68) attached to a sp2-carbon (δC 152.0) showing HMBC correlations with two vinylic protons at δH 4.58 and d 4.70. The double bond and three carbonyl groups observed in the 13C NMR of 1 (δ 172.5, 177.4 and 180.1) accounted for four degrees of unsaturation thus indicating that the five remaining unsaturation sites corresponded to a pentacyclic structure. The spectroscopic data of 1 proved to be very similar to those reported for ceanothic acid (2), a ceanothane triterpene also known as emmolic acid, originally isolated from Ceanothus americanus20 and later identified from Colubrina granulosa.13 However, the presence of a low-field carbinol proton (δ 5.07) and an acetyl methyl singlet (δ 2.03) in the 1H NMR spectrum of 1, both showing strong HMBC correlations with the ester carbonyl carbon (δ 172.5), suggested that 1 was the 3-O-acetyl derivative of ceanothic acid, which was confirmed when acetylation of 2 produced 1 as the only product. The presence of 1 in the original root extract of C. greggii, as detected by TCL (Figure S2), ruled out its being an artifact of the isolation procedure.
Discarine B (5), chrysophanein (6) and ceanothenic acid (3) were identified from comparison of their spectroscopic data (Tables S1, S2 and S3) with those reported in literature.21-23 Betulinic acid (4) was identified by comparison with an authentic sample.24
All of the isolated metabolites and the semisynthetic esters acetyl-dimethyl ceanothate (1a), dimethyl ceanothate (2a) and chrysophanein peracetate (6a) were evaluated for their in vitro antiprotozoan (leishmanicidal, trypanocidal, and antiplasmodial), cytotoxic and antiproliferative activities (Table 1). The results showed a moderate leishmanicidal activity (IC50 values of 20-28 µg mL-1) for natural ceanothanes 1 and 3 and semisynthetic derivatives 2a and 6a, whereas a low trypanocidal activity (IC50 of 30-70 µg mL-1) was observed for the natural products 3-5 and the semisynthetic esters 1a and 6a. Betulinic acid (4) appeared to contribute to the antiplasmodial activity of the crude extract of C. greggii, with an IC50 of 9.7 µg mL-1.25,26
Although the crude root extract, together with the low and medium polarity fractions showed cytotoxic activity against HEp-2 cells, none of the isolated metabolites displayed this type of activity (Table S4). Furthermore, none of the metabolites tested, with the exception of the peracetylated chrysophanein (6a), showed antiproliferative activity against KB cells (Table S5). It is interesting to point out that the cytotoxic activity of C. macrocarpa and C. texensis has been attributed to the presence of colubrinol and its acetate,11,27 however these metabolites were not detected in the root extract of C. greggii.
To date, ceanothane triterpenes have only been reported to occur in species of the Rhamnaceae family,20,23,28-36 and particularly in those belonging to the ziziphoids in the tribal classification reported by Richardson et al.38 These results, together with our finding of ceanothanes in the root extract of C. greggii, support the possible use of this class of triterpenes as chemotaxonomic markers for a classification of Rhamnaceae based on a phylogenetic analysis.
Experimental
General
Analytical TLC (thin layer chromatography) was carried out on aluminum-backed silica gel (60F254) plates (E.M. Merck, 0.2 mm thickness) and the chromatograms visualized using a solution of phosphomolybdic acid (20 g) and ceric sulfate (2.5 g) in 500 mL of sulfuric acid (5%). Flash chromatography purifications were performed using silica gel (Aldrich, 200-400 mesh), while TLC-grade silica gel 60GF254 (E.M. Merck) was used for vacuum liquid chromatography (VLC). Prep-TLC purifications were carried out using glass plates impregnated with silica gel 60 F254 (E.M. Merck, 0.25 mm thickness, 20 × 20 cm). Melting points (uncorrected) were determined from a Mel-Temp II apparatus (Laboratory Devices Inc.). The optical rotations were measured in CHCl3 using a Perkin Elmer 341 polarimeter. FTIR (Fourier transform infrared) spectra were recorded in CHCl3 or MeOH (film) using an FT-Nicolet Magna Protégé 460 spectrophotometer. 1H NMR (400 and 600 MHz) and 13C NMR (100 and 150 MHz) spectra were acquired on a Bruker Avance 400 spectrometer or a Bruker Avance 600 spectrometer with CHN cryoprobe, using the residual solvent resonances as internal references, calibrated to TMS. Electrospray high-resolution mass spectra (ESI-HRMS) were determined by direct injection on a Waters Q-TOF microsystem (using 0.1% phosphoric acid in a 1:1 water/acetonitrile mixture as reference), or using an Orbitrap MS (Thermo) connected to a Surveyor HPLC (high-performance liquid chromatography, Thermo) for sample injection.
Plant material
Roots of Colubrina greggii S. Watson var. yucatanensis M. C. Johnst. were collected in Abalá, Yucatán, México. A voucher specimen (P. Simá-D. Domínguez 2916) was deposited in the Herbarium of Centro de Investigación Científica de Yucatán.
Extraction of plant material and purification of crude extract
Dry roots of C. greggii (365 g) were extracted three times with ethanol (4 L) at room temperature. Evaporation of the solvent yielded the corresponding crude extract (CG-1, 54.5 g, 14.9%), which was suspended in 1.8 L of a H2O/MeOH (3:2, v/v) mixture. The suspension was fractionated by successive liquid-liquid partition with hexane (three times; 2:1, 1:1, 1:1; v:v of solvent:aqueous suspension), ethylacetate (three times; 2:1, 1:1, 1:1) and water-saturated butanol (1:2; v:v solvent:aqueous suspension) to yield the corresponding low (CG-2A), medium (CG-2B) and high (CG-2C) polarity fractions. Purification of the medium polarity fraction (CG-2B, 9.6 g) by VLC, using a gradient elution with CH2Cl2/Me2CO (99:1 to 94:6) followed by CHCl3/hexane/MeOH (70:25:5), yielded fractions CG-5A-N. Fraction CG-5E (550 mg) was purified by flash chromatography using an isocratic elution with hexane/Me2CO 8:2, to produce pure 3-O-acetyl-ceanothic acid (1, 172.1 mg), and fraction CG-6I (50 mg) which was further purified by column chromatography [ether/hexane (1:1)] to yield ceanothic acid (2, 16.5 mg). Additional purification of fraction CG-5C (414 mg) by flash chromatography, using a gradient elution with hexane/Me2CO (8:2-7:3), resulted in the isolation of discarine B (5, 108 mg). Finally, chrysophanein (6, 168.5 mg) was collected as a yellow precipitate by filtration from fraction CG-5G. Purification of the low polarity fraction (CG-2A, 1.5 g) by VLC, using a gradient elution with mixtures of hexane/Me2CO/MeOH (95:3:2 to 60:38:2), produced eleven fractions (CG-3A-K). Fractions CG-3E-F were combined (500 mg) and purified by flash chromatography (hexane/ether 7:3), to yield ceanothenic acid (3, 20.8 mg) and betulinic acid (4, 20.4 mg).
3-O-acetyl-ceanothic acid (1)
White amorphous solid; mp: 271.5-273.1 ºC; [α]D20 +33.7º (c 0.01, Me2CO); FTIR (film) νmax cm-1: 3073 (CH=C), 1731 (ester), 1685 (carboxyl); 1H NMR (CD3OD, 400 MHz) and 13C NMR (CD3OD, 100 MHz) data: see Table S1; ESI-HRMS m/z 551.3524 [M + Na]+ (calc. for C32H48NaO6: 551.3584).
Ceanothic acid (2)
White amorphous solid; [α]D20 + 30.8º (c 0.003, MeOH); FTIR (film) νmax cm-1: 3411 (OH), 1697 (>C=O); 1H NMR (CD3OD, 400 MHz): δ 0.88 (s, 3H, H-24), 0.90 (s, 3H, H-26), 0.97 (s, 3H, H-27), 1.01 (s, 3H, H-25), 1.35 (s, 3H, H-23), 1.67 (s, 3H, H-29), 2.45 (s, 1H, H-1), 3.06 (m, 1H, H-19), 4.06 (s, 1H, H-3), 4.57 (br s, 1H, H-30a), 4.69 (br s, 1H, H30-b); ESI-HRMS m/z 487.3418 [M + H]+ (calc. for C30H47O5: 487.3423).
3-O-acetyl-dimethyl-ceanothate (1a)
A mixture of 1 (5.1 mg), K2CO3 (80 mg), CH3I (300 µL) and acetone (1 mL) was stirred for 72 h at room temperature. The reaction mixture was poured over distilled water (14 mL) and the resulting suspension was extracted twice with EtOAc (4:1, v/v). The organic layer was dried over anhydrous Na2SO4 and evaporated to produce 4.6 mg of the crude esterified product, which was purified by column chromatography (hexane/Me2CO 9:1) to give 1a (4.2 mg, 91.3% yield) as a white powder. 1H NMR (CDCl3, 600 MHz) and 13C NMR (CDCl3, 150 MHz) data: see Table S1; LR-MS m/z 557 [M + H]+.
Dimethyl-ceanothate (2a)
A fraction containing ceanothic acid as the main product (8.5 mg), was mixed with K2CO3 (220 mg), CH3I (800 µL) and acetone (1 mL), and then stirred for 72 h at room temperature. The reaction mixture was poured over distilled water (13 mL) and the resulting suspension was extracted twice with EtOAc (4:1, v/v). The organic phase was dried over anhydrous Na2SO4 and evaporated to produce 9.6 mg of the crude esterified product, which was purified by multiple-elution (5 ×) prep-TLC (hexane/ether 7:3) to give 5.4 mg of 4 (56.2%) as a white solid. 1H NMR (CD3OD, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data: see Table S1; ESI-HRMS m/z 515.3731 [M + H]+ (calc. for C32H51O5: 515.3736).
Ceanothenic acid (3)
White powder; FTIR (film) νmax/cm-1: 3067 (CH=C), 1721 (carboxyl), 1685 (carboxyl); 1H NMR (CDCl3/CD3OD 9:1, 400 MHz) and 13C NMR (CDCl3/CD3OD 9:1, 100 MHz) data: see Table S1; ESI-HRMS m/z 455.3156 [M + H]+ (calc. for C29H43O4: 455.3161).
Betulinic acid (4)
Colorless needles; ESI-HRMS m/z 440.3690 [M-H2O + 2H].+ (calc. for C30H48O2: 440.3654).
Discarine B (5)
White amorphous solid; νmax/cm-1: 3267 (NH), 3027 (CH=C), 1634 (>C=O); 1H NMR (CD3OD, 30 ºC, 400 MHz) and 13C NMR (CD3OD, 30 ºC, 100 MHz) data: see Table S3; ESI-HRMS m/z 574.3393 [M + H].+ (calc. for C33H44N5O4: 574.7336).
Chrysophanein (6)
Yellow powder; FTIR (film) νmax/cm-1: 3344 (OH), 1634 (>C=O); 1H NMR (DMSO-d6, 400 MHz) and 13C NMR (DMSO-d6, 100 MHz) data: see Table S2; ESI-HRMS m/z 255.2302 [M-C6H10O5 + H]+ (calc. for C15H11O4: 255.0657).
Chrysophanein peracetate (6a)
A mixture of 6 (10 mg), acetic anhydride (1 mL) and pyridine (0.5 mL) was stirred at room temperature for 72 h. The reaction mixture was poured over distilled water (20 mL) and the resulting suspension was extracted twice with ethylacetate (2:1 v/v). The organic layer was washed successively with equal volumes of HCl (5%), NaOH (3%), H2O, and NaCl saturated, and then dried over anhydrous MgSO4. Filtration and evaporation of the solvent yielded 13.9 mg (92.4%) of crude acetylated product obtained as a yellow powder; FTIR (film) νmax cm-1: 3021 (CH=C), 1757 (ester), 1680 (>C=O); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 100 MHz) data: see Table S2; ESI-HRMS m/z 649.1499 [M + Na]+ (calc. for C31H30NaO14: 649.1533).
Bioassays
Leishmanicidal assay
The growth inhibition of promastigotes was carried out following the procedure previously reported by Muñoz et al.,39 and Inchausti et al.40 Briefly, a strain of L. amazonensis (IFLA/BR/75/PH8) was grown in Schneider culture medium with 10% fetal bovine serum (FBS), penicillin (100 IU mL-1) and streptomycin (100 mg mL-1) at 25 ºC; parasites in the log phase of their growth cycle were then transferred to a microplate (96 wells; 1 × 105 parasites/well). Stock solutions of DMSO (blank), pentamidine (positive control), crude extract, semipurified fractions and pure metabolites were diluted in Schneider medium at < 100 µg mL-1, added to the plate, and incubated for 72 h. The percentages of inhibition were obtained by directed observation of each well with an inverted phase microscope. All the assays were carried out in triplicate.
Trypanocidal assay
Epimastigotes of Trypanosoma cruzi strain Tulahuen parasites were maintained in liver infusion tryptose (LIT) medium supplemented with 5% FBS, following the procedure modified by Chataing et al.41 Briefly, parasites in the log phase of growth cycle were transferred to a microplate (96 wells; 1 × 106 parasites/well) together with stock solutions of benznidazole, DMSO (positive control and blank respectively), extract, semipurified fractions or pure metabolites prepared at different concentrations (< 100 µg mL-1). The microplates were incubated at 26 ºC for 72 h.
Antiplasmodial assay
Plasmodium falciparum strain F32 was grown at 37 ºC in RPMI medium with 10% of human serum and 4% of hematocrit (O, Rh+), under anaerobic conditions, according to a reported method.41 Cultures with 1% parasitemic and 2% hematocrite (100 µL) were transferred to a 96 well plate. Stock solutions of chloroquine (positive control), DMSO (blank), extract, semipurified fractions or pure metabolites were diluted in RPMI medium to a concentration of < 10 µg mL-1 and added to each well. The plate was then incubated at 37 ºC for 48 h.
Cytotoxicity assay
Human laryngeal carcinoma (HEp-2), human cervical adenocarcinoma (HeLa), human nasopharyngeal carcinoma (KB), and green monkey Vero kidney cells (VERO) were grown in DMEM (Gibco) media supplemented with 10% (v/v) FBS (Gibco), penicillin (100 IU mL-1), and streptomycin (100 mg mL-1). All the cell lines were maintained at 37 ºC in a 5% CO2 atmosphere with 95% humidity. The cytotoxicity assay was performed according to a method described by Rahman et al.43 Briefly, cell lines were transferred to a microplate (1.5 × 104 viable cells of each cell line) and incubated at 37 ºC, with 95% humidity and 5% CO2 in DMEM medium supplemented with 10% of FBS, penicillin (10000 IU), streptomycin (10 mg mL-1), and amphotericine B (5 mg mL-1). After 24 h, the medium was replaced by fresh medium with 0.05% DMSO (blank) or different concentrations of docetaxel (positive control, Sigma), crude extract, semipurified fractions or pure metabolites dissolved in DMSO (100, 50, 25, 12.5 and 6.25 µg mL-1), and the cells were incubated for 72 h under the conditions already described. The medium was removed and 200 µL of a 0.5% MTT (Sigma) solution in PBS (pH 7.2) were added to each well, and left to stand for 4 h at 37 ºC. Then 100 µL of acidified isopropanol (0.4 mol L-1 HCl) were added to each well and the optical density (OD) measured at 540 nm using a Bioassay reader (Bio-Rad). The experiment was carried out in triplicate and each concentration was tested in duplicate.
Antiproliferative assay
The sulforhodamine B (SRB) assay was carried out according to the method reported by Rahman et al.,43 using DMEM medium with 10% FBS to induce cell proliferation. After 48 h of incubation, the medium was discarded and 100 µL of ice-cold 40% trichloroacetic acid (TCA, Aldrich) were added to fix the cells, incubating for 1 h at 4 ºC. The cells were washed five times with water, left to dry, and then 50 µL of SRB stain (10 mg 1% acetic acid, Sigma) were added to each well and left to stand for 30 min. Finally, the cells were washed with 50 mL 1% acetic acid, and rinsed four times with water. The OD was measured at 540 nm using an ELISA reader (Bio-Rad model 450). The experiment was carried out in triplicate.
Statistical analysis
Data were analyzed with commercial software (GraphPad 4.0, Software Inc., San Diego, CA). The dose-response curves (variable slope) to obtain the inhibitory concentration (in µg mL-1) of 50% of parasites (IC50), the growth inhibition of 50% of cells (IG50), and the cytotoxic concentration of 50% of cells (CC50), were fitted to the algorithm: Y = Emin + [(Emax - Emin)/(1 + 10(log ED50 - log D) hill slope)].
Supplementary Information
Supplementary data (Figures S1-S2, Tables S1-S5) are available free of charge at http://jbcs.sbq.org.br as PDF file.
Acknowledgements
The authors wish to thank to Paulino Simá for the identification of plant material. This work was supported by Program CYTED (Projects X.5 and RIBIOFAR) and Project FOMIX-Yucatán (66262).
Submitted: December 14, 2010
Published online: March 11, 2011
Supplementary Information
Figure S2 - Click to enlarge
Figure S4 - Click to enlarge
Table S1 - Click to enlarge
Table S2 - Click to enlarge
Table S3 - Click to enlarge
Table S4 - Click to enlarge
Table S5 - Click to enlarge
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Publication Dates
-
Publication in this collection
22 July 2011 -
Date of issue
July 2011
History
-
Received
14 Dec 2010 -
Accepted
11 Mar 2011