Antioxidant and antimicrobial capacity Cecropia mutisiana Mildbr. (Cecropiaceae) leave extracts

Emirates Journal of Food and Agriculture. 2017. 29(1): 25-35 doi: 10.9755/ejfa.2016-07-915 http://www.ejfa.me/ REGULAR ARTICLE Antioxidant and antimicrobial capacity Cecropia mutisiana Mildbr. (Cecropiaceae) leave extracts Andrés Eduardo Ortíz-Ardila1, Jennifer Paola Correa-Cuadros2,3, Crispín Astolfo Celis-Zambrano1, María Ximena Rodríguez-Bocanegra2, Jorge Robles-Camargo1, Luis Gonzalo Sequeda-Castañeda1,4* 1 Departamento de Química, Facultad de Ciencias, Pontiicia Universidad Javeriana, Bogotá, Colombia, 2Unidad de Investigaciones Agropecuarias, Facultad de Ciencias, Pontiicia Universidad Javeriana, Bogotá, Colombia, 3Programa de Doctorado en Ciencias Biológicas mención en Ecología, Facultad de Ciencias Biológicas, Pontiicia Universidad Católica de Chile, Santiago, Chile, 4Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia, Bogotá, Colombia ABSTRACT Background: Given the lack of knowledge in Cecropia mutisiana Mildbr phytochemical and pharmacological properties, the objective of this work was to determine the leaves antioxidant and antimicrobial capacities, taking into account its wide use by Colombian communities. For this study Rosmarinus oficinalis Govaerts extracts were used as experimental standards for comparisons, since there are no studies of Cecropia mutisiana Mildbr biological activities. Methods: Extraction was performed by dried leaves obtaining extracts of different polarity (petroleum ether, ethyl acetate, dichloromethane, and ethanol). To identify Cecropia mutisiana Mildbr functional groups and main compounds preliminar phytochemical analysis was run. Likewise, antioxidant capacity for both plants was determined by colorimetric assays; followed by phenol quantiication correlation by Folin Ciocalteu reagent. Last, its antimicrobial capacity was evaluated by the Kirby-Bauer test. Results: Cecropia mutisiana Mildbr ethanol extract had the highest antioxidant capacity expressed as IC50 (165.47 ± 3.0 ppm), as well as the dichloromethane Rosmarinus oficinalis Govaerts extract (272.63 ± 4.9 ppm), without any correlation with total phenols. Additionally, antimicrobial activity was observed for Cecropia mutisiana Mildbr in the ethyl acetate extract and for Rosmarinus oficinalis in the ethanol extract. Conclusion: Regarding both plant comparison Cecropia mutisiana Mildbr ethanol extract had the highest antioxidant capability, whereas Rosmarinus oficinalis Govaerts ethanol extract had the greatest antimicrobial activity. Keywords: Antioxidant; Antimicrobial; DPPH; ABTS; ORAC; Cecropia mutisiana; Rosmarinus oficinalis INTRODUCTION Ample vegetation species diversity in the Neotropic makes it indispensable to describe, study and characterize plants with high phytotherapeutic potential and possible industrial use, where its ethnobotany knowledge is fundamental for its identiication and classiication as a promissory species (Bernal et al., 2011). In Colombia approximately 2,404 plant species with ethnobotanical reports are used, of which 1,656 are cultivated in the country. Despite this fact few have been scientiically studied regarding their phytochemical, toxicological, and pharmacognostic characteristics (Bernal et al., 2011). Therefore, it is necessary to perform studies to validate the etnobotanical knowledge elucidating their active compounds, biosynthesis pathways, and pharmacological activities that deine their phytotherapeutic and industrial potential for traditional medical use. Cecropia mutisiana Mildbr., is a Colombian species (Berg et al., 2005; Bernal et al., 2011) with Vademecum medicinal plant etnobotanical registration, classified as phytotherapeutic and pharmacological promissory (Minprotección, 2008; Manosalva-Moreno, 2011). This plant is known etnobotanically for its effectiveness against nosocomial diseases, hypertension, as a cardiac tonic, and nervous system depressant (Sequeda-Castañeda et al., 2015). However, pharmacological and chemical determinations are somewhat unspeciic. ZambranoOspina described Cecropia mutisiana Mildbr aqueous leaf extract use as an antianxiety and anticonvulsant treatment (Zambrano-Ospina, 2000). In addition, Ahumada perfor med a chromatographic analysis identifying lavonoid type compounds, tannis, coumarins, steroids, and terpene lactones (Ahumada, 2006). None the less, currently no scientiic registry has documented *Corresponding author: Luis Gonzalo Sequeda-Castañeda, Departamento de Química, Facultad de Ciencias. Pontiicia Universidad Javeriana. Carrera 7 #43-82. Ediicio 52. Oicina 110. Bogotá, Colombia / Departamento de Farmacia, Facultad de Ciencias, Universidad Nacional de Colombia. Carrera 30 #45-03. Ediicio 450. Bogotá, Colombia. E-mail: lsequeda@javeriana.edu.co Received: 22 August 2016; Revised: 28 December 2016; Emir. J. Food Agric ● Vol 29 ● Issue 1 ● 2017 Accepted: 30 December 2016; Published Online: 05 January 2017 25 Ortíz-Ardila, et al.: Cecropia mutisiana antioxidant and antimicrobial this Colombian plant antimicrobial and/or antioxidant capacity to shed light on its pharmacological and phytotherapeutic use. Due to its Vademecum indexing as a medicinal plant that can be employed for traditional medicinal use, it is therefore important to study Cecropia mutisiana Mildbr. Hence, studies validating this understanding at the experimental level are essential (Bernal et al., 2011; Manosalva-Moreno, 2011). Given Cecropia mutisiana Mildbr antioxidant and antimicrobial activities have not been addressed, we proposed to use Rosmarinus officinalis Govaerts, a widely studied plant as a comparison standard with known phytochemical and pharmacognostic characteristics (Borras-Linares et al., 2014; Hay et al., 2015; Abkhoo and Jahani, 2016; Habtemariam, 2016; Moore et al., 2016). MATERIALS AND METHODS Extract preparation Cecropia mutisiana plant material was purchased in Mogambo Environmental Trail in the Municipality of Viotá (Cundinamarca, Colombia) and Rosmarinus oficinalis in the Marketplace Municipality of Chía (Cundinamarca, Colombia). Plants without mechanical (trauma, damage, and defoliations), biological (leaf damage caused by herbivores) or microbiological (phytopathogen signs or symptoms) lesions were purchased. Cecropia mutisiana was identiied by taxonomic classiication in National Herbarium of Universidad Nacional de Colombia, Bogotá campus, under voucher number: COL 575453. Leaves were separated and dried at 20°C. Metabolic compounds were obtained by solvent extraction with increasing polarity using petroleum ether (PE), ethyl acetate (EtOAc), dichloromethane (CH2Cl2), and ethanol (EtOH) as the solvent with the maximum polarity, shaking at 100 rpm for seven days for all solvents. Extracts were iltered and then concentrated by rotary-evaporation at 40°C to prevent damaging thermolabile compounds, and inally the excess solvent was removed by drying under extraction hood for six hours.(Rodríguez-Rojo et al., 2012). Phytochemical assays for each Rosmarinus oficinalis and Cecropia mutisiana extracts were performed to qualitatively identify compounds and associate them with their biological activity identifying main compounds through LiebermannBurchard (Steroids and sterols), Salkowski (Terpenes), Baljet (Terpenes and sterols), ferric hydroxamate (Sesquiterpene lactones), Shinoda (Flavonoids and phenolics), ferric chloride (Flavonoids and phenolics), anthrone (Flavonoid glycosides), Dragendroff (Alkaloids), and froth (saponins) tests (Tiwari et al., 2011; Dos-Santos et al., 2014). 26 Antioxidant capacity characterization Antioxidant capacity was characterized by 2,2-diphenyl1-picrylhydrazyl radical (DPPH); 2,2-azinobis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Oxygen Radical Absorbance Capacity (ORAC). As antioxidant comparison standards, ascorbic acid, 6-hydroxy-2,5,7,8tetramethylchroman-2-carboxylic acid (Trolox), and gallic acid were used. A statistical correlation was carried-out to evaluate an association between antioxidant capacity and total phenolic content. DPPH method For DPPH assay 2,2-diphenyl-1-picrylhydrazyl radical chromogen was used according to Asadujjaman (Asadujjaman et al., 2013). Percentage free radical scavenging was determined by sample concentration inhibiting 50% of radical production (IC50) with a 1:39 µL, sample: radical ratio. Spectrophotometric UV-VIS (Cary 100 CONC-Varian instruments) delta of absorbance was determined with kinetics every two minutes at 515 nm until stabilization tendency was observed, indicating maximum analyte-radical reaction capacity (Karadag et al., 2009; Asadujjaman et al., 2013). Ascorbic acid and trolox were used as standards. Plant and control half maximal inhibitory capacity (IC50) were determined nine times (n=9) to have statistical representativeness. ABTS method ABTS methodology was performed with 2,2-azinobis(3ethylbenzothiazoline-6-sulfonic acid) chromogen with radical production by potassium persulphate (2.5 mM K2O8S2) addition. Results are presented as IC50, since they correspond to total extract measurement at different polarities. Spectrophotometric UV-Vis at 734 nm absorbance change was determined (Cary 100 CONCVarian instruments) with the same sample: radical ratio as for DPPH every three minutes until a stabilization tendency was observed indicating a maximum reaction analyte/radical capacity (Nilsson et al., 2005; Karadag et al., 2009). Ascorbic acid and trolox were used as standards; plant and control half maximal inhibitory capacity (IC50) were established nine times (n=9) to have statistical representativeness. ORAC method Oxygen Radical Absorbance Capacity (ORAC) was determined by using 2,2’-azobis(2-aminido-propane) dihydrochloride (AAPH) and a sodium luorescent salt such as luorescein by which the antioxidant protective capacity was evidenced (IP50) against radical attack, by determining a luorometric delta difference between 485 nm excitation and 520 nm emission (FLUOstar Optima BMG Labtech). 96 well plates containing luorescein blanks in phosphate buffer, ascorbic acid and trolox positive controls and extracts to be tested in a 1:11.5 µL sample: radical ratio Emir. J. Food Agric ● Vol 29 ● Issue 1 ● 2017 Ortíz-Ardila, et al.: Cecropia mutisiana antioxidant and antimicrobial were employed. Obtained data was graphed as third order adjusted polynomial curves using polynomial orthogonal test to ensure all data met such distribution. Area under the curve (AUC) was used as a comparable measurement between the extracts and controls to which statistical analysis was performed (Dudonné et al., 2009; Karadag et al., 2009; Armstrong3, 2010). Total phenolic content (Folin-Ciocalteu reagent) For this method a gallic acid standard curve was used at the following concentrations 30, 60, 90, 120, and 150 ppm (r2 = 0.9998; p = 0.000). 250 µL of 1N Folin-Ciocalteu reagent + 250µL of 20% Na2CO3 + 2mL distilled water at a ratio 1:25 sample: mix ratio were employed (Cicco et al., 2009). Sample was analyzed at 760 nm and results were expressed as mg of gallic acid equivalent per g of extract (at different polarities). 24 h and 48 h after incubation. 1,000, 100 and 10 ppm were established as extract treatment concentrations for each plant species extract. Microorganism sensibility was determined by inhibition halo caused by the antibacterial, determining minimum inhibitory concentration (MIC) against the microorganism (Bonev et al., 2008). Likewise, inhibition diameter relative percentage was determined according to Rojas formula (Rojas et al., 2006). Since it is important to specify plant extract concentration for each of the treatments performed, as the weight that inhibits organism growth, inoculated concentrations are expressed as the net quantity applied 10 mL on the disk with the real extract quantity, which was 10 mg, 1 mg and 0.1 mg for each treatment (1) X mg 10 3 mg 1L × × × 10 mL = X mg extract L 106 mL 1mg (1) Antimicrobial capacity determination Statistical analysis Staphylococcus aureus CMPUJ 080, Bacillus cereus CMPUJ 251, Salmonella sp. CMPUJ 302 and Pseudomonas aeruginosa CMPUJ 055 bacterial strains obtained from Pontiicia Universidad Javeriana Microorganism Collection Bogotá Campus (CMPUJ Certiication: National collection registry No. 148, WFFC and WDMC No.857) were used. A 25% glycerol bank was established and kept at -80°C, from which all antimicrobial assays were carried. Antioxidant level response comparison for Rosmarinus officinalis and Cecropia mutisiana was performed by a completely randomized design. First, Normality distribution was assessed by the Kolmogorov-Smirnof and Shapiro-Wilk test. Additionally, variance homogeneity was determined by the Levenne test. Comparison among groups was established by ANOVA with HSD Tukey post hoc tests to identify antioxidant capacity signiicant differences among groups. Transformations were performed when required (square root, natural logarithm, base 10 logarithm and reciprocal). P value < 0.05 was considered as signiicant. Data not following a normal distribution were assessed by Kruskal-Wallis statistical analysis to determine signiicant differences among group means followed by mean comparison post hoc tests. SPSS Statistics 20 (IBM, Armonk, New York USA) and SigmaPlot V11 (Systat Software Inc, London UK) were used. Statistical correlation was determined by Pearson correlation to establish an association between IC50 values per plant extract obtained by the ABTS method and phenolic content for obtained concentrations deined as mg of gallic acid/kg extract (ppm). Completely randomized design was performed for Rosmarinus oficinalis and Cecropia mutisiana antimicrobial response applying the same statistical tests previously described for antioxidant level comparison. Antimicrobial capacity determination for four Rosmarinus officinalis and Cecropia mutisiana extracts was verified using the Kirby-Bauer test with modiications according to Clinical Laboratory Standard Institute (M02-A12 document) (Klancnik et al., 2009; CLSI, 2015). Each assay was performed nine times in time (n=9). Muller-Hinton medium (pH 7.2-7.4) was used adjusting the inoculum to the 0.5 McFarland nephelometer standard (1x108 cells/mL) for Staphylococcus aureus CMPUJ 080, Bacillus cereus CMPJU 251, Salmonella sp. CMPUJ 302, and Pseudomonas aeruginosa CMPUJ 055. An initial antimicrobial susceptibility screening was performed – antibiogram employing vancomycin, streptomycin, gentamycin, and chloramphenicol to establish a positive control. As a negative control, paper disks were loaded with dimethyl sulfoxide (DMSO) and 90% ethanol (EtOH) (1:1 ratio) and analytical-reagent grade extraction solvents (PE, EtOAc, CH2Cl2 and EtOH). (Rojas et al., 2006; Klancnik et al., 2009). For treatments paper discs with 10 µL for each extract and control at established concentrations were applied to each Petri dish. All Petri dishes were kept between 2 - 4°C for 12 h to overnight (O/N) to allow for proper diffusion, followed by 37°C 24 h incubation. Inhibition halo was veriied at Emir. J. Food Agric ● Vol 29 ● Issue 1 ● 2017 RESULTS AND DISCUSSION Extract characterization Four extracts of different polarities were obtained for each of the plants from 200 g dried Rosmarinus oficinalis and 1,000 g Cecropia mutisiana yields are detailed in Table 1. Extraction yield for each polarity in Rosmarinus oficinalis ranged between 1.0 % and 2.8 % extract per g of dried 27 Ortíz-Ardila, et al.: Cecropia mutisiana antioxidant and antimicrobial plant material. In comparison to Rodríguez-Rojo results yields were low (Rodríguez-Rojo et al., 2012). Moreover, Cecropia mutisiana yields ranged between 1.2% and 1.9%. Showing similarities among extract percent yield, given the extraction methodology for each one. Preliminary phytochemical analysis The tests identiied for both plants triterpenes, terpenoids, sesquiterpene lactones, flavonoids and phenols and lavonoids glycosides (Table 2). Liebermann-Bouchard test identified triterpenes are derived from squalene cyclization that in some cases can be found in a free form or glycosylated (anthrone test) (Sanabria-Galindo, 1999). For all Cecropia mutisiana extracts these compounds were identified, as well as for Rosmarinus officinalis medium polarity extracts. Oliveira et al., established these types of compounds have pharmacological properties such as antimicrobial, hypocholesterolemic, anti-inlammatory and cytotoxic against cancer cell lines (Oliveira et al., 2005). Taking into account no compound identiication studies have been performed for Cecropia mutisiana a correlation with other species of the same genus could be established, where terpenes and glycosides have been identiied (Table 3). Salkowski and Shinoda tests conirmed sterol presence, lavonoids, and derivatives of these (lavanols, isolavones, lavanes, among others) for both plants. According to Uchoa et al., for Cecropia species sterols and lavonoids are secondary metabolites that are not involved in the plant’s development and growth (Uchoa et al., 2009). This type Table 1: Extract yield/plant species Extract Percent yield (%Y)* Rosmarinus oficinalis Cecropia mutisiana Petroleum ether 2.8±0.8 1.9±0.6 Ethyl acetate 1.0±0.2 1.4±0.4 Dichloromethane 2.6±0.8 1.5±0.5 Ethanol 1.7±0.5 1.2±0.3 Total yield 8.1±1.3 6.0±0.9 *n=3 of compound has been investigated in other Cecropia species (Table 4). Presence of phenols was observed, as well as alkaloid type compounds, saponins, sesquiterpene lactones, lactones, and coumarins. Studies in other species have demonstrated these compounds, mainly chlorogenic acid in Cecropia glaziovii, Cecropia obtusifolia, Cecropia pachystachya, and Cecropia peltata (Andrade-Cetto and Wiedenfeld, 2001; Lacaille et al., 2001; Herrera-Arellano et al., 2004; Andrade-Cetto et al., 2007; Rocha et al., 2007a; Nicasio-Torres et al., 2009; Andrade-Cetto and Vázquez, 2010; Aragão et al., 2010; Arend et al., 2011; Mora Izquierdo et al., 2011; NicasioTorres et al., 2011; Petronilho et al., 2012; Beringhs et al., 2013; Cruz et al., 2013). In addition, for Cecropia glaziovii caffeic acid (Arend et al., 2011; Beringhs et al., 2013); protocathechuic acid in Cecropia glaziovii (Lacaille et al., 2001); alkaloids in Cecropia pachystachya, Cecropia glaziovii, and Cecropia peltata (King and Haddock, 1959; Consolini and Migliori, 2005; Ninahuaman et al., 2007). Furthermore, other phenolic compounds in Cecropia obtusifolia (Guerrero et al., 2010), in addition to saponins in Cecropia pachystachya (Consolini and Miglori, 2005), have been speciied. Given the lack of studies for primary or secondary metabolites and evaluation of the closest phylogenetic species must be perfomed. Therefore identification and comparison of all compounds present in species of the Cecropia genus are valid as an approximation potentia Cecropia mutisiana activities. Rocha et al. (2007) described typical chemical constituents such as catechins, different classes of lavonoids and procyanidins, terpenes, triterpenoids and other compounds for this genus, thus, suggesting possible promising pharmacological activities for Cecropia mutisiana an approximation of possible activities (Rocha et al., 2002; Rocha et al., 2007a; Rocha et al., 2007b). Antioxidant capacity characterization Currently no studies have reported antioxidant capacity for Cecropia mutisiana, and are scarce for other species of Table 2: Preliminary phytochemical analysis for Rosmarinus oficinalis and Cecropia mutisiana extracts Metabolite (test) Extract Rosmarinus oficinalis Cecropia mutisiana PE EtOAc CH2Cl2 EtOH PE EtOAc CH2Cl2 Steroids and sterols (Liebermann-Burchard) + + + + + Terpenoids (Salkowski) + + + + Terpenoids and sterols (Baljet) + + + + + + Sesquiterpene lactone (Ferric hydroxamate) + + + + + + + Flavonoids and phenols (Shinoda) + Flavonoids and phenols (Ferric chloride) + + + + + + Flavonoid glycosides or terpene (Anthrone) + + + + + + Alkaloids (Dragendroff) + + + + Saponins (Froth tests) + - EtOH + + + + + + - PE: Petroleum ether extract, EtOAc: Ethyl acetate extract, CH2Cl2: Dichloromethane extract, EtOH: Ethanol extract 28 Emir. J. Food Agric ● Vol 29 ● Issue 1 ● 2017 Ortíz-Ardila, et al.: Cecropia mutisiana antioxidant and antimicrobial Table 3: Cecropia genus terpene and glycosidic compounds Plant Described compound Cecropia catharinensis Tormentic acid, euscapic acid, pomolic acid, ursolic acid, oleanolic acid, maslinic acid, 2-O-acetyl-tormentic acid, 2α-acetoxy-3β-19α-dihydroxy-11α-12α-epoxi-ursan-28-13β-olide, 3β-acetoxy-2α,19α-dihydroxy-11α,12α-epoxi-ursan-28-13β-olide Cecropia glaziovii Other terpenes and triterpenes Cecropia lyratiloba Cecropia obtusifolia Cecropia pathystachya Cecropia peltata Cecropia schreberiana Tormentic acid, isoarjunolic acid, euscaphic acid, 3-acetyl tormentic acid Other terpenes and triterpenes Tormentic acid, pomolic acid, α-amyrin, other terpenes and triterpenes Glycans Tormentic acid, ursolic acid, pomolic acid, α-amyrin Reference Machado et al. (2008), Li et al. (2013) Ninahuaman et al. (2007), Sarris et al. (2013) Oliveira et al. (2005), Li et al. (2013) Morton (1981) Hikawczuk et al. (1998), Consolini and Migliori (2005), Schinella et al. (2008), Teixeiria-Uchoa et al. (2010), Li et al. (2013) Marshall and Rickson (1973) Schinella et al. (2008), Li et al. (2013) Table 4: Cecropia genus compounds of sterol, lavonoids, and sterol/lavonoid derivatives Plant Described compound Reference Cecropia catharinensis Isoorientin, orientin, isovitexin, vitexin Machado et al. (2008) Cecropia glaziovii Isoorientin, orientin, isovitexin, vitexin, catechin, Lacaille-Dubois et al. (2001), Rocha et al. (2002), Rocha epicatechin, procyanidine B2, B3, B5 and C1, et al. (2007), Lima-Landman et al. (2007), Delarcina other lavonoids and sterols. et al. (2007), Silva et al. (2010), Petronilho et al. (2012), Beringhs et al. (2012), Sarris et al. (2013), Costa et al., (2014) Cecropia lyratiloba Isoorientin, orientin, isovitexin, apigenin Oliveira et al. (2003) 6-C-galactosyl-6”-O-β-galactopyranoside. Cecropia obtusifolia Isoorientin, orientin, isovitexin, vitexin, other Andrade-Cetto and Wiedenfeld (2001), Herrera-Arellano lavonoids and sterols. et al. (2004), Nicasio-torres et al. (2009), Andrade-Cetto and Cardenas-Vazquez (2010), Aragao et al. (2010), Guerrero et al. (2010), Nicasio-Torres et al. (2012) Cecropia pachystachya Isoorientin, orientin, isovitexin, catechin, Consolini and Migliori (2005), Teixeiria-Uchoa epicatechin, procyanidine B2, B3, B5 and C1, et al. (2010), Aragao et al. (2010), Mello-Cruz isoquercetin, β-sitosterol, luteolin, sitosterol, et al. (2013), Oliveira-Aragao et al. (2013) other lavonoids and sterols Cecropia peltata Isoorientin, other lavonoids and sterols Pardo-Concepción et al. (2000), Andrade-Cetto et al. (2007), Nicasio-Torres et al. (2009), Andrade-Cetto and Cardenas-Vazquez (2010), Aragao et al. (2010), Mora-Izquierdo et al. (2011), Ospina-Chávez et al. (2013) Cecropia schreberiana Isoorientin, orientin, vitexin, catechin, Li et al. (2013) epicatechin, cinchonain 1a and 1b this same genus (Aragão et al., 2010; Mora Izquierdo et al., 2011; Petronilho et al., 2012). Therefore, for this study a comparison with Rosmarinus oficinalis, a plant broadly known for its antioxidant activities was proposed to establish as a biological referral. Summary of antioxidant capacities for both plants by different test are summarized in Table 5. Signiicant differences (ANOVA) were observed when comparing between DPHH, ABTS, and ORAC for each Rosmarinus oficinalis extraction method. DPPH (F = 3790.49, p < 0.001), ABTS (F = 3044.86, p < 0.01), and ORAC (F = 3582.36, p < 0.01). It was evidenced dichloromethane extract had the highest antioxidant capacity for this plant (Tukey p < 0.01). Similarly, signiicant differences were also observed for each method in Cecropia mutisiana extracts: DPPH (F = 93588.817, p < 0.01), ABTS (F = 84358.817, p < 0.01), and ORAC (F = 90327.01, p < 0.01). Ethanol Emir. J. Food Agric ● Vol 29 ● Issue 1 ● 2017 extraction had the highest antioxidant and protective capacity (ORAC) for this plant (Tukey p < 0.01). Analysis of standardized IC50 values for both plants evidenced Rosmarinus oficinalis dichloromethane extract and Cecropia mutisiana ethanol extract had the greates antioxidant capacity (IC50) against DPPH, ABTS, and AAPH free radicals, comparable to trolox and ascorbic acid control IC50 values. To establish if extract antioxidant capacity was associated with total phenolic content, these compounds were quantified for both plants (Table 6). No significant correlation for Rosmarinus oficinalis and Cecropia mutisiana extracts was found between total phenol content and IC50 concentration (r = -0.815, p = 0.185) and (r = -0.580, p = 0.420), respectively. 29 Ortíz-Ardila, et al.: Cecropia mutisiana antioxidant and antimicrobial Table 5: Antioxidant capacity determination by DPPH, ABTS and ORAC – IC50 or IP50 [ppm] Method Extract EP CH2Cl2 AcOEt EtOH I II I II I II I II DPPH (IC50) 1813±68 5578±6 558±9* 14597±90 718±38 3843±54 3505±168 631±11* ABTS (IC50) 1303±25 2544±21 439±12* 2333±8 481±14 414±16 2083±168 253±2* ORAC (IP50) 915±17 1695±31 273±5* 2040±37 273±5 2075±37 1279±23 165±3* Control A 163±8 134±7 88±2* B 124±9* 96±10* 107±2 I: Rosmarinus oficinalis, II: Cecropia mutisiana, Control A: Trolox, Control B: Ascorbic acid. (*) Extracts with highest antioxidant capacity (p<0.05) Table 6: Total phenolic content/plant extract Total phenols/plant (mg GA/g Ext)* Extract Rosmarinus oficinalis Cecropia mutisiana PE 27.3±1.4 24.6±1.5 EtOAc 135.1±6.4 60.6±2.9 CH2Cl2 239.8±10.3 35.8±1.7 EtOH 28.9±1.6 169.6±6.9 *Total phenolic content was established by Folin-Ciocalteu and is expressed as mg gallic acid per g plant extract (n=3). PE: Petroleum ether, EtOAc: Ethyl acetate, CH2Cl2: Dichloromethane, EtOH: Ethanol Petronilho et al., (2012) performed in vitro and in vivo antioxidant capacity assays from Cecropia glaziovii hydroethanolic crude extracts through lipid peroxidation enzyme activity quantiication. Their indings revealed a signiicant activity at low concentrations (2 ppm). In contrast, the minimum activity obtained in our study was in ethanol extract (253.2 ppm). An approximation can be therefore established to the type of antioxidant present in the plant species as a possible “scavenger”, interrupting lipid peroxidation through iron chelation, directly inluencing lipid solubility and preventing ROS generation. IC50 antioxidant capacity quantiication deines the concentration required to obtain 50% inhibition/ sequestration of the free radical to attain its chemical stabilization, as determined by DPPH and ABTS tests. The highest IC50 observed were for high polarity ethanol extract, suggesting presence of phenolic and lavonoid compounds, phytochemically characterized for Cecropia mutisiana including chlorogenic acid, orientin, isoorientin, isovitexin reported by other authors (Nicasio-Torres et al., 2009; Aragão et al., 2010; Petronilho et al., 2012; Cruz et al., 2013). Aragao et al. (2010) performed Cecropia pachystachya antioxidant capacity for the methanol extract using the DPPH test establishing an IC50 of 10.8 ± 0.7 ppm. Moreover, Mora-Izquierdo et al. (2011) characterized Cecropia peltata antioxidant capacity in function of chlorogenic acid (CGA) standard by the ABTS methodology inding a stabilization capacity at 13.8 ± 2.2 mg CGA/g dry weight (13,780 ppm) for the methanol extract. For Cecropia mutisiana ethanol extracts the following IC50 were established for DPPH (630 ± 11.1 ppm) and ABTS (253.2 ± 2.0 ppm). Demonstrating the nature of the antioxidant molecule is of high polarity, typical behavior of previously described polyphenols and lavonoids (Aragão et al., 2010; Mora Izquierdo et al., 2011; Petronilho et al., 2012). 30 ORAC methodology allows a more thorough approximation of the antioxidant type, its nature and possible mechanism of action for the species in question. Additionally, a positive correlation between the protective capacity determined by ORAC and the antioxidant inhibitory capacity (ABTS and DPHH) was evidenced (r = 0.968, r = 0.949, p < 0.01). Demonstrating the presence of plant antioxidant compounds particularly of polar nature, acting as free radical “scavengers”. In addition, they can act as quenchers in vitro sequestering lipid ROS production. Given its antioxidant activity this bivalent behavior could be due to complex interaction between majority and minority compounds present in Cecropia mutisiana extracts. For Cecropia mutisiana they have not been totally identiied, in contrast to other species where majority compounds responsible for antioxidant capacity have been described, such as glycosylated lavonoids. In comparison to other Cecropia species a greater antioxidant capacity was observed for Cecropia mutisiana compared with Cecropia peltata, and to lesser extent when compared with Cecropia pachystachya, due to environmental conditions to which the Colombian species is submitted in comparison to other species in the Neo-tropic, such as light exposure and nitrate supply or different quantities and types of compounds between the methanolic and ethanolic extracts. Mora-Izquierdo et al. (2011) have established the aforementioned factors are fundamental for natural antioxidant production, since high photosynthesis conditions and reduction of available nitrate increase ROS production, and with them the production of defensive compounds, such as antioxidant molecules based on carbon compounds. Different authors have studied Rosmarinus officinalis antioxidant capacity characterizing plant extracts with findings evidencing medium polarity extract result in the best IC 50 values, specifically for acetone and dichloromethane extractions (Yesil-Celiktas et al., 2007), given the tripenic nature of carnosic acid (CA), the main molecule responsible for the antioxidant capacity. Rodríguez-Rojo et al. (2012) established a Rosmarinus oficinalis IC50 value determined by the DPPH scavenging assay between 69 and 45 ppm from a bioassay guided fractionation extraction. Moreover, Chang et al. (2008), Emir. J. Food Agric ● Vol 29 ● Issue 1 ● 2017 Ortíz-Ardila, et al.: Cecropia mutisiana antioxidant and antimicrobial obtained with a supercritical luid extraction an IC50 of 5 mg/mL, representing 5,000 ppm with extraction yields higher than solvent extraction, yet a marked decrease in antioxidant capacity (Chang et al., 2008). Likewise, Jordán et al. (2013b), compared different locations for Rosemary extractions in the Mediterranean inding on average an IC50 of 565.9 ppm for DPPH and 533.9 ppm for ABTS assays (Jordán et al., 2013b). Both values are comparable to our indings, where the best IC50 value was 558.3 ± 8.6 ppm observed with a DPPH assay from a CH2Cl2 extract. In addition to an IC50 value of 439.1 ± 11.9 ppm with an ABTS test. Cecropia mutisiana and Rosmarinus officinalis antioxidant activity of each obtained plant extract was determined by using DPPH and ABTS radical tests. A signiicant IC 50 value difference (p < 0.05) was observed for Cecropia mutisiana ethanol and EtOAc extracts for their antioxidant capacity determined by ABTS in comparison with Rosmarinus oficinalis. For Rosmarinus oficinalis DPPH antioxidant capacity from the dichloromethane extract was signiicantly higher compared (p < 0.05) with EtOAc or EtOH extracts from the same plant. Additionally, for Cecropia mutisiana ethanol extract ABTS antioxidant activity was also signiicantly higher compared to other extracts (p < 0.05). These results are likely due to the types of molecules that are known. Some authors have established CA (antioxidant molecule in Rosmarinus oficinalis) acts as a proton donor and “scavenger” of free radicals (Masuda et al., 2001; Yesil-Celiktas et al., 2007; Rodríguez-Rojo et al., 2012). Thus, Karadag et al. (2009) described for DPPH and ABTS test results similar in vitro behavior. DPPH identiies antioxidant capacity with proton/electron donor capacity, and ABTS determines molecules of donating and or quenching capacity. Rosmarinus oficinalis, DPPH and ABTS results for this study had appreciable IC50 differences, yet of low magnitude. This inding is supported by the lack of correlation between antioxidant capacity and total phenol quantiication, given CA triterpenic nature. In contrast, antioxidant capacity molecule or molecules for Cecropia mutisiana were different. For other Cecropia species chlorogenic acid (phenol compound) and/or lavonoids such as orientin, isoorientin, and isovetexin were described by Aragao et al. (2010), Mora-Izquierdo et al. (2011) and Petronilho et al. (2012) as the molecules responsible for antioxidant capacity. In this regard, our data evidenced a greater antioxidant capacity through the ABTS methodology compared with DPPH test. Cecropia mutisiana antioxidant activity could be the result of a possible proton donor or radical “scavenging-quenching” compound, as described by Karadag et al. (2009). This, in part supported by Cecropia mutisiana lack of correlation between antioxidant capacity and total phenol quantiication (Folin Ciocalteu reagent). Emir. J. Food Agric ● Vol 29 ● Issue 1 ● 2017 Thus, it could be inferred Cecropia mutisiana antioxidant capacity could be mostly accounted by lavonoid type of compounds with chain-blocking activity. Last, given antioxidant capacities attained stem from different solvent extraction at distinct polarities direct comparisons cannot be established. However, Rosmarinus oficinalis data grants an approximation to the nature and possible compound mechanisms of antioxidant capacity action in Cecropia. Gold standard trolox and ascorbic acid antioxidant capabilities were signiicantly higher compared with both plant extracts (p < 0.05). A better IC50 was observed for ascorbic acid in both DPPH and ABTS assays; most likely due to the molecule’s purity and proton/electron donor mechanism and latter radical inactivation and destruction. Antimicrobial capacity determination To determine Rosmarinus oficinalis and Cecropia mutisiana extract antimicrobial properties and Minimal Inhibitory Concentration (MIC) 10 mg, 1 mg or 0.1 mg extract/disc was used. Data is summarized in Table 7. No signiicant differences were observed for Rosmarinus oficinalis percentage of relative MIC among treatments (p = 0.395), thus responses at the inhibition level among extracts were not different. Likewise, no significant differences were observed for Cecropia mutisiana (p = 0.601). In addition, no significant differences were attained for comparisons between both plants (ANOVA, p = 0.660). Despite no statistically signiicant differences Rosmarinus oficinalis ethanol extract and Cecropia mutisiana dichloromethane extract were capable of inhibiting a greater number of microorganisms at the lowest concentrations (10 ppm and 100 ppm) respectively. Furthermore, Rosmarinus oficinalis ethanol extract had the highest antimicrobial activity. At present no studies have addressed antimicrobial activity for members of the Cecropia species. Cecropia mutisiana extracts were capable of inhibiting Gram positive and Gram negative bacteria, within a gamut of distinctive compounds and routes of action, likely due to variations in extract polarity. Even though this study was a irst attempt to characterize Cecropia mutisiana antioxidant and antimicrobial properties in comparison to a widely studied plant Rosamarinus oficinalis future studies should also include other species such as Cecropia pachystachya, Cecropia glaziovii, and Cecropia peltata against Leishmania spp., and Plasmodium falciparum parasites (Uchoa et al., 2009; Cruz et al., 2013). In addition, comparison studies could include their antiviral properties, as case in point herpes (Silva et al., 2010), pathogenic bacteria: β hemolytic Streptococcus, Escherichia coli, and Candida albicans yeast (Rojas et al., 2006). 31 Ortíz-Ardila, et al.: Cecropia mutisiana antioxidant and antimicrobial Table 7: Relative percentage inhibition of the minimum inhibitory concentrations (in bold number)* Microorganism µg extract/disk Rosmarinus oficinalis Govaerts Cecropia mutisiana Mildbr PE EtOAc CH2Cl2 EtOH PE EtOAc CH2Cl2 EtOH Pseudomonas 10 5.8 ± 3.2 10.1 ± 7.5 16.6 ± 4.8 10.5 ± 8.9 aureuginosa 1.0 5.4 ± 4.1 0.1 2.2 ± 0.4 Staphylococcus 10 1.6 ± 0.6 5.2 ± 2.3 17.6 ± 6.6 aureus 1.0 9.8 ± 5.3 0.1 Salmonella sp. 10 22.4 ± 4.8 16.0 ± 4.1 22.4 ± 6.9 2.7 ± 0.8 28.5 ± 4.5 22.4 ± 2.7 11.7 ± 5.3 1.0 10.8 ± 3.0 9.6 ± 4.6 9.7 ± 2.0 16.0 ± 5.4 12.8 ± 3.0 8.0 ± 1.8 0.1 4.3 ± 2.9 6.9 ± 4.7 5.3 ± 3.0 Bacillus cereus 10 13.0 ± 3.9 12.4 ± 4.2 4.5 ± 2.6 15.6 ± 6.2 16.1 ± 4.9 6.3 ± 1.3 5.6 ± 2.3 1.0 6.8 ± 1.1 7.4 ± 2.5 7.4 ± 3.5 0.1 1.9 ± 0.6 *Positive control, gentamycin (100±2%). PE: Petroleum ether, EtOAc: Ethyl acetate, CH2Cl2: Dichloromethane, EtOH: Ethanol Rojas et al. (2006) described for Cecropia peltata an important antimicrobial activity against Staphylococcus aureus and Bacillus cereus, mainly in their ethanol extract, with greater than 78% inhibition for both bacteria. In this study Cecropia mutisiana had a 9.8% inhibition against Staphylococcus aureus, and was not capable of inhibiting Bacillus cereus, with a MIC > 1,000 ppm. These results could be due to differences in plant variability. Moreover, such contrasting results could also be attributed to the microbial strains utilized in this study (Staphylococcus aureus CMPUJ 080, Bacillus cereus CMPJU 251, Salmonella sp. CMPUJ 302, and Pseudomonas aeruginosa CMPUJ 055). Rosmarinus officinalis and Cecropia mutisiana inhibition percentage comparison for each extract, as previously described, was not statistically signiicant. Never the less, biologically differences in percentage magnitude, as well as the number of microorganisms sensitive to the extracts were observed. The highest antimicrobial activity was for Rosmarinus oficinalis ethanol extract followed by Cecropia mutisiana EtOAc extract. At present, there are no conclusive Cecropia genus antimicrobial molecule studies. It has been described they are achieved through lavonoids and steroids (Rojas et al., 2006; Uchoa et al., 2009; Silva et al., 2010; Cruz et al., 2013). In contrast, phenolic compounds and terpenes have been speciied as the main antimicrobial molecules for Rosmarinus officinalis (Celiktas et al., 2007; Klancnik et al., 2009; Jordán et al., 2013a; Zampini et al., 2013; Gemeda et al., 2015). This in part could account for Rosmarinus oficinalis superior activity in comparison with Cecropia mutisiana. Polyphenols are more soluble in lipids and have better membrane permeability in comparison with lavonoids (Yi et al., 2010). The extract can penetrate bacteria more feasibly, thus having a direct antimicrobial effect (Varela and Ibañez, 2009). 32 CONCLUSIONS Cecropia mutisiana Mildbr ethanol extract presented the best antioxidant capacity, as determined by DPPH and ABTS IC50 values. Additionally, dichloromethane extract for Rosmarinus oficinalis Govaerts had the leading antioxidant activity. Furthermore, regarding antimicrobial activity Cecropia mutisiana Mildb EtOAc extract had the greatest antimicrobial capacity. For Rosmarinus oficinalis Govaerts the ethanol extract was responsible for the highest microorganism growth inhibition. When comparing both plants Cecropia mutisiana Mildbr ethanol extract had the highest antioxidant capacity, while Rosmarinus oficinalis Govaerts presented the highest antimicrobial activity. ACKNOWLEDGEMENTS The authors are grateful for the support by the Administrative Department of Science, Technology and Innovation -COLCIENCIAS-, and Academic ViceRectory and Vice-Rectory for Research of the Pontiicia Universidad Javeriana (Projects 0027 and 5757). In addition, acknowledge the collaboration and time to Diego Alberto Villota Erazo of the Phytochemistry Research Group Javeriana University -GIFUJ-, and María Lucía Gutiérrez Gómez, Professor at the Pontiicia Universidad Javeriana. All the authors state that they have no conlicts of interest. Author's contributions All authors contributed substantially to the writing and revising of the manuscript. LGSC and AEOA designed the work, acquired, analyzed, and interpreted data. AEOA and JPCC obtained Cecropia mutisiana and Rosmarinus oficinalis extracts and fractions in different solvents, and statistical analysis. AEOA, CACZ, and LGSC (corresponding author) develop and standardized antioxidant methods. Emir. J. 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