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

doi: 10.9755/ejfa.2016-07-915


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 identification and classification 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 scientifically 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 define 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 unspecific.Zambrano-Ospina described Cecropia mutisiana Mildbr aqueous leaf extract use as an antianxiety and anticonvulsant treatment (Zambrano-Ospina, 2000).In addition, Ahumada performed a chromatographic analysis identifying flavonoid type compounds, tannis, coumarins, steroids, and terpene lactones (Ahumada, 2006).None the less, currently no scientific registry has documented 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).

Extract preparation
Cecropia mutisiana plant material was purchased in Mogambo Environmental Trail in the Municipality of Viotá (Cundinamarca, Colombia) and Rosmarinus officinalis 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 identified by taxonomic classification 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 (CH 2 Cl 2 ), and ethanol (EtOH) as the solvent with the maximum polarity, shaking at 100 rpm for seven days for all solvents.Extracts were filtered and then concentrated by rotary-evaporation at 40°C to prevent damaging thermolabile compounds, and finally the excess solvent was removed by drying under extraction hood for six hours.(Rodríguez-Rojo et al., 2012).

ABTS method
ABTS methodology was performed with 2,2-azinobis(3ethylbenzothiazoline-6-sulfonic acid) chromogen with radical production by potassium persulphate (2.5 mM K 2 O 8 S 2 ) addition.Results are presented as IC 50, since they correspond to total extract measurement at different polarities.Spectrophotometric UV-Vis at 734 nm absorbance change was determined (Cary 100 CONC-Varian 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 (IC 50 ) 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 fluorescent salt such as fluorescein by which the antioxidant protective capacity was evidenced (IP 50 ) against radical attack, by determining a fluorometric delta difference between 485 nm excitation and 520 nm emission (FLUOstar Optima BMG Labtech).96 well plates containing fluorescein blanks in phosphate buffer, ascorbic acid and trolox positive controls and extracts to be tested in a 1:11.5 µL sample: radical ratio 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).
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 verified at 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)

Extract characterization
Four extracts of different polarities were obtained for each of the plants from 200 g dried Rosmarinus officinalis and 1,000 g Cecropia mutisiana yields are detailed in Table 1.
Extraction yield for each polarity in Rosmarinus officinalis ranged between 1.0 % and 2.8 % extract per g of dried 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.
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 flavonoids 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  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 officinalis and Cecropia mutisiana extracts was found between total phenol content and IC 50 concentration (r = -0.815,p = 0.185) and (r = -0.580,p = 0.420), respectively.Demonstrating the nature of the antioxidant molecule is of high polarity, typical behavior of previously described polyphenols and flavonoids (Aragão et al., 2010;Mora Izquierdo et al., 2011;Petronilho et al., 2012).
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 identified, in contrast to other species where majority compounds responsible for antioxidant capacity have been described, such as glycosylated flavonoids.
In   obtained with a supercritical fluid extraction an IC 50 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 finding on average an IC 50 of 565.9 ppm for DPPH and 533.9 ppm for ABTS assays (Jordán et al., 2013b).Both values are comparable to our findings, where the best IC 50 value was 558.3 ± 8.6 ppm observed with a DPPH assay from a CH 2 Cl 2 extract.In addition to an IC 50 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 significant 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 officinalis.For Rosmarinus officinalis DPPH antioxidant capacity from the dichloromethane extract was significantly 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 significantly 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 officinalis) 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 identifies antioxidant capacity with proton/electron donor capacity, and ABTS determines molecules of donating and or quenching capacity.Rosmarinus officinalis, DPPH and ABTS results for this study had appreciable IC 50 differences, yet of low magnitude.This finding is supported by the lack of correlation between antioxidant capacity and total phenol quantification, 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 flavonoids 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 quantification (Folin Ciocalteu reagent).
Thus, it could be inferred Cecropia mutisiana antioxidant capacity could be mostly accounted by flavonoid 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 officinalis 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 significantly higher compared with both plant extracts (p < 0.05).A better IC 50 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 officinalis 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 significant differences were observed for Rosmarinus officinalis 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 significant differences Rosmarinus officinalis 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 officinalis 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 first attempt to characterize Cecropia mutisiana antioxidant and antimicrobial properties in comparison to a widely studied plant Rosamarinus officinalis 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).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 significant.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 officinalis 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 flavonoids 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 specified 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 officinalis superior activity in comparison with Cecropia mutisiana.Polyphenols are more soluble in lipids and have better membrane permeability in comparison with flavonoids (Yi et al., 2010).The extract can penetrate bacteria more feasibly, thus having a direct antimicrobial effect (Varela and Ibañez, 2009).

Table 2
(Uchoa et al., 2009)no compound identification 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 identified (Table3).Salkowski and Shinoda tests confirmed sterol presence, flavonoids, and derivatives of these (flavanols, isoflavones, flavanes, among others) for both plants.According to Uchoa et al., for Cecropia species sterols and flavonoids are secondary metabolites that are not involved in the plant's development and growth(Uchoa et al., 2009).This type of compound has been investigated in other Cecropia species (Table4).

Table 2 : Preliminary phytochemical analysis for Rosmarinus officinalis and Cecropia mutisiana extracts Metabolite (test)
Petronilho et al., 2012).Therefore, for this study a comparison with Rosmarinus officinalis, 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 Table5.

Table 6 : Total phenolic content/plant extract
Cecropia mutisiana Mildbr ethanol extract presented the best antioxidant capacity, as determined by DPPH and ABTS IC 50 values.Additionally, dichloromethane extract for Rosmarinus officinalis Govaerts had the leading antioxidant activity.Furthermore, regarding antimicrobial activity Cecropia mutisiana Mildb EtOAc extract had the greatest antimicrobial capacity.For Rosmarinus officinalis 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 officinalis Govaerts presented the highest antimicrobial activity.AEOA, JPCC, MXRB, JRC, and LGSC standardized microbiological methods and preliminar phytochemical analysis.