Evaluation of antibacterial and antioxidant activity of purple araçá essential oil (Psidium rufum, Myrtaceae)

ABSTRACT The aim of this study was to evaluate the antibacterial and antioxidant activities of essential oil (EO) from fresh leaves of Psidium rufum. The EO was extracted by hydrodistillation and identified by gas chromatography coupled to mass spectrometry. The antibacterial activity was evaluated by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Antioxidant activity was determined by β-carotene/linoleic acid co-oxidation system, 2,2-diphenyl-1-picrylhydrazyl radical scavenging and iron reduction methods. Hydrocarbon sesquiterpenes were the predominant class, indicating 1,8 cineole, α-longipinene as major. The EO was tested against the bacteria Staphylococcus aureus and Pseudomonas aeruginosa (MIC = 2,500 µg/mL and MBC = 20,000 µg/mL); Enterococcus faecalis (MIC = 2,500µg/mL and MBC > 20,000µg/mL) and Escherichia coli (MIC > 20,000µg/mL and MBC > 20,000µg/mL). The EO showed antioxidant potential due to β-carotene/linoleic acid co-oxidation system, with 76.63% of oxidation inhibition (1.0mg/mL) and due to the iron reduction power (5,38 μmol Fe 2+ /mg sample). The results are promising in recommending this species for the development of food, cosmetic and pharmaceutical products.


INTRODUCTION
Brazil, a country of rich biodiversity, has in its territory an invaluable asset of resources, however, very little is known of this diversity (Bresolin and Cechinel, 2003).The Myrtaceae family has approximately 140 genera and more than 3,000 species worldwide, being considered complex from the taxonomic point of view (Gomes et al., 2009).This family has leaves with a large number of volatile constituents (Souza and Lorenzi, 2005;Stieven et al., 2009), where they almost always have proanthocyanins, ellagic and gallic acids, as well as produce saponins and less cyanogenic compounds characteristics of native fruit, to which the Araçazeiros belong.They assume prominence because the oils produced by species of this family exhibit insecticide activity, pesticide, nematicide, antifungal, antibacterial, antioxidant and even antiallergic properties , and the presence of flavonoids aids in immunological activity, determining chronic responses in inflammatory processes (Batish et al., 2012;Amarante and Santos, 2013).
From a pharmacological point of view, studies with gross and compound extracts have proven anti-inflammatory, analgesic, antifungal, antipyretic, hypotensive, antidiabetic and antioxidant activities (Oliveira et al., 2006;Armstrong, 2011).Simonetti et al. (2016) observed antimicrobial activities in Psidium plant statements against Escherichia coli and Listeria monocytogenes.
According to Daikos et al. (2021), in recent years there has been an increase in bacterial species with a high degree of resistance to conventional therapy, such as P. aeruginosa, which can present multiresistant phenotypes.Therefore, there is an interest in increasing the knowledge about the inhibitory concentrations of essential oils, in search of a balance between the acceptability and the effectiveness of the antimicrobial action.It is estimated that 20 to 50% of antimicrobial use in humans and 40 to 80% in animals is unnecessary or highly questionable, which leads to a high rate of resistance by bacteria (Beovic, 2006).
The development of natural antioxidants is interesting due to their role in the protection of human cells from damage caused by free radicals and natural compounds rather than synthetic antioxidants, which appear to be preferred in the industry (Mutlu-Ingok et al., 2020).Practical uses of these activities are suggested in humans and animals, as well as in the food industry.Since medicinal plants produce a variety of substances with antimicrobial (Alvarenga, et al., 2007) and antioxidant properties (Jerônimo et al., 2021), research with Psidium essential oil is of interest.
The genus Psidium originates from the tropical and subtropical Americas and consists of about 100 tree species and shrubs (Landrum and Kawasaki, 1997), of which the most important is the guava (P.guajava L.).The genre also includes numerous other species producing edible, logging, and ornamental fruits, with great potential for commercial exploitation.Among these species, araçazeiros, Psidium ruffum, are deserving of greater attention, especially due to some specific characteristics of their fruits, such as exotic flavor, high vitamin C content and good acceptance by consumers (Manica et al., 2000;Pires et al., 2002), beyond the presence of αtocopherol (Barcia et al., 2010).
However, research is focused on the extract of fruits and peels, few studies refer to the essential oil (EO) obtained from its leaves, so the proposed study can be a promising field for new discoveries.The objective in this study was to evaluate the antimicrobial and antioxidant activity of the essential oil of the fresh leaves of P. ruffum.P. ruffum fresh leaves EO was extracted by the hydrodistillation process for two hours using the modified Clevenger device (Gazim et al., 2010(Gazim et al., , 2011;;Armstrong, 2011).The EO was removed Arq.Bras.Med.Vet.Zootec., v.75, n.4, p.612-622, 2023 from the device with n-hexane, filtered with anhydrous sodium sulfate (NA2SO4) (Simões and Spitzer, 2002), packed in amber bottles, kept under refrigeration at 4ºC until complete evaporation of the solvent (Omolo et al., 2004).

Psidium
EO analysis was by gas chromatography coupled to mass spectrometry -(GC/MS).Using an Agilent 19091S-433 chromatograph.The DB5 Capillary Column (30 m x 0.25 mm x 0.25μm).The column temperature programming was 60°C, remaining for 3 min to 250°C with a heating ramp of 5.0°C/min, remaining at 250ºC for 15 min.The carrier gas was the helium used at a constant pressure of 80 kPa and with linear speed of 1 mL/min to 210°C and with a pressure flow of 25 kPa.Injector temperature: 250°C, the injection was in splitless mode.The conditions for the mass (MS): Source temperature 200°C; interface temperature 250°C; Detection system was by electronic impact to 70 eV; Mass Scan Range, 40-350 amu.In addition to the results obtained by GC/MS, the identification of the compounds was also based on comparing their retention rates (RR) obtained using a homologous series of N-alkans (C7-C30).The mass spectra were compared to the Wiley 275 Libraries library and the literature (Adams, 2017).
EO was evaluated against Gram-positive bacteria Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212 and Gramnegative Pseudomonas aeruginosa ATCC 27853 and Escherichia coli ATCC 25922, the test was performed using the microdilution method in broth with alterations for natural products (Methods…, 2009).
The bacteria were grown in Petri plates containing blood agar at 36°C for 24 hours.A saline suspension (NaCl at 0.85%) was prepared, with standardization according to the 0.5 scale of MacFarland.Subsequently, these inoculants were diluted 1:10 in saline to obtain a final concentration of 10 7 UFC mL -1 .
They were added to the sterile microplate containing 96 wells 100µl of the middle brain heart infusion (BHI) and 100µL of essential oil, prepared at the concentration of 20,000μg.ml-¹,EO was dissolved in 2% Tween 80.Serial dilutions were performed obtaining essential oil concentrations ranging from 20,000μg.ml-¹ to 1.207μg.ml-¹.Then the inoculum (50μL) was added to microlate.The controls: medium (only BHI), negative (BHI and essential oil) and positive (BHI and bacteria) were evaluated.After the microplates were incubated at 35°C for 24 hours.The reading was performed with the addition of 10 μL of TTC aqueous solution (Triphenyl tetrazolium chloride) to 10% in all wells and after 30 minutes to 35°C, the presence or not of reddish coloration was verified.The presence of reddish coloration was considered bacterial growth.
Bacteria colonies were cultivated separately to assess whether there was growth, determining the MBC individually by the subculture of each well.MIC was determined as the lowest concentration in which growth is inhibited and MBC was determined as the lowest concentration capable of inhibiting bacterial growth in subculture.
To determine the free radical scavenging capacity of DPPH, the methodology was evaluated according to Rufino et al. (2007).An aliquot of 0.1mL of the different concentrations of the EO of purple araçá (10.0; 7.5; 5.0; 2.5 and 1.0mg.mL - ), with 3.9mL of methanolic solution of DPPH (60µM) were freshly prepared for the activity.For the negative control, 0.1 mL of methanol was used in the DPPH solution (60µM).The mixture was kept in the dark at room temperature for 30 minutes.The absorbance reduction was measured at 515nm in a UV/VIS spectrophotometer.The total antioxidant capacity of extracts and fractions was calculated using a standard solution of quercetin (60µM), as a reference of 100%.From the correlation between absorbance versus concentration of the antioxidant sample, the concentration necessary to reduce 50% of free radicals (IC50) was determined.
The antioxidant capacities of the samples (EO) of purple araçá were evaluated according to Rufino et al. (2006b).The reaction can be monitored by spectrophotometry, loss of βcarotene staining at 470nm.20µL of linoleic acid, 265µL of Tween 40, 25µL of β-carotene solution (20 mg.mL -1 ) and 0.5 mL of chloroform were added to a beaker protected from light (wrapped in aluminum foil).The solvent was removed using a dryer.Then, the mixture was dissolved in 20mL of deionized water and oxygenated (by oxygen for 30 min) under vigorous stirring to form an emulsion.The emulsion had the absorbance adjusted to 0.7 at 470nm.The antioxidant activity of the samples was determined by mixing 280µL of emulsion with 20 µL of samples at different concentrations (1.0; 0.75; 0.50 and 0.25mg.mL -1 ) plotted on 96well flat-bottomed microplates.The samples were placed in the SpectraMax Plus384 Microplate Reader device, maintained at 40 ºC for 120 minutes with readings taken every five minutes, and the absorbance measured at 470nm.A 615rolox solution (0.2mg.mL -1 ) was used as a reference standard.Results were expressed as percentage of oxidation inhibition, following Eq. 1 the reduction in the absorbance of the antioxidant system was considered as 100% oxidation.From the absorbance following Eq.2, the percentage of oxidation was calculated correlated with the absorbance of the sample decreasing with the absorbance of the system, the percentage of oxidation of each sample was subtracted from 100 (Eq.3) for the percentage of inhibition of oxidation (%).
All tests were performed in triplicate.The results were submitted to analysis of variance (ANOVA), and the differences between the means determined by Duncan's test (p ≤ 0.05) by the SPSS Statistics 22 program.

RESULTS
The chemical identification of the EO of the fresh leaves of P. rufum revealed the presence of 41 compounds, considering only the compounds with a relative area above 0.30% (Table 1).The major classes were hydrocarbon sesquiterpenes (70.01%) followed by oxygenated monoterpenes (28.08%),where the main compounds identified were 1,8-cineole (19.36%) and α-longipinene (19.00%).
Regarding the antioxidant activity by the DPPH method, the EO of P. rufum was able to reduce the stable DPPH radical to yellow diphenylpicrylhydrazine, where the concentration required to reduce 50% of free radicals was 4.74±1.82mgmL -1 , about 474 times higher than quercetin, positive control, which makes evident the reduced antioxidant activity of this oil by this method (Table 2), presenting an antioxidant activity of 17.73 % at a concentration of 1.00 mg mL -1 .
The result of the antioxidant activity by the reduction of iron for the EO of P. rufum (Table 3) was calculated from the equation of the straight line obtained by the standard curve of ferrous sulfate (R 2 = 0.999), where it was possible to quantify the concentration of Fe 2+ present in solution, being 5.38±0.63µMferrous sulfate mg -1 of essential oil, a value only 0.6 times lower than the positive control (Trolox) 9.17µM ferrous sulfate mg -1 .70.01 a Compounds listed in order of elution by HP-5MS column (5% phenylmethylsiloxane).b RI= Retention index calculated using C7 -C30 n-alkanes in HP-5MS UI column.c MS= identification based on the comparison of mass spectra found in NIST 11.0 libraries (Adams, 2017).Relative area (%): percentage of the area occupied by the compounds in the chromatogram.t=trace.The most expressive result in the antioxidant activities was obtained by the method of oxidation inhibition by the β-carotene/linoleic acid co-oxidation system.In Figure 1, the lack of antioxidant potential can be accompanied by a reduction in the absorbance of the negative control (without antioxidant) which went from 0.633 to 0.110 after 120 minutes.On the other hand, the EO at a concentration of 0.25mg.mL - , showed a final absorbance 1.35 times lower than the EO at a concentration of 1.00 mg.mL -1 (Figure 1).The different P. rufum EO dilutions were higher than the positive control (Trolox), which reduced the absorbance to 0.504 at the end of the test.By measuring the percentage of oxidation inhibition by the β-carotene/linoleic acid cooxidation system (Table 4 and Fig. 1), it is verified that the EO of P. rufum leaves was effective at all concentrations, maintaining its antioxidant potential throughout the experiment 76.63% (1mg.mL - ) and 57.02% (0.25mg.mL -1 ).

DISCUSSION
The characteristics of the essential oil of P. rufum obtained in this study were compared with the obtained in the literature (Table 5).The production and characteristics of essential oil can vary depending on the genetic, regional, and climatic characteristics of the plant (Baser and Buchbauer, 2010;Jeribi et al., 2014).In recent years, there has been an increase in bacterial species with a high degree of resistance to conventional therapy (Daikos et al., 2021;Qin et al., 2022).Therefore, there is an interest in increasing the knowledge about the inhibitory concentrations of essential oils, in search of a balance between the acceptability and the effectiveness of the antimicrobial action.It is estimated that 20 to 50% of antimicrobial use in humans and 40 to 80% in animals is unnecessary or highly questionable, which leads to a high rate of resistance by bacteria (Beovic, 2006).
Plant essential oils have been shown to be effective in controlling the growth of a wide variety of microorganisms, including filamentous fungi, yeasts, and bacteria.Practical uses of these activities are suggested in humans and animals, as well as in the food industry, since medicinal plants produce a variety of substances with antimicrobial properties (Alvarenga, et al., 2007).
According to the MIC results, it was observed that E. coli was not inhibited up to the highest concentration tested, while S. aureus, E. faecalis and P. aeruginosa showed inhibitory activity at 2,500μg.mL -1 .Regarding the bactericidal concentration, for E. faecalis and E. coli it was not possible to determine the MBC up to the maximum concentration tested.S. aureus and P. aeruginosa did not show bacterial growth in their cultures, being defined as MBC = 20,000μg.mL -1 .
Studies have shown how E. coli is increasingly resistant to antibiotics, the pathogenicity of E. coli strains is related to the expression of virulence factors found in genetic elements called plasmids, which may explain the fact that this microorganism has been the only one with MIC above 20,000μg.mL - .In Canada, in a study with 2,483 cattle, it was found that 2.1% transmitted E. coli resistant to cefoxitin (Mulvey et al., 2009) and in the United States, a change in the resistance pattern of these microorganisms isolated from mastitis was observed of cows, with resistance to two or more antimicrobials in different combinations (Srinivisan et al., 2007).
There are numerous examples of the increase in antimicrobial resistance in veterinary medicine, in several animal species, and many of the microorganisms are resistant to antimicrobials for human use, which is worrying, since isolated bacteria can be a reservoir of resistant genes, with a role in dissemination of this resistance to pathogenic and commensal bacteria (Srinivisan et al., 2007).
According to Sartonatto et al. (2004), MICs between 50-500μg.mL - are considered to have high activity; moderate activity for MICs between 600-1500μg.mL - and weak activity for MICs above 1500μg.mL - .According to the results obtained by the MIC, it was possible to verify that the EO of P. rufum showed high antibacterial activity against the Gram-positive bacteria S. aureus (ATCC 29213), E. faecalis (ATCC 29212) and P. aeruginosa (ATCC 27853) (Gram-negative).
The antioxidant efficiency of an essential oil is mainly attributed to its major components, although it can also be caused by the synergistic effect of the minor components, as well as the possible interaction between the compounds (Tian et al., 2014).
P. rufum EO showed greater antioxidant activity by the FRAP method (5.38 µM ferrous sulphate mg of sample -1 ) (Table 3).This method evaluates the ferric ion reduction capacity of a given sample (Rufino et al., 2006a), being used to measure the antioxidant capacity of fruits (Sucupira et al., 2012).Rufino et al. (2010), using the method of oxidation inhibition (%) by the βcarotene/linoleic acid co-oxidation system, classified the antioxidant activity as being high when the percentage of oxidation inhibition is greater than 70%, intermediate when is between 40 and 70% and low when the oxidation inhibition percentage is less than 40%.Based on this classification, this study found a high antioxidant capacity for EO of purple araçá at a concentration of 1 mg/mL (76.63%) (Table 4).According to Sucupira et al. (2012), this method has been used to analyze various food matrices, mainly fruits and seeds rich in lipids.
The purple araça EO showed low antioxidant potential by the DPPH method with (IC 50 = 4.74 mg mL -1 ) when compared to the positive control quercetin (IC 50 = 0.001mg mL -1 ) (Table 2).EO are generally poorly soluble in aqueous and methanolic solutions such as those used in DPPH method.Thus, the lower molecular movement of Arq.Bras.Med.Vet.Zootec., v.75, n.4, p.612-622, 2023 these solvents probably reduces the reactive antioxidant capacity of EO when compared to the antioxidant activity in non-polar solvents, such as those used by the BCLA method.Thus, chemical interactions between essential oil and solvents may explain the lower antioxidant activity of P. rufum EO by DPPH methods and the higher activity by BCLA.The results found in this study suggest the use of EO in the food industry.

Figure 1 .
Figure 1.Antioxidant activity of essential oil from fresh leaves of Psidium rufum by the method of inhibition of oxidation (%) by the co-oxidation system β-carotene/linoleic acid.

Table 2 .
Antioxidant activity (%) and IC 50 (mg mL -1 ) by the Radical 2,2 diphenyl-1-picrylhydrazyl (DPPH) method, from purple Araçá EO Values are the mean ± standard deviation of the experiment performed in triplicate.The statistical analysis used was analysis of variance (ANOVA), and the differences between the means determined by Duncan's test (p ≤ 0.05) by the SPSS Statistics 22 program.Different letters in the same column indicate that there was a significant difference between the results (p ≤ 0.05).

Table 4 .
Antioxidant activity of essential oil from fresh leaves of Psidium rufum by the method of inhibition of oxidation (%) by the co-oxidation system β-carotene/linoleic acid (BCLA)Values are the mean ± standard deviation of the experiment performed in triplicate.The statistical analysis used was analysis of variance (ANOVA), and the differences between the means determined by Duncan's test (p≤0.05)by the SPSS Statistics 22 program.Different letters on the same line indicate that there was a significant difference between the results (p≤0.05);Positive control : Trolox (0.2mg.mL-1).