Chemical Composition, Antimicrobial Activity, and Antioxidant Activity of Ocotea minarum (Nees & Mart.) Mez.

Ocotea minarum is a native plant from Brazil, popularly known as “canelinha” or “canela vassoura.” The objective of this study was to investigate the chemical composition of the extracts of the bark and the leaves of O. minarum and to evaluate its antimicrobial and antioxidant activities. The phenolic compounds, flavonoids and tanins, were quantified with the reagents Folin-Ciocalteu, aluminium chloride, and vanillin. The chemical profile was performed by HPLC-DAD. The minimum inhibitory concentration was evaluated by the microdilution in a broth method. The antioxidant activity was measured by the capture of free radicals 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid). In addition, protection against oxidative hemolysis and generation of malondialdehyde were evaluated in human erythrocytes. The composition of the extracts included the caffeic acid, p-coumaric acid, and rosmarinic acid, besides the flavonoids quercetin and luteolin. The EEL showed bacteriostatic action of 1000 μg/mL for all evaluated Salmonella Typhimurium, Salmonella Enteritidis, Pseudomonas aeruginosa, and Proteus mirabilis, and the EHEB had a moderate antifungal action against Candida krusei and Cryptococcus gattii (250 μg/mL). IC50 values of 8.19 (EEL) and 4.51 μg/mL (EEB) in the assay with DPPH and 6.25 (EEL) and 2.87 μg/mL (EEB) in the assay with ABTS were obtained. Up to the 3rd hour of oxidative hemolysis testing induced by AAPH, the EEB and EEL had a protective action, reducing the malondialdehyde. In conclusion, the data indicate that the O. minarum extracts can be evaluated as bioactive supplies for the development of new drugs for the prevention and treatment of diseases related to oxidative stress and microbial infections.


Introduction
The use of medicinal plants as raw material to develop products for therapeutic purposes is common. It has been estimated that from the 60,000 species discovered, the biological potential of 800 species has been evaluated [1,2]. It is known that plants with secondary metabolites, such as alkaloids, coumarins, tannins, terpenes, and flavonoids, are sources of several bioactive compounds that are beneficial to human health [3].
These compounds are described as having various biological activities, including antimicrobial action. Microbial infections are growing at an alarming level [4,5] owing to the ability of bacteria and yeasts to acquire and transmit genetic resistance material, leading to the constant evolution of multidrug-resistant microorganisms that threaten public health [6][7][8]. In this milieu, medicinal plants are receiving special attention for the development of new antimicrobial agents due to their therapeutic activity, low toxicity, and economic viability when compared with those of traditional allopathic compounds [9,10].
Another problem that affects public health is the degeneration of tissues and biomolecules caused by the imbalance of oxidative substances and antioxidants in the body, leading to oxidative stress. Oxidative stress is an important factor that triggers cardiovascular and neurodegenerative diseases, inflammation, diabetes, and premature aging, among others [11,12]. An approach to reduce this condition is the use of antioxidant substances that can act in the neutralization or even in the capture of reactive species, thus, minimizing oxidative tissue damage [13,14].
Compounds such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) are the widely used synthetic antioxidants, but are questioned due to their nutritional value and possible mutagenic, carcinogenic, and toxic effects, including hepatotoxicity [15][16][17]. Generally, natural antioxidants have low toxicity and are easily biodegradable, making them an effective alternative to reduce oxidative stress [18,19].
Additionally, tropical and subtropical plant species are of increasing research interest, as they have diverse properties, such as potent antimicrobial and antioxidant activities [13,20]. In this context, the members of the genus Ocotea stand out, which are among the most expressive of the family Lauraceae. Among these, approximately 300-400 species are found in the tropical and subtropical regions [21,22], and it has been estimated that 120-160 species are found in Brazil [23] dispersed in the biomes of Atlantic Forest and Cerrado [24]. Studies on species of the genus Ocotea have demonstrated several biological activities, such as antimicrobial [25][26][27][28][29], antioxidant [25,26,[29][30][31][32], antiinflammatory [30,33], and antiprotozoal activities [34].

Preparation of Extracts.
The barks (246.80 g) and leaves (237.28 g) were sterilized and dried in a laminar flow cabinet at 40°C for 10 and 6 days, respectively. Subsequently, the samples were pulverized using a cutting mill. The sample was extracted with 800 mL of 80% ethyl alcohol at ambient temperature with regular shaking at every 24 h. After 72 h, the mixture was filtered using a filter paper (0.45 μm), rotoevaporated at 35°C and lyophilized to obtain the ethanolic extract of barks (EEB) and leaves (EEL) of O. minarum.
Solvents with increasing polarity (hexane and ethyl acetate) were used to prepare the liquid-liquid partitions to obtain hydroalcoholic extract (HYEB and HYEF), hexane extract (HEEB and HEEL), and ethyl acetate extract (EAEB and EAEL) from the barks and leaves of O. minarum, respectively [38].

Determination of Phenolic
Compounds. The content of phenolic compounds was determined as described by Djeridane et al. [39]. To obtain 100 μL of the EEB and EEL at a concentration of 100 μg/mL, 500 μL of Folin-Ciocalteu reagent and 1 mL of distilled water were added and incubated at ambient temperature for 1 min. Subsequently, 1.5 mL of 20% sodium carbonate was added to this solution and incubated for 2 h in the dark at ambient temperature. The absorbance was read at 760 nm. The concentration of phenolic compounds was calculated by preparing an analytical curve using gallic acid as standard at concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μg/mL. With the data, the linear regression was developed and the equation of line was obtained, with a linear correlation coefficient of R 2 = 0 997, slope a = −0 018, and linear coefficient b = 0 0016. The results are expressed as milligrams of gallic acid equivalent (mg/GAE) per gram of extract. All analyses were performed in triplicate.
2.4. Determination of Flavonoids. The content of flavonoids was determined by the method described by Lin and Tang [40]. Five hundred microliters of EEB and EEL at a concentration of 100 μg/mL was added to 1.5 mL of 95% ethyl alcohol, 0.1 mL of 10% aluminum chloride hexahydrate (AlCl 3 ), 0.1 mL of 1 M potassium acetate (CH 3 COOK), and 2.8 mL of deionized water. The reaction mixture was incubated at ambient temperature for 40 min, and the absorbance was read using a spectrophotometer at a wavelength of 415 nm. To determine the concentration of flavonoids, an analytical curve was prepared using quercetin as standard at concentrations of 10, 20, 30, 40, and 50 μg/mL, and the respective absorbance was read. With the obtained data, the linear regression and the equation of the line with R 2 = 0 999, a = 0 0019, and b = 0 0105 were obtained. The results are expressed as milligrams of quercetin equivalent (mg/QE) per gram of extract. All analyses were performed in triplicate.
2.5. Determination of Tannins. The tannins were quantified by the vanillin reaction according to the method of Broadhurst and Jones [41], adapted by Agostini-Costa et al. [42]. Five milliliters of freshly prepared vanillin reagent (vanillin-HCL-methanol, 4 : 10 : 86) was taken in a test tube and 1 mL of EEB or EEL, at a concentration of 100 μg/mL, was added and agitated for 30 s. The reaction mixture was undisturbed for 15 min, and the absorbance was read at 490 nm. The quantification was done using an external calibration curve with catechin as standard, at concentrations of 2.5, 5, 10, 20, 30, and 40 μg/mL, and their absorbance was read, to obtain the equation of line with R 2 = 0 998, a = 0 006, and b = 0 0018. The results are expressed as milligrams of catechin equivalent (mg/CAE) per gram of extract. All analyses were performed in triplicate.

Determination of Minimum Inhibitory Concentration
(MIC) of the Extract. The susceptibility assay was performed by the broth microdilution technique, according to the guidelines of the Clinical and Laboratory Standards Institute. The standards of document M27-A3 [44] and document M07-A9 [45] with some modifications for the use of plant extracts were used for yeasts and bacteria, respectively.
The EEB, EEL, HYEB, HEEB, EAEB, HYEF, HEEL, and EAEL were dissolved in dimethylsulfoxide (DMSO) and then diluted (1 : 2) to concentrations of 1.9 and 1000 μg/mL. As a control, 100 μL of culture medium and 50 μL of extracts were added to the wells of column 1 of a 96-well microplate. The amount of inoculum added to the plates was 10 and 100 μL for bacteria and yeasts, respectively. The culture medium used was RPMI-1640 for yeasts and Müller Hinton broth for bacteria. The microplates were incubated at 35°C for 48 h for Candida sp. and 72 h for Cryptococcus sp. and Rhodotorula sp. and at 37°C for 24 h for the bacteria. Fluconazole was used as a control antifungal agent and ampicillin as a control antibiotic. The experiments were performed in triplicate at three different times.

Minimal Fungicidal Concentration (MFC) and
Minimal Bactericidal Concentration (MBC). Aliquots from a microdilution plate (of MIC assay) were transferred to a Petri dish containing Sabouraud Dextrose agar or Müller Hinton agar to the fungicidal and bactericidal activities, respectively. The plates were incubated at 35°C for 48 h for Candida sp. and 72 h for Cryptococcus sp. and Rhodotorula sp. and at 37°C for 24 h for bacteria. The MFC and MBC were defined as the lowest concentration capable of inhibiting colony growth [46].

Antioxidant Activity
The antioxidant activity of EEB and EEL was evaluated by the method of Sharma et al. [47] using the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). Ethanolic solution of DPPH (0.11 mM, 1800 μL) was incubated with 200 μL of the positive controls, ascorbic acid, and hydroxybutyltoluene (BHT) or EEB and EEL at concentrations of 0.1-2000 μg/mL in 80% ethanol. After 30 min of incubation at ambient temperature (protected from light), the absorbance was read using a spectrophotometer (Quimis, Diadema, SP, Brazil) at 517 nm; the absorbance of each sample was transformed into free radical inhibition percent using formula (1). Three independent experiments were performed in triplicate.

Radical scavenging % =
Abs control − Abs sample Abs control × 100 The free radical inhibition capacity of the EEB and EEL was evaluated according to the method described by Re et al. [48] using 2,2 ′ -azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Ascorbic acid and BHT were used as positive controls. The stock solution of ABTS was prepared using potassium persulfate at 12-16 h prior to the experiment and maintained at ambient temperature, protected from light. The ABTS radical was diluted in ethanol PA until the absorbance reached 0 70 ± 0 05 at 734 nm and was incubated for 6 min with the extracts or controls at the concentrations used in the antioxidant assay. The volume of ABTS and extract used was 1980 μL and 20 μL, respectively. The absorbance was read using a spectrophotometer (Quimis, Diadema, SP, Brazil) at 734 nm. The results were expressed as % of free radical inhibition using formula (2). Three independent experiments were performed in triplicate.
Percent inibition of ABTS % = Abs control − Abs sample Abs controle × 100 2 2.8.3. Hemolysis Oxidative Test Induced by 2,2 ′ -Azobis(2amidinopropane) (AAPH). The protection effect of EEB and EEL against lipid peroxidation was evaluated by the method of oxidative hemolysis induced by 2,2 ′ -azobis(2amidinopropane) dihydrochloride (AAPH, Sigma-Aldrich), described by Campos et al. [49]. After the approval of the Research Ethics Committee (REC) of the Federal University of Grande Dourados (UFGD) with the process number 5160, 25 mL of blood was collected from a single, healthy, nonsmoking adult individual and stored in tubes containing the anticoagulant sodium citrate. From this sample, a 10% solution of red blood cells was prepared using 0.9% NaCl solution.
The extracts (final concentrations of 10-125 μg/mL) were preincubated with the erythrocyte solution at 37°C for 30 min. After this period, 0.9% NaCl solution or 50 mM AAPH solution was added to investigate whether the extracts promote hemolysis in erythrocytes or inhibit oxidative hemolysis, respectively. The samples were incubated in a water bath at 37°C for 4 h, and the mixture was homogenized steadily during this period. After 120, 180, and 240 min, the samples were removed, centrifuged at 1500 rpm/10 min, and then, the absorbance of the supernatant using a spectrophotometer (Quimis, Diadema, SP, Brazil) at 540 nm was read. The positive control ascorbic acid was maintained under the same conditions in assays. As a solvent control, the erythrocytes were incubated with ethanol at a final concentration of 0.4%. Three independent experiments were performed in duplicates. The percent of hemolysis was determined using formula 3, where the absorbance of total hemolysis represents erythrocytes incubated with distilled water.

Determination of Malondialdehyde (MDA).
A byproduct of lipid peroxidation, MDA, was measured to assess the ability of extracts to protect erythrocytes against oxidative stress. For this, the ascorbic acid or extracts (final concentrations of 10-125 μg/mL) were preincubated with erythrocyte suspension at 37°C for 30 min. After this period, the oxidizing agent AAPH, at 50 mM concentration, was added and the mixture was incubated for 4 h, under periodic agitation. After 120, 180, and 240 min, the samples were centrifuged at 1500 rpm for 10 min and 500 μL of the remaining supernatant was added to 1 mL of 10 nM thiobarbituric acid (TBA; Merck), solubilized in monosodium potassium phosphate buffer (75 mM) at pH 2.5. The control was prepared by the mixture of TBA and 20 mM MDA solution. The samples were incubated in a water bath at 96°C for 45 min.
Subsequently, the reaction mixture was placed on an ice bath for 15 min to cease the reaction, and then, 4 mL of butanol was added to the mixture, with subsequent vortexing followed by centrifugation at 3000 rpm for 5 min, according to the method described by Campos et al. [50]. The absorbance of the supernatant was recorded using a spectrophotometer (Quimis, Diadema, SP, Brazil) at 532 nm. The content of MDA is expressed as nmol/mL, obtained using MDA nmol/mL = Abs sample 20 × 220 32 Abs control 4 2.9. Statistical Analyses. The results are presented as the mean ± standard error of the mean. The analysis of variance (ANOVA) followed by Dunnett's test was performed for the comparison between more than two experimental groups. The comparison between two experimental groups was performed by the Newman-Keuls test. The statistical analyses were performed using the statistical program GraphPad Prism 5 (GraphPad Software Inc., USA). The data were considered significant when the P value was <0.05.

Chemical Composition.
The content of phenolic compounds and tannins in the EEB was higher than those in the EEL. The level of flavonoids in the EEL was higher than that in the EEB ( Table 1). The compounds were identified by HPLC and by the analysis of ultraviolet spectra of each peak. Caffeic acid (15 min) and rosmarinic acid (53.7 min) were identified in the EEB (Figures 1 and 2). In the EEL, caffeic acid (15.1 min), p-coumaric acid (25.2 min), rosmarinic acid (53.7 min), quercetin (59.1 min), and luteolin (59.7 min) were identified (Figures 2 and 3).

Antimicrobial Activity.
We evaluated the antimicrobial activity of different extracts against yeasts, gram-positive bacteria, and gram-negative bacteria (Tables 2 and 3). The HEEB was effective against all evaluated yeast strains, except against R. glutinis and R. mucilaginosa. The EEL, HYEL, and HEEB exhibited fungistatic and fungicidal action against C. krusei ( Table 2).

Protection against Oxidative Hemolysis.
After assessing the hemolytic activity of extracts in human erythrocytes, the protective activity of EEB and EEL was observed in the erythrocytes subjected to oxidative hemolysis by AAPH ( Figure 5). At 120 and 180 min (Figures 5(a) and 5(b)), the extracts exhibited protective action against oxidative hemolysis at the lowest concentrations of 10 to 100 μg/mL. At 240 min, only the EEB at a concentration of 25 μg/mL maintained this activity compared with that of the AAPH control ( Figure 5(c)).

Generation of Malondialdehyde.
The capacity of the extracts to protect erythrocytes incubated with the oxidizing agent AAPH against lipid peroxidation was assessed by the generation of MDA. At 120 min, the erythrocytes incubated with EEB at concentrations of 10 and 25 μg/mL showed a reduction in the generation of MDA by approximately 83%, when compared with that of the AAPH control group (Figure 6(a)). When incubated with the EEL at concentrations of 10 to 100 μg/mL, there was a reduction in the formation of MDA by approximately 85%.
In addition, at 180 min ( Figure 6(b)), only the EEL reduced the formation of MDA by approximately 40% at concentrations of 10 to 100 μg/mL and, at 240 min, reduced in 47% the formation of MDA, at a concentration of 100 μg/mL, which was significantly different from that of the AAPH group (Figure 6(c)).

Discussion
Medicinal plants are sources of important biomolecules, including phenolic compounds, which are described as having various biological activities as antibacterial, antifungal, anti-inflammatory, antioxidant, and antitumor. The phenolic compounds caffeic acid and p-coumaric acid, identified by HPLC in the extracts of O. minarum, have already been related to antioxidant and antimicrobial activities in plants [51,52].  whereas gram-negative cells have a double layer of lipopolysaccharide-phospholipid, hindering the passage of molecules into the bacterial cell [53]. There are also mechanisms for the efflux of small molecules, inactivation of enzymes, and loss of porins in the plasma membrane [54,55]. Furthermore, some yeasts, such as Cryptococcus sp. and Rhodotorula sp., are encapsulated, which confer them greater protection [56].
According to Morales et al. [57] different parameters of antimicrobial activity were considered: MIC < 100 μg/ mL, active; MIC 100-500 μg/mL, moderate; MIC 500-1000 μg/mL, weak antimicrobial activity; and MIC > 1000 μg/mL, inactive. Based on the adopted criteria, the EHEB showed good antifungal activity against C. neoformans and moderate activity against C. tropicalis, C. gattii, and C. krusei. Cryptococcus neoformans might have gained resistance to fluconazole, and C. krusei is intrinsically resistant to the same antifungal agent and is often isolated from opportunistic and invasive infections caused by Candida species [58]. Therefore, a search for new techniques for the development of new antimicrobials is important to improve the treatment of infections. With respect to gram-positive bacteria, in the present study, the EAEL and HYEB showed good antibacterial activity against Bacillus cereus and moderate activity against S. aureus and S. epidermidis.
Bruni et al. [29] showed the antibacterial activity of the essential oil of Ocotea quixos flowers against S. aureus and E. faecalis. Gil et al. [27] demonstrated that the essential oil of Ocotea caudata leaves has antibacterial action against B. subtilis and S. aureus. In the present study, the EEB and EEL of O. minarum did not present active antibacterial action against gram-negative bacteria. This suggests that the extracts probably act on the cell wall and peptidoglycan bonds of gram-positive bacteria and do not have good activity against the gram-negative bacteria due to the presence of a double layer of lipopolysaccharide-phospholipid, conferring less sensitivity.
The antimicrobial activity exhibited by the EEB and EEL of O. minarum is probably due to the presence of sesquiterpenes: lanceolic acid, curcumen-12-oic acid, (+)-Eexo-abergamoten-12-oico acid, loliolide terpene lactone, and nor-sesquiterpene [37]. The flavonoids quercetin and luteolin, present in EEB and EEL, have been associated with antimicrobial activity, as they can penetrate cellular phospholipid membranes due to their hydrophobicity [59,60]. To the best of our knowledge, the present study is the first to report the antibacterial activity of O. minarum.
Further, the phenolic acids caffeic acid and p-coumaric acid and the flavonoid quercetin identified are also associated with antioxidant activity, due to their direct action in the capture of free radicals [61][62][63]. The EEB and EEL exhibited promising antioxidant activity in the DPPH and ABTS assay, when compared with that of ascorbic and BHT, reference antioxidants. In particular, EEB presented a higher antioxidant potential than EEL in both the tests.
Other authors describe the antioxidant activity of species of the genus Ocotea [25,29]. Comparatively, in the present study, the EEB and EEL of O. minarum showed a better antioxidant potential than other species of the same genus.
The redox balance of the body is regulated by endogenous and exogenous mechanisms, in which the excess of free radicals is a fundamental factor causing numerous diseases, such as diabetes, atherosclerosis, cardiovascular diseases, cancer, and premature aging, among others [11]. Therefore, the search for natural antioxidants has increased in order to replace the synthetic compounds that might pose harmful effects on human health [64].
When evaluating the antioxidant properties of extracts in human erythrocytes, although the EEB and EEL at the highest concentrations induced high hemolysis rates, both promoted the reduction of AAPH-induced lipid peroxidation at lower doses and during the early evaluation times, protecting the erythrocytes from cell lysis. The peroxidation of the phospholipid membrane is induced by reactive species, especially oxygen and nitrogen species, causing cell lysis and death. Furthermore, a byproduct of this reaction is malondialdehyde [65,66], which is a toxic product leading to the release of unsaturated fatty acids and the fragmentation of phospholipids, causing mutations and cellular rupture [19]. The EEB and EEL reduced the generation of malondialdehyde during the early evaluation times, contributing to the maintenance of a cellular structure, at low concentrations. Thus, protection against this type of cellular damage is considered a positive effect [11], as it is associated with the reduction of damage caused by oxidative stress and its complications.
Luteolin, a flavonoid present in the extract of the leaves of O. minarum, has been described by modulating the expression of antioxidant enzymes superoxide dismutase and catalase, besides reducing the levels of malondialdehyde generated in an oxidative stress model in vivo [67]. The flavonoid quercetin and the polyphenol rosmarinic acid, also identified in extracts of the O. minarum, have already been described by activation of the transcriptional factor Nrf-2 (nuclear factor erythroid 2), an important cell antioxidant mechanism [68,69].
In conclusion, the data indicate that the O. minarum extracts can be evaluated as bioactive supplies for the development of new drugs for the prevention and treatment of diseases related to microbial infections and oxidative stress.

Data Availability
The chemical composition, antimicrobial, and antioxidant data used to support the findings of this study are included within the article.

Conflicts of Interest
The authors declare that there is no conflict of interest regarding the publication of this paper.