Piper cernuum Vell.: Chemical profile and antimicrobial potential evaluation

Industrial Crops & Products 140 (2019) 111577

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Piper cernuum Vell.: Chemical profile and antimicrobial potential evaluation a

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Antonio Linkoln Alves Borges Leal , Camila Fonseca Bezerra , Janaína Esmeraldo Rocha , Antonia Thassya Lucas dos Santosa, Rafael Pereira da Cruzb, Joara Nályda Pereira Carneirob, Débora Lima Salesa, Thiago Sampaio de Freitasa, Saulo Relison Tintinoa, Waltécio de Oliveira Almeidab, Wanderlei do Amarald, Luiz Everson da Silvae, Aurea Portes Ferrianie, Beatriz Helena Lameiro de Noronha Sales Maiae, Maria Flaviana Bezerra Morais-Bragaa, Humberto Medeiros Barretoc, ⁎ Henrique Douglas Melo Coutinhoa, a

Department of Biological Chemistry, Regional University of Cariri, Crato, CE, Brazil Department of Biological Sciences, Regional University of Cariri, Crato, CE, Brazil c Department of Health Sciences, Federal University of Piaui, Teresina, PI, Brazil d Department of Chemical Engineering, Federal University of Paraná, Paraná, PR, Brazil e Department of Chemistry, Federal University of Paraná, Paraná, PR, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Piper cernuum Piper Antibacterial Antifungal Essential oil Chemical composition

In this study, the Piper cernuum Essential Oil (EOPC) was tested against bacterial and fungal strains using the microdilution method to evaluate its minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC), as well as its antibiotic modifying effect, in addition to observing the oils ability to cause fungal dimorphism and to generate an IC50 cell viability curve. The EO was obtained through hydrodistillation using a Clevenger-type apparatus with the dry leaves and analyzed by GC-FID, allowing the identification of 14 compounds, with the main compound being 4-epi-cis-dihydromarofuran (28.97%). An EOPC intrinsic activity was identified only against Candida albicans 4127 (IC50 56.851 μg/mL). Given the MIC results from the EOPC intrinsic activity against Staphylococcus aureus 10 at the concentration of 406 μg/mL, its association with fluconazole potentiated it’s antifungal action. The action of gentamicin was potentiated by the addition of the EOPC to Staphylococcus aureus 10 and Escherichia coli 06 strains, both with MIC < 40 μg/ml, however, when the EOPC was combined with other antibiotics against Staphylococcus aureus 10 they presented an antagonistic response. When the EOPC was tested with the antibiotic norfloxacin against the Gram-negative Escherichia coli 06 strain an antagonistic response was also found. The EOPC inhibited fungal dimorphism, reducing the virulence of these strains, with this effect being better than that of fluconazole. These results highlight the species as a promising source of active antimicrobial compounds.

1. Introduction Pharmaceutical development has led to the discovery of new and more efficient drugs for the treatment of bacterial infections (Silveira et al., 2006). However, with the disseminated and uncontrolled use of antibiotics as well as other drugs, drug resistance emerged in the last three decades for drugs considered as controls for several infections. Microbial resistance is a reality, which presents the capacity to be genetically transmitted, thus microorganisms are able to acquire resistance to drugs commonly used as therapeutic agents (Nascimento

et al., 2000). Microbial resistance is the main factor responsible for several public health problems. The therapy, with the administration of antibiotics without prior knowledge of which microorganism is being fought, favors the growth and genetic availability of resistant microorganisms, this being dramatically increased in the last years and is considered today one of the main threats to global health in the 21 st century (O’Neill, 2016). The Piperaceae family is known to present plant-producing essential oils with many bioactive compounds against insects, bacteria and fungi

⁎ Corresponding author at: Laboratory of Microbiology and Molecular Biology, Department of Biological Chemistry, Regional University of Cariri – URCA, Rua Cel. Antônio Luís 1161, Pimenta, 63105-000, Crato, CE, Brazil. E-mail address: [email protected] (H.D. Melo Coutinho).

https://doi.org/10.1016/j.indcrop.2019.111577 Received 22 January 2019; Received in revised form 7 July 2019; Accepted 15 July 2019 Available online 05 September 2019 0926-6690/ © 2019 Elsevier B.V. All rights reserved.

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in 1% dichloromethane which was injected with a split ratio of 1:20 in an Agilent 6890 gas chromatograph (Palo Alto, CA) coupled to a Agilent 5973 N selective mass detector. The injector temperature was maintained at 250 °C. Separation of the constituents was obtained with a HP5MS capillary column (5% phenyl – 95% – dimethylpolysiloxane, 30 m × 0.25mm × 0.25 μm) using helium as the carrier gas (1.0 mL min−1). The incubation temperature was programmed from 60 to 240 °C at a rate of 3 °C min−1. The mass detector was operated in the electronic ionization mode (70 eV) at a rate of 3.15 scan s−1 and a mass range from 40 to 450 u. The transfer line was maintained at 260 °C, the ion source at 230 °C and the analyzer (quadrupole) at 150 °C. For quantification, the diluted sample was injected into an Agilent 7890A chromatograph equipped with a flame ionization detector (FID), operated at 280 °C. The same column and analytical conditions described above were employed, using hydrogen at a flow rate of 1.5 mL min−1 as the carrier gas. The percentage composition was obtained by electronic integration of the FID signal with the division area of each component in the total area. The chemical constituent’s identification was obtained by comparing their mass spectra with libraries (23–24) and by linear retention indices, calculated after the injection of a homologous series of hydrocarbons (C7–C26) and compared with literature data. An analyses of variance for the essential oil yield as well as the Scott-Knott test (P < 0.05) of the mean comparison procedures were performed using ASSISTAT, release 7.6 Beta.

(Silva et al., 2014). According to Alves et al. (2016), the Piper genus is known for its extracts being popularly used as antifungal. Piper cernuum (pariparoba), its leaves and roots, used in decoctions or infusions are used for the treatment of renal and hepatic disorders, ulcers, colds, bronchitis, diuretics, febrile, depurative, energetic, jaundice, syphilis, leukorrhea, urinary tract infections, boils, and burns (Mariot et al., 2003). Fungi are capable of causing diseases which are hard to be controlled in humans. Some fungal species cause diseases in considerably healthy as well as in immunosuppressed patients. Additionally, fungi provoke various damages through mycoses, as well as intervening in the success rates of the latest medical advances in cancer treatment, solid organ transplants and hematopoietic cells, autoimmune diseases and sophisticated surgeries (Bezerra et al., 2018). Premature morbidity and mortality in cancer patients have increased due to systemic infections caused by fungi, with this fact being due to the immunosuppressive effect of patient treatments, enhancing the risk of fungal infections, especially candidiasis (Sorendino et al., 2017). Fungal infections are not the only infections associated with resistant hospital infections, bacteria are also responsible for 25,000 deaths per annum according to the WHO (2012). According to Owen and Laird (2018), 700,000 people die each year due to resistant diseases worldwide, with this number being predicted to become 10 million by 2050 if no intervention occurs (O’neill, 2016). This occurs especially because of failures in infectious disease treatment, caused by bacteria with high virulence and transmissibility, which are associated with resistance markers. Some bacterial cells possess virulence attributes such as extracellular toxins in addition to capsules, peptidoglycan and teichoic acids (Davies and Davies, 2010). Essential oils (OE) are constantly used in the pharmaceutical segment due to their antimicrobial properties. Many reports addressing EO action on bacterial wall degradation, plasma membrane, membrane protein, electron flux and cytoplasmic coagulation alterations exist (Canton and Onofre, 2010). The extracts and oils evaluated are also used as agents capable of modifying the effects of drugs in order to increase antimicrobial action. Moreover, chemical compounds such as phenothiazines and other natural products have an indirect effect against bacteria, increasing the activity of certain antibiotics, reversing the natural resistance of specific bacteria to antibiotics (Chaves-López et al., 2018; Coutinho et al., 2010). This study aims to analyze the chemical composition and antimicrobial potential of the Piper cernuum essential oil, against bacterial and fungal strains and evaluate the possible interactions between the oil compounds with antibiotics and antifungals, as well as to verify reduction of fungal virulence due to morphological yeast alterations.

2.2. Strains The experiments were carried out with clinical E. coli 06 (EC06) and S. aureus 10 (SA10) isolates resistant to the antibiotics gentamicin, norfloxacin and erythromycin. The E. coli ECATCC2592 and S. aureus SA-ATCC25923 strains were used as positive controls. All bacterial cultures were maintained at 4 °C in Heart Infusion Agar (HIA, Difco). Prior to the assays, all cells were grown overnight at 37 °C in Brain Heart Infusion (BHI, Difco). 2.3. Drugs Gentamicin, erythromycin and norfloxacin were obtained from Sigma Chemical Corp., St. Louis, MO, USA. All the drugs were dissolved in sterile water to obtain the appropriate concentrations and decrease their toxicity. 2.4. Direct antibacterial test (MIC) and antibiotic activity modulation MIC (Minimal Inhibitory Concentration) was determined in a microdilution assay as according to Javadpour et al. (1996). The final oil concentrations varied from 1024–0.5 μg/mL. For the evaluation of the oil as modulator of antibiotic resistance, the MIC of the antibiotics was determined in the presence and absence of the EOPC at subinhibitory concentrations (ranging from 128 to 64 μg/mL) and the plates were incubated for 24 h at 37 °C. Each antibacterial assay for MIC determination was carried out in triplicates.

2. Materials and methods 2.1. Plant material The leaves were collected from The Bom Jesus Biological Reserve, in the municipality of Guaraqueçaba – PR (years 2015/2016), located at 25°14.346′S and 48°33.081′ W, Parana State, South of Brazil. Plant material collection in the reserve was carried out under a license from the Environmental Institute of the State of Paraná, number 284/11. Sample specimens were sent to the “Municipal Botanical Museum Herbarium (MBM)” with number Nº 396416 (UPCB)”.

2.5. Antifungal assays 2.5.1. Strains and culture media The standard strains utilised were obtained from the Oswaldo Cruz Culture Collection (FIOCRUZ) of the Brazilian Institute of Health Quality Control (INCQS), these being Candida albicans 40006 and Candida tropicalis 40042. The strains were inoculated in Sabouraud Dextrose Agar (SDA, KASVI) and incubated for 24 h at 37 °C. Thereafter, yeast aliquots were transferred to test tubes each containing 3 mL sterile (0.9%) sodium chloride solution. The inoculum concentration was standardized by comparing to the 0.5 McFarland scale. The double concentrate Sabouraud Dextrose broth (SDB, HIMEDIA), was used in

2.1.1. Essential oil isolation and analysis methodology The essential oil was obtained from 100 g of fresh or 50 g of the dried sample by hydrodistillation over 4 h using a Clevenger-type apparatus. The dried samples were obtained after drying the plant material for 24 h in an electric dryer FANEM (320 SE Mod) with air circulation at 40 °C. The extracted oil was stored in dark vials at −20 °C until analysis. GC–MS analysis was performed using 1.0 L of the samples 2

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the Petri dishes to make two parallel streaks on the solid medium (PDA), which were then covered with a sterile coverslip. The chambers were placed in an incubator for 24 h (37 °C) and were inspected and the results recorded using an optical light microscope (AXIO IMAGER M23525001980-ZEISS- Germany) with a 20X objective. A control was performed for yeast growth (hyphae stimulated in nutrient-lacking medium) as well as a control with the conventional antifungal fluconazole for comparative purposes. The assays were performed according to Sidrin and Rocha (2010) and Mendes (2011), with modifications with respect to concentrations and media used.

microdilution assays. Depleted Potato Dextrose Agar (PDA) with bacteriological agar was used in the micromorphological analysis. 2.5.2. Drugs, reagents and solution preparation Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany) was used for the dilution of the fractions, while the antifungal fluconazole (Capsule - Prati donaduzzi) was diluted in water and used as a reference medicine. The product solution was prepared by weighing 0.05 g of the fractions and then diluting this into 1 mL of DMSO. To obtain the desired concentrations for the assays, the fractions were further diluted in sterile distilled water (4096 μg/mL) so the DMSO (0.5%) concentration would not exert an activity on the tested cells.

2.5.7. Measurement of the extension of filamentous structures It was possible to measure hyphae and pseudohyphae extensions, as well as any reductions in values following the action of the fractions using the Zen software in conjunction with an optical microscope (AXIO IMAGER M2-3525001980- ZEISS- Germany). Five photos from each slide were taken, according to the concentrations, and from each photo ten hyphae/pseudohyphae were randomly chosen to be measured, after which the arithmetic mean of the length of each slide was calculated where in the end the means were combined.

2.5.3. IC50 determination and obtaining the cellular viability curve In this test we used the fractions in isolation and in combination with each other using the 96-well plate broth microdilution method. Each well was filled with 100 μL of CSD containing 10% fungal inoculum, then 100 μL of the natural product (4096 μg/mL) or fluconazole (antifungal reference; at the same concentration) were added to the first well followed by serial dilution. The concentrations in the wells ranged from 2 to 2048 μg/mL. The last well was used as a growth control (Javadpour et al., 1996). Controls for the product diluents (with 0.9% sodium chloride solution instead of the inoculum) and sterility control of the media were also prepared. All tests were performed in quadruples. The plates were incubated at 37 °C for 24 h and subsequently read on an ELISA spectrophotometer (Thermoplate®) at a wavelength of 630 nm. The results obtained in the ELISA assays were used to construct the cell viability curve and determine the IC50 of the fractions (Morais-Braga et al., 2016).

2.6. Statistical analysis For statistical analysis, the software Graphpad Prism, v. 5.0 was used. The data obtained were checked for their normal distribution and analyzed by a two-way ANOVA (P < 0.05; *P < 0.1; ****P < 0.0001), comparing the values for each concentration of the extract, point by point, using Bonferroni's post hoc test. The IC50 values were obtained by non-linear regression with interpolation of standard curve unknowns obtained from fungal growth as a function of extract concentration and expressed in μg/mL. To analyze their values, multiple t-tests were used, one per line, whose statistical significance was determined by the Holm-Sidak method, with alpha ≤0.05. The results of the antibacterial assays were performed in triplicate and expressed as geometric mean. Statistical analysis was applied using a two-way ANOVA followed by Bonferroni’s post-hoc test.

2.5.4. Determination of the minimum fungicidal concentration (MFC) For this test, the tip of a sterile rod was inserted into each well of the microdilution test plate after the 24 h incubation period (except for the sterility control). After mixing the medium in each well, the rod was taken to a Petri dish containing SDA, with the aid of a guide bracket attached to the bottom of the plate for yeast subculture. After 24 h of incubation, the plates were inspected for the formation of any Candida colonies. The concentration where no fungal colony growth was observed was considered the MFC of the natural product.

3. Results

2.5.5. Evaluation of the modifying effect over fluconazole action Initially, the action of the fractions and fluconazole were verified against the growth of yeasts individually, where in this test the effect of the isolated fractions and their combination with the reference drug was evaluated in order to verify whether or not potentiation of the antifungal action by the fractions occurred. For this, the natural product was used at a sub-inhibitory concentration (MFC/16), according to the methodology used by Coutinho et al. (2008. If the fraction potentiates the action of the antifungal, the verified effect is considered of the synergic type, if the action of the antifungal is interfered, an antagonistic effect would be verified. The plates were filled with 100 μL of medium + inoculum + fraction and then microdiluted with 100 μL of the 4096 μg/mL fluconazole concentration added to the first well of each of the columns to undergo serial dilution at concentrations ranging from 2 to 2048 μg/mL. The plates were incubated at 37 °C for 24 h. The reading was performed on an ELISA spectrophotometric apparatus (Thermoplate®).

3.1. Chemical composition The essential oil was extracted from dry Piper cernuum leaves using the hydrodistillation with a Clevenget type apparatus, using 100 g of dry leaves per litre of distilled water. Chemical analysis proceeded by gas chromatography coupled to mass spectrometry GC–MS identified 14 constituents (Table 1), including the major constituent, 4-epi-cisdihydroagarofuran, representing 28.97%. Studies reported the 4-epiTable 1 Essential oil composition (%) of Piper cernuum Vell. EOPC

2.5.6. Effect of the EOPC and fluconazole on fungal morphology To determine if the individual and combined fractions caused changes in fungal morphology by inhibiting morphological transition, considered to be an important fungal virulence factor, humid sterile micro-culture chambers were prepared for yeast observation. Three milliliters of depleted PDA medium were poured in a lamina containing a Superior Concentration Assay - SCA x2 (4096 μg/mL), SCA (2048 μg/ mL) and SCA/4 (512 μg/mL). Subculture aliquots were withdrawn from 3

Piper cernuum

Composition

IR

%

α – Pinene Camphene Sabinene Caryophyllene < (E)- > Selineno < α- > Dihidroagarofuran < 4-epi-cis- > Cadinene < δ- > Agarofuran Elemol Hinesol Eudesmol < γ- > Eudesmol < dihydro- > Eudesmol < 7-epi-α- > Eudesmol < α- >

935 951 967 1426 1505 1508 1523 1555 1558 1624 1631 1662 1664 1655 TOTAL

4,88 2,71 1,55 3,15 11,47 28,97 1,85 1,44 8,91 2,9 17,1 2,51 4,24 1,61 84,38

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potentiation did not occur against the tested strains. On the other hand, norfloxacin displayed an antagonistic effect against E. coli 06 (Fig. 6), while erythromycin showed no alteration, thus establishing the permanence of the antibiotic without any specific action when associated with the EOPC (Figs. 7–10). The EOPC results presented bacterial inhibition against S.A 10 at concentrations below 512 μg/mL. This EOPC antibacterial effect, independent of not having been observed against other strains, was effective for gentamicin potentiation against both tested bacterial strains.

Table 2 Minimum inhibitory concentration (MIC) of the Essential Oil of Piper cernuum – EOPC (μg/mL). Substance

EOPC

Bacteria S.A. ATCC

E.C. ATCC

S.A. 10

E.C. 06

≥1024

≥1024

406

≥1024

cis-dihydroagarofuran isomers present in the Piper amplum essential oil comprised 48% of the total constituents analyzed, being identified as the major compound. However, in this study neither the oil’s nor its isolated major constituent’s antimicrobial activity were evaluated (Table 2).

3.4. Fungal dimorphism effect A positive result for the direct EOPC action against one of the virulence factors, compared to the antifungal used as a control, can be observed in each graph. The EOPC fungistatic effect against CA 40006 and CA 4127, at concentrations ≤ 2.048 μg/mL, were greater than the antifungal with the presence of hyphae or pseudohyphae not being observed. The EOPC completely inhibited the CT URM 40042 virulence factor up to a MFC/8 concentration, where the presence of small hyphae were identified, which still presented a lower quantity than those obtained with fluconazole. The oil was also more efficient against CT URM 4262 than the fluconazole. The results were effective in comparison to fluconazole’s fungistatic activity, which behaved inferiorly to the oil with respect to virulence inhibition against all strains.

3.2. Fungal minimal inhibitory concentration (MIC) determination and antifungal modifying activity evaluation Results obtained from the fungal assays revealed an intrinsic activity for the EOPC from the lowest concentration, showing increasing activity up to the highest concentration. The EOPC reached 50% (IC50) C.A 4127 elimination from the 56.85 μg/mL concentration and complete strain elimination from a concentration below 256 μg/mL. IC50 values at concentrations of 479.63 μg/mL, 541.76 μg/mL and 744.41 μg/mL for the C.A 40006, CT 40042, CT 4262 strains, respectively, were observed without achieving complete inhibition at concentrations below 1000 μg/mL. Results from the natural product association with antifungal drugs demonstrated excellent results. This biological activity was identified in the tested strains, except against C.A 40006, where no positive combined action was identified with the drug. With respect to antifungal associations, a significant synergism was observed for the MICs with the antifungal IC50 values decreasing 6-fold against CT 400042, 2-fold against CT 4262 and 1-fold against CA 4127 (Figs. 1–4).

4. Discussion 4.1. EOPC antifungal effects This research was carried out with the aim of discovering new antimicrobial agents derived from plant extracts, raw materials, essential oils and other compounds exposed to pharmaceutical formulations (Ostrosky et al., 2008). According to Oliveira et al. (2016), Piper cernuum has a chemical composition which, according to Duarte et al. (2016), shows antifungal activity through alterations in membrane permeability, after performing microdilution assays against Candida using a test capable of detecting this action with violet crystal (Oliveira et al., 2016; Donadu et al., 2019). According to Devi et al. (2010), the crystal hardly penetrates the outer membrane, however, when the outer membrane is damaged the crystal easily penetrates it and concentrates intracellularly. Burt (2004) reinforces the alteration in membrane permeability, pH and nutrient transport changes are the main mechanisms of action of essential oils. Moreover, the chemical composition of the OEPC is very important for clarifying its presented biological activities, where the presence of the 4-epi-cis-dihydroagarofuran isomer of the P. cernuum essential oil

3.3. Bacterial minimum inhibitory concentration (MIC) determination and antibiotic modifying activity evaluation The EOPC did not show intrinsic antibacterial activity against standard and resistant EC 06 strains, except for SA 10 where an intrinsic activity producing a geometrical average of 406 μg/mL (Table 1) was observed. The EOPC potentiated the antibiotic effect against the resistant S. aureus 10 strain, as seen in Fig. 5. The essential oil associated with the antibiotic reduced the MIC by two times that of the isolated drug. However, for norfloxacin and erythromycin a significant

Fig. 1. Antifungal effect (μg/mL) of the Essential Oil of Piper cernuum alone and in association with Fluconazol as a control against Candida albicans 40006 strain. ns – p > 0,005; * – p < 0,005; ** – p < 0,01; *** - p < 0,001; **** – p < 0,0001. Fluconazole IC50 – 91.974 μg/mL; EOPC -479.635 μg/mL; EOPC + FCZ – 92.210 μg/mL. 4

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Fig. 2. Antifungal effect (μg/mL) of the Essential Oil of Piper cernuum alone and in association with Fluconazol as a control against Candida albicans 4127 strain. ns – p > 0,005; * – p < 0,005; ** – p < 0,01; *** – p < 0,001; **** – p < 0,0001. Fluconazole IC50 - 11.814 μg/mL; EOPC – 56,851 μg/mL; EOPC + FCZ – 5,625 μg/ mL.

destruction of fungal specimens. In the case of fluconazole, an imidazole derivative, its entry into the cell was facilitated, increasing its ability to inhibit ergosterol acetate incorporation, which inhibits lanosterol demethylase by interfering with yeast cytochrome P-450, thus resulting in fungal cytoplasmic membrane fluidity and permeability changes, disrupting nutrient uptake or by fungal growth inhibition causing morphological changes resulting in cellular necrosis (Richardson and Warnock, 1993; Alves et al., 1999).

found in some studies including de Soletti (2015), was identified as a major compound with biological activity. In a comparative study performed by Costantin et al. (2001), the Piper cernuum oil and Eugenol isolate antifungal action were evaluated against C. albicans strains showing antifungal activity with oil obtaining 12.2 ± 0.6 mm of diameter and the Eugenol isolate obtaining 28.9 ± 1.3 mm, thus suggesting the compound needs the presence of other substances to obtain greater efficiency against fungal strains. Fluconazole is a triazole drug designed from the first-generation imidazole, this being one of the main first generation compounds. The main triazole antifungals are from the first and second generation. First generation drugs are primarily used to treat superficial mycoses, with second-line drugs being used to treat systemic infections. The class is distributed within the first and second generation with the first generation being ketoconazole and itraconazole (imidazole), fluconazole (triazolic first generation), voriconazole and posaconazole (second generation triazolic) (Menozzi et al., 2017). The main mechanism of action of fluconazole is steroid synthesis inhibition without altering ergosterol synthesis in mammals, being less toxic and better absorbed than other azoles. Ergosterol biosynthesis inhibition plays an important role in the integrity and maintenance of the fungal cell membrane (Nobre et al., 2002). In the present study, the association of FCZ with the EOPC was evaluated, observing a synergism, which can take into account assumptions by Burt (2004) and Oliveira et al. (2016), which report EOs acting through membrane wall damage, facilitating the entry of antifungals into the cell, causing the

4.2. EOPC antibacterial effects According to Tortora et al. (2012), some bacteria possess virulence factors which hinder the action of substances capable of destroying their cell wall or chromosomal inhibition. The antimicrobial properties of essential oils extracted from aromatic plants (Santurio et al., 2011) is possibly due to aromatic classes as well as monoterpene, sesquiterpene, phenylpropanoid, aldehyde, ketone and long chain alcohol compounds (Donadu et al., 2018; Amorese et al., 2018). Essential oils, as mentioned by Burt (2004), present as a mechanism of action cell membrane permeability alterations and have the capacity to break or penetrate the lipid structure present in Gram-negative bacteria (Bertini et al., 2005). However, from a chemical perspective, a study by Da Silva et al. (2017) addressing the Piperaceae family correlation, revealed essential oil biological activity would be due to monoterpene hydrocarbon compounds (MH), oxygenated monoterpenes (OM), sesquiterpene

Fig. 3. Antifungal effect (μg/mL) of the Essential Oil of Piper cernuum alone and in association with Fluconazol as a control against Candida tropicalis 40042 strain. ns – p > 0,005; * – p < 0,005; ** - p < 0,01; *** – p < 0,001; **** – p < 0,0001. Fluconazole IC50 – 1421.633 μg/mL; EOPC – 541.766 μg/mL; EOPC + FCZ – 33,461 μg/mL. 5

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Fig. 4. Antifungal effect (μg/mL) of the Essential Oil of Piper cernuum alone and in association with Fluconazol as a control against Candida tropicalis 4262 strain. ns – p > 0,005; * – p < 0,005; ** – p < 0,01; *** – p < 0,001; **** – p < 0,0001. Fluconazole IC50 – 114.467 μg/mL; EOPC – 744.411 μg/mL; EOPC + FCZ – 53.704 μg/mL.

combined with conventional antibiotics, these often present direct activity against many bacterial species modifying or increasing the activity of specific antibiotic, reversing the bacteria's natural resistance. Activity potentiation or antibacterial resistance reversal allows the classification of these compounds as antibiotic activity modifiers. These antibiotics may suffer changes in their chemical structure when associated with other substances, a modification which may potentiate their biological activity leading to positive results at low concentrations. Studies with this methodology are widely used, aiming to provide new methods of combating bacterial and fungal virulence and resistance mechanisms (Dos Santos et al., 2007). The action of quinolone, macrolide and aminoglycoside antibiotics have been observed, where Norfloxacin was the chosen quinolone being able to cause negative DNA helicoidation through topoisomerase II inhibition, by blocking bacterial metabolism leading to the elimination of the microorganism. Quinolone antibiotics possess two ring types: a naphthyridine nucleus, with two nitrogen atoms at positions 1-C and 8C and a quinoline nucleus, with only one nitrogen atom at position 1-C. Both quinolones and naphthyridines contain a keto-oxygen at C-4 and a carboxylic acid side chain at C-3, both considered essential for exerting their biological activity (Perianu et al., 2019). During the antibiotic modifying activity tests, an antagonism between norfloxacin and the EOPC can be observed, a fact which may possibly be explained by an interaction with the carboxylic acid side chain as this is essential for drug activity; or pH alteration; or possibly by preventing the quinolone from binding to its active site. With respect to the chosen macrolide, erythromycin, this associate acts through protein synthesis inhibition by reversibly binding the ribosomal/50S subunits of sensitive microorganisms (National Cancer Institute - NCI, 2018). However, the drug was not efficient against the E. coli 06 strain, since the clinical isolate was resistant against this drug

hydrocarbons (SH), oxygenated sesquiterpenoids (OS) and phenylpropanoids (PP) classes. Feng and Zheng (2007) and Knaak and Fiuza (2010) highlight compounds, such as terpene and phenolic compounds, present in essential oils are responsible for the observed antimicrobial activity, presenting a mechanism associated with the lipophilic character of the compounds. The EOPC presents a variation of these MH, OM, SH, OS compounds which may be associated with their intrinsic biological activity. Soletti (2015) reports the presence of the 4-epi-cis-dihydroagarofuran isomer in the P. cernuum essential oil in a seasonality study from winter 2012 to autumn 2013, where the aforementioned compound was identified at greater concentrations in autumn. As for the antimicrobial activity of the same oil, this presented better results at a concentration of 780 parts per million (ppm) against S. aureus during the winter period, during which time the compound was present at lower concentrations, with the compound Trans-dihydroagarofuran being found at higher concentrations. Moreover, the oil remained active at 780 ppm during all collection periods (winter, spring, summer and fall) against S. pyogenes. According to Wolff (2017), sesquiterpenes with a β-dihydroagarofuran skeleton may present insecticidal action, antifeedant action and insect narcosis for pests. These actions are due to the presence of compounds with structures similar to β-dihydroagarofuran which has resonance in the furan ring, which favors its biological activity. The furan ring, in addition to presenting high volatility suffers resonance, which can be seen from undergoing an inductive effect, known as one of the factors that explains why certain groups activate the benzene ring during electrophilic substitution while others deactivate it, thus when in association with drugs these compounds undergo electron attack by other structures increasing the spectrum of action of the drug. According to Coutinho et al. (2009), when natural products are

Fig. 5. Modulatory effect of essential oil from P. cernuum in the antibiotic activity of Norfloxacin, Erythromycin and Gentamicin against multidrug resistant strain of S. aureus 10. The bar accompanied by four stars means p < 0.0001 when compared to the control. 6

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Fig. 6. Modulatory effect of essential oil from P. cernuum in the antibiotic activity of Norfloxacin, Erythromycin and Gentamicin against multidrug resistant strain of E. coli 06. The bar accompanied by four stars means p < 0.0001 when compared to the control.

Fig. 7. Measurement of the hyphae extension under the action of the EOPC against C. albicans 40006 strain. a4: p < 0.0001 vs control of growth; #: no growth.

Fig. 8. Measurement of the hyphae extension under the action of the EOPC against C. albicans 4127 strain. a4: p < 0.0001 vs control of growth; #: no growth.

bacilli (Coutinho et al., 2015). Gentamicin is commonly used in association with other antibiotics, usually beta-lactams or cephalosporins, for spectrum increase. Its action is given by the decoding region such as the mr-rnr1 gene, which presents its sequence differently in bacteria and humans. Gentamicin binds to the ribosomal 30S subunit which interacts with the decoding site. At this point, the ribosome needs to select tRNA precisely, according to the appropriate mRNA codon. Thus, errors in this region have led to inadequate mRNA codon translation, with incorrect amino acids being inserted into the polypeptide chain, leading to peptide chain elongation interruption and bacterial death (Pratt et al., 2018). The gentamicin association with the EOPC was potentiated against both studied strains, which may be due to drug facilitation at 30S subunits and increased porins increasing drug sensitivity causing bacterial inactivation.

type, nor was it efficient when associated with the product. As for the S.A 10 strain, the association effect was antagonistic. Some of the mechanisms in which bacterial strains acquire resistance to macrolides involve changes in the drug target, efflux and inactivation. Modifications to the drug target confer a broad spectrum of macrolide resistance, while efflux and inactivation affect only some types of these antimicrobials (Coutinho et al., 2015). When considering oil action, which aims to favor membrane permeability and the increased concentration of substances in the cell’s interior, Bertini et al. (2005), report what may have happened during the antagonism involving S.A 10 was the loss of erythromycin sensitivity, decreased outer membrane permeability, known as porin loss, and/or inactivation of its chemical structure or binding sites. Gentamicin, the chosen aminoglycoside, is a broad-spectrum clinical bactericide capable of inhibiting protein synthesis in Gram-negative 7

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Fig. 9. Measurement of the hyphae extension under the action of the EOPC against C. tropicalis 40042 strain. a4: p < 0.0001 vs control of growth; #: no growth.

Fig. 10. Measurement of the hyphae extension under the action of the EOPC against C. tropicalis 4262 strain. a4: p < 0.0001 vs control of growth; #: no growth.

5. Conclusion

F.A., Cavalcanti, E.S.B., 2005. Perfil de Sensibilidade de Bactérias Frente a Óleos Essenciais de algumas Plantas do Nordeste do Brasil. Infarma 17, 80–83. Bezerra, C.F., Rocha, J.E., Silva, M.K.N., De Freitas, T.S., De Sousa, A.K., Dos Santos, A.T.L., Da Cruz, R.P., Ferreira, M.H., Da Silva, J.C.P., Machado, A.J.T., Carneiro, J.N.P., Sales, D.L., Coutinho, H.D.M., Ribeiro, P.R.V., De Brito, E.S., Morais-Braga, M.F.B., 2018. Analysis by UPLC-MS-QTOF and antifungal activity of guava (Psidium guajava L). Food Chem. Toxicol. 119, 113–122. Burt, S.A., 2004. Essential oils: their antibacterial properties and potential applications in foods – a review. Int. J. Food Microbiol. 94, 223–253. Canton, M., Onofre, S.B., 2010. Interference from extracts of Baccharis dracunculifolia DC, Asteraceae, on the activity of antibiotics used in the clinic. Rev. Bras. Farmacogn. 20, 348–354. Chaves-López, C., Usai, D., Donadu, M.G., Serio, A., González-Mina, R.T., Simeoni, M.C., Molicotti, P., Zanetti, S., Pinna, A., Paparella, A., 2018. Potential of BorojoapatinoiCuatrecasas water extract to inhibit nosocomial antibiotic resistantbacteria and cancer cell proliferation in vitro. Food Funct. 23, 2725–2734. Costantin, M.B., Sartorelli, P., Limberger, R., Henriques, M.T., Steppe, M., Ferreira, M.J.P., Ohara, M.T., Emerenciano, V.P., Kato, M.J., 2001. Essential oils from Piper cernuum and Piper regnellii: antimicrobial activities and analysis by GC/MS and 13CNMR. Planta Med. 67, 771–773. Coutinho, H.D.M., Costa, J.G.M., Siqueira, J.R., Lima, J.P.E.O., 2008. In vitro antistaphylococcal activity of Hyptis martiusii Benth against methicillin resistant Staphylococcus aureus-MRSA strains. Rev. Bras. Farmacogn. 18, 670–675. Coutinho, H.D.M., Costa, J.G.M., Lima, O.E., Falcão-Silva, V.S., Junior-Siqueira, J.P., 2009. In vitro interference of Momordica charantia in the resistance to aminoglycosides. Pharma. Biol. 47, 1056–1059. Coutinho, H.D.M., Costa, J.G.M., Falcão-Silva, V.S., Siqueira-Júnior, J.P., Lima, E.O., 2010. Effect of Momordica charantia L. in the resistance to aminoglycosides in methicilin-resistant Staphylococcus aureus. Comp. Immunol. Microbiol. Infect. Dis. 33, 467–471. Coutinho, H.D.M., Brito, S.M.O., Leite, F.N., Vandesmet, V.C.S., Oliveira, M.T.A., Martins, G.M.A.B., Silva, A.R.P., Costa, M., Do, S., 2015. Comparative evaluation of the modulation of antibiotic-activity against strains of Escherichia coli and Staphylococcus aureus. Rev. Cienc. Salud. 13, 345–354. Da Silva, J.K., Da Trindade, R., Alves, N.S., Figueiredo, P.L., Maia, J.G.S., Setzer, W.N., 2017. Essential oils from neotropical Piper species and their biological activities. Int. J. Mol. Sci. 18, 2571. Davies, J., Davies, D., 2010. Origins and evolution of antibiotic resistance. Rev. Microbiol. Mol. Biol. 74, 417–433. Devi, K.P., Nisha, S.A., Sakthivel, R., Pandian, S.K., 2010. Eugenol (an essential oil of

Fungal findings revealed the EOPC possesses strong intrinsic activity against some of the tested strains, in addition to acting synergistically when associated with strains resistant to the antifungal. The EOPC was capable of inhibiting the fungal virulence mechanism, with even better results than the tested antifungal, where the analysis revealed hyphae or pseudohyphae absence at almost all tested concentrations. This growth inhibition may be due to lipophilic substances in the EOPC which have made themselves present in the cell membranes preventing the fungi from branching out in search of nutrients for life. The EOPC acted positively against only one gram-positive clinical isolate, S.A 10, while for other strains the oil did not present an intrinsic activity at the tested concentrations. For the drug-modifying effect, gentamicin was the antibiotic which best acted synergistically. This synergistic result is possibly due to the EOPC facilitating membrane ruptures, favoring drug action at 30S subunits, increasing porins and consequently, the sensitivity of the drug, causing bacterial inactivation. In this study the EOPC or compounds present in it have been shown to be promising sources for combating fungal and bacterial infections. References Alves, H.S., Rocha, W.R.V., Fernandes, A.F.C., Nunes, L.E., Pinto, D.S., Costa, J.I.V., Chaves, M.C.O., Mayer, R., Catão, R., 2016. Antimicrobial activity of products obtained from Piper species (Piperaceae). Rev. Cubana Pl. Med. 21, 168–180. Alves, S.H., Lopes, J.O., Cury, A.E., 1999. Susceptibility Tests of Antifungals: Why, When and as Perform (in Portuguese). Available at http://www.newslab.com.br/antifung. htm, Accessed at 05/April/2019. . Amorese, V., Donadu, M., Usai, D., Sanna, A., Milia, F., Pisanu, F., Molicotti, P., Zanetti, S., Doria, C., 2018. In vitro activity of essential oils against Pseudomonas aeruginosa isolated from infected hip implants. J. Infect. Dev. Ctries 12, 996–1001. Bertini, L.M., Pereira, A.F., Oliveira, C.L., De, L., Menezes, E.A., De Morais, S.M., Cunha,

8

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A.L. Alves Borges Leal, et al.

ncitbrowser/ConceptReport.jsp?dictionary=NCI_Thesaurus&ns=NCI_Thesaurus& code=C476. Accessed at 10/August1./2018. Nobre, M.O., Nascente, P., da, S., Meireles, M.C., Ferreiro, L., 2002. Antifungical drugs for small and large animals. Ciência Rural 32, 175–184. O’Neill, J., 2016. Tackling Drug-resistant Infections Globally: Final Report and Recommendations. HM Government and the Wellcome Trust, London, UK. Oliveira, L.B.S., Batista, A.H.M., Fernandes, F.C., Sales, G.W.P., Nogueira, N.A.P., 2016. Antifungal activity and potential action mechanisms of essential oil of Ocimum gratissimum (Linn.) leaves against Candida species. Rev. Bras. Pl. Med. 18, 511–523. Ostrosky, E.A., Mizumoto, M.K., Lima, M.E.L., Kaneko, T.M., Nishikawa Freitas, B.R., 2008. Methods for evaluation of the antimicrobial activity and determination of Minimum Inhibitory Concentration (MIC) of plant extracts. Rev. Bras. Farmacogn. 18, 301–307. Owen, L., Laird, K., 2018. Synchronous application of antibiotics and essential oils: dual mechanisms of action as a potential solution to antibiotic resistance. Crit. Rev. Microbiol. 44, 414–435. Perianu, E., Rau, I., Vijan, L.E., 2019. DNA influence on norfloxacin fluorescence. Spectrochimica Acta Part A 206, 8–15. Pratt, V., Mcleod, H., Rubinstein, W., Dean, L., Malheiro, A., 2018. Medical Genetics Summaries. National Center for Biotechnology Information, Bethesda, MD, USA. Richardson, M.D., Warnock, D.W., 1993. Fungal Infection – Diagnosis and Management. Blackwell, London, UK. Santurio, D.F., Costa, M.M., Grazieli, M., Cavalheiro, C.P., Sá, M.F., Dal Pozzo, M., Alves, S.H., Fries, L.L.M., 2011. Antimicrobial activity of spice essential oils against Escherichia coli strains isolated from poultry and cattle. Ciência Rural 41, 1051–1056. Sidrin, J.J.C., Rocha, M.F.G., 2010. Micologia médica à luz de autores contemporâneos. Guanabara Koogan, Rio de Janeiro, RJ, Brasil. Silva, J.A., Oliveira, F.F., Guedes, E.S., Bittencourt, M.A.L., Oliveira, R.A., 2014. Antioxidant activity of Piper arboreum, Piper dilatatum, and Piper divaricatum. Rev. Bras. Pl. Med. 16, 700–706. Silveira, G.P., Nome, F., Gesser, J.C., Sá, M.M., Terenzi, H., 2006. Recent achievements to combat bacterial resistance. Quím. Nova 29, 844–855. Soletti, A.G., 2015. Effects of the Seasonality on the Chemical Composition, Antimicrobial, Cytotoxic and Mutagenic Potential of Essential Oils and Fractions of Dichloromethane and Ethyl Acetate of Piper amplum and Piper cernuum. (in Portuguese). PhD. Thesis. Univali, Itajaí-SC, Brazil. Sorendino, G., Sampaio, L.E., Gonçalves, B., Krelling, A., Vasco, J.F., De, M., Rodrigues, L.S., 2017. Incidência de Candida spp. Em Hemoculturas de Pacientes AtendidoS em Hospital Oncológico no Sul do Brasil. Anais do EVINCI – UniBrasil. 3, 16–25. Tortora, G.J., Funke, B.R., Case, L., 2012. Microbiologia. Artmed, São Paulo, SP, Brasil. WHO – World health Organization, 2012. The Evolving Threat of Antimicrobial Resistance: Options for Action. WHO, Geneva, Switzerland. Wolff, F.R., 2017. Toxicological Evaluation of the Extracts Obtained From Piper cernuum Leaves and Stems. (in Portuguese). MSc. Thesis. Univali, Itajaí-SC, Brazil.

clove) acts as antibacterial agent against Salmonella typhi by disrupting the cellular membrane. J. Ethnopharmacol. 130, 107–115. Donadu, M., Usai, D., Pinna, A., Porcu, T., Mazzarello, V., Fiamma, M., Marchetti, M., Cannas, S., Delogu, G., Zanetti, S., Molicotti, P., 2018. In vitro activity of hybrid lavender essential oils against multidrug resistant strains of Pseudomonas aeruginosa. J. Infect. Dev. Ctries 12, 9–14. Donadu, M.G., Usai, D., Marchetti, M., Usai, M., Mazzarello, V., Molicotti, P., Montesu, M.A., Delogu, G., Zanetti, S., 2019. Antifungal activity of oils macerates of North Sardinia plants against Candidaspecies isolatedfrom clinical patients with candidiasis. Nat. Prod. Res. 24, 1–5. Dos Santos, A.L., Santos, D.O., De Freitas, C.C., Ferreira, B.L.A., Afonso, I.F., Rodrigues, C.R., Castro, H.C., 2007. Staphylococcus aureus: visiting a strain of clinical importance. Rev. Bras. Patol. Med. Lab. 43, 413–423. Duarte, A.E., De Menezes, I.R.A., Morais-Braga, M.F.B., Leite, N.F., Barros, L.M., Waczuk, E.P., da Silva, M.A.P., Boligon, A., Rocha, J.B.T., Souza, D.O., Kamdem, J., Coutinho, H.D.M., Burger, M.E., 2016. Antimicrobial activity and modulatory effect of essential oil from the leaf of Rhaphiodon echinus (Nees & mart) Schauer on some antimicrobial drugs. Molecules 21, 743. Feng, W., Zheng, X., 2007. Essential oils to control Alternaria alternate in vitro and in vivo. Food Control 18, 1126–1130. Javadpour, M.M., Juban, M.M., Lo, W.C., Bishop, S.M., Alberty, J.B., Cowell, S.M., Becker, C.L., McLaughlin, M.L., 1996. Antimicrobial peptides with low mammalian cell toxicity. J. Med. Chem. 39 (3107-), 3113. Knaak, N., Fiuza, L.M., 2010. Potencial of essential plant oils to control insects and microorganisms. Neotrop. Biol. Cons. 5, 120–132. Mariot, A., Mantovani, A., Reis, M.S., 2003. Use and conservation of Piper cernuum Vell. (Piperaceae) in Atlantic Tropical Forest: I. Reproductive phenology and seed dispersal. Rev. Bras. Pl. Med. 5, 1–10. Mendes, J.M., 2011. Investigation of Antifungal Activity of Essential oil Eugenia caryophyllata Thunb. on strains of Candida tropicalis. MSc. Thesis. UFPB, João Pessoa – PB. Menozzi, C.A.C., Castelo-Branco, F.S., França, R.R.F., Domingos, J.L.O., Boechat, N., 2017. Optimization of fluconazol synthesis: an important azole antifungal drug. Rev. Virtual Quim. 9, 1216–1234. Morais-Braga, M.F.B., Carneiro, J.N.P., Machado, A.J.T., Sales, D.L., Brito, D.I.V., Albuquerque, R.S., Boligon, A.A., Athayde, M.L., Júnior, J.T.C., Souzad, S.L., Lima, E.O., Menezes, I.R.A., Costa, J.G.M., Ferreira, F.S., Coutinho, H.D.M., 2016. Highperformance liquid chromatography-diodic array detector, fungistatic, and antimorphogenical analysis of extracts from Psidium brownianum Mart. ex DC. against yeasts of the genus Candida. Int. J. Food Prop. 19, 1837–1851. Nascimento, G.G.F., Locatelli, J., Freitas, P.C.S., Giuliana, L., 2000. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz. J. Microbiol. 31, 247–256. National Cancer Institute - NCI (2018). Available at: https://ncit.nci.nih.gov/

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