**3. Discussion**

The genus *Piper* L. presents a high number of species with medicinal, insecticidal and condiment applications, since the representatives are sources of volatile oils produced by the secondary metabolism [14]. Some species of this genus present antifungal activities, such as *Piper amalago* L. [15], *Piper aduncum* L. and *Peperomia pelúcida* (L.) Kunth [16], and antibacterial activity, among them *Piper betle* L. [17]. Thus, the selection strategy of *P. caldense* for the investigation of the antimicrobial activities of this study was based on chemotaxonomy, since there is a phylogenetic relationship between *P. caldense* and other species of the genus and possibly in the evolutionary historical branch the biosynthetic routes are similar [18].

The phytochemicals (monoterpenes and sesquiterpenes) of the essential oil of the species under study were elucidated by Rocha et al. [19], however, there were marked differences in the constitution, the first is that in our study, the caryophyllene oxide was the major constituent (13.9%), whereas in the results of the aforementioned study this sesquiterpene is not present in the oil of leaves, but in the stem essential oil (6.2%). In addition, Rocha et al. [19] states that the constituent in higher percentage is the α-cadinol, reaching compose 19% of the total composition, and in our study this sesquiterpeno is in low percentages (4.2%). This variation is justified by several factors, both intrinsic and genetic, as well as extrinsic factors such as geographic origin of the plant, cultivation, collection form, and especially the period of the year that was collected [20–22].

Although the essential oil of *P. caldense* did not present antifungal activity in low concentrations (≤500 μg/mL), it presented a modulating e ffect for fluconazole against strains of *C. albicans* URM 4387. So that this finding is relevant, since the introduction of azole class antibiotics (miconazole, econazole, ketoconazole, fluconazole and triazonazole) for the treatment of infections caused by *Candida* species, a growing emergence of resistant *Candida* species has been observed [23,24].

The activity of caryophyllene oxide, an oxygenated terpenoid, was tested in the laboratory against dermatophyte fungi, showing significant results, and their activity has been compared with antifungals such as cyclopiroxolamine and sulconazole [12].

Silva [25], evaluating the antifungal activity of 2-geranyl-3,4-dihydroxybenzoic acid and 3-geranyl-4-hydroxyxidozoic acid, both substances isolated from the fruits of *P. caldense*, demonstrated, respectively, a moderate and high activity against *C. albicans* strains (LM-86 and LM-111), so that the first substance showed a MIC of 512 μg/mL for both strains and the second one 32 μg/mL, also for the two strains. It is important to highlight that in our study the essential oil was used as the product, and this is a mixture of mono and sesquiterpenes, whereas in the study mentioned above the substances were derived from the benzoic acid prenylate, so that the chemical structures are quite di fferent.

Inhibition of virulence of *C. albicans* strains by natural products was also shown by other scientists, among them Santos et al. [26], who evaluated the essential oil of *Eugenia uniflora* L. (Myrtaceae) and demonstrated that at concentrations of 8192 μg/mL there is inhibition of hyphal projection. Two other species also from the same family that have the ability to inhibit the virulence of *C. albicans* are *Psidium brownianum* Mart. ex DC. And *Psidium guajava* L., the former having medicinal properties and is used to combat infections caused by fungi of the genus *Candida* [6,27].

The activities of the natural products, concerning the antimicrobial agents act by diverse mechanisms of action such as the disintegration of the cytoplasmic membranes, destabilization of the motor proton force (MPF), altering the polarization of the membrane, and the coagulation of the cellular content [28–30].

#### **4. Materials and Methods**

#### *4.1. Botanical Material*

The collection of leaves of *Piper caldense* was performed in the municipality of Piraquara in the state of Paraná, Brazil, under the coordinates 25◦29.693 S and 49◦00.844 W at 528 m elevation (Figure 5). An exsicata was identified and deposited in the Herbarium of the Faculdades Integradas Espírita under voucher 9.103.

#### *4.2. Extraction of Volatile Terpenes and Determination of the Chemical Composition*

Healthy *P. caldense* leaves were selected and dried in a incubator at 40 ◦C. After dehydration, the leaves were crushed to increase their contact surface and maximize the extraction of their volatile components. For such extraction, the hydrodistillation system was used, in which 50 g of the plant material was placed in a volumetric flask with 1000 mL of distilled water, being constantly heated for 4.5 h, until the oil extraction [21].

For the chemical characterization of *P. caldense* essential oil, it was made by gas chromatography-mass spectrometry (GC/MS). Initially, the essential oil was diluted in dichloromethane to 1% concentration, then 1 μL of this solution was injected (1:20) into Agilent 6890 chromatograph, coupled to Agilent 5973N mass selective detector, wherein the injector temperature was 250 ◦C. For the separation of the constituents, helium gas was used as a carrier (1 mL/min) and an HP-5MS capillary column with the following specifications: 5% phenyl-95%-dimethylpolysiloxane, 30 m × 0.25 mm × 0.25 μm. For the heating ramp, the temperature started at 60 ◦C with a heating rate of 3 ◦C/min to

240 ◦C, totaling 60 min. The mass detector was operated in the electronic ionization mode (70 eV), at a rate of 3.15 s<sup>−</sup><sup>1</sup> sweeps and a mass range of 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.

**Figure 5.** Location of the species *Piper caldense* in the municipality of Piraquara in the state of Paraná, Brazil.

For quantification, the diluted samples were injected into an Agilent 7890A chromatograph equipped with a flame ionization detector (FID), operated at 280 ◦C. We employed the same column and analytical conditions described above except for the carrier gas used, which was hydrogen at a flow rate of 1.5 mL/min. The percentage composition was obtained by the electronic integration of the FID signal by dividing the area of each component by the total area (area%).

For the determination of chemical constituents, the mass of the constituents was compared with the library (NIST and Wiley) e and also by their linear retention indexes, calculated from the injection of a homologous series of hydrocarbons (C7-C26) and compared with data from the literature [31].

#### *4.3. Drugs, Reagents, Solution Preparation and Fungal Strains*

For the antifungal test, the essential oil stock solution was prepared from 0.15 g and diluted in 1 mL of dimethyl sulfoxide (DMSO). To obtain the initial concentration of 16,384 μg/mL, the stock solution was diluted in sterile distilled water so that the DMSO concentration in the natural product had no activity in the cells tested. The reference antifungal was fluconazole (Capsule-FLUCOMED), diluted in sterile water at the same oil concentration [27]. For microbiological assays, two *Candida albicans* strains were used: CA INCQS 40006 (standard strain), obtained from the Oswaldo Cruz Culture Collection of the National Institute for Quality Control in Health (INCQS) and CA URM 4387 (clinical isolate), provided by mycology collection of the Federal University of Recife (URM - University Recife Mycology). For the antifungal activity test the culture media were used: Sabouraud Dextrose Agar (SDA) and Sabouraud Dextrose Broth (SDB). For the fungal micromorphology evaluation test the nutrient-poor Potato Dextrose Agar (PDA) culture medium prepared with solidification Agar was used. The media were prepared according to the supplier's guidelines (Difco®) and autoclaved at 121 ◦C within 15 min.

#### *4.4. Determination of Minimum Inhibitory Concentration (MIC)*

To perform this experiment the broth microdilution method was used according to Javadpour. First the yeasts were inoculated in ASD and kept incubated at 37 ◦C for 24 h. Subsequently, the inoculum were prepared by transferring small aliquots of the strains to tubes containing sterile saline, the inoculum were compared to McFarland scale resulting in a concentration of 1 × 10<sup>5</sup> cells/mL. 96-well plates were filled containing in each well 100 μL of SDB and 10% fungal inoculum. The plates were then microdiluted with 100 μL of the essential oil of P. caldense, where the well concentrations were from 8192 μg/mL to 64 μg/mL. The last well was not diluted as a growth control. Media sterility controls and substance dilution controls were also used, using only saline without fungal inoculum. The plates were incubated at 37 ◦C for 24 h and subsequently read on a 630 nm wavelength ELISA spectrophotometer (Thermoplate ®). All assays were performed in triplicate and the results obtained were used to construct the cell viability curve and the IC50 of essential oil and fluconazole [6,32]. MIC was defined as the lowest concentration able to reduce the fungal growth curve.

#### *4.5. Evaluation of Modulating Activity of Natural Product*

The essential oil was tested at sub-inhibitory concentration (MIC/16) according to the method proposed by Coutinho et al., 2008 [33]. The plates were filled with a solution containing SDB, fungal inoculum and the essential oil, then 100 μL of fluconazole was mixed into the first well and serially microdiluted at a ratio of 1:1 to the penultimate well, at 1 μg/mL. Control of culture media sterility and antifungal dilution control were performed, and the MIC of fluconazole was also determined. The tests were performed in triplicate and the plates were incubated at 37 ◦C for 24 h. The reading was performed in an ELISA spectrophotometer (Termoplate ®). The modulatory activity is defined when used in combination. The natural product enhances the action of the antifungal, showing synergism. If the opposite occurs and the natural product interferes with the action of the drug, the e ffect is considered antagonistic.

#### *4.6. Determination of Minimum Fungicidal Concentration (MFC)*

After the MIC test, a sterile stem was placed in each well of the plates, first the stem was used to mix the solutions contained in the wells, then small aliquots with medium, inoculum and essential oil were transferred to Petri dishes containing solid medium SDA, for yeas<sup>t</sup> subculture and verification of cell viability. After 24 h of incubation, the plates were analyzed and the concentration at which no fungal colony growth was observed is considered the Minimum Fungicidal Concentration of the essential oil [34].

#### *4.7. E*ff*ect of Natural Product on Fungal Morphology*

To verify whether P. caldense volatile terpenes cause any change in fungal morphology by inhibiting hyphae emission, micromorphological sterile chambers were mounted for yeas<sup>t</sup> observation. On the blade chamber (sterile) were poured 3 mL of medium PDA, poor nutrient for dilution, containing the natural product in CFM/4 concentrations CFM/8 and CFM/16. Aliquots of fungal inoculum were taken from SDA-containing Petri dishes to make two parallel strips in the solidified solid medium (PDA) and then covered by a sterile coverslip. The chamberwere taken to the incubator and after 24 h (37 ◦C) the culture was visualized under optical microscopy using a 400 X objective. A camera was attached to the microscope for image capture. A control for yeas<sup>t</sup> growth (with hyphal emission stimulated by depletion of the medium) was performed, as well as a control with the reference antifungal fluconazole was also used for comparative purposes [35].
