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Article

Antibiotic Potential and Chemical Composition of the Essential Oil of Piper caldense C. DC. (Piperaceae)

by
José Weverton Almeida Bezerra
1,
Felicidade Caroline Rodrigues
1,
Rafael Pereira da Cruz
2,
Luiz Everson da Silva
3,
Wanderlei do Amaral
3,
Ricardo Andrade Rebelo
3,
Ieda Maria Begnini
3,
Camila Fonseca Bezerra
2,
Marcello Iriti
4,*,
Elena Maria Varoni
5,
Henrique Douglas Melo Coutinho
6 and
Maria Flaviana Bezerra Morais-Braga
2
1
Postgraduate Program in Plant Biology, Federal University of Pernambuco—UFPE, Recife 50670-901, PE, Brazil
2
Laboratory of Applied Mycology of Cariri, Regional University of Cariri—URCA, Crato 63105-000, CE, Brazil
3
Research Laboratory of Natural Products, Federal University of Paraná—UFPR, Matinhos 83260-000, PR, Brazil
4
Department of Agricultural and Environmental Sciences, Milan State University, 20133 Milan, Italy
5
Department of Biomedical, Surgical and Dental Sciences, Milan State University, 20142 Milan, Italy
6
Laboratory of Microbiology and Molecular Biology—LMBM, Regional University of Cariri—URCA, Crato 63105-000, CE, Brazil
*
Author to whom correspondence should be addressed.
Appl. Sci. 2020, 10(2), 631; https://doi.org/10.3390/app10020631
Submission received: 17 December 2019 / Revised: 8 January 2020 / Accepted: 10 January 2020 / Published: 15 January 2020
(This article belongs to the Special Issue Biological Activity and Applications of Natural Compounds)
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Versions Notes

Abstract

:
Infections by multiresistant microorganisms have led to a continuous investigation of substances acting as modifiers of this resistance. By following this approach, the chemical composition of the essential oil from Piper caldense leaf and its antimicrobial potential were investigated. The antimicrobial activity was determined by broth microdilution method providing values for minimum inhibitory concentration (MIC), IC50, and minimum fungicidal concentration (MFC). The essential oil was tested as a modulator for several antibiotics, and its effect on the morphology of Candida albicans (CA) strains was also investigated. The chemical characterization revealed an oil composed mainly of sesquiterpenes. Among them are caryophyllene oxide (13.9%), spathulenol (9.1%), δ-cadinene (7.6%) and bicyclogermacrene (6.7%) with the highest concentrations. The essential oil showed very low activity against the strains of CA with the lowest values for IC50 and MFC of 1790 μg/mL and 8192 μg/mL, respectively. The essential oil modulated the activity of fluconazole against CA URM 4387 strain, which was demonstrated by the lower IC50 obtained, 2.7 μg/mL, whereas fluconazole itself presented an IC50 of 7.76 μg/mL. No modulating effect was observed in the MFC bioassays. The effect on fungal morphology was observed for both CA INCQS 40006 and URM 4387 strains. The hyphae projection was completely inhibited at 4096 μg/mL and 2048 μg/mL, respectively. Thus, the oil has potential as an adjuvant in antimicrobial formulations.

1. Introduction

Infections caused by fungi, are the major problem of hospital infections, mainly due to the emergence of new infections and the generalized resistance to antibiotics [1]. Due to the rapid resistance acquired by microorganisms the search for natural products with antimicrobial activity has been constant in recent years and drugs derived from plants have contributed largely to human health, historically representing a source for the creation of new drugs [2].
One way of combating microbial resistance is the investigation of substances that can act as resistance modifiers by combining common antimicrobial drugs with some phytochemicals, and in some cases observed synergism [3,4].
Yeasts of the genus Candida are found as commensals in the human organism, however, because of factors that disturb the dynamics of the host can favor the growth of these fungi, from superficial infections to systemic infections [5]. Among the yeasts of this genus, Candida albicans stands out as the main cause of infections, being a species with great ability to change its morphology, a necessary factor for its virulence and pathogenicity [6,7].
For the treatment of infectious diseases, populations without access to medicines, especially those from underdeveloped and developing countries, use medicinal plants to combat disease. Such use of these vegetables is due to a number of advantages, such as their availability, low purchasing power and being in popular knowledge for several generations [2,8]. Among the species of the Brazilian flora, the genus Piper L. is one of the largest in the Piperaceae family, with 290 species and 45 varieties occurring in Brazil [9]. Some species of the genus are used as a flavoring of food and treatment of diseases [10]. A scientific study evidenced the antimicrobial potential of four species of the genus against Staphylococcus aureus and three strains of Candida, including C. albicans [11]. Among the species of the genus, Piper caldense C. DC., is popularly known in Brazil as “pimenta d’água” or “pimenta d’arda”, being commonly used for the treatment of snake bites, sedative and stomach problems [10]. Research using P. caldense essential oil has revealed promising antimicrobial potential [12,13].
In view of the above problem, this study aimed to evaluate the antibiotic and modulator potential of volatile terpenes of P. caldense against Candida albicans, as well as to determine if the natural product is capable of reducing pleomorphism. Finally, it was evaluated by gas chromatography which terpenes were present in the essential oil.

2. Results

2.1. Chemical Composition

The chromatographic analysis of the essential oil of P. caldense identified 25 phytochemical constituents, corresponding to 94.3% of the total composition. Sesquiterpenes are the main class observed, reaching a percentage of 89.7% of the sample. Monoterpenes were found in very low concentration (4.6%). There were no major constituents (>20%) and no constituent in trace concentration (<1%). All the identified phytochemicals are secondary metabolites, with caryophyllene oxide (13.9%), spathulenol (9.1%), δ-cadinene (7.6%), and bicyclogermacrene (6.7%) as constituents with the highest concentrations (Table 1).

2.2. Antifungal Activity

2.2.1. Cell Viability Curve and IC50

Regarding the antifungal activity of the essential oil of P. caldense, it was found that it has a low antifungal effect against strains of C. albicans because the IC50 values were high, 2256.24 and 1790.24 μg/mL for the CA INCQS 40006 and CA URM 4387, respectively (Table 2) (Figure 1 and Figure 2). However, it was observed that for the C. albicans URM 4387 strains, the oil potentiated the effect of the drug used, fluconazole, since it had an IC50 of 7.73 μg/mL alone, and when associated with the essential oil, the value of evaluated parameter dropped to 2.7 μg/mL, i.e., a significant reduction. It is noteworthy that for C. albicans 40006 strains, no potentiating effect was found for fluconazole but an antagonistic effect, since there was an increase in the IC50 of the oil associated with fluconazole. Thus, based on the behavior of the curve through non-linear regression, the IC50 values of P. caldense oil were clinically irrelevant when evaluated alone.

2.2.2. Cell Viability Curve and Minimum Fungicidal Concentration (MFC)

In determining MFC it was considered those samples capable of inhibiting the growth of the fungal colonies. Thus, for the CA INCQS 40006 line, none of the products alone or in combination were able to totally inhibit colony growth, since MFC was ≥ 16,384 μg/mL (Table 3). However, there was an MFC for the oil against CA URM 4387, in which fluconazole alone and combined with P. caldense oil showed a much lower MFC, 16 μg/mL, so that the oil did not modulate the fungicidal effect of fluconazole for this lineage (Table 3).

2.2.3. Activity of the Piper caldense in the Control of Virulence of Candida albicans

In the evaluation of the activity of the oil in the morphology of the yeasts of C. albicans, the effect was caused by impoverishment of the culture medium, so that the yeasts project the hyphae and pseudohyphae in search of nutrients. For the growth control of CA INCQS 40006 (Figure 3, Slides 1–7), it is possible to observe the formation of several hyphae (S1), whereas in the treatments with fluconazole, there is a significant decrease in the hyphae projection (S2–4), resulting in a complete inhibition of hyphae at concentration of 4096 μg/mL (S2). The EOPc showed a similar result, being as effective as fluconazole.
For the CA URM 4387 strain, fluconazole at a concentration as low as 8 μg/mL could diminish the virulence (Figure 4, S2). The oil at higher concentrations, such as 2048 μg/mL (S3), was able to inhibit the hyphae projection; on the other hand, at concentration of 512 μg/mL (S5), there was a significant decrease in hyphae projections.

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 [20,21,22].
Although the essential oil of P. caldense did not present antifungal activity in low concentrations (≤500 μg/mL), it presented a modulating effect 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 different.
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 [28,29,30].

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−1 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.
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 × 105 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 effect 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 yeast 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. Effect 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 yeast 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 yeast 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].

4.8. Statistical Analysis

The IC50 was calculated by means of linear regression. Subsequently, data from P. caldense antifungal assays were investigated by one-way analysis of variance (ANOVA), using the Bonferroni test and considered significant when p < 0.05. All analyzes were performed on the GraphPad Prism 6.0 software.

5. Conclusions

The essential oil of P. caldense presented mono- and sesquiterpene components, though not presented a major constituent. In addition, it showed low antifungal activity for C. albicans strains but was able to modulate the effect of standard drug (fluconazole) and decreased the virulence of these strains. In this way, the oil has in its composition constituents that are promising in the formulation of drugs used in the treatment of infectious diseases.

Author Contributions

Conceptualization, J.W.A.B., M.I., H.D.M.C. and M.F.B.M.-B.; Data curation, J.W.A.B., F.C.R., R.P.d.C., L.E.d.S., E.M.V., H.D.M.C. and M.F.B.M.-B.; Formal analysis, J.W.A.B., F.C.R., R.P.d.C. and L.E.d.S.; Methodology J.W.A.B., F.C.R., R.P.d.C., L.E.d.S., W.d.A., R.A.R., I.M.B., and E.M.V.; Supervision, M.I., H.D.M.C. and M.F.B.M.-B.; Methodology, C.F.B.; Writing—original draft, J.W.A.B., F.C.R., R.P.d.C. and L.E.d.S.; Writing—review & editing, M.I., E.M.V., H.D.M.C. and M.F.B.M.-B. All authors have read and agreed to the published version of the manuscript.

Funding

The authors thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) by research grants, Fundação Cearense de Apoio ao Desenvolvimento Científico e Tecnológico (FUNCAP) by the concession of equipment and projects, and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for supporting research.

Conflicts of Interest

The authors declare no conflict of interest.

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Applsci 10 00631 g001 550
Figure 1. Anti-Candida potential of essential oil of Piper caldense (EOPc) against strains of Candida albicans 40006 INCQS.
Figure 1. Anti-Candida potential of essential oil of Piper caldense (EOPc) against strains of Candida albicans 40006 INCQS.
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Applsci 10 00631 g002 550
Figure 2. Anti-Candida potential of essential oil of Piper caldense (EOPc) against strains of Candida albicans URM 4387.
Figure 2. Anti-Candida potential of essential oil of Piper caldense (EOPc) against strains of Candida albicans URM 4387.
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Applsci 10 00631 g003 550
Figure 3. Effects of essential oil of Piper caldense on the dimorphism of Candida albicans INCQS 40006. Slide (S1): Growth control; S2–4: Effect of fluconazole at concentrations 4096 μg/mL (S2), 2048 μg/mL (S3), 1024 μg/mL (S4). S5–7: Effect of essential oil at concentrations of 4096 μg/mL (S5), 2048 μg/mL (S6), 1024 μg/mL (S7). Display 400× increased.
Figure 3. Effects of essential oil of Piper caldense on the dimorphism of Candida albicans INCQS 40006. Slide (S1): Growth control; S2–4: Effect of fluconazole at concentrations 4096 μg/mL (S2), 2048 μg/mL (S3), 1024 μg/mL (S4). S5–7: Effect of essential oil at concentrations of 4096 μg/mL (S5), 2048 μg/mL (S6), 1024 μg/mL (S7). Display 400× increased.
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Applsci 10 00631 g004 550
Figure 4. Effects of essential oil of Piper caldense on the dimorphism of Candida albicans URM 4387. Slide (S1): Growth control; S2: Effect of fluconazole at the concentration of 8 μg/mL. S3–5: Effect of the essential oil at 2048 μg/mL (S3), 1024 μg/mL (S4), 512 μg/mL (S5).
Figure 4. Effects of essential oil of Piper caldense on the dimorphism of Candida albicans URM 4387. Slide (S1): Growth control; S2: Effect of fluconazole at the concentration of 8 μg/mL. S3–5: Effect of the essential oil at 2048 μg/mL (S3), 1024 μg/mL (S4), 512 μg/mL (S5).
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Figure 5. Location of the species Piper caldense in the municipality of Piraquara in the state of Paraná, Brazil.
Figure 5. Location of the species Piper caldense in the municipality of Piraquara in the state of Paraná, Brazil.
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Table
Table 1. Chemical composition of the essential oil of leaves from Piper caldense.
Table 1. Chemical composition of the essential oil of leaves from Piper caldense.
Compounds%AIlitMF
α-Pinene2.5935C10H16
Camphene2.1952C10H16
α-copaene21381C15H24
(E)-Caryophyllene2.61426C15H24
Aromadendrene2.71445C15H24
γ-muurolene41478C15H24
β-selinene3.21490C15H24
Bicyclogermacrene6.71492C15H24
Germacrene D5.31493C15H24
α-muurolene4.21498C15H24
δ-Cadinene7.61520C15H24
γ-Cadinene3.31521C15H24
α-Calacorene2.21551C15H20
β-Calacorene1.91572C15H20
Spathulenol9.11576C15H24O
Caryophyllene oxide13.91580C15H24O
Globulol2.31594C15H26O
Rosifoliol1.31597C15H26O
Humulene epoxide II11619C15H24O
1.10-di-epi-Cubenol1.61624C15H26O
1-epi-Cubenol3.41638C15H26O
epi-alfa-muurolol3.11640C15H26O
α-cadinol4.21650C15H26O
α-Muurolol2.11651C15H26O
Cadalene21667C15H18
Total sesquiterpenes89.7
Total monoterpenes4.6
Total identified (%)94.3
AIlit—Arithmetic Retention Indices from literature; MF: Molecular Formula.
Table
Table 2. IC50 of the essential oil of Piper caldense (EOPc) against Candida albicans.
Table 2. IC50 of the essential oil of Piper caldense (EOPc) against Candida albicans.
Products TestedYeast
Candida albicans INCQS 40006Candida albicans URM 4387
Fluconazole (FCZ)7.76 µg/mL7.73 µg/mL
EOPc2256.24 µg/mL1790.24 µg/mL
EOPc + FCZ12.37 µg/mL2.7 µg/mL
INCQS: National Institute for Health Quality Control. URM: University Recife Mycology.
Table
Table 3. Minimum fungicidal concentration (MFC) of essential oil of Piper caldense (EOPc) and fluconazole associated and isolated against strains of Candida albicans.
Table 3. Minimum fungicidal concentration (MFC) of essential oil of Piper caldense (EOPc) and fluconazole associated and isolated against strains of Candida albicans.
Products TestedYeast
Candida albicans INCQS 40006Candida albicans INCQS 40006
Fluconazole (FCZ)≥16,384 μg/mL16 μg/mL
EOPc≥16,384 μg/mL8192 μg/mL
EOPc + FCZ≥16,384 μg/mL16 μg/mL
INCQS: National Institute for Health Quality Control. URM: University Recife Mycology.

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MDPI and ACS Style

Bezerra, J.W.A.; Rodrigues, F.C.; Pereira da Cruz, R.; Silva, L.E.d.; do Amaral, W.; Andrade Rebelo, R.; Begnini, I.M.; Fonseca Bezerra, C.; Iriti, M.; Varoni, E.M.; et al. Antibiotic Potential and Chemical Composition of the Essential Oil of Piper caldense C. DC. (Piperaceae). Appl. Sci. 2020, 10, 631. https://doi.org/10.3390/app10020631

AMA Style

Bezerra JWA, Rodrigues FC, Pereira da Cruz R, Silva LEd, do Amaral W, Andrade Rebelo R, Begnini IM, Fonseca Bezerra C, Iriti M, Varoni EM, et al. Antibiotic Potential and Chemical Composition of the Essential Oil of Piper caldense C. DC. (Piperaceae). Applied Sciences. 2020; 10(2):631. https://doi.org/10.3390/app10020631

Chicago/Turabian Style

Bezerra, José Weverton Almeida, Felicidade Caroline Rodrigues, Rafael Pereira da Cruz, Luiz Everson da Silva, Wanderlei do Amaral, Ricardo Andrade Rebelo, Ieda Maria Begnini, Camila Fonseca Bezerra, Marcello Iriti, Elena Maria Varoni, and et al. 2020. "Antibiotic Potential and Chemical Composition of the Essential Oil of Piper caldense C. DC. (Piperaceae)" Applied Sciences 10, no. 2: 631. https://doi.org/10.3390/app10020631

APA Style

Bezerra, J. W. A., Rodrigues, F. C., Pereira da Cruz, R., Silva, L. E. d., do Amaral, W., Andrade Rebelo, R., Begnini, I. M., Fonseca Bezerra, C., Iriti, M., Varoni, E. M., Melo Coutinho, H. D., & Bezerra Morais-Braga, M. F. (2020). Antibiotic Potential and Chemical Composition of the Essential Oil of Piper caldense C. DC. (Piperaceae). Applied Sciences, 10(2), 631. https://doi.org/10.3390/app10020631

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