In Vitro Effect of Cinnamomum zeylanicum Blume Essential Oil on Candida spp. Involved in Oral Infections

Rangel, Marianne de Lucena; Aquino, Sabrina Garcia de; Lima, Jefferson Muniz de; Castellano, Lúcio Roberto; Castro, Ricardo Dias de

doi:https://doi.org/10.1155/2018/4045013

Evidence-Based Complementary and Alternative Medicine

On this page

Abstract Introduction Materials and Methods Results Discussion Conclusion Data Availability Conflicts of Interest Acknowledgments References Copyright Related Articles

Research Article | Open Access

Volume 2018 | Article ID 4045013 | https://doi.org/10.1155/2018/4045013

In Vitro Effect of Cinnamomum zeylanicum Blume Essential Oil on Candida spp. Involved in Oral Infections

Marianne de Lucena Rangel,¹Sabrina Garcia de Aquino,¹Jefferson Muniz de Lima,¹Lúcio Roberto Castellano,^1,2and Ricardo Dias de Castro¹

Academic Editor: Nativ Dudai

Received06 Sept 2018

Accepted04 Oct 2018

Published17 Oct 2018

Abstract

The present study demonstrates the antifungal potential of chemically characterized essential oil (EO) of Cinnamomum zeylanicum Blume on Candida spp. biofilm and establishes its mode of action, effect on fungal growth kinetics, and cytotoxicity to human cells. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) values varied from 62.5 to 1,000 μg/mL, and the effect seems to be due to interference with cell wall biosynthesis. The kinetics assay showed that EO at MICx2 (500 μg/mL) induced a significant (p < 0.05) reduction of the fungal growth after exposure for 8 h. At this concentration, the EO was also able to hinder biofilm formation and reduce Candida spp. monospecies and multispecies in mature biofilm at 24 h and 48 h (p < 0.05). A protective effect on human red blood cells was detected with the EO at concentrations up to 750 μg/mL, as well as an absence of a significant reduction (p > 0.05) in the viability of human red blood cells at concentrations up to 1,000 μg/mL. Phytochemical analysis identified eugenol as the main component (68.96%) of the EO. C. zeylanicum Blume EO shows antifungal activity, action on the yeast cell wall, and a deleterious effect on Candida spp. biofilms. This natural product did not show evidence of cytotoxicity toward human cells.

1. Introduction

Candida spp. are commensal versatile microorganisms that colonize up to 70% of dental prosthesis users. However, local or systemic alterations might cause an imbalance in the host, predisposing the host to the development of disease, which might range from superficial and localized involvement to a fatal condition when it disseminates across the body of immunocompromised individuals [1]. The oral manifestations of the disease are considered signs indicative of an immune response imbalance, particularly among patients with acquired immunodeficiency syndrome [2].

Essentially four classes of drugs exist for the treatment of fungal infections with Candida: polyenic agents, azoles, echinocandins, and antimetabolites. As oral infections are superficial, treatment includes topical antifungals, such as nystatin and miconazole. Despite the number of available therapeutic options, microbial resistance to antifungals from different classes is increasing [3–7]. One of the mechanisms strongly associated with such resistance is the ability of certain microorganisms to form biofilms. Microorganisms in these communities undergo genetic changes that increase their resistance to drugs and the immune system. In the case of Candida, its ability to form biofilm on biotic and abiotic surfaces represents a relevant virulence factor and is thus an interesting target of study of the development of antifungal agents against candidiasis [8–11].

Within this context, the study of the therapeutic properties of plants that may represent an alternative therapeutic resource has increased [12–14]. One of the plants investigated over time for exhibiting significant biological properties is Cinnamomum zeylanicum Blume, commonly known as cinnamon. Several parts of the plant, such as the bark, leaves, flowers, fruits, and roots, have medicinal and culinary applications. The chemical composition of the materials obtained from the different parts of a plant exhibit considerable variation, resulting in different pharmacological effects [15–18].

Regarding the biological activity of C. zeylanicum Blume, its analgesic, antiseptic, anticancer, antispasmodic, coagulant, neuroprotective, hepatoprotective, gastroprotective, cardioprotective, and antimicrobial potentials, as well as its action for control of the scenic and lipid levels and reduction of the blood cholesterol concentration, have been described [15, 16, 18, 19]. Several studies presented further evidence of this plant’s antimicrobial potential against fungi and gram-positive and gram-negative bacteria [20–24]. Despite demonstrations of the antifungal activity of C. zeylanicum, most studies have analyzed products extracted from the bark and used microorganisms in their planktonic phase.

Therefore, the aims of the present study were to describe the phytochemical profile of C. zeylanicum Blume and to investigate the in vitro antifungal activity, the mode of action, and the effect on the growth kinetics of Candida spp. in planktonic form and in biofilms of the essential oil (EO) extracted from leaves. In addition, the cytotoxic effect of this natural product on human peripheral blood cells and its phytochemical profile are described.

2. Materials and Methods

2.1. Test Product

Essential oil from the leaves of C. zeylanicum Blume (Ferquima Ind. and Com.,Vargem Grande, São Paulo, Brazil) is extracted using the hydrodistillation method and exhibiting a characteristic appearance, color and odor, specific gravity 1.043, and refractive index 1.533.

2.2. Microorganisms

The present study used Candida spp. reference strains C. albicans ATCC 60193, C. albicans ATCC 90029, C. krusei ATCC 34135, and C. tropicalis ATCC 750 from the American Type Culture Collection (ATCC) and C. albicans CBS 562 and C. tropicalis CBS 94 from the Centraalbureau voor Schimmelcultures (CBS). In addition, the following clinical strains were used, which were collected from the oral cavity, isolated, identified, and provided by the Laboratory of Clinical Mycology, Center of Health Sciences, Federal University of Paraíba: C. albicans LM01, C. albicans LM03, C. albicans LM04, C. tropicalis LM05, C. tropicalis LM07, and C. tropicalis LM08.

2.3. Phytochemical Analysis by Gas Chromatography-Mass Spectrometry (GCMS-QP2010 Ultra)

The volatile components profile was established using a Shimadzu GCMS-QP2010 gas chromatography-mass spectrometry device with Rtx-5MS capillary column, a stationary phase with 5% phenyl 95% dimethylpolysiloxane, 30 m of length, 0.25 mm of internal diameter, and 0.25 μm of film thickness.

The initial temperature was set to increase from 60°C to 240°C (3°C/min). The run time was set to 60 minutes, and the temperature of the injector furnace was set to 250°C. Helium was used as the carrier gas (mobile phase) with a flow rate of 1.0 mL/min, a split ratio of 1:20, and an injection volume of 1 μL. Component ionization was performed by means of electron impact at 70 eV with a 1.25 kV detector voltage. The spectrometer was used in the SCAN mode with a 40–500 a.m.u. mass range. The ion source temperature was 250°C, and the compounds were identified by comparing their mass spectra to the ones contained in the device database.

The C. zeylanicum Blume EO sample was injected in a concentration of 2 ppm, and hexane was used as the solvent. Chromatogram and mass spectra analysis were performed using the device library with integration parameters of width 3 and slope 2000.

2.4. Minimum Inhibitory Concentration (MIC)

The MIC was estimated by means of the microdilution technique on 96-well plates. For this purpose, 100 μL of Sabouraud Dextrose Broth (SDB) medium (KASVI®, Kasv Imp e Dist de Prod p/Laboratorios LTDA, Curitiba, Brazil) was added to each well. C. zeylanicum Blume EO was added (100 μL) to the first wells on the plates, following a serial dilution from 2,000 to 15.62 μg/mL. The fungal strain inoculums were prepared and adjusted under the spectrophotometer to a concentration of 5.0 x 10⁶ colony forming units (CFU)/mL (absorbance 0.08 to 0.10 at 530 nm) and then diluted until reaching a final concentration of 2.5 x 10³ CFU/mL after having added 100 μL to each well. Nystatin (Sigma-Aldrich, São Paulo–SP, Brazil) was used as a positive control in concentrations from 12 to 0.09 μg/mL. Strain viability was assessed, as well as the sterility of the culture medium and of the Tween 80 (diluent of EO emulsion). The plates were incubated in an oven at 37°C for 24 h and then subjected to a visual reading [25]. To confirm the presence of viable microorganisms, 50 μL of 2,3,5-triphenyltetrazolium chloride (TTC) was added to each well, and the plates were incubated for 24 h [26]. The test was performed in triplicate and repeated at three different time points.

2.5. Minimal Fungicidal Concentration (MFC)

Based on the results of the MIC assay, 50 μL of culture from the wells that achieved the MIC and the two concentrations above it (MICx2 and MICx4) were used to seed Petri dishes containing Sabouraud Dextrose Agar (SDA) medium (KASVI®, Kasv Imp e Dist de Prod p/Laboratorios LTDA, Curitiba, Brazil). The dishes were incubated at 37°C for 48 h and a visual reading of the fungal growth was performed. The MFC was defined as the lowest concentration able to inhibit visible subculture growth. This test was performed in triplicate. To establish whether the tested EO had fungicidal or fungistatic activity, the ratio of the MFC to the MIC was calculated; activity was considered to be fungistatic when the MFC/MIC ≥ 4 and fungicidal when the MFC/MIC < 4 [27].

2.6. Essential Oil Mechanism of Action

2.6.1. Sorbitol Test

The microdilution technique described in Section 2.4 was performed with and without sorbitol. The fungal inoculums were prepared using SDB medium (KASVI®, Kasv Imp e Dist de Prod p/Laboratorios LTDA, Curitiba, Brazil) with added sorbitol (D-sorbitol anhydrous-INLAB, São Paulo, SP, Brazil) to a final concentration of 0.8 M. Sorbitol acts as an osmotic protector; therefore, a reduction in the action of the tested substance in its presence is indicative of action on the fungal cell wall. The plates were incubated at 37°C for 48 h before being read [28, 29]. Caspofungin (caspofungin diacetate, Sigma-Aldrich, São Paulo, SP, Brazil) was used as a positive control [30, 31]. This test was performed in triplicate. The mechanism of action was established as involving the cell wall when the MIC attained by the test substance in the presence of sorbitol was higher than the one attained in the absence of sorbitol.

2.6.2. Ergosterol Test

To establish whether the mechanism of action of the tested substance was related to the ergosterol present in the cell membrane, the microdilution technique described in Section 2.4 was performed with and without exogenous ergosterol (Sigma-Aldrich, São Paulo, SP). The C. albicans ATCC 60193 and C. tropicalis ATCC 750 inoculums were prepared in SDB medium with added ergosterol (at 400 μg/mL). The plates were incubated at 37°C for 48 h before being read [28, 29]. Nystatin (Sigma-Aldrich, São Paulo, SP) and amphotericin B (Sigma-Aldrich, São Paulo, SP) were used as positive controls [30, 31]; the involvement of the cellular membrane was established as the mechanism of action when the MIC attained by the test substance in the presence of ergosterol was higher than the one attained in the absence.

2.7. Assessment of the Inhibition Kinetics of C. zeylanicum Blume EO on Fungal Growth

The interference of the test substance with the growth and multiplication of C. albicans ATCC 60193 cells was studied by means of the CFU counting method [32, 33]. The effect of the test substance concentrations corresponding to MIC, MICx2, and MICx4 on fungal growth was assessed at various time points: T0 (baseline), T1 (1 hour), T2 (2 hours), T6 (6 hours), T8 (8 hours), T12 (12 hours), and T24 (24 hours).

The same protocol of microdilution on 96-well plates was used to grow the fungus under the effect of the test substance [25]. Next, Petri dishes containing SDA medium (KASVI®, Kasv Imp e Dist de Prod p/Laboratorios LTDA, Curitiba, Brazil) were seeded with 10 μL of the well contents at the present time points. The dishes were incubated at 37°C for 24 h before the CFU counting, which was determined visually, and the values were transformed into log₁₀ CFU/mL and presented as a fungal cell death curve. Nystatin was used as positive control; the growth of the tested strains and sterility of the culture medium were assessed.

2.8. Assessment of the Antimicrobial Activity of C. zeylanicum Blume EO on Candida spp. Monospecies and Multispecies Biofilm

This assay investigated the effect of C. zeylanicum Blume EO on two stages of biofilm development: during formation and at maturity (48 h). Three types of biofilm were used in these cases: C. albicans ATCC 60193 monospecies, C. tropicalis ATCC 750 monospecies, and C. albicans ATCC 60193 and C. tropicalis ATCC 750 multispecies.

2.8.1. Assessment of the Antimicrobial Activity of C. zeylanicum Blume EO on Candida spp. Biofilm Formation

The effect of the test EO on biofilm formation was assessed on 96-well flat-bottom plates (CRAL Artigos para Laboratório LTDA, Cotia-SP, Brazil). Each well received 100 μL of SDB medium (KASVI®, Kasv Imp e Dist de Prod p/Laboratorios LTDA, Curitiba, Brazil). C. zeylanicum Blume EO solution was added (100 μL) to the first wells on the plates and subjected to serial dilution from 1,000 to 7.81 μg/mL. Nystatin was used as the positive control in concentrations from 100 to 0.78 μg/mL. Next, the inoculums, which were prepared in the SDB medium with 2% sucrose and adjusted under the spectrophotometer (BioPet Technologies–722G) to a concentration of 2.5 x 10⁵ CFU/mL, were transferred to the plates. A volume of 100 μL of inoculum was used for the monospecies biofilm, and a volume of 50 μL of each strain (C. albicans ATCC 60193 and C. tropicalis ATCC 750) was used for the multispecies biofilm. The plates were incubated at 37°C for 48 h.

2.8.2. Biofilm Biomass Quantification

Once the incubation period was complete, the culture medium was taken from the plates, and the nonadhered cells were removed by rinsing the wells twice with 200 μL of phosphate buffered saline (PBS), followed by drying at room temperature for 45 minutes. A total of 100 μL of 0.4% crystal violet solution was added to the wells and left in contact with the biofilm for 45 minutes. The wells were then rinsed three times with 200 μL of distilled water and immediately destained with 200 μL of 95% ethanol for 45 minutes. Finally, 100 μL of the destained solution was transferred to another plate, and the absorbance was read using a GloMax®-Multi Detection System–Promega plate reader at 600 nm [34].

The EO absorbance and growth control values were used to calculate the percent of biofilm formation inhibition; growth control was considered 100% of the fungal formation. The test was performed in triplicate. The samples used for the sterility assessment were not added to the cell suspension, and the ones used for growth control, as well as the fungal strains corresponding to each biofilm type, were added to the culture medium.

2.8.3. Assessment of the Antimicrobial Activity of C. zeylanicum Blume EO on Reduction of C. albicans Mature Biofilm

A total of 200 μL of the fungal suspension corresponding to each type of the assessed biofilm grown in the SDB medium (KASVI®, Kasv Imp e Dist de Prod p/Laboratorios LTDA, Curitiba, Brazil), which was supplemented with 2% of sucrose and adjusted to a concentration 2.5 x 10⁵ CFU/mL, was added to the 96-well flat-bottom plates. The plates were incubated at 37°C for 48 h to allow for mature biofilm formation. Next, the culture medium was removed from the plates, and the wells were rinsed with 200 μL of PBS so that only adhered biofilm remained in the plates. The C. zeylanicum Blume EO solution (100 μL) was added in concentrations ranging from 1,000 to 7.81 μg/mL to the plates containing the mature biofilm. Nystatin was used as a positive control in concentrations from 100 to 0.78 μg/mL. The samples were divided into two groups according to the duration of the incubation period and also to the duration of their exposure to the EO: G1 (24-h incubation in an oven at 37°C) and G2 (48-h incubation in an oven at 37°C). Once the time allotted for incubation was over, the mature biofilm reduction was quantified using the technique described in Section 2.8.2.

2.9. Assessment of C. zeylanicum Blume EO Cytotoxicity on Human Peripheral Blood Cells

2.9.1. Ethical Issues and Volunteers

The study was approved by the research ethics committee of Federal University of Paraíba, ruling no. 1869951. Blood for isolation of the peripheral blood mononuclear cells (PBMCs) was donated by five volunteers, who gave written consent by signing an informed consent form. The inclusion criteria were healthy individuals, who had no history of infectious or metabolic diseases and were not using immunomodulating drugs.

2.9.2. Hemolysis Test

The hemolytic activity of C. zeylanicum Blume EO was investigated using the method of [35]. EO solution was prepared using four solvents: water, water with neutralized pH, serum, and serum with neutralized pH. One hour after the EO was added to the wells containing the red blood cells, 70 μL of the supernatant was transferred to a 96-well flat-bottom plate, and the absorbance was read using a GloMax®-Multi device at 560 nm. Red blood cells with added distilled water and normal saline were used as positive and negative controls, respectively. The percent hemolysis was calculated using the following equation:

where AA, AP, and AN correspond to the sample absorbance, positive control absorbance, and negative control absorbance, respectively. According to the index recommended by [36], percentages under 5% should be considered as acceptable hemolysis.

2.9.3. PBMC Cell Viability against the C. zeylanicum Blume EO Assay

Heparinized total blood was collected through venipuncture and centrifuged for 8 h with Ficoll-Paque ™ specific gravity gradient 1077 (GE Healthcare). The PBMCs were collected, rinsed three times with phosphate buffer saline, and counted via trypan blue exclusion (Sigma-Aldrich, St. Louis, USA) in a Neubauer chamber. The cells were resuspended in aliquots of 2 x 10⁶ PBMCs/mL in RPMI 1640 medium (GIBCO, Life Technologies, UK) containing 10% of inactivated fetal bovine serum (GIBCO, USA),followed by the addition of phytohemagglutinin in a concentration of 5 μg/mL (PHA-P, Sigma-Aldrich, St. Louis, MO, USA) [37].

Black, 96-well, polystyrene plates (Greiner Bio-One, USA) were used to incubate 100 μL of PBMC suspension and C. zeylanicum Blume EO (31.25–2,000 μg/mL) at a ratio of 1:1 in culture medium at 37°C, in a humidified atmosphere, and with 5% CO₂ for 24 h. Next, the cell viability was measured using the alamarBlue® fluorescence protocol (Bio-Rad, Hercules, CA, USA). The percent viability was calculated as follows:where FI 590 = the fluorescence intensity at 590-nm emission (560-nm excitation).

2.10. Statistical Analysis

Exploratory analysis was performed first to establish the most adequate statistical test. Significant differences between the controls and test substance in the kinetic, biofilm, and PBMC cytotoxicity assays were assessed with the Kruskal-Wallis test followed by Dunn’s post hoc test (α = 0.05) using GraphPad Prism version 7.0 (San Diego, CA, USA).

3. Results

3.1. Chromatographic Profile and Identification of Compounds in C. zeylanicum Blume EO

The chemical composition of C. zeylanicum Blume EO is described in Table 1. Twenty-six components were identified, corresponding to 100% of the EO composition. Eugenol was the largest component (68.96%), whereas the concentration of all others was under 3.5%. Analysis demonstrated the presence of volatile compounds, particularly aromatic and terpenic compounds.

3.2. Determination of the MIC and MFC

The MIC and MFC values corresponding to the C. zeylanicum Blume EO and the standard antifungal nystatin are described in Table 2. Relative to the EO, the MIC and MFC ranged from 125 to 1,000 μg/mL, with C. krusei ATCC 60193 being the most resistant strain. The vehicles used for the EO emulsion (distilled water and Tween 80) did not affect the growth of the fungal strains. The MFC/MIC ratio showed that the EO had fungicidal effect on all the Candida species tested [27].

3.3. Cinnamomum zeylanicum Blume EO Mechanism of Action

The values described in Table 3 show that the antifungal properties of the EO are related to the fungal cell wall biosynthesis pathways, as the MIC in the presence of sorbitol was higher compared to the MIC in the absence in all the tested strains, except for C. albicans LM04. For caspofungin, which was used as a positive control, the MIC was at least four times higher in the presence of sorbitol in all the tested strains.

The results of the test that assessed the MIC in the presence of the ergosterol suggest that, in the case of species C. tropicalis, EO has weak action on the ion permeability of the cell membrane. This effect was not detected in the C. albicans strain, as the presence of ergosterol was not associated with an increase in the MIC. The nystatin and amphotericin B controls confirmed the previously established mechanism of action (Table 4).

3.4. Assessment of the Inhibition Kinetics of C. zeylanicum Blume EO on Fungal Growth

Cinnamomum zeylanicum Blume EO interfered with the growth of C. albicans ATCC 60193 in all the tested concentrations (250 μg/mL, 500 μg/mL, and 1,000 μg/mL), inducing a significant reduction in fungal growth, as shown by the Kruskal-Wallis test followed by Dunn’s post hoc test (α = 0.05). At the MIC level, the fungus was completely eliminated after 24-h exposure to EO; at the CIMx2 and CIMx4 levels, the same effect was achieved after 8 h (Figure 1).

(a)

(b)

(c)

3.5. Cinnamomum zeylanicum Blume EO’s Effect on Candida spp. Monospecies and Multispecies Biofilm

Cinnamomum zeylanicum Blume OE inhibited the formation of the Candida spp. monospecies and multispecies biofilm and reduced the mature biofilm in both tested groups (24 h and 48 h). The best results concerning mature biofilm reduction were detected after 24 h of exposure, as the lowest concentration of EO (250 μg/mL) was able to significantly reduce C. tropicalis ATCC 750 monospecies biofilm (p = 0.0017) and Candida spp. multispecies biofilm (p = 0.0081). A concentration of 500 μg/mL induced a significant reduction in all phases of biofilm development in all biofilm types, except for the Candida tropicalis ATCC 750 mature biofilm after 48 h of exposure (Figure 2). The results corresponding to nystatin are shown in Figure 3.

(a)

(b)

(c)

(a)

(b)

(c)

3.6. Cytotoxicity

The percent hemolysis induced by EO is depicted in Figure 4. Up to a concentration of 750 μg/mL, the EO diluted in the serum and neutralized serum exhibited a protective effect on human red blood cells. In concentrations of 875 μg/mL and 1,000 μg/mL, the highest percentages of hemolysis were 2% and 8%, respectively, which correspond to the EO solution in nonneutralized water.

(a)

(b)

The C. zeylanicum Blume EO did not induce a significant reduction of the human PBMC viability in concentrations of up to 1,000 μg/mL; neither was dose-dependency detected up to this concentration. A significant reduction compared to the control was detected with concentrations of 2,000 μg/mL only (Figure 4).

4. Discussion

The increasing limitations of the antifungals currently used for treatment of candidiasis in association with the high prevalence of disease and the demonstrated antimicrobial potential of plant essential oils [38–40] led to the present study. In this study, the antifungal effect of C. zeylanicum Blume EO on Candida spp. planktonic cells and biofilm and the possible mechanisms of action involved in this effect were investigated.

The results of the chemical analysis of the EO extracted from C. zeylanicum Blume leaves in the present study agree with the findings by previous authors, which indicate that eugenol is its largest component (68.96%) [38, 41], whereas the other 25 chemical compounds appear in smaller concentrations.

Natural products are considered powerful inhibitors of microbial activity when the MIC is equal to or lower than 500 μg/mL [42]. Consistently, the results of the present study show that C. zeylanicum Blume EO had powerful antifungal activity on all the tested strains. The only exception was C. krusei ATCC 3413, for which the effect might be rated as moderate, which is also the case with the available antifungals [43]. The MFC/MIC ratio suggests that the tested EO had a fungicidal effect [27] on both the reference (ATCC and CBS) and clinical strains. Reports in the literature corroborate the effect of C. zeylanicum EO on Candida spp. [39, 41, 44–46]. However, these studies exhibit significant variability as to the Cinnamomum species and plant material used and the methods used to investigate fungal sensitivity, whereas some of them do not indicate from which plant part the EO was extracted. These differences make comparison difficult, as one plant might produce oils with different main components as a function of the plant part used, and different species of the same genus produce EOs with variable chemical composition, resulting in different biological effects [15, 16]. In addition, clinical Candida strains that are multiresistant to the drugs that are widely employed in clinical practice demonstrated susceptibility to C. zeylanicum in other studies [47, 48].

The antifungal activity detected at the MIC and MFC levels might be attributed to the complex combination of volatile components, particularly to the EOs, which account for the different biological activities in humans, animals, and plants. Such activity might correspond to two or three main components, i.e., the ones with higher concentration compared to other components [49, 50].

Notably, previous studies corroborate the antifungal activity of eugenol, the largest component of the EO used in the present study, on Candida spp. planktonic cells, biofilm under formation, and mature biofilm [51–53]. One study that assessed the correlation between the antifungal activity and chemical components of C. zeylanicum Blume EO attributed the strong antifungal effect of this EO on C. albicans, C. parapsilosis, C. tropicalis, and C. glabrata strains (MIC from 0.31 to 0.63 μg/mL) to the eugenol [41]. However, when the same authors compared the MIC of EO and eugenol individually, they found that the MIC was slightly higher for the latter, which suggests that other EO components, even if in small concentrations, act synergically, thus contributing to the antifungal effect of the EO. Therefore, eugenol can be inferred to be the main component responsible for the antifungal effect detected in the present study, but that other components, such as cinnamaldehyde, linalool, benzyl benzoate, alpha-pinene, and beta-pinene, must have also contributed to that effect, as the antifungal activity of each individual compound has been demonstrated [41, 46, 54, 55].

To understand better the antifungal effect detected with the MIC and MFC tests, the effect of the EO on the fungal growth kinetics was assessed. This test showed that for the three concentrations assessed (MIC, MICx2, and MICx4), the effect of the EO on C. albicans began to be perceptible after 8-h exposure and that a 24-h exposure was sufficient for the antifungal effect of the EO to occur. No studies assessing the effect of EO extracted from C. zeylanicum Blume leaves on Candida growth and development could be found in the literature.

The study of the mechanism of action of antifungals is a relevant strategy for limiting the emergence of resistance to the drugs currently available, as well as for developing more powerful and safe drugs against infection [56, 57]. However, no studies assessing the mechanism of action of C. zeylanicum Blume EO against Candida spp. could be found in the literature. The results of the present study indicate that the EO antifungal action seems to be related to the fungal cell wall biosynthesis pathways. However, the tested substance cannot be concluded to interfere with the cell membrane ion permeability through binding to ergosterol because the MIC of only one of the assessed species (C. tropicalis ATCC 750) increased twice in the presence of exogenous ergosterol. Considering the discrete increase of MIC detected in the presence of an osmotic protector (sorbitol) and the complex composition of EO, the antifungal activity of EO might involve other mechanisms of action, in addition to the ones addressed in the present study. Because the antifungal effect of eugenol, the main component of the tested EO, on Candida spp. is related to ergosterol biosynthesis, a study of this pathway might provide promising hypotheses for future studies [58].

In addition to the ability of the C. zeylanicum Blume EO to act on Candida spp. planktonic cells, an interesting antifungal potential was detected relative to the Candida spp. biofilm in the various stages of formation. At concentrations of 500 μg/mL, the EO reduced the Candida spp. biofilm formation by 34.94% to 49.42%. For the mature biofilm, the EO at a concentration of 500 μg/mL reduced all the tested biofilm types by at least 50%, except for the C. tropicalis biofilm, which exhibited a decrease of 30.2% after 48-h exposure to EO. These findings suggest that the potential of C. zeylanicum Blume EO to reduce mature biofilm is superior to its ability to inhibit the formation of the microbial community as biofilm. On these grounds, the tested EO might be used as an antifungal for patients affected with candidiasis, i.e., already exhibiting formed biofilm, rather than as a preventive agent, in which case it would play a role in the inhibition of the microorganisms’ adherence.

Notably, at a concentration of 250 μg/mL and after 24 h of exposure, the EO induced a statistically significant (p < 0.05) reduction of the mature biofilm in two out of the three types of investigated biofilms (C. tropicalis monospecies and Candida spp. multispecies). Similarly, all the stages of Candida biofilm formation were sensitive to EO at concentrations of 500 μg/mL, suggesting the use of this concentration in the development of medicines based on C. zeylanicum Blume EO.

Few studies exist in the literature on the antimicrobial effect of C. zeylanicum Blume EO. A single study assessed its action on Candida, and no study using C. zeylanicum Blume EO extracted from the plant leaves could be located [24, 38, 59, 60]. [59] analyzed the effect of C. zeylanicum Blume EO on C. parapsilosis and C. albicans biofilm formation and reduction and found the full inhibition of biofilm formation at concentrations of 250 μg/mL and 125 μg/mL, respectively. Mature biofilm reduction (after 24-h formation) was analyzed based on the minimum biofilm reduction concentration values, which were 1,000 μg/mL and 2,000 μg/mL for C. parapsilosis and C. albicans, respectively. Although the authors did not mention the plant part used for the EO production, the extraction was presumably made from the bark because phytochemical analysis showed that cinnamaldehyde was the main component [15, 16]. This component might account for the differences found relative to the present study, in which the EO was extracted from the plant leaves, and eugenol was the largest component. Eugenol was assessed for its ability to inhibit C. albicans biofilm formation and reduce the mature biofilm (after 24-h formation); the results showed that it could inhibit biofilm development by 70% when used at a concentration of 500 μg/mL and that it could reduce the mature biofilm at concentrations of 1,000 μg/mL [52].

Susceptibility of the biofilms of other microorganisms, such as Enterococcus faecalis [24], Escherichia coli, Staphylococcus aureus [38], and Pseudomonas aeruginosa [60], is also reported in the literature. Although very few, the studies that assessed the effect of C. zeylanicum EO on microbial biofilm had promising outcomes vis-à-vis its possible use in infectious diseases.

In the part of the study on the EO cytotoxicity on human red blood cells, the percent hemolysis found in the samples diluted in the neutralized and nonneutralized serum was dismissible in all the tested concentrations according to the reference values recommended by [36], according to whom values under 5% are dismissible. Based on the same criterion, only the EO diluted in the distilled water at a concentration of 1,000 μg/mL induced more than 5% hemolysis. In all the tested concentrations, the neutralized solvent induced less hemolysis, which points to the relevance of the neutralization of solutions, as the occurrence of hemolysis in the living body is undesirable. No study assessing the hemolytic effect of C. zeylanicum Blume EO could be found in the literature for comparison with the results of the present study.

The viability of human peripheral blood cells had no dose-dependency relation with the EO effect up to a concentration of 1,000 μg/mL, with the cell viability reduction being approximately 30%. The EO at concentrations of 2,000 μg/mL significantly reduced cell viability; however, the results of the present study indicate that the antifungal effect of the EO occurred in concentrations at least four times lower than the one that induced a toxic effect on the PBMCs. In addition, other studies that assessed the cytotoxic effect of C. zeylanicum found compatibility among the various cell lines, with the percent cell viability being at least 80% [24]. However, those studies used the plant bark, whereas the present study used leaves as the source of the EO extraction, which once again emphasizes the originality of the latter.

Although countless in vitro studies have used natural products for the treatment of candidiasis, with optimistic results, currently, the level of scientific evidence is low due to significant methodological differences in the design of the studies, the choice of extracts, and the concentrations used in the tests [61]. That is, much research has been conducted, but little advances have been achieved regarding the treatment of the disease. The C. zeylanicum Blume EO used in the present study is a natural product with potential for clinical application in the treatment of candidiasis. More thorough studies, however, including in vitro investigations of toxicity and clinical trials, are needed to develop an antifungal agent based on C. zeylanicum Blume EO.

5. Conclusion

The EO extracted from C. zeylanicum Blume leaves has eugenol as its largest component, has antifungal action on Candida spp. planktonic cells and monospecies and multispecies biofilm, acts on the cell wall biosynthesis, and is poorly cytotoxic against human red blood cells and PBMCs. The evidenced properties of the EO extracted from C. zeylanicum Blume leaves encourage the performance of phase I and II clinical studies to validate its use in oral fungal infections.

Data Availability

All the datasets used/or analyzed during the current study are available from the corresponding author on reasonable request.

Conflicts of Interest

The authors declare that they have no conflicts of interest in this work.

Acknowledgments

This study was funded by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) of Brazilian Government.

References

A. Singh, R. Verma, A. Murari, and A. Agrawal, “Oral candidiasis: An overview,” Journal of Oral and Maxillofacial Pathology, vol. 18, no. 5, pp. 81–85, 2014.
View at: Publisher Site | Google Scholar
K. M. Vidya, U. K. Rao, W. Nittayananta, H. Liu, and F. J. Owotade, “Oral mycoses and other opportunistic infections in HIV: Therapy and emerging problems - a workshop report,” Oral Diseases, vol. 22, pp. 158–165, 2016.
View at: Publisher Site | Google Scholar
K. Pianalto and J. Alspaugh, “New Horizons in Antifungal Therapy,” Journal of Fungi, vol. 2, no. 4, p. 26, 2016.
View at: Publisher Site | Google Scholar
J. R. Perfect, ““Is there an emerging need for new antifungals?”,” Expert Opinion on Emerging Drugs, vol. 21, no. 2, pp. 129–131, 2016.
View at: Publisher Site | Google Scholar
P. C. Anibal, J. D. C. O. Sardi, I. T. A. Peixoto, J. J. D. C. Moraes, and J. F. Höfling, “Conventional and alternative antifungal therapies to oral candidiasis,” Brazilian Journal of Microbiology, vol. 41, no. 4, pp. 824–831, 2010.
View at: Publisher Site | Google Scholar
S. Paul and W. S. Moye-Rowley, “Multidrug resistance in fungi: regulation of transporter-encoding gene expression,” Frontiers in Physiology, vol. 5, article 143, 2014.
View at: Publisher Site | Google Scholar
E. B. Nunes, N. B. Nunes, J. C. Monteiro, and A. L. Paes, “Perfil de sensibilidade do gênero Candida a antifúngicos em um hospital de referência da Região Norte do Brasil,” Revista Pan-Amazônica de Saúde, vol. 2, no. 4, pp. 23–30, 2011.
View at: Publisher Site | Google Scholar
G. Morace, F. Perdoni, and E. Borghi, “Antifungal drug resistance in Candida species,” Journal of Global Antimicrobial Resistance, vol. 2, no. 4, pp. 254–259, 2014.
View at: Publisher Site | Google Scholar
G. Ramage, S. Culshaw, B. Jones, and C. Williams, “Are we any closer to beating the biofilm: Novel methods of biofilm control,” Current Opinion in Infectious Diseases, vol. 23, no. 6, pp. 560–566, 2010.
View at: Publisher Site | Google Scholar
C. J. Seneviratne, L. Jin, and L. P. Samaranayake, “Biofilm lifestyle of Candida: a mini review,” Oral Diseases, vol. 14, no. 7, pp. 582–590, 2008.
View at: Publisher Site | Google Scholar
D. Cirasola, R. Sciota, L. Vizzini, V. Ricucci, G. Morace, and E. Borghi, “Experimental biofilm-related Candida infections,” Future Microbiology, vol. 8, no. 6, pp. 799–805, 2013.
View at: Publisher Site | Google Scholar
D. J. Newman and G. M. Cragg, “Natural products as sources of new drugs over the 30 years from 1981 to 2010,” Journal of Natural Products, vol. 75, no. 3, pp. 311–335, 2012.
View at: Publisher Site | Google Scholar
S. J. Doddanna, S. Patel, M. A. Sundarrao, and R. S. Veerabhadrappa, “Antimicrobial activity of plant extracts on Candida albicans: an in vitro study,” Indian Journal of Dental Research, vol. 24, no. 4, pp. 401–405, 2013.
View at: Publisher Site | Google Scholar
J. F. Höfling, P. C. Anibal, G. A. Obando-Pereda et al., “Antimicrobial potential of some plant extracts against Candida species,” Brazilian Journal of Biology, vol. 70, no. 4, pp. 1065–1068, 2010.
View at: Publisher Site | Google Scholar
P. Ranasinghe, S. Pigera, G. S. Premakumara, P. Galappaththy, G. R. Constantine, and P. Katulanda, “Medicinal properties of “true” cinnamon (Cinnamomum zeylanicum): a systematic review,” BMC Complementary and Alternative Medicine, vol. 13, article 275, 2013.
View at: Publisher Site | Google Scholar
S. F. Nabavi, A. Di Lorenzo, M. Izadi, E. Sobarzo-Sánchez, M. Daglia, and S. M. Nabavi, “Antibacterial effects of cinnamon: From farm to food, cosmetic and pharmaceutical industries,” Nutrients, vol. 7, no. 9, pp. 7729–7748, 2015.
View at: Publisher Site | Google Scholar
M. Vangalapati, N. Sree Satya, D. V. Surya Prakash, and S. Avanigadda, “A review on pharmacological activities and clinical effects of Cinnamon species,” Research Journal of Pharmaceutical, Biological and Chemical Sciences, vol. 3, no. 1, pp. 653–663, 2012.
View at: Google Scholar
P. V. Rao and S. H. Gan, Cinnamon: a multifaceted medicinal plant. Evidence-Based Complementary and Alternative Medicine, a multifaceted medicinal plant. Evidence-Based Complementary and Alternative Medicine, Cinnamon, 2014.
B. E. Al-Dhubiab, “Pharmaceutical applications and phytochemical profile of Cinnamomum burmannii,” Pharmacognosy Reviews, vol. 6, no. 12, pp. 125–131, 2012.
View at: Publisher Site | Google Scholar
R. Naveed, I. Hussain, A. Tawab et al., “Antimicrobial activity of the bioactive components of essential oils from Pakistani spices against Salmonella and other multi-drug resistant bacteria,” BMC Complementary and Alternative Medicine, vol. 13, no. 1, 2013.
View at: Publisher Site | Google Scholar
R. Sleha, P. Mosio, M. Vydrzalova, A. Jantovska, V. Bostikova, and J. Mazurova, “In vitro antimicrobial activities of cinnamon bark oil, anethole, carvacrol, eugenol and guaiazulene against Mycoplasma hominis clinical isolates,” Biomedical Papers, vol. 158, no. 2, pp. 208–211, 2014.
View at: Publisher Site | Google Scholar
A. Gupta, J. Duhan, S. Tewari et al., “Comparative evaluation of antimicrobial efficacy of Syzygium aromaticum, Ocimum sanctum and Cinnamomum zeylanicum plant extracts against Enterococcus faecalis: A preliminary study,” International Endodontic Journal, vol. 46, no. 8, pp. 775–783, 2013.
View at: Publisher Site | Google Scholar
F. Q. S. Guerra, J. M. Mendes, J. P. D. Sousa et al., “Increasing antibiotic activity against a multidrug-resistant Acinetobacter spp by essential oils of Citrus limon and Cinnamomum zeylanicum,” Natural Product Research (Formerly Natural Product Letters), vol. 26, no. 23, pp. 2235–2238, 2012.
View at: Publisher Site | Google Scholar
A. Abbaszadegan, S. Dadolahi, A. Gholami et al., “Antimicrobial and cytotoxic activity of cinnamomum zeylanicum, calcium hydroxide, and triple antibiotic paste as root canal dressing materials,” Journal of Contemporary Dental Practice, vol. 17, no. 2, pp. 105–113, 2016.
View at: Publisher Site | Google Scholar
N. C. f. C. L. Standards, Reference Methods for Broth Dilution Antifungal Susceptibility Testing of Yeast: Approved Standar, National Committee for Clinical Laboratory Standards, 2002.
D. P. Deswal and U. Chand, “Standardization of the tetrazolium test for viability estimation in ricebean (Vigna umbellata (Thunb.) ohwi and ohashi) seeds,” Seed Science and Technology, vol. 25, no. 3, pp. 409–417, 1997.
View at: Google Scholar
Z. N. Siddiqui, F. Farooq, T. N. M. Musthafa, A. Ahmad, and A. U. Khan, “Synthesis, characterization and antimicrobial evaluation of novel halopyrazole derivatives,” Journal of Saudi Chemical Society, vol. 17, no. 2, pp. 237–243, 2013.
View at: Publisher Site | Google Scholar
I. O. Lima, F. De Oliveira Pereira, W. A. De Oliveira et al., “Antifungal activity and mode of action of carvacrol against Candida albicans strains,” Journal of Essential Oil Research, vol. 25, no. 2, pp. 138–142, 2013.
View at: Publisher Site | Google Scholar
A. Escalante, M. Gattuso, P. Pérez, and S. Zacchino, “Evidence for the mechanism of action of the antifungal phytolaccoside B isolated from Phytolacca tetramera Hauman,” Journal of Natural Products, vol. 71, no. 10, pp. 1720–1725, 2008.
View at: Publisher Site | Google Scholar
D. S. Perlin, “Current perspectives on echinocandin class drugs,” Future Microbiology, vol. 6, no. 4, pp. 441–457, 2011.
View at: Publisher Site | Google Scholar
C. G. Pierce, A. Srinivasan, P. Uppuluri, A. K. Ramasubramanian, and J. L. López-Ribot, “Antifungal therapy with an emphasis on biofilms,” Current Opinion in Pharmacology, vol. 13, no. 5, pp. 726–730, 2013.
View at: Publisher Site | Google Scholar
R. D. Castro, E. D. Lima, I. D. Freires, and L. A. Alves, “Combined effect of Cinnamomum zeylanicum blume essential oil and nystatin on Candida albicans growth and micromorphology,” Revista de Ciências Médicas e Biológicas, vol. 12, no. 2, p. 149, 2013.
View at: Publisher Site | Google Scholar
M. C. A. Leite, A. P. de Brito Bezerra, J. P. de Sousa, F. Q. S. Guerra, and E. de Oliveira Lima, “Evaluation of antifungal activity and mechanism of action of citral against Candida albicans,” Evidence-Based Complementary and Alternative Medicine, vol. 2014, Article ID 378280, 9 pages, 2014.
View at: Publisher Site | Google Scholar
D. Djordjevic, M. Wiedmann, and L. A. McLandsborough, “Microtiter plate assay for assessment of Listeria monocytogenes biofilm formation,” Applied and Environmental Microbiology, vol. 68, no. 6, pp. 2950–2958, 2002.
View at: Publisher Site | Google Scholar
J. P. Singhal and A. R. Ray, “Synthesis of blood compatible polyamide block copolymers,” Biomaterials, vol. 23, no. 4, pp. 1139–1145, 2002.
View at: Publisher Site | Google Scholar
R. C. Hartmann, D. E. Jenkins Jr., and A. B. Arnold, “Diagnostic specificity of sucrose hemolysis test for paroxysmal nocturnal hemoglobinuria,” Blood, vol. 35, no. 4, pp. 462–475, 1970.
View at: Google Scholar
S. Kutscher, C. J. Dembek, S. Deckert et al., “Overnight Resting of PBMC Changes Functional Signatures of Antigen Specific T- Cell Responses: Impact for Immune Monitoring within Clinical Trials,” PLoS ONE, vol. 8, no. 10, p. e76215, 2013.
View at: Publisher Site | Google Scholar
A. F. Millezi, R. H. Piccoli, J. M. Oliveira, and M. O. Pereira, “Anti-biofim and Antibacterial Effect of Essential Oils and Their Major Compounds,” Journal of Essential Oil Bearing Plants, vol. 19, no. 3, pp. 624–631, 2016.
View at: Publisher Site | Google Scholar
S. M. Bersan, L. C. Galvão, V. F. Goes et al., “Action of essential oils from Brazilian native and exotic medicinal species on oral biofilms,” BMC Complementary and Alternative Medicine, vol. 14, no. 1, 2014.
View at: Google Scholar
I. S. Rana, A. Singh, and R. Gwal, “In vitro study of antibacterial activity of aromatic and medicinal plants essential oils with special reference to cinnamon oil,” International Journal of Pharmacy and Pharmaceutical Sciences, vol. 3, no. 4, pp. 376–380, 2011.
View at: Google Scholar
I. B. Jantan, B. A. Karim Moharam, J. Santhanam, and J. A. Jamal, “Correlation between chemical composition and antifungal activity of the essential oils of eight Cinnamomum species,” Pharmaceutical Biology, vol. 46, no. 6, pp. 406–412, 2008.
View at: Publisher Site | Google Scholar
M. C. Duarte, E. E. Leme, C. Delarmelina, A. A. Soares, G. M. Figueira, and A. Sartoratto, “Activity of essential oils from Brazilian medicinal plants on Escherichia coli,” Journal of Ethnopharmacology, vol. 111, no. 2, pp. 197–201, 2007.
View at: Publisher Site | Google Scholar
M. A. Pfaller, S. A. Messer, L. Boyken et al., “Cross-resistance between fluconazole and ravuconazole and the use of fluconazole as a surrogate marker to predict susceptibility and resistance to ravuconazole among 12,796 clinical isolates of Candida spp.,” Journal of Clinical Microbiology, vol. 42, no. 7, pp. 3137–3141, 2004.
View at: Publisher Site | Google Scholar
M. Unlu, E. Ergene, G. V. Unlu, H. S. Zeytinoglu, and N. Vural, “Composition, antimicrobial activity and in vitro cytotoxicity of essential oil from Cinnamomum zeylanicum Blume (Lauraceae),” Food and Chemical Toxicology, vol. 48, no. 11, pp. 3274–3280, 2010.
View at: Publisher Site | Google Scholar
H. Parthasarathy and S. Thombare, “Evaluation of antimicrobial activity of Azadirachta indica, Syzygium aromaticum and Cinnamomum zeyalnicumagainst oral microflora,” Asian Journal of Experimental Sciences, vol. 27, no. 2, pp. 13–16, 2013.
View at: Google Scholar
H. Ferhout, J. Bohatier, J. Guillot, and J. C. Chalchat, “Antifungal activity of selected essential oils, cinnamaldehyde and carvacrol against malassezia furfur and candida albicans,” Journal of Essential Oil Research, vol. 11, no. 1, pp. 119–129, 1999.
View at: Publisher Site | Google Scholar
R. Khan, B. Islam, M. Akram et al., “Antimicrobial activity of five herbal extracts against multi drug resistant (MDR) strains of bacteria and fungus of clinical origin,” Molecules, vol. 14, no. 2, pp. 586–597, 2009.
View at: Publisher Site | Google Scholar
P. Pozzatti, L. A. Scheid, T. B. Spader, M. L. Atayde, J. M. Santurio, and S. H. Alves, “In vitro activity of essential oils extracted from plants used as spices against fluconazole-resistant and fluconazole-susceptible Candida spp,” Canadian Journal of Microbiology, vol. 54, no. 11, pp. 950–956, 2008.
View at: Publisher Site | Google Scholar
F. Bakkali, S. Averbeck, D. Averbeck, and M. Idaomar, “Biological effects of essential oils—a review,” Food and Chemical Toxicology, vol. 46, no. 2, pp. 446–475, 2008.
View at: Publisher Site | Google Scholar
B. Adorjan and G. Buchbauer, “Biological properties of essential oils: an updated review,” Flavour and Fragrance Journal, vol. 25, no. 6, pp. 407–426, 2010.
View at: Publisher Site | Google Scholar
C. D. Churchill, M. Klobukowski, and J. A. Tuszynski, “The unique binding mode of laulimalide to two tubulin protofilaments,” Chemical Biology ##hssm###38; Drug Design, vol. 86, no. 2, pp. 190–199, 2015.
View at: Publisher Site | Google Scholar
S. K. Doke, J. S. Raut, S. Dhawale, and S. M. Karuppayil, “Sensitization of candida albicans biofilms to fluconazole by terpenoids of plant origin,” The Journal of General and Applied Microbiology, vol. 60, no. 5, pp. 163–168, 2014.
View at: Publisher Site | Google Scholar
T. B. de Souza, M. Orlandi, L. F. L. Coelho et al., “Synthesis and in vitro evaluation of antifungal and cytotoxic activities of eugenol glycosides,” Medicinal Chemistry Research, vol. 23, no. 1, pp. 496–502, 2014.
View at: Publisher Site | Google Scholar
S. B. Rajput and S. M. Karuppayil, “Small molecules inhibit growth, viability and ergosterol biosynthesis in Candida albicans,” SpringerPlus, vol. 2, article 26, 2013.
View at: Publisher Site | Google Scholar
A. C. R. Da Silva, P. M. Lopes, M. M. B. De Azevedo, D. C. M. Costa, C. S. Alviano, and D. S. Alviano, “Biological activities of α-pinene and β-pinene enantiomers,” Molecules, vol. 17, no. 6, pp. 6305–6316, 2012.
View at: Publisher Site | Google Scholar
G. Lopes, E. Pinto, P. B. Andrade, and P. Valentão, “Antifungal activity of phlorotannins against dermatophytes and yeasts: approaches to the mechanism of action and influence on Candida albicans virulence factor,” PLoS ONE, vol. 8, no. 8, Article ID e72203, 2013.
View at: Publisher Site | Google Scholar
I. Freires, C. Denny, B. Benso, S. de Alencar, and P. Rosalen, “Antibacterial activity of essential oils and their isolated constituents against cariogenic bacteria: a systematic review,” Molecules, vol. 20, no. 4, pp. 7329–7358, 2015.
View at: Publisher Site | Google Scholar
A. Ahmad, A. Khan, N. Manzoor, and L. A. Khan, “Evolution of ergosterol biosynthesis inhibitors as fungicidal against Candida,” Microbial Pathogenesis, vol. 48, no. 1, pp. 35–41, 2010.
View at: Publisher Site | Google Scholar
R. H. Pires, L. B. Montanari, C. H. G. Martins et al., “Anticandidal efficacy of cinnamon oil against planktonic and biofilm cultures of Candida parapsilosis and Candida orthopsilosis,” Mycopathologia, vol. 172, no. 6, pp. 453–464, 2011.
View at: Publisher Site | Google Scholar
Y.-G. Kim, J.-H. Lee, S.-I. Kim, K.-H. Baek, and J. Lee, “Cinnamon bark oil and its components inhibit biofilm formation and toxin production,” International Journal of Food Microbiology, vol. 195, pp. 30–39, 2015.
View at: Publisher Site | Google Scholar
G. L. S. Ferreira, A. L. A. L. Pérez, I. M. Rocha et al., Does Scientific Evidence for the Use of Natural Products in the Treatment of Oral Candidiasis Exist? A Systematic Review, Evidence-Based Complementary and Alternative Medicine, 2015.

Copyright

Copyright © 2018 Marianne de Lucena Rangel et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

PDF Download Citation

Download other formats

Order printed copies

Views

2783

Downloads

1176

Citations