Pimenta pseudocaryophyllus inhibits virulence factors and promotes metabolic changes in Candida yeast

Introduction: This is the fi rst study to examine the in vitro susceptibility and the expression of virulence factors in Candida species in the presence of Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae), a Brazilian plant known as paucravo. Additionally, the mechanisms of action of the crude ethanol extract and the ethyl acetate and aqueous fractions of this plant were investigated. Methods: The in vitro susceptibility of Candida was tested using the broth microdilution method, whereas an XTT reduction assay was used for biofi lms. Adherence was determined by counting the number of yeast cells that adhered to 100 oral epithelial cells, and hyphal formation was verifi ed in the hyphal induction medium M199. Flow cytometry with propidium iodide and FUN-1 was performed to assess the mechanism of action. Results: The results revealed that the crude ethanol extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus inhibited the growth of Candida isolates at a minimal inhibitory concentration (MIC) ranging from 64 to 256μg/mL, whereas the 50% sessile minimal inhibitory concentration (SMIC50) ranged from 512 to >1,024μg/mL. Adherence and hyphal formation were signifi cantly reduced in the presence of the crude ethanol extract and both fractions. Although cell membrane injury was detected, the predominant mechanism of action appeared to be the alteration of yeast metabolism, as demonstrated by fl ow cytometry. Conclusions: Our results indicated that antifungal activity reduced the expression of virulence factors in yeast via the alteration of yeast metabolism, suggesting that the crude extract of P. pseudocaryophyllus and its fractions may contain novel antifungal agents.

Fungal infections have increased signifi cantly in recent years due to an increase in the number of immunocompromised individuals.Candida species are responsible for the majority of yeast infections in humans 1 .Resistance in yeast and many side effects associated with antifungal agents have been reported 2,3 , and thus, alternative therapeutics lacking these limitations are under investigation.In particular, medicinal plants are being explored for novel treatments.Pimenta pseudocaryophyllus (Gomes) L.R. Landrum (Myrtaceae), popularly known as paucravo, craveiro-do-mato, louro-cravo or chá-de-bugre, is found in the Brazilian Atlantic Forest and Cerrado regions, especially in cerradão; it is a tree with a height of approximately four meters or a small bush in some regions, and it displays antifungal activity in vitro.According to Paula et al. 4,5 , the crude extract of this plant exerts antiedematogenic and antinociceptive effects on mice.
Biofilm formation, adherence to epithelial cells and fi lamentation are virulence mechanisms associated with yeast species of the Candida genus 6 .The ability to inhibit or reduce the expression of virulence factors is a desirable characteristic of an antifungal agent 7 .
In the present study, we examined the in vitro susceptibility, biofi lms, adherence to epithelial cells and hyphal formation of Candida species in the presence of the crude extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus.Furthermore, we investigated the basic mechanisms of action of these extracts against these yeast species.

Pimenta pseudocaryophyllus extract and fractions
The leaves of P. pseudocaryophyllus were collected in São Gonçalo do Abaeté in the State of Minas Gerais, Brazil at 18º20'58.4"south, 45°55'23.4"west at an altitude of 864m.The plant was identifi ed by Carolyn Elinore Barnes Proença, and the voucher specimen was deposited in the herbarium of the Universidade Federal de Goiás (UFG) under number 27159.The crude ethanol extract and fractions obtained from the leaves of P. pseudocaryophyllus (citral chemotype) were prepared according to Paula et al 5 .Four fractions were obtained: hexane, dichloromethane, ethyl acetate, and aqueous fractions.Previous experiments had demonstrated that the crude ethanol extract and the ethyl acetate and aqueous fractions provide the best results based on in vitro susceptibility tests against Candida yeast species.Therefore, these extracts were selected for the present study.Fluconazole (Pfi zer Pharmaceutical Group, New York, NY) was used in all assays.

Chemical components of the extract
High-performance liquid chromatography (HPLC) was performed to characterize and quantify the primary constituents of the crude extract.Quercitrin (quercetin 3-O-α-L-rhamnopyranoside; Sigma), catechin (Sigma), and gallic acid (Sigma) were used as reference standards.

Organisms
Bioassays were performed on 12 Candida isolates obtained from blood and nail samples and stored at -70°C in yeast extract potato dextrose (YEPD) agar (Difco®) containing 10% glycerol in the Laboratory of Mycology at the Institute of Tropical Diseases and Public Health of the Federal University of Goiás.These isolates were obtained from a previous study that was approved by the Bioethics Committee of the Hospital das Clínicas in Goiânia, Goiás.Candida parapsilosis ATCC 22019 was used as the standard strain.

Antimicrobial activity
Antifungal susceptibility tests using the crude extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus leaves were performed using the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) document M27-A3 8 .The crude extract and the fractions were dissolved in 0.1% dimethyl sulfoxide (DMSO) (0.1mL DMSO in of 9.9mL Roswell Park Memorial Institute [RPMI] medium), which exerted a negligible effect on the growth kinetics of the yeast species 9 .Then, the extract and the fractions were diluted in RPMI medium buffered to pH 7.0 using morpholinepropanesulfonic acid (MOPS).The fi nal concentrations of these samples ranged from 2.0 to 1,024µg/mL.
The inoculum of Candida isolates was suspended in sterile saline (0.85%) that was adjusted to a cell density of 0.5 McFarland standards using a spectrophotometer at a wavelength of 530nm.This suspension was diluted 1:50, followed by a 1:20 dilution in RPMI-1640 medium to obtain a fi nal concentration from 1 × 10 3 to 5 × 10 3 cells/mL.Aliquots of 100μL of the fungal suspension and 100μL of plant extract were inoculated in the wells of a microtiter plate.To determine the minimal inhibitory concentration (MIC), the plates were incubated at 35ºC and examined after 72h.Each assay was performed in duplicate.The MICs for the P. pseudocaryophyllus crude extract and fractions were defi ned as the lowest concentrations at which there was an absence of fungal growth.The MIC of fl uconazole was defi ned as the lowest concentration that achieved 80% growth inhibition compared to the growth of the drug control.
The minimal fungicidal concentrations (MFCs) of the P. pseudocaryophyllus crude extract and its fractions were determined as described by Torres-Rodriguez et al 10 .Briefl y, 10µL of the samples were collected from optically clear wells and seeded on Sabouraud dextrose agar.The plates were incubated at 35 o C for 72h, and the MFC was defi ned as the lowest concentration at which no fungal colony growth was detected.

In vitro susceptibility of Candida biofi lms
The biofi lm was formed in pre-sterilized polystyrene 96-well microtiter plates as described by Pierce et al. 11 .Briefl y, 100µL of the standardized suspension (RPMI-1640 broth) containing 10 6 cells/mL (counted using a hemocytometer) of C. albicans (n=6) or C. parapsilosis (n=6) isolates were placed in the selected wells of the microtiter plates and incubated for 48h at 37°C.The crude extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus, which varied from 1,024 to 1µg/mL (according to two-fold serial dilutions), were added to the formed biofi lms and incubated for 48h at 35°C.The 50% and 80% sessile minimal inhibitory concentrations (SMIC50 and SMIC80, respectively) relative to the control were determined using a 2,3-bis(2-methoxy-4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide (XTT) reduction assay and were measured spectrophotometrically using a microtiter plate reader at 550nm.

Yeast adherence
The adherence of Candida isolates was conducted according to Kimura and Pearsall 12 , with some modifi cations: 0.5mL of the yeast suspension (10 isolates) containing 2.5 x 10 7 yeast/ mL with or without the crude extract or the ethyl acetate or aqueous plant fraction at a concentration of 1/4, 1/2 or 1 x MIC (as previously determined) was mixed with 0.5mL of a pooled oral epithelial cell suspension collected from healthy adult volunteers.Adherence was determined by counting the number of yeast cells that adhered to 100 epithelial cells via microscopy 13 .

Hyphal induction
The hyphal induction assay was performed according to a previously published method by Ha and White 14 .Candida albicans isolates (n=5) grown in YEPD medium for 48h were subcultured in 5mL of yeast nitrogen without amino acids supplemented with ammonium sulfate and dextrose (YAD) growth medium with or without the crude ethanol extract or the ethyl acetate or aqueous fraction at a concentration of 1/4, 1/2 or 1 x MIC.The cells in the YAD culture medium were transferred to 5mL of the hyphal induction medium M199 (containing L-glutamine and Earle's salts, without bicarbonate; Gibco) with or without the crude ethanol extract or the ethyl acetate or the aqueous fraction at the same concentrations described above.This medium was incubated at 37°C for 3h with shaking (240rpm), and 250 cells (yeast + hyphae) were counted using a hemocytometer.

Mechanism of action
The mechanisms of action of the crude ethanol extract and the ethyl acetate and aqueous fractions against C. parapsilosis (ATCC 22019) and two clinical isolates of C. albicans were analyzed via flow cytometry (Accuri C6 cytometer, Becton Dickinson) according to the approach developed by Vale-Silva et al. 15 and Pina-Vaz and Rodrigues 16 .Two markers were used: propidium iodide (PI), which is a marker of membrane integrity due to its ability to permeate only damaged membranes, and 2-chloro-4-(2,3-dihydro-3-methyl-[benzo-1,3-thiazol-2-yl]methylidene)-1phenylquinolinium iodide (FUN-1), which is an indicator of yeast metabolic activity.The inoculum of 400µL containing 2x10 6 cells/ mL was added to 100µL of the crude ethanol extract or either fraction at a concentration of 1/4, 1/2, 1, 2 or 4 x MIC.Amphotericin B (2mg/ mL) was used as a control.Controls included the following: an autofl uorescence control, consisting of untreated yeast, and a labeled cell control, consisting of yeast that were treated with markers but not exposed to the crude ethanol extract or either fraction.

Data analysis
All the assays were performed in triplicate.Microsoft® Excel 2007 was used to analyze the data, and statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS®) for Windows® version 16.0.The infl uence of the crude ethanol extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus on Candida adhesion and hyphal formation were evaluated using the Mann-Whitney U-test.P values <0.05 were considered to be signifi cant.
Comparison of the retention times (RT), which was based on the HPLC profi les, between the reference standards and the samples revealed the presence of catechin and quercetin in the crude ethanol extract of P. pseudocaryophyllus.These substances were identifi ed in the extracts and fractions obtained from the leaves of the citral chemotype of P. pseudocaryophyllus.
The in vitro antifungal susceptibility of the crude extract of P. pseudocaryophyllus leaves, as well as the ethyl acetate and aqueous fractions, inhibited the growth of Candida at a MIC range of 64 to 256µg/mL (Table 1) and at a MFC range of 256 to >1,024µg/mL.The aqueous fraction inhibited the growth of 41% of the Candida isolates at a MIC of 64µg/mL and a MFC of 256µg/mL.

Of the 12 Candida isolates, biofi lm formation was only detected in 10 isolates (6 C. albicans and 4 C. parapsilosis).
In vitro susceptibility assays demonstrated high SMIC values for the crude ethanol extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus, as well as fl uconazole, against the yeast in the biofi lms.The SMIC 50 values ranged from 512 to >1,024µg/mL and the SMIC 80 values were greater than 1.02µg/mL for all of the Candida isolates evaluated.
The crude extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus, at their respective MIC, signifi cantly reduced the number of Candida yeast that adhered to oral epithelial cells compared to the control.The aqueous fraction was the most active and displayed a level of performance similar to that of fl uconazole in terms of the MIC.The number of yeast cells that adhered at 1/2 x MIC of the aqueous fraction was similar to that in the presence of 1 MIC of the crude extract.The means and standard deviations (SDs) of adherence are shown in Table 2.
The crude extract and fractions of P. pseudocaryophyllus, at their respective MIC, signifi cantly reduced hyphal formation compared to the control.At a concentration of 1/2 x MIC, the crude extract and the aqueous fraction also caused a signifi cant reduction in hyphal formation (Table 2).
Flow cytometric analysis demonstrated that the crude extract and the ethyl acetate and aqueous fractions affected the metabolism of C. albicans and C. parapsilosis, while the crude extract exhibited an additional capacity to injure the membranes of these cells.The metabolism of the yeast treated with these components was impaired in a dose-dependent manner compared to the control yeast, and this effect was intensifi ed at higher concentrations.Figure 1 shows the alterations in Candida metabolism in the presence of various concentrations of the aqueous fraction of P. pseudocaryophyllus.The crude extract and the ethyl acetate fraction induced lesions in the plasmalemma of Candida spp. at approximately 1 x MIC (Figure 2) and 2 x MIC, respectively.The aqueous fraction did not damage the yeast membranes at any of the examined concentrations.Medicinal plants are emerging as sources of new antifungal drugs that display greater efficiency, wider availability and lower toxicity 17,18 .The growth inhibition of yeast caused by P. pseudocaryophyllus and the antiedematogenic and antinociceptive effects observed in mice in previous investigations by our group 4,5 have increased interest in this plant.According to Scorzoni et al 19 , plant compounds displaying antifungal activity at an MIC of 250µg/mL are considered to be useful for therapeutic purposes.Paula et al. 5 determined that the MIC of the crude extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus was <256µg/mL for isolates of Candida spp.and was ≤128µg/mL for isolates of Cryptococcus neoformans complex species.In the current study, the MIC of the aqueous fraction was 64µg/mL for 42% of the examined isolates.

DISCUSSION
The antifungal activity of P. pseudocaryophyllus may be attributed to fl avonoids, which are constituents of the crude ethanol extract of this plant and some of its fractions.Narayana et al. 20 have reported the biological activity of fl avonoids, specifi cally quercetin, against yeast.Furthermore, the antifungal activity of catechin has been demonstrated against C. albicans and T. mentagrophytes.Masatomo and Kazuko 21 demonstrated the inhibitory activity of catechins against C. albicans, and Toyoshima et al. 22 examined the mechanism underlying the effects of the green tea of catechin on T. mentagrophytes and suggested that catechin attacked the cell membrane and caused the lysis of conidia and hyphae.
Antifungal agents that act by reducing the expression of yeast virulence factors are expected to be more effi cient.According to Zuzarte et al. 23 , inhibition of fi lamentation appears be suffi cient to treat candidiasis.In the present study, the P. pseudocaryophyllus crude extract and aqueous fraction (which displayed performance similar to that of the fl uconazole control) reduced hyphal formation and decreased adherence to buccal epithelial cells, signifi cantly affecting these Candida virulence mechanisms.Similar results obtained by Zuzarte et al. 23 and Vale-Silva et al. 24 verifi ed that hyphal formation in Candida was reduced in the presence of essential oils from Thymus x viciosoi and Lavandula multifi da L. Our results indicate that further studies of P. pseudocaryophyllus should be conducted to assess its therapeutic potential for the treatment of Candida infections.
Elucidating the mechanism of action of each antifungal agent is an important step toward their appropriate use and may help to reduce toxicity.In the present study, the examined fractions of P. pseudocaryophyllus altered the metabolism of the target yeasts, as shown in Figure 1.The results obtained by Evensen and Braun 25 indicated that polyphenols from Camellia sinensis inhibited biofi lm formation by C. albicans via the alteration of yeast protein metabolism.In addition to changes in metabolism, we observed injury to the fungal cell membrane in response to the P. pseudocaryophyllus crude extract and ethyl acetate fraction.It is diffi cult to explain why the aqueous fraction was not able to injure the yeast membranes.However, we suggest that this fi nding may be due to the absence of polar compounds from the aqueous fraction, causing it to act preferentially on fungal metabolism but preventing it from damaging the cell membrane.High concentrations (2 x MIC) of the ethyl acetate fraction were necessary to damage the cell membrane; however, at a concentration equivalent to 1 x MIC, the crude extract caused similar damage.These results indicate that the crude extract likely contains constituents that act synergistically.Although both mechanisms of action have been observed in the crude ethanol extract as well as the ethyl acetate and aqueous fractions of P. pseudocaryophyllus, the metabolic effects on yeast appear to predominate.The altered metabolism and plasma membrane injury of yeast observed in this study indicated that the constituents of P. pseudocaryophyllus might inhibit yeast development and most likely induce the destruction of the microorganism.
These results demonstrated that the antifungal activity of the crude extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus of the citral chemotype inhibited Candida virulence mechanisms, including adherence and hyphal induction (Table 2).Flavonoids, such as quercetin and catechin, found in P. pseudocaryophyllus are considered to be antimicrobials and explain this antifungal activity.In conclusion, our results indicate that the crude extract and the ethyl acetate and aqueous fractions of P. pseudocaryophyllus display antifungal activity, induce metabolic changes, cause membrane injury and reduce virulence factor expression in yeast, suggesting that these components may lead to the development of novel antifungal agents.

Plot 13 :FIGURE 2 -
FIGURE 2 -Histogram showing the red fl uorescence in relative units (red fl uorescence: FL3 channel, FL3-A log) of Candida parapsilosis (ATCC 22019) stained with propidium iodide.Control consisting of stained untreated cells (in black).Stained cells treated with the crude extract of the leaves of Pimenta pseudocaryophyllus (in red).