Evaluation of antimicrobial , cytotoxic and chemopreventive activities of carvone and its derivatives

1Laboratory of Pharmacognosy, School of Pharmaceutical Sciences, São Paulo State University (Unesp), Araraquara, 2Laboratory of Biotechnology, School of Pharmaceutical Sciences, São Paulo State University (Unesp), Araraquara, 3Laboratory of Cell Biology, School of Pharmaceutical Sciences, São Paulo State University (Unesp), Araraquara, 4Departament of Pharmaceutical Sciences, Health Sciences Center, UFPB – Univ Federal da Paraíba


INTRODUCTION
Natural products diversity is an important source in the search for bioactive compounds.Plants have a wide range of secondary metabolites that are important for the discovery of drugs for various diseases such as cancer and infections by microorganisms.Essential oils are generally a mixture of secondary metabolites including terpenes and phenolic compounds, mainly monoterpenes, sesquiterpenes, phenylpropanoids and other low molecular weight volatile compounds.Essential oils are responsible for the characteristic odor of aromatic plants which is important for attracting pollinators and seed dispersers, moreover, it acts as a defense mechanism against predators and diseases.Many of them present pharmacological activity and may be used therapeutically (De Sousa, 2015;Bakkali et al., 2008;Wagner, Elmadfa, 2003).Carvone and related metabolites are monoterpene constituents of essential oils that presented interesting activities including antibacterial, antifungal, anticonvulsivant and cytotoxic to cancer cell lines (Carvalho, Fonseca, 2006;Chen et al., 2006;Freire;Costa, 2006;Stammati et al., 1999).
Considering the reported activities of carvone in the literature, this study aimed to evaluate the antimicrobial, cytotoxic and chemopreventive activities of (+)-and (-)-carvone, (+)-and (-)-hydroxydihydrocarvone and α,β-epoxycarvone.Although enantiomers have similar physicochemical properties, they have different behavior in chiral environments, such as endogenous drug receptors, membrane proteins and enzymes.The antifungal activity was evaluated against Candida parapsilosis, Candida tropicalis, Candida krusei and Candida albicans and the antimicrobial activity against Escherichia coli and Staphylococcus aureus.The cytotoxicity assays were performed with human cancer cell lines HepG-2 (hepatocellular carcinoma) and SiHa (cervical tumor cell) and normal strain MRC-5 (normal lung fibroblast) through sulphor rodamine B assay.
Regarding potential chemopreventive action, the compounds were also evaluated in the induction of quinone reductase assay.Cancer quimioprevention involves the use of natural or synthetic agents to prevent, reverse or retard the carcinogenesis process.

Microorganisms
Strains of bacteria and yeast used in this study were Bacillus subtilis (ATCC 9362), Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 10536), Candida albicans (ATCC 64548), Candida parapsilosis (ATCC 22019), Candida tropicalis (ATCC 750), and Candida krusei (ATCC 6258).A bacterial colony of each strain was inoculated in brain heart infusion (BHI) broth and incubated at 37 °C for 24 h.The final concentration of cells in suspension was adjusted to 0.5 McFarland standard (approximately 1.5 x 10 8 cells/mL).Yeast colonies were incubated for 48 h at 35 °C and suspended in 5 mL of 0.9% sterile saline solution.Yeast cells were counted in Neubauer chamber and adjusted to a final concentration of 2.5 -5.0 x 10 3 cells/mL.

Positive controls
Ampicillin was used as positive control in antibacterial assays with stock solution concentration of 50 mg/mL (deionized water).Amphotericin B was used as a positive control for Candida strains with stock solution concentration of 32 mg/mL (dimethylsulfoxide: RPMI-1640 medium 1:5; v/v).

Determination of Minimum Inhibitory Concentration (MIC) by microplate dilution Method and Minimum Fungicidal Concentration (MFC)
MIC was determined using microplate dilution assays according to reference document M27-A3 of the CLSI with modifications (CLSI, 2008).RPMI-1640 containing glutamine and phenol red was used in Candida strains maintenance, the medium, without sodium bicarbonate, was supplemented with 3-(N-morphin)propanesulfonic acid (MOPS) at 0.165 M, pH 7.0.In each well of a microplate, 100 μL of sterilized RPMI-1640 medium was added.Then, 100 μL of carvone samples were added to the first well of each column (columns 1-8).In the column 9, 100 μL of amphotericin B was added to the first well.Serial dilution was performed by transferring 100 μL from the first to the next until the end of the line.Finally, 100 μL of yeast was added to each well to a final concentration of 2.5 x 10 3 cells/mL.Controls: yeast growth control (medium and yeast), solvent control (100 μL of 1% Tween 80 with and without yeast), sterility control (only medium), compound control (compound and medium) and positive control (amphotericin B).The microplates were incubated for 48 h at 35 °C under agitation.Fungal growth inhibition was assessed by the addition of 20 μL of 2% aqueous solution 2,3,5-triphenyltetrazolium chloride (TTC) that was read after incubation at 35 °C for 1 h.The lowest concentration which no visible growth was observed was considered the minimum inhibitory concentration (MIC).Tests were performed in triplicate.The minimal fungicidal concentration (MFC) was determined by plating the contents of each sample from MIC test into Petri dish containing Saboraund agar.The plates were incubated for 48 h at 35 °C.MFC was evaluated by absence or presence of yeast growth.

Determination of Minimum Inhibitory Concentration (MIC) by microplate dilution method and Minimum Bactericidal Concentration (MBC)
MIC was determined using dilution microplate method, according to reference document M7-A9 of the CLSI with modifications (CLSI, 2012).For that, 100 μL of BHI (brain heart infusion) was added to the wells.Then, 100 μL of carvone (or derivatives) sample was added to the first well of each column (columns 1-8).In the column 9, 100 μL of ampicillin was added in the first well.Serial dilution was performed transferring 100 μL from the first well to the subsequent until the end of each line.Finally, 100 μL of bacterial suspension (1:200; BHI) was added to each well.The final concentration of bacteria in each well was 2.5 x 10 5 cells/mL.Controls: solvent control (Tween 80 1% with and without bacteria), growth control (bacteria and medium), sterility control (only medium), compound control (compound and medium) and positive control (ampicillin).Microplates were incubated for 24 h at 37 °C.The growth inhibition was detected by adding 20 μL of a 0.01% aqueous solution of resazurin after incubation at 37 °C for 1 h.Tests were performed in triplicate.The minimal bactericidal concentration (MBC) was determined by plating each sample of the MIC test into Petri dishes containing agar Mueller-Hinton.Petri dishes were incubated for 24 h at 37 °C.MBC was evaluated by absence or presence of bacterial growth.

Cell lines and cell culture
The cell lines used in cytotoxicity assays was HepG2 (human hepatocellular liver carcinoma, ATCC®HB-8065™), courtesy of Dra.Dayse Maria Favero Salvadori (Department of Patology, UNESP), SiHa (human cancer of cervix, ATCC®HB-35™), courtesy of Dra.Luisa Lina Villa (Laboratory of Virology, Ludwig Institute) and MRC-5 (human lung fibroblast, ATCC®CCL-171™).In the quinone-reductase assay, the cell line used was Hepa c1c7 (murine hepatocellular carcinoma, ATCC®CRC-2026™), courtesy of Dr. John M. Pezzuto of College of Pharmacy, University of Hawaii.The medium used for in vitro culture of HepG2, SiHa and MRC-5 was Dulbecco´s Modified Eagle Medium (DMEM) and for Hepa c1c7 was Alfa Modification of Eagle Medium (α-MEM).DMEM and α-MEM were supplemented with fetal bovine serum for cells maintenance.The cells were cultured in bottles at 5% CO 2 atmosphere and 37 °C until the cell monolayer reached confluence (80%).After, cells were washed with Hanks and trypsin-EDTA solutions to detach cells and counting.

Sulforhodamine B (SRB) assay
SRB is used to measure the protein content in adherent cell cultures using 96 wells microplate.The plates were pre-incubated with culture medium (DMEM supplemented with 10% fetal bovine serum, 100 μL/well) for 24 h at 37 °C in incubator with 5% CO 2 .Wells without cells were used as controls, doxorrubicine as positive control (36.79 μM) and dimethyl sulfoxide (DMSO 1%) as vehicle control.After 24 h of incubation cells adhesion and growth were observed.Then, the medium was discarded and treatments, controls and DMEM without fetal bovine serum were added in each well.After 24 h of incubation, the cells were fixed with trichloroacetic acid (100 μL/well) and placed under refrigeration for 1 h at 4 °C.TCA was removed and plates were washed in low-flow of water three times and dried.Following, plates were stained for 20 min at room temperature with SRB 0.4% (50 μL/well).In mildly acidic conditions the colorant binds to basic amino acid residues in fixed cells.This reaction provides an estimate of the total protein mass.The plates were washed with 1% acetic acid solution and dried at room temperature.The unbound dye was removed after washing, the dyestuff bound to the protein was solubilized in a basic medium (Tris base, 10 mM, pH 10.5) to determine the optical density in a plate reader at 570 nm.The colorimetric evaluation gives an estimate of total protein mass.The assay was conducted as described in Skehan et al. (1990) with modifications (Voigt, 2005).Cellular concentration used was 1.4 x 10 4 cells/mL.
NQO1 induction ratio (IR) was represented by specific enzyme activity of cells treated compared to control (DMSO 0.5%).Compounds were considered active if IR > 2.0.4'-bromoflavone, a potent NQO1 inducer, was used as positive control for the assay.

Statistical analysis
One-way analysis of variance (ANOVA) with Tukey´s post-test was employed for the statistical analysis for cytotoxicity and chemoprevention results.Data were expressed as the mean ± SD.

Antimicrobial assays
Compounds C-and HC-only presented antibacterial activity against S. aureus at the highest concentration (MIC/MBC: 2.5 mg/mL) (Table 1).C+, HC+ and EP did not show antibacterial activity.Data from literature showed activity of EP against S. aureus using a diffusion method in plates with solid medium (Arruda et al., 2006).Also from literature, C-and C+ inhibited the growth of E. coli at concentrations of 9.6 mg/mL (Naigre et al., 1996) and 1.5 mg/mL (MIC) (Helander et al., 1998), respectively.All these literature data differ from results found in our study.
Table I shows the results of MIC for antifungal activity and MFC.Carvones presented weak activity against C. tropicalis with values of MIC/MFC of 2.5 mg/mL and low inhibitory action against C. parapsilosis was also observed (MFC: 1.25 mg/mL).C+ presented intermediate activity to C. Krusei (0.625 mg/mL).Carvones presented activity against C. albicans in this study, C+ and C-were the most active compounds with MIC/MFC values of 0.312 and 0.625 mg/mL, respectively.Scientific literature reports that essential oils rich in C+ inhibited the growth of this fungus (Jirovetz et al., 2003) and that carvone inhibited the transformation of C. albicans to its pathogenic form (Mcgeady, Wansley, Logan, 2002).Similarly to carvone and its derivatives, several monoterpenes also showed activity against C. albicans, including linalool, citral, citronellol, thymol, eugenol and carvacrol (Zore et al., 2001;Alvarez et al., 2012).All samples inhibited C. krusei and EP and C+ were the most active compounds against this yeast (MIC/ MFC: 0.625 mg/mL).

Cytotoxicity assay
All samples presented low cytotoxicity in the cell lines tested at the concentrations of 0.62 to 50.00 μg/mL and did not reach IC 50 or IC 20 values (Figure 2).In vitro assays have been used to evaluate toxicity, determine activity and mechanism of action of natural antitumor products (Nagle et al., 2004).Despite carvone and its derivatives did not show cytotoxicity in this study, previous studies showed cytotoxic activity against prostate and breast cancer cell lines, P-815 (murine mastocytoma), K-562 (human chronic myeloid leukemia), CEM (acute lymphoblastoid leukemia T) and MCF-7 (human breast adenocarcinoma) (Stammati et al., 1999;Chen et al., 2006;Jaafari et al., 2012).Natural carvones are present in low concentrations in food and have applications in industry and agriculture, increasing the human exposure to these compounds (Carvalho, Fonseca, 2006).The low toxicity observed in this study suggests safety in the use of carvones.

Quinone reductase assay
Three independent experiments were performed to evaluate the carvone and derivatives ability to induce quinone reductase enzyme.The cell strain used was Hepa c1c7 and 4'-bromoflavone was used as positive control.Carvones did not show induction of quinone reductase at tested concentrations (Figure 3).The percentage of living cells by crystal violet assay also demonstrated that these compounds did not present cytotoxic activity.

CONCLUSIONS
Plants' secondary metabolites are an important source to development of new drugs.This study evaluated the activity of carvone and its derivatives against strains of bacteria and fungi, the cytotoxicity in human cells and the inhibition of quinone reductase in murine carcinoma cells.
As conclusions, C-and HC-showed low activity against E. coli.However, EP, C+ and HC+ did not inhibit the growth   Candida yeasts and the most significant result was found with C+, C-and EP.These results suggest the potential use of carvones and its derivatives as antifungal agents against Candida yeasts.In citotoxicity SRB assay none of the compounds showed activity in HepG2, SiHa and MRC-5 cell lines and also did not inhibit the enzyme quinone reductase.The absence of cytotoxicity in both cancer and normal cell lines indicates the safe use of these compounds.
of the bacteria strains tested.Compounds presented weak antifungal activity against C. tropicalis and C. parapsilosis.EP and C+ showed moderate activity against C. krusei similar to C+ and C-against C. albicans.All tested samples demonstrated fungistatic and fungicidal activity against

TABLE I -
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of carvones against S. aureus and E. coli