Inhibitory Effect of Eugenol and Trans‐Anethole Alone and in Combination with Antifungal Medicines on Candida Albicans Clinica

Uniwersytet Medyczny w Łodzi Medical University of Lodz https://publicum.umed.lodz.pl

Inhibitory Effect of Eugenol and Trans-Anethole Alone and in Combination with Antifungal Medicines on Candida albicans Clinical Isolates, Publikacja / Publication Dąbrowska Marta, Zielińska-Bliźniewska Hanna, Kwiatkowski Paweł, Łopusiewicz Łukasz, Prus Agata, Kostek Mateusz, Kochan Ewa, Sienkiewicz Monika DOI wersji wydawcy / Published http://dx.doi.org/10.1002/cbdv.202000843 version DOI Adres publikacji w Repozytorium URL / Publication address in https://publicum.umed.lodz.pl/info/article/AML3aacc66138914656916b7a87814195e4/ Repository Data opublikowania w Repozytorium 2021-03-27 / Deposited in Repository on Rodzaj licencji / Type of licence Other open licence Dąbrowska Marta, Zielińska-Bliźniewska Hanna, Kwiatkowski Paweł, Łopusiewicz Łukasz, Prus Agata, Kostek Mateusz, Kochan Ewa, Sienkiewicz Monika: Inhibitory Cytuj tę wersję / Cite this version Effect of Eugenol and Trans-Anethole Alone and in Combination with Antifungal Medicines on Candida albicans Clinical Isolates, Chemistry & Biodiversity, vol. 18, no. 5, 2021, pp. 1-11, DOI:10.1002/cbdv.202000843 Title: Inhibitory Effect of Eugenol and Trans-Anethole Alone and in Combination with Antifungal Medicines on Candida albicans Clinical Isolates

Authors: Marta Dąbrowska, Hanna Zielińska-Bliźniewska, Paweł Kwiatkowski, Łukasz Łopusiewicz, Agata Pruss, Mateusz Kostek, Ewa Kochan, and Monika Sienkiewicz

This manuscript has been accepted after peer review and appears as an Accepted Article online prior to editing, proofing, and formal publication of the final Version of Record (VoR). This work is currently citable by using the Digital Object Identifier (DOI) given below. The VoR will be published online in Early View as soon as possible and may be different to this Accepted Article as a result of editing. Readers should obtain the VoR from the journal website shown below when it is published to ensure accuracy of information. The authors are responsible for the content of this Accepted Article.

To be cited as: Chem. Biodiversity 10.1002/cbdv.202000843

Link to VoR: https://doi.org/10.1002/cbdv.202000843

Pobrano z https://publicum.umed.lodz.pl / Downloaded from Repository of Medical University of Lodz 2021-10-07 Chemistry & Biodiversity 10.1002/cbdv.202000843

Chem. Biodiversity

Inhibitory Effect of Eugenol and Trans-Anethole Alone and in Combination with Antifungal Medicines on Candida albicans Clinical Isolates

Marta Dąbrowskaa, Hanna Zielińska-Bliźniewskaa, Paweł Kwiatkowskib, Łukasz Łopusiewiczc, Agata Prussd, Mateusz Kostekc, Ewa Kochane, Monika Sienkiewicza*

a Department of Allergology and Respiratory Rehabilitation, Medical University of Lodz, Żeligowskiego 7/9 Str., 90-752 Lodz, Poland; [email protected] b Department of Diagnostic Immunology; Chair of Microbiology, Immunology and Laboratory Medicine; Pomeranian Medical University in Szczecin; 72 Powstańców Wielkopolskich Avenue; 70-111 Szczecin, Poland c Center of Bioimmobilisation and Innovative Packaging Materials; Faculty of Food Sciences and Fisheries; West Pomeranian University of Technology in Szczecin; Janickiego 35; 71-270 Szczecin, Poland d Department of Laboratory Medicine; Chair of Microbiology, Immunology and Laboratory Medicine; Pomeranian Medical University in Szczecin; 72 Powstańców Wielkopolskich Avenue; 70-111 Szczecin, Poland e Pharmaceutical Biotechnology Department, Medical University of Lodz, Muszyńskiego 1, 90-151 Łódź, Poland

Abstract: One of the most common pathogens among yeasts is Candida albicans, which presents a serious health threat. The study aimed to check the antifungal properties of trans-anethole and eugenol with selected antifungal medicines (AMs) against C. albicans clinical isolates. The checkerboard method was used to tests of interactions between these compounds. Achieved results indicated that eugenol showed synergistic and additive activities with miconazole and econazole against investigated clinical isolates, respectively. Moreover, the combination — trans-anethole — miconazole also showed an additive effect against two clinical isolate. We tried to relate the results to changes in C. albicans cell sheaths under the influence of essential oils compounds (EOCs) performing the Fourier transform infrared spectra analysis to confirm the presence of particular chemical moieties in C. albicans cells. Nevertheless, no strong relationships was observed between synergistic and additive actions of used EOC-AMs combinations and chemical moieties in C. albicans cells.

Keywords: Candida spp., trans-anethole, eugenol, synergistic activity, miconazole

Introduction Vulvovaginal candidiasis (VVC) is the second most common abnormality associated with vaginal biocenosis.[1] In most cases, there are endogenous infections, and the reason for the disease process is the disturbance of the balance between the yeast and the host.[2] It is estimated that about 75% of women will experience at least one episode of VVC over a lifetime, and approximately 40-45% will have two or more episodes. In 5-10% of patients, the recurrent form of mycosis will develop. Asymptomatic colonization affects 10-15% of women. [3] According to available data, the most common pathogen responsible for VVC is Candida albicans.[4] Nevertheless, complicated VVC is defined as a severe or recurrent disease (more than four episodes of symptomatic VVC within one year), infection due to Candida species other than [5]

C. albicans, and/or VVC in an abnormal host. Several topical antifungal agents are effective therapy for VVC, and no agent is clearly Manuscript superior.[6,7] Oral and topical antimycotics achieve entirely equivalent results.[8] For recurring VVC, therapy with a topical or oral azole, followed by fluconazole is recommended.[9] The literature showed that several mechanisms were found to fluconazole resistance among Candida's genus, and an increase in azole resistance may have a clinical impact.[10]

The evolution and increasing factor of antimicrobial resistance in pathogens have prompted extensive research to find alternative therapeutics; for example, some essential oils (EOs) and their main compounds.[11] EOs obtained from lemongrass (Cymbopogon citratus), geranium (Pelargonium asperum),[12] tea tree (Melaleuca alternifolia),[13,14] lavender (Lavandula angustifolia),[15] and thyme (Thymus vulgaris)[16] are reported to inhibit Candida mycelial growth in vitro. Pietrella et al.[17], show that the EO of a Moroccan plant Mentha suaveolens is candidastatic and candidacidal and has a degree of anticandidal activity in a model of vaginal infection. In vivo studies are very limited; Maruyama et al.[18], examine the efficacy of vaginal application of geranium EO, or its main compound, geraniol, against murine experimental candidiasis was investigated. In the study, there was shown that the application of geranium oil or its component, geraniol, suppressed Candida cell growth in the vaginal cavity only when it was combined with vaginal washing. Natural bioactive substances can be used in combination therapy. Mertas et al.[19], showed that combining natural substances such as tea tree EO and conventional drugs such as fluconazole may help treat difficult yeast infections.

In our research, we focused on examining the antifungal properties of trans-anethole and eugenol against C. albicans clinical isolates come from vaginal secretions. Both oil components show biological activity and seem to be promising ingredients in the fight against pathogenic microorganisms, especially those showing resistance to standard medications in medical practice.[20,21] Therefore, the study aimed to assess the antifungal activity of trans-anethole and eugenol alone and in combination with selected antifungal medicines against C. albicans of clinical origin. Accepted

Results The Effects of Phenylpropenes and Antifungal Medicines The results of the MICs of tested phenylpropenes and antifungal medicines against C. albicans strains are summarized in Table 1 and Table 2, respectively. The results showed that all yeast strains were susceptible to tested EOCs. The highest inhibiting activity against isolates was observed for eugenol (MIC = 1.96 ± 0.00 mg/mL — 3.92 ± 0.00 mg/mL; MFC = 3.92 ± 0.00 mg/mL — 7.72 ± 0.00 mg/mL). Moreover, only fungicidal power of eugenol against C. albicans strains was exhibited. On the contrary, the lower inhibition for trans-anethole was detected (MIC = 61.75 ± 0.00 mg/mL — 247.00 ± 0.00 mg/mL; MFC = 247.00 ± 0.00 mg/mL — 494.00 ± 0.00 mg/mL).

Both the control and clinical isolates were susceptible to tested antifungal medicines. Furthermore, all tested drugs showed fungicidal power against C. albicans strains. The highest and lowest inhibiting activity against isolates were observed for clotrimazole (MIC = 0.01 ± 0.00 mg/mL — 0.02 ± 0.00 mg/mL; MFC = 0.02 ± 0.00 mg/mL — 0.03 ± 0.00 mg/mL), and econazole (MIC = 0.02 ± 0.00 mg/mL — 0.06 ± 0.00

Pobrano z https://publicum.umed.lodz.plThis article is protected / Downloaded by copyright. from AllRepository rights reserved. of Medical University of Lodz 2021-10-07 Chemistry & Biodiversity 10.1002/cbdv.202000843

Chem. Biodiversity mg/mL; MFC = 0.04 ± 0.00 mg/mL — 0.13 ± 0.00 mg/mL), respectively. Moreover, it has been also found that the highest inhibiting activity against C. albicans ATCC 10231 strain was to miconazole, econazole and clotrimazole.

Table 1. Minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC), ratio MFC/MIC and power of the essential oil compounds against Candida albicans strains

MIC (MFC) of trans- Number of Ratio MIC (MFC) of Ratio Eugenol trans-Anethole Anethole strain MFC/MIC eugenol [mg/mL] MFC/MIC power [mg/mL] power Control strain 123.50 ± 0.00 1.96 ± 0.00 (3.92 ± (Candida albicans 2 fungicidal 2 fungicidal (247.00 ± 0.00) 0.00) ATCC 10231) 247.00 ± 0.00 1.96 ± 0.00 (3.92 ± 1 2 fungicidal 2 fungicidal (494.00 ± 0.00) 0.00) 61.75 ± 0.00 1.96 ± 0.00 (3.92 ± 2 4 fungistatic 2 fungicidal (247.00 ± 0.00) 0.00) 247.00 ± 0.00 3.92 ± 0.00 (7.72 ± 3 1 fungicidal 2 fungicidal (247.00 ± 0.00) 0.00) 61.75 ± 0.00 1.96 ± 0.00 (3.92 ± 4 4 fungistatic 2 fungicidal (247.00 ± 0.00) 0.00) 247.00 ± 0.00 1.96 ± 0.00 (3.92 ± 5 2 fungicidal 2 fungicidal (494.00 ± 0.00) 0.00) MIC — minimal inhibitory concentration; MFC — minimal fungicidal concentration. Values are expressed as means ± standard deviation; the test was repeated twice (n = 2). Manuscript Accepted

Chem. Biodiversity

Table 2. Minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC), ratio MFC/MIC and power of the antifungal medicines against Candida albicans strains

MIC MIC MIC (MFC) (MFC) (MFC) Clotri MIC (MFC) of Ratio Micon of Ratio Econa of Ratio of Ratio Nystat mazol Number of strain miconazole MFC/M azole econa MFC/M zole clotrim MFC/M nystati MFC/M in e [mg/mL] IC power zole IC power azole IC n IC power power [mg/m [mg/m [mg/m L] L] L] 0.03 ± 0.03 ± 0.13 ± 0.03 ± 0.01 (0.06 ± fungicid 0.00 fungicid 0.00 fungicid 0.00 fungicid Control strain (Candida albicans ATCC 10231) 2 2 2 2 0.00) al (0.06 ± al (0.06 ± al (0.25 ± al 0.00) 0.00) 0.00) 0.06 ± 0.02 ± 0.13 ± 0.13 ± 0.00 (0.25 ± fungicid 0.00 fungicid 0.00 fungicid 0.00 fungicid 1 2 2 1.5 2 0.00) al (0.13 ± al (0.03 ± al (0.25 ± al 0.00) 0.00) 0.00) 0.02 ± 0.01 ± 0.06 ± 0.06 ± 0.00 (0.13 ± fungicid 0.00 fungicid 0.00 fungicid 0.00 fungicid 2 2 2 2 2 0.00) al (0.04 ± al (0.02 ± al (0.13 ± al 0.00) 0.00) 0.00) 0.06 ± 0.02 ± 0.13 ±

0.25 ± 0.00 (0.25 ± fungicid 0.00 fungicid 0.00 fungicidManuscript 0.00 fungicid 3 1 1 1.5 2 0.00) al (0.06 ± al (0.03 ± al (0.25 ± al 0.00) 0.00) 0.00) 0.06 ± 0.02 ± 0.13 ± 0.06 ± 0.00 (0.13 ± fungicid 0.00 fungicid 0.00 fungicid 0.00 fungicid 4 2 2 1.5 2 0.00) al (0.13 ± al (0.03 ± al (0.25 ± al 0.00) 0.00) 0.00) 0.06 ± 0.01 ± 0.06 ± 0.13 ± 0.00 (0.25 ± fungicid 0.00 fungicid 0.00 fungicid 0.00 fungicid 5 2 1 2 2 0.00) al (0.06 ± al (0.02 ± al (0.13 ± al 0.00) 0.00) 0.00) MIC — minimal inhibitory concentration; MFC — minimal fungicidal concentration. Values are expressed as means ± standard deviation; the test was repeated twice (n = 2). Accepted Combination of EOCs and Antifungal Medicines

The results for checkerboard assay against C. albicans ATCC 10231 and clinical isolates are presented in Figure 1 and as Supporting information for Figure 1. The study indicated that eugenol with miconazole showed synergistic activity (FICI = 0.2 — 0.4) against studied clinical isolates compared to the reference strain (indifferent effect; FICI = 1.6). Moreover, synergistic and additive effects in a combination of eugenol with econazole were also reported for clinical isolate no. 1 (FICI = 0.4) and for clinical isolates no. 2, no. 3, no. 4, and no. 5 (FICI = 0.6 — 1.0), respectively. The additive effect was also exhibited in trans-anethole — miconazole combination (for strain no. 2 and no. 5; FICI = 1.0). However, no additive activity in reference strain in all pairs was noted. Eugenol presented an antagonistic

Pobrano z https://publicum.umed.lodz.pl / ThisDownloaded article is protected by copyright. from All rights Repository reserved. of Medical University of Lodz 2021-10-07 Chemistry & Biodiversity 10.1002/cbdv.202000843

Chem. Biodiversity effect when used with nystatin against all tested strains. Overall, numerous combinations (trans-anethole — econazole/clotrimazole/nystatin) showed antagonistic effects against both reference strain and clinical isolates. Manuscript Accepted

Chem. Biodiversity

Figure 1. Fractional inhibitory concentration indices (FICI) of essential oil compounds-antifungal medicines pairs against Candida albicans strains: (a) C. albicans ATCC 10231, (b) — strain no. 1, (c) — strain no. 2, (d) — strain no. 3, (e) — strain no. 4, (f) — strain no. 5. tA — trans- anethole, E — eugenol. Manuscript Accepted

FTIR Analysis

Fourier transform infrared (FTIR) spectroscopy analyses were performed to corroborate the presence of chemical moieties in C. albicans cells (both reference strain and clinical strains). FTIR spectroscopy is a powerful technique in studying biological macromolecules and 5

Chem. Biodiversity complex biological systems, such as tissues and cells.[22] The complete FTIR spectra of the C. albicans cells samples are shown in Figure 2. In the FTIR spectrum of all tested strains, peaks at approx. 3286/3278 cm-1 corresponds to OH vibration from water and amide A from proteins.[23- 26] -1 Peaks at approx. 2926 cm observed from strains 1, 2, 3, 4 and 5 originate from asymmetric stretching of C-H bond of the -CH 2 groups -1 combined with that of -CH3 groups. However, a significant shift and strong peak at 2970 cm were observed in the reference strain sample.

Those changes may suggest some quantitative and qualitative differences in -CH2 and -CH3 groups of references strain in comparison with clinical isolates (strains 1, 2, 3, 4 and 5), resulting presumably in different electric potential of fungal cell walls and efficacy of antifungal drugs, as shown for observed indifferent effect. The peaks at approx. 1640 cm-1 and approx 1538 cm-1 observed in all tested strains correspond to amide I and amide II regions (attributed to the asymmetric bending of methyl groups in proteins and stretching C-N vibrations of cytosine- [22] -1 guanine, respectively). Peaks at approx. 1402 cm originate from C=O and COO- symmetric stretching vibration in proteins and CH2 wagging vibrations in lipids and β(1-3)glucans and stretching C-N vibrations of cytosine-guanine pairs. The peaks at approx. 1240 cm -1 originate from C-O asymmetric stretching vibrations and PO2- in phospholipids.[25-26] Peaks at wavenumbers approx. 900 cm−1 and approx. 1030 cm−1 are corresponding to C-O, C-O-H, and C-O-C deformation and C-C stretching vibrations of carbohydrates and β(1-3)glucans of the fungal cell wall, nucleic acids and glycogen and PO2- symmetric skeletal stretching vibrations mainly from RNA and DNA, respectively. [23-26] The additional strong peak about 991 cm-1 of C. albicans (strains no. 1, 2 and 4) samples might be assignable to change of C-O-C and C-O-P symmetric stretching vibrations in cell wall oligosaccharides and polysaccharides.[26] Manuscript

Figure 2. Fourier transform infrared spectra of Candida albicans strains.

Discussion

The widespread use of a limited number of antifungal agents, particularly azoles drugs, has led to the development of drug resistance in treating C. albicans infections,[27,28] so there is a need to develop new treatment methods other than antifungal agents. Biological activities of essential oil compounds and their combinations with antimicrobial agents may prove more useful in antifungal therapy than such compounds used alone. Therefore, some natural substances are investigated for synergism with azoles, polyenes, or echinocandins. Trinh et al.[29], in their study on mice, found the antimicrobial activity of α-terpineol (which is extracted from essential oils) to be comparable to that of clotrimazole. Martes et al.[19], in the study, exposed clinical strains of fluconazole-resistant C. albicans to sub-lethal concentrations of tea tree oil or its main bioactive component, terpinen-4-ol. Terpinen-4-ol was found to be more active than tea tree oil and strongly enhanced fluconazole activity against fluconazole-resistant C. albicans strains. This study demonstrates that combining natural substances such as tea tree oil or their main constituent — terpinen-4-ol and conventional drug such as fluconazole may help treat difficult yeast infections. Our research also shows that the constituent of Eugenia caryophyllata essential oil — eugenol — enhances miconazole and econazole action. The latest research of Accepted Sharifzadeh and Shokri[30], confirm the ability of eugenol to synergistic action with antimycotic agents against yeast form Candida genus. The authors showed that eugenol in combination with voriconazole possess synergistic potential against C. tropicalis and C. krusei isolates (FICI = 0.19—0.88). This combination was effective for 83.3% of C. tropicalis and 77.7% of C. krusei isolates. In turn, the research described by Ahmad et al.[31], indicated that both eugenol and methyleugenol show great antifungal activity in combination with fluconazole against both fluconazole-resistant and fluconazole-susceptible clinical isolates of C. albicans. What's more, the authors did not observe antagonistic effect. Samber et al.[32], demonstrated the synergistic interaction between carvone — fluconazole (FICI = 0.28—0.48) and menthol — fluconazole (FICI = 0.26—0.49) against both C. albicans susceptible and resistant to fluconazole. The authors stated that use of carvone affected extensive cell damage and leakage of the intracellular content in large quantities using scanning electron microscopy (SEM). Moreover, they showed that this compound decreased the ergosterol content of Candida spp. strains in a dose-dependent fashion. This allowed them to conclude that these compounds penetrate the cell membrane and target ergosterol biosynthesis pathway. Latifah-Munirah’s et al. [33], research also proved that eugenol can the release of cellular material, to increase cell permeability and causes visible changes in the ultrastructure and cell surface morphology. Currently, scientific research is focused on the search for effective and safe ingredients for antifungal preparations. 6

Chem. Biodiversity Nevertheless, the effective combinations of antimycotic agents with chemical constituents have already been patented. For instance, patent WO 2019/015975 describes that miconazole combined with quaternary ammonium salts exhibits synergistic antibiofilm activity and may help treat difficult yeast infections such as recurrent oral and vulvovaginal candidiasis.[34] Data of literature suggest that eugenol and trans-anethole as natural product of plants are considered safe agents. Available toxicology data indicated that the lethal dose (LD50) of trans-anethole, administered orally in laboratory mammalians, was between 2090 mg/kg and 3050 mg/kg. [35,36] Moreover, Abraham found that the naturally occurring flavoring agents, trans-anethole and eugenol have antigenotoxic effects in mice.[37] Eugenol propertied antimicrobial and anti- inflammatory action has long been used in dentistry. According to the literature data, eugenol is not acutely toxic, with an oral LD50 of 2650 mg/kg in the rat. Acute oral LD50 values in mammalian were greater than 1190 mg/kg.[38]

Conclusions

Our investigations showed that miconazole or econazole with eugenol could be a good candidate as an ingredient of anti-Candida preparations. The FTIR analysis showed that trans-anethole's additive action with miconazole might be connected with the quantitative and qualitative differences in C. albicans cells' chemical moieties. Nevertheless, no strong relationships was observed between synergistic and additive actions of used EOC-AMs combinations and chemical moieties in C. albicans cells. However, we can assume that those differences may induce some changes in fungal cell walls' electric potential, influencing antifungal agents' diffusion into fungal cell compartments. It indicates that the efficacy of antifungal drugs against C. albicans may largely be a strain trait resulting from biochemical changes in yeast cells. However, further in depth studies (including involvement of other spectroscopic methods, as well as molecular and biochemical analyses) should be carried out to determine this mechanisms. Understanding the mechanism of the occurrence of beneficial interactions of EOCs with antifungal drugs is an important element in research into their possible use.

Experimental Section

Identification of Yeast Strains and Growth Condition

The yeast strains investigated in this study were isolated from women's vaginal secretions treated for recurrent candidiasis. The yeast strains were cultivated in Sabouraud Gentamicin Chloramphenicol 2 agar (SGC2, bioMérieux, Poland) and incubated for 48 h at 37 °C in an aerobic atmosphere. According to the manufacturer protocol, all yeast strains were identified with VITEK 2 YST ID Card (bioMérieux, Poland). The investigated clinical strains, highly resistant were chosen among the thirty eight clinical isolates after investigations of susceptibility to recommended antimycotic agents (Miconazole, Econazole, Clotrimazole, Nystatin) by the use of disc-diffusion method and minimal inhibitory concentration assays. The standard C. albicans ATCC 10231 strain used as control came from the collection of Chair of Microbiology, Immunology and Laboratory Medicine, Pomeranian Medical University in Szczecin (Poland). The Bioethics Committee approved the study of Medical University in Lodz, No: RNN/360/18/KE.

Chemicals

Following antifungal medicines: miconazole (≥99% purity), econazole (≥99% purity), and clotrimazole (≥98% purity) were obtained from Pol-Aura (Roznowo, Poland), whereas nystatin was purchased from Teva (Warsaw, Poland) manufacturer. Concentrations of all antifungal medicines from 1000 to 2.44 µg/mL were prepared with dimethyl sulfoxide (DMSO, Loba Chemie, Germany) (2%, v/v) and diluted Manuscript by Sabouraud broth (SAB, Sigma-Aldrich, Darmstadt, Germany). Concentrations of the following essential oil compounds (EOCs): eugenol (≥98% purity, Ernesto Ventos S.A, Barcelona, Spain) and trans-anethole (99% purity, Sigma-Aldrich, Germany) from 500 to 0.12 µL/mL were dissolved in Tween 80 (Sigma-Aldrich, Germany) (1%, v/v) and diluted by SAB.

Determination of Minimal Inhibitory Concentration (MIC) and Minimal Fungicidal Concentration (MFC) of Chemicals

The MIC values of the chemicals against C. albicans strains were determined by broth microdilution method according to Clinical and Laboratory Standards Institute (CLSI document M27-A3),[39] with the following modifications: a final concentration of 1% (v/v) autoclaved Tween 80 (for EOCs)[40] and 2% (v/v) autoclaved DMSO (for antifungal medicines) were incorporated into the SAB medium to enhance chemicals solubility. Then, two-fold dilutions (1000 µg/mL — 2.44 µg/mL for antifungal medicines, and 500 - 0.02 µL/mL for EOCs) were performed. Briefly, each well contained 50 μL of tested EOCs or antifungal medicines and 50 μL of yeast suspension at the final concentration of 1 × 103 - 5 × 103 CFU/mL. All assays were performed in duplicate. The MIC value was estimated after 24 h of incubation at 37 °C in SAB medium. Turbidity was evaluated using a microscope (Olympus Life Science). The lack of growth also confirmed antifungal activity after transferring approx. 10 μL of the sample from the clear wells onto SGC2 medium and incubating for 24 h at 37 °C. To exclude an inhibitory effect of surfactants (Tween 80 and DMSO) on the C. albicans growth, control tests with SAB and SAB supplemented with Tween 80 (1%, v/v) and DMSO (2%, v/v) were performed. Using the known densities of eugenol and trans-anethole, the result of MIC was expressed in mg/mL.

The MFC values of the chemicals were determined by transferring about 20 μL of cultures (culture + tested chemical) in higher or equal to MIC concentrations on a 96-well microplate contained a sterile SAB medium (100 μL) in each well. Then, all plates were incubated for the next 24 h at 37 °C. After this time, the MFC was observed, and the concentration on which transparent SAB medium could be found was Accepted identified.

The MFC/MIC ratio was calculated to analyse whether all tested chemicals have a fungicidal (MFC/MIC <4) or fungistatic (MFC/MIC ≥4) activities.[41]

Checkerboard Method

The checkerboard assay was conducted against the yeast strains as previously described [42] with a slight modification: a final concentration of 1% (v/v) autoclaved Tween 80 (for EOCs) and 2% (v/v) autoclaved DMSO (for antifungal medicines) were incorporated into the SAB medium to enhance chemicals solubility. Finally, each well contained the following compounds: 25 μL of the appropriate concentration of eugenol or trans-anethole, 25 μL of the appropriate antifungal medicine (miconazole or econazole or clotrimazole or nystatin) concentration, and 50 µL of yeast suspension containing a final concentration of 1 × 103 - 5 × 103 CFU/mL in each well. The plates were incubated for 24 h at 37 °C. All assessments were performed in duplicates. The MIC of both EOCs and antifungal medicines in combination was determined as

Chem. Biodiversity described in Section "Determination of Minimum Inhibitory Concentration (MIC) of chemicals". The combined effects of chemicals were calculated and expressed in terms of a fractional inhibitory concentration index (FICI) using the following formula:

FICI = FIC of EOC + FIC of antifungal medicine

Interpretation of results: synergy (FICI < 0.5), addition (0.5 ≤ FICI ≤ 1.0), indifference (1.1 ≤ FICI ≤ 4.0) or antagonism (FICI > 4.0).[42]

Fourier Transform Infrared (FTIR) Spectroscopy Measurements

After the cultivation of these strains at 37 °C for 24 h on SAB agar, the yeast cells were washed three times using PBS (5 mL), centrifuged for 5 min at 5000 rpm, and dried at 37 °C for 24 h. The FTIR spectra of dry samples were received at 21 °C by attenuated total reflection with a FTIR spectrometer (Perkin Elmer Spectrophotometer 100, Waltham, MA, USA). The dry samples (approx. 100 mg) were then scanned (range between 650 cm-1 and 4000 cm-1; 64 scans and 1 cm-1 resolution). Finally, the received spectra were normalized, corrected, and analyzed in detail using software (SPECTRUM v10, Perkin Elmer Spectrophotometer, Waltham, MA, USA).

Statistical Analysis

All data were expressed as mean ± standard deviation (SD).

Author Contribution Statement

M.D.: conceptualization, data curation, investigation, writing-original draft, funding acquisition; H.Z-B.: formal analysis; P.K.; Ł.Ł.; A.P.: data curation, methodology, visualization, writing-original draft; M.K.: investigation, methodology, E.K.: formal analysis M.S.: supervision, writing- original draft, methodology, data curation, formal analysis. All authors have read and agreed to the published version of the manuscript.

References

[1] A. Ahmed, A. Azim, A.K. Baronia, S.K. Marak, M. Guriar, 'Invasive candidiasis in non neutropenic critically ill — need for region-specific management guidelines', Indian J. Crit. Care. Med. 2015, 19(6), 333–339.

[2] M.A. Pfaller, D.J. Diekema, 'Epidemiology of invasive candidiasis: a persistent public health problem', Clin. Microbiol. Rev. 2007, 20, 133– 163.

[3] Sexually Transmitted Diseases Treatment Guidelines, 2015. https://www.cdc.gov/std/tg2015/candidiasis.htm Manuscript [4] A. Bitew, Y. Abebaw, 'Vulvovaginal candidiasis: species distribution of Candida and their antifungal susceptibility pattern', BMC Womens Health 2018, 18, 98. doi: 10.1186/s12905-018-0607-z .

[5] J.S. Berek, 'Berek and Novak's gynecology', 15th ed. Philadelphia: Lipincott, Williams & Wilkins, c2012.

[6] J.M. Achkar, B.C. Fries, 'Candida infection of the genitourinary tract', Clin. Microbiol. 2010, 23, 253-73.

[7] L. Sekhavat, A. Tabatabaii, F.Z. Tezerjani, 'Oral fluconazole 150 mg single dose versus intra-vaginal clotrimazole treatment of acute vulvovaginal candidiasis', J. Infect. Public Health 2011, 4, 95-99.

[8] P.G. Pappas, C.A.Kauffman, D.R. Andes, C.J. Clancy, K.A. Marr, L. Ostrosky-Zeichner, A.C. Reboli, M.G. Schuster, J.A. Vazquez, T.J. Walsh, T.E. Zaoutis, J.D. Sobel, 'Executive summary: clinical practice guideline for the management of Candidiasis: 2016 Update by the Infectious Diseases Society of America' Clin. Infect. Dis. 2016, 62(4), 409–417.

[9] J.D. Sobel, H.C. Wiesenfeld, M. Martens, P. Danna, T.M. Hootno, A. Rompalo, M. Sperling, C. Livengood, B. Horowitz, J. Von Thron, L. Edwards, H. Panzer, T.C. Chu, 'Maintenance fluconazole therapy for recurrent vulvovaginal candidiasis', N. Engl. J. Med. 2004, 351, 876-883. Accepted [10] S.G. Whaley, E.L. Berkow, J.M. Rybak, A.T. Nishimoto, K.S. Barker, P.D. Rogers, 'Azole antifungal resistance in Candida albicans and emerging non-albicans Candida species', Front. Microbiol. 2016, 7, 2173.

[11] S. M. Mandal, A. Roy, A. K. Ghosh, T. K. Hazra, A. Basak, O. L. Franco, 'Challenges and future prospects of antibiotic therapy: from peptides to phages utilization', Front. Pharmacol. 2014, 5, 105.

[12] S. Abe, Y. Sato, S. Inoue, H. Ishibashi, N. Maruyama, T. Takizawa, H. Oshima, H. Yamaguchi, 'Anti-Candida albicans activity of essential oils including lemongrass (Cympopagon citrus) oil and its component citral', Jpn J. Med. Mycol. 2003, 44, 285 – 291.

[13] F. D. Auria, L. Laino, V. Strippoli, M. Tecca, G. Salvatore, L. Battinelli, G. Mazzanti, 'In vitro activity of tea tree oil against Candida albicans mycelia conversion and other pathogenic fungi', J. Chemother. 2001, 13, 377 – 383. 8

Chem. Biodiversity [14] K. A. Hammer, C. F. Carson, T. V. Riley, 'Melaleuca alternifolia (tea tree) oil inhibits germ tube formation by Candida albicans', Med. Mycol. 2000, 38, 355 – 362.

[15] F. D. D’Auria, M. Tecca, V. Strippoli, G. Salvatore, L. Battinelli, G. Mazzanti, ‘Antifungal activity of Lavandula angustifolia essential oil against Candida albicans yeast and mycelial form', Med. Mycol. 2005, 43, 391 – 396.

[16] F. Nazzaro, F. Fratianni, L. De Martino, R. Coppola, V. De Feo, 'Effects of essential oils on pathogenic bacteria', Pharmaceuticals 2013, 6, 1451 – 1474.

[17] D. Pietrella, L. Angiolella, E. Vavala, A. Rachini, F. Mondello, R. Ragno, F. Bistoni, A. Vecchiarelli, ‘Beneficial effect of Mentha suaveolens essential oil in the treatment of vaginal candidiasis assessed by real-time monitoring of infection’, BMC Complement. Altern. Med. 2011, 11, 18.

[18] N. Maruyama, T. Takizawa, H. Ishibashi, T. Hisajima, S. Inouye, H. Yamaguchi, S. Abe, 'Protective activity of geranium oil and its component, geraniol, in combination with vaginal washing against vaginal candidiasis in mice', Biol. Pharm. Bull. 2008, 31(8), 1501 – 1506.

[19] A. Mertas, A. Garbusińska, E. Szliszka, A. Jureczko, M. Kowalska, W. Krol, ‘The Influence of tea tree oil (Melaleuca alternifolia) on fluconazole activity against fluconazole-resistant Candida albicans strains’, Biomed. Res. Int. 2015, 590470. doi: 10.1155/2015/590470.

[20] M. Nejad, H. Ozgunez, N. Basaran, 'Pharmaclogycal and toxicological properties of eugenol', Turk. J. Pharm. Sci. 2017, 14(2), 201-206. doi: 10.4274/tjps.62207.

[21] V. Marinov, S. Valcheva-Kuzmanova, 'Review on the pharmacological activities of anethole', Script. Sci. Pharm. 2008, 2(2), 14-19.

[22] Ł. Łopusiewicz, K. Mazurkiewicz-Zapałowicz, C. Tkaczuk, ‘Chemical changes in spores of the entomopathogenic fungus Metarhizium robertsii after exposure to heavy metals, studied through the use of FTIR spectroscopy’, J. Elem. 2019, 25, 487–499.

[23] L. Potocki, J. Depciuch, E. Kuna, M. Worek, A. Lewińska, M. Wnuk, 'FTIR and Raman Spectroscopy-Based Biochemical Profiling Reflects Genomic Diversity of Clinical Candida Isolates That May Be Useful for Diagnosis and Targeted Therapy of Candidiasis', Int. J. Mol. Sci. 2019, 20, 988.

[24] M. Taha, M. Hassan, S. Essa, Y. Tartor, 'Use of Fourier transform infrared spectroscopy (FTIR) spectroscopy for rapid and accurate identification of yeasts isolated from human and animals', Int. J. Vet. Sci. Med. 2013, 1(1), 15 – 20. Manuscript

[25] W. Mihoubi, E. Sahli, A. Gargouri, C. Amiel, 'FTIR spectroscopy of whole cells for the monitoring of yeast apoptosis mediated by p53 over- expression and its suppression by Nigella sativa extracts', PLoS One 2017, 12(7), e0180680. doi:10.1371/journal.pone.0180680.

[26] P. Kwiatkowski, A. Pruss, B. Wojciuk, B. Dołęgowska, A. Wajs-Bonikowska, M. Sienkiewicz, M. Mężyńska, Ł. Łopusiewicz, 'The influence of essential oil compounds on antibacterial activity of mupirocin-susceptible and induced low-level mupirocin-resistant MRSA strains', Molecules 2019, 24, 3105.

[27] A. Zida, S. Bamba, A. Yacouba, R. Ouedraogo-Traore, R. T. Guiguemde, 'Anti-Candida albicans natural products, sources of new antifungal drugs: A review', J. Mycol. Méd. 2017, 27, 1 – 19.

[28] R. Ben-Ami, 'Treatment of invasive candidiasis: A narrative review', J. Fungi 2018, 4(3), 97. doi: 10.3390/jof4030097.

[29] H. T. Trinh, I. A. Lee, Y. J. Hyun, D. H. Kim, 'Artemisia princeps Pamp. essential oil and its constituents eucalyptol and α-terpineol ameliorate bacterial vaginosis and vulvovaginal candidiasis in mice by inhibiting bacterial growth and NF-κB activation', Planta Med. 2011,

77(18), 1996 – 2002. doi: 10.1055/s-0031-1280094. Accepted

[30] A. Sharifzadeh, H. Shokri, ‘In vitro synergy of eugenol on the antifungal effects of voriconazole against Candida tropicalis and Candida krusei strains isolated from the genital tract of mares’, Equine Vet. J. 2021, 53(1), 94 – 101. doi: 10.1111/evj.13268.

[31] A. Ahmad, A. Khan, L. A. Khan, N. Manzoor, ‘In vitro synergy of eugenol and methyleugenol with fluconazole against clinical Candida isolates’, J. Med. Microbiol. 2010, 59(10), 1178 – 1184. doi: 10.1099/jmm.0.020693-0.

[32] N. Samber, A. Khan, A. Varma, N. Manzoor, ‘Synergistic anti-candidal activity and mode of major components’, Pharm. Biol. 2015, 53 (10), 1496 – 1504. doi: 10.3109/13880209.2014.989623. 9

Chem. Biodiversity [33] B. Latifah-Munirah, W. H. Himratul-Aznita, N. Mohd Zain, ‘Eugenol, an essential oil of clove, causes disruption to the cell wall of Candida albicans (ATCC 14053)’, Front. Life Sci. 2015, 8(3), 231 – 240. doi: 10.1080/21553769.2015.1045628.

[34] WO 2019/015975, ‘Compositions against Candida infections’, PCT/EP20 18/068 192.

[35] P. M. Jenner, E. C. Hagan, J. M. Taylor, E. L. Cook, O. G. Fitzhugh, 'Food flavourings and compounds of related structure I. Acute oral toxicity', Food Cosmet. Toxicol. 1964, 2, 327 – 43.

[36] J. R. Boissier, P. Simon, B. Le Bourhis, 'Experimental psychotropic action of cis and trans isomers of anethole', Therapie 1967, 22, 309 – 23.

[37] S. K. Abraham, 'Anti-genotoxicity of trans-anethole and eugenol in mice', Food Chem. Toxicol. 2001, 39, 493 – 8.

[38] S. Kegley, E. Konlisk, M. Moses, 'Marin Municipal Water District. Herbicide Risk Assessment', Chapter 6—Clove Oil (Eugenol), Draft Final January 1, 2010.

[39] P. A.Wayne, Clinical and Laboratory Standards Institute [CLSI], 'Reference method for broth dilution antifungal susceptibility testing of yeasts; approved standard', 3rd Edn., 2008, CLSI document M27-A3.

[40] D. Kalemba, A. Kunicka, 'Antibacterial and antifungal properties of essential oils', Curr. Med. Chem., 2003, 10, 813.

[41] Z. N. Siddiqui, F. Farooq, T. N. M. Musthafa, A. Ahmad, A. U. Khan, ‘Synthesis, characterization and antimicrobial evaluation of novel halopyrazole derivatives’, J. Saudi Chem. Soc. 2013, 17, 237 – 43.

[42] P. S. Yap, S. H. Lim, C. P. Hu, B. C. Yiap, 'Combination of essential oils and antibiotics reduce antibiotic resistance in plasma-conferred multidrug resistant bacteria', Phytomed. 2013, 20, 710 – 713. Manuscript Accepted

Pobrano z https://publicum.umed.lodz.plThis article is protected / Downloaded by copyright. from AllRepository rights reserved. of Medical University of Lodz 2021-10-07