Manuscript accepted on :September 25, 2017
Published online on: --
Alessandro Leite Cavalcanti1, Yêska Paola Costa Aguiar1, Fábio Gomes Dos Santos1, Alidianne Fabia Cabral Cavalcanti1 and Ricardo Dias De Castro2
1School of Dentistry, State University of Paraiba, Campina Grande, Brazil.
2School of Dentistry, Federal University of Paraiba, Joao Pessoa, Brazil.
Corresponding Author E-mail: firstname.lastname@example.org
Candida albicans is the most prevalent species in fungal infections. Substances contained in plants may have effects similar to synthetic drugs against pathogenic microorganisms. The essential oils (EO) of five plants (Cymbopogon winterianus, Mentha arvensis, Pimpinella anisum, Eucalyptus citriodora and Baccharis trimera) were tested against standard Candida albicans, C. tropicalis and C. krusei strains. The antifungal activity was determined based on the Minimum Inhibitory Concentration (MIC) and Minimum Fungicide Concentration (MFC) performed by the microdilution method using 96-well microplates and sowing in Petri dishes, respectively. EOs presented antifungal activity, with MIC between 625 and 10,000μg / mL, especially EO obtained from Cymbopogon winterianus, which presented better performance, representing a potential natural product with anti-Candida activity.
Candida Albicans; Plants; Medicinal; PhytotherapyDownload this article as:
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Cavalcanti A. L, Aguiar Y. P. C, Santos F. G. D, Cavalcanti A. F. C, Castro R. D. D. Susceptibility of Candida Albicans and Candida Non-Albicans Strains to Essential Oils. Biomed Pharmacol J 2017;10(3).
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Cavalcanti A. L, Aguiar Y. P. C, Santos F. G. D, Cavalcanti A. F. C, Castro R. D. D. Susceptibility of Candida Albicans and Candida Non-Albicans Strains to Essential Oils. Biomed Pharmacol J 2017;10(3). Available from: http://biomedpharmajournal.org/?p=16807
Candidosis is characterized as an opportunistic infection that can affect skin and mucosa, being caused by yeast of the genus Candida, having C. albicans as the main pathogen that affects humans1. Antibiotic therapy with broad spectrum agents, use of corticosteroids, immunosuppression, parenteral nutrition and exposure to invasive medical procedures such as intravascular catheter insertion, hemodialysis and abdominal surgery are considered risk factors for the progressive increase of their frequency2.
Several studies have indicated that although C. albicans is the most prevalent species in fungal infections, there has been an increase in the infection rates by non-albicans species such as C. parapsilosis, C. tropicalis, C. glabrata, and C. krusei3-6. However, the change in the proportion of infections between C. albican and C. non-albican species is still unclear7.
A global public health concern is the increased resistance of bacteria and fungi to antimicrobial drugs8,9 and the increasing number of immunocompromised patients undergoing fungal infections10,11, which are a major cause of morbidity and mortality in the general population6. In this sense, the production of new drugs by the pharmaceutical industry has been stimulated12,13.
Evidence on the biological properties of essential oils and extracts from various plants has led to the search for potentially active compounds as alternative solutions for the treatment of infectious diseases9. Numerous compounds present in plants are capable of promoting protection especially against pathogenic microorganisms12,14. In addition, the enormous production of drugs from biomolecules present in plants is an important economic factor15. Thus, the use of essential oils for the control of yeast growth has gained importance due to the resistance acquired by pathogens to a series of widely used drugs16.
This study aimed to evaluate the antifungal activity of five essential oils against different Candida albicans and Candida non-albicans species.
Materials and Methods
Essential oils of five plant species belonging to five distinct botanical families: Baccharis trimera (Family Asteraceae), Cymbopogon winterianus (Family Poaceae), Eucalyptus citriodora (Family Myrtaceae), Mentha arvensis (Family Lamiaceae) and Pimpinella anisum (Family Apiaceae) were commercially obtained (Quinarí Fragrâncias e Cosméticos Ltd., Ponta Grossa, PR, Brazil).
The antifungal activity was tested against Candida albicans (ATCC 289065), Candida Krusei (ATCC 40042) and Candida tropicalis (ATCC 40147) species. Microorganisms were provided by the Laboratory of Oral Microbiology of the Department of Tropical Medicine – Health Sciences Center – Federal University of Paraíba, Brazil. The storage and viability of strains were obtained by preservation under refrigeration at 4°C and periodic peaks.
Solutions of Essential Oils
The density of each essential oil, calculated from the quotient between weight (in g) and volume (in mL), was verified using a precision digital scale Kern® model PCB 1000- 2 (Ziegelei, Balingen, Germany). Then, an emulsion of oils was obtained in the proportion of 0.4 mL of essential oil, 5 mL sterile distilled water and 0.04 mL TWEEN 8017. The mixtures were homogenized for 5 minutes with the aid of a PHOENIX® Vortex type tube shaker model AP 56 (Araraquara, São Paulo, Brazil).
Determination of Minimum Inhibitory Concentration (MIC) and Minimum Fungicide Concentration (MFC)
The Minimal Inhibitory Concentration (MIC) was determined by microdilution technique13,18 in 96-well plates (ALAMAR®, Diadema, São Paulo, Brazil) divided into eight columns (A to H) and 12 lines. Each column corresponded to an essential oil, columns F, G and H were represented by positive control (Nystatin), sterility control and microbial growth control, respectively. The concentration of plant products ranged from 40,000 μg / mL (line 1) to 19.5 μg / mL (line 12).
Each well was added of 100μL of doubly concentrated Sabouraud-Dextrose broth (DIFICO®, Detroit, MI, USA), 100μL of essential oil emulsion and 10μL of fungal inoculum. Plates were then placed in bacteriological oven at 37°C for 24 hours. MIC was determined by the visual method with the aid of the addition of 10μL of 2, 3, 5 triphenyl chloride tetrazolium dye (Sigma-Aldrich®, St. Louis, MO, USA), where the formation of agglomerates of cells in the well concavity was considered. Thus, the lowest concentration of the test product capable of producing visible inhibition on the growth of yeast strains used in microbiological assays was considered as MIC19.
Aliquots of 10μL corresponding to MIC and the two previous concentrations were sown in Petri dishes containing SD agar medium (DIFICO®, Detroit, MI, USA) in order to obtain the Minimum Fungicide Concentration (MFC). Subsequently, they were incubated for 24 hours in bacteriological oven at 37°C. Concentrations capable of completely preventing microbial growth or less than three colony forming units (CFU) were considered as fungicides. Assays were performed in triplicate.
The susceptibility of strains to the essential oils is presented in Table 1.
Table 1: Minimum Inhibitory Concentration (MIC) and Minimum Fungicide Concentration (MFC) of the test substances against Candida albicans and Candida non-albicans species.
|Essential Oils and Positive Control||C. albicans||C. krusei||C. tropicalis|
The extensive use of antifungal agents may select Candida species that are less sensitive to these substances10. The number of fungal infections by species of the genus Candida has increased and the widespread use of synthetic antifungals seems to be associated with the increased resistance of yeasts to these important agents20.
This panorama supports the conduction of studies aimed to evaluate the antifungal activity of alternative substances, since it is necessary to scientifically investigate plants that have been indicated in traditional medicine to improve the quality of therapies13,18,21,22, as the potential of plants as an alternative in the development of new drugs is still little explored, although substances used in the treatment of various diseases are part of the composition of these plants23.
In this context, the antimicrobial activity of essential oils has been studied by several researchers to evaluate the viability of their use in human models13,14,24-27. However, the observation of the antimicrobial activity of essential oils is influenced by the physicochemical properties of molecules, such as solubility and volatility, which may facilitate or hinder the chemical interaction with their probable pharmacological receptors28.
Based on the results observed in the present study, the essential oil of Cymbopogon winterianus was considered, among those evaluated, as the compound with the highest anti-yeast potential. Regarding the action of the essential oil of Cymbopogon winterianus and Mentha arvensis on Candida albicans species, MICs of 625 μg / mL and 1,250 μg / mL, respectively, have been observed. Other authors also confirmed the anti-candida activity of Cymbopogon winterianus (MIC = 600 μg / mL) and Mentha arvensis (MIC = 1,100 μg / mL), corroborating our findings.
The biological activity of natural products is influenced by their chemical composition, which may present variability due to the use of different methods for collecting the botanical material, plant part, climatic conditions of the collection region, type of technique for extracting the essential oil, and the method used to verify their pharmacological activity.
Although MIC values between 0.6 and 1.5 mg / mL are classified as moderate inhibitory effect29, the results obtained suggest an advance in scientific research in order to identify the chemical compounds responsible for this effect. When evaluated in isolation, they may exhibit potent effects on Candida strains.
From the point of view of the systemic effects of Cymbopogon winterianus, some researchers observed hypotensive and vasorelaxant effect on rat30, but doses of 20 mg / kg induced bradycardia and transient arrhythmia. In another study, essential oil extracted from the leaf of the same species was able to produce effects on the Central Nervous System (CNS) of mice at doses of 25, 50 and 100 mg / kg31.
Mentha arvensis was identified as a plant with antifungal activity that represents an interesting alternative in efforts to combat infectious diseases such as candidiasis32. MFC similar to Nystatin against C. tropicalis presented by the essential oil from this plant found in the present study confirms this condition. Some researchers have isolated Menthol33, also present in Mentha Arvensis, which presented MICs against Candida albicans of 125.0 μg / mL, which suggests that isolated compounds may offer an antimicrobial activity with greater potential, since in the present study, the essential oil of this plant showed MIC for the same species of fungus ten times higher than that observed by that study. Most of the times, isolated substances have less action potential, which justifies the use of extracts, since synergisms among phytoconstituents are considered.
In the present study, the essential oil of Eucalyptus citriodora showed MIC against Candida Krusei, corroborating the results described in previous studies that reported antifungal activity of this essential oil34,35.
Some researchers found susceptibility of C. albicans to the methanolic extract of Pimpinella anisum (MIC = 16mg / mL and MFC = 256mg / mL)36 and also to clinically isolated (MIC = 25mg / mL) C. albicans, C. Glabrata and C. krusei37. Alcoholic extracts obtained from a specific part of the same botanical species may exhibit different biological activities, since the type of solvent used in the extraction procedure also influences the pattern of the chemical composition of the products obtained due to differences in solubility. In the present study, although the MIC and MFC results were considerably higher when compared to the other substances analyzed, Pimpinella anisum presented antifungal action against C. krusei and C. tropicalis.
The antiseptic activity is one of the popular indications of Baccharis trimera38. Monotherpenic and sesquiterpene compounds are the main secondary metabolites present in the essential oils of plants of the genus Baccharis39.
The results of this research pointed that only B. trimera showed no activity against all Candida species studied. However, some researchers who used other parts of the same plant38,40 obtained positive results against some bacteria and fungi, including C. albicans. Therefore, in addition to the analysis of the chemical composition of this oil, it is necessary to verify the susceptibility to other microorganisms, of clinical origin or not, as well as to consider the same part of the investigated plant, making possible comparisons among studies and advancements in the scientific knowledge related to the medicinal properties of plant species.
The essential oil of citronella (Cymbopogon winterianus) is a potential anti-yeast agent that causes oral infections. However, cytotoxicity analyzes and clinical trials are required to ensure its use in humans.
This study was supported by the National Council for Scientific and Technological Development (CNPq) – Fellowship of Research Productivity (PQ).
Conflicts of Interest
The authors declare no conflict of interest.
- Raz-Pasteur A, Ullmann Y, Berdicevsky I. The pathogenesis of Candida infections in a human skin model: scanning electron microscope observations. ISRN Dermatol.; 2011:150642. (2011). doi: 10.5402/2011/150642.
- Pfaller MA, Diekema DJ. Epidemiology of invasive mycoses in North America. Crit Rev Microbiol.; 36(1):1-53. (2010). doi: 10.3109/10408410903241444.
- Trick WE, Fridkin SK, Edwards JR, Hajjeh RA, Gaynes RP; National Nosocomial Infections Surveillance System Hospitals. Secular trend of hospital-acquired candidemia among intensive care unit patients in the United States during 1989-1999. Clin Infect Dis.; 35(5):627-30. (2002).
- Wisplinghoff H, Bischoff T, Tallent SM, Seifert H, Wenzel RP, Edmond MB. Nosocomial bloodstream infections in US hospitals: analysis of 24,179 cases from a prospective nationwide surveillance study. Clin Infect Dis.; 39(3):309-17. (2004).
- Rodríguez D, Almirante B, Cuenca-Estrella M, Rodríguez-Tudela JL, Mensa J, Ayats J, et al. Predictors of candidaemia caused by non-albicans Candida species: results of a population-based surveillance in Barcelona, Spain. Clin Microbiol Infect.; 16(11):1676-82. (2010). doi: 10.1111/j.1469-0691.2010.03208.x.
- Bassetti M, Taramasso L, Nicco E, Molinari MP, Mussap M, Viscoli C. Epidemiology, species distribution, antifungal susceptibility and outcome of nosocomial candidemia in a tertiary care hospital in Italy. PLoS One.; 6(9):e24198. (2011). doi: 10.1371/journal.pone.0024198.
- Morgan J. Global trends in candidemia: review of reports from 1995-2005. Curr Infect Dis Rep.; 7(6):429-39. (2005).
- Levy SB, Marshall B. Antibacterial resistance worldwide: causes, challenges and responses. Nat Med.; 10(12 Suppl):S122-9. (2004).
- Kurdelas RR1, Lima B, Tapia A, Feresin GE, Gonzalez Sierra M, Rodríguez MV, Zacchino S, Enriz RD, Freile ML. Antifungal activity of extracts and prenylated coumarins isolated from Baccharis darwinii Hook & Arn. (Asteraceae). Molecules.; 15(7):4898-907. (2010). doi: 10.3390/molecules15074898.
- Montravers P, Jabbour K. Clinical consequences of resistant Candida infections in intensive care. Int J Antimicrob Agents.; 27(1):1-6. (2006).
- Tempone AG, Sartorelli P, Teixeira D, Prado FO, Calixto IARL, Lorenzi H, Melhem MSC. Brazilian flora extracts as source of novel antileishmanial and antifungal compounds. Mem Inst Oswaldo Cruz.; 103(5):443-9. (2008).
- Silva NCC, Fernandes Junior A. Biological properties of medicinal plants: a review of their antimicrobial activity. J Venom Anim Toxins incl Trop Dis.; 16(3):402-413. (2010).
- Rocha EALSS, Medeiros ACD, Castro RC, Rosalen PL, Saraiva KLA, Godoy GP, Silva LRA, Aleixo CSS, Silva PG, Costa EMMB. Antifungal activity, phytochemical characterization and thermal profile of Anadenanthera colubrina (Vell.) Brenan. Pesq Bras Odontoped Clin Integr.; 17(1): e3389. (2017).
- Kamazeri TS, Samah OA, Taher M, Susanti D, Qaralleh H. Antimicrobial activity and essential oils of Curcuma aeruginosa, Curcuma mangga, and Zingiber cassumunar from Malaysia. Asian Pac J Trop Med.; 5(3):202-9. (2012). doi: 10.1016/S1995-7645(12)60025-X.
- Cardoso FL, Murakami C, Mayworm MAS, Marques LM. Seasonal analysis from the antimicrobial potency and flavonoid and quinone content from Aloe arborescens Mill., Xanthorrhoeaceae, leaf extracts. Rev Bras Farmacogn.; 20(1):35-40. (2010).
- TyagiAK, Malik A. Liquid and vapour-phase antifungal activities of selected essential oils against candida albicans: microscopic observations and chemical characterization of cymbopogoncitratus. BMC Complement Altern Med.; 10:65. (2010). doi: 10.1186/1472-6882-10-65.
- Allegrini J, Bouchberg MS, Mailols H. Emulsions d’huiles essentielles fabrication et applications en microbiologie. Soc Pharm Montpellier.; 33:73-86. (1973).
- Prabuseenivasan S, Jayakumar M, Ignacimuthu S. In vitro antibacterial activity of some plant essential oils. BMC Complement Altern Med.; 6:39. (2006).
- NCCLS. Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically; Approved Standard. 6.th. ed. NCCLS document M7-A6.
- Akortha EE, Nwaugo VO, Chikwe NO. Antifungal resistance among Candida species from patients with genitourinary tract infection isolated in Benin City, Edo state, Nigeria. African J Microbiol Res.; 3(11):694-699. (2009).
- Nóbrega DRM, Santos RL, Soares RSC, Alves PM, Medeiros ACD, Pereira JV. A randomized, controlled clinical trial on the clinical and microbiological efficacy of Punica granatum Linn mouthwash. Pesq Bras Odontoped Clin Integr.; 15(1):301-308. (2015).
- Jahan E, Amiridelui M, Nodehi M. S, Haghighat A. Investigating the effects of Thymus Vulgaris products and clotrimazole lotion to prevent the growth of Candida Albicans Fungus. Biomed Pharmacol J.;8(2). (2015).
- Oliveira WA, Pereira FO, Luna GCDG, Lima IO, Wanderley PA, Lima RB, Lima EO. Antifungal activity of Cymbopogon winterianus jowitt ex bor against Candida albicans. Braz J Microbiol.; 42(2):433-441. (2011).
- Akın M, Saraçoğlu HT, Demirci B, Başer KHC, Küçüködük M. Chemical composition and antibacterial activity of essential oils from different parts of Bupleurum rotundifolium L. Rec Nat Prod.; 6(3):316-320. (2012).
- Angienda PO, Onyango DM, Hill DJ. Potential application of plant essential oils at sub-lethal concentrations under extrinsic conditions that enhance their antimicrobial effectiveness against pathogenic bacteria. African J Microbiol Res.; 4(16):1678-1684. (2010).
- Silva ICG, HBP, Cavalcanti YW, Nonaka CFW, Sousa SA, Castro RD. Antifungal activity of eugenol and its association with nystatin on Candida albicans. Pesq Bras Odontoped Clin Integr.; 17(1): e3235. (2017).
- Ferreira Filho JCC, Gondim BLC, Cunha DA, Figueiredo CC, Valença AMG. Physical properties and antibacterial activity of herbal tinctures of Calendula (Calendula officinalis L.) and Cashew Tree (Anacardium occidentale L.). Pesq Bras Odontoped Clin Integr.; 14(1):49-53. (2014).
- Nascimento GGF, Locatelli J, Freitas PC, Silva GL. Antibacterial activity of plant extracts and phytochemicals on antibiotic-resistant bacteria. Braz J Microbiol.; 31(1):247-256. (2000).
- Aligiannis N, Kalpotzakis E, Mitaku S, Chinou IB. Composition and antimicrobial activity of the essential oils of two Origanum species. J Agric Food Chem.; 49(9):4168-70. (2001).
- de Menezes IA, Moreira IJ, de Paula JW, Blank AF, Antoniolli AR, Quintans-Júnior LJ, Santos MR. Cardiovascular effects induced by Cymbopogon winterianus essential oil in rats: involvement of calcium channels and vagal pathway. J Pharm Pharmacol.; 62(2):215-21. (2010). doi: 10.1211/jpp.62.02.0009.
- Leite BLS, Souza TT, Antoniolli AR, Guimarães AG, Siqueira RS, Quintans JSS, Bonjardim LR, Alves PB, Blank AF, Botelho MA, Almeida JRGS, Lima JT, Araújo AAS, Quintans-Júnior LJ. Volatile constituents and behavioral change induced by Cymbopogonwinterianus leaf essential oil in rodents. African J Biotechnol.; 10(42):8312-8319. (2011).
- Santos KK1, Matias EF, Souza CE, Tintino SR, Braga MF, Guedes GM, Nogueira LF, Morais EC, Costa JG, Menezes IR, Coutinho HD. Anti-Candida activity of Mentha arvensis and Turnera ulmifolia. J Med Food.; 15(3):322-4. (2012). doi: 10.1089/jmf.2011.0128.
- Al-Bayati FA. Isolation and identification of antimicrobial compound from Mentha longifolia L. leaves grown wild in Iraq. Ann Clin Microbiol Antimicrob.; 8:20. (2009). doi: 10.1186/1476-0711-8-20.
- Fiori ACG, Schwan-Estrada KRF, Stangarlin JR, Vida JB, Scapim CA, Cruz MÊS, Pascholati SF. Antifungal activity of leaf extracts and essential oils of some medicinal plants against Didymellabryoniae. J Phytopathol.; 48(7):483-487. (2000).
- Ramezani H, Singh HP, Batish DR, Kohli RK. Antifungal activity of the volatile oil of Eucalyptus citriodora. Fitoterapia.; 73(3):261-262. (2002).
- Yazdani D, Rezazadeh SH, Amin GH, ZainalAbidin MA, Shahnazi S, Jamalifar H. Antifungal activity of dried extracts of Anise (Pimpinellaanisum L.) and Star anise (Illiciumverum Hook. f.) against dermatophyte and saprophyte fungi. J Med Plants.; 8(5):24-29. (2009).
- Darwish RM, Aburjai TA. Antimicrobial activity of some medicinal plants against different Candida species. Jordan J Pharmaceutical Sci.; 4(1):70-80. (2011).
- Davicino R, Mattar MA, Casali YA, Correa SG, Pettenati EM, Micalizz B. Actividad antifungica de extractos de plantas usadas en medicina popular em Argentina. Rev Peru Biol.; 14(2):247-251. (2007).
- Souza SP, Cardoso MG, Souza PE, Guimarães LGL, Andrade J, Mallet ACT, Nelson DL. Baccharis tridentata Vahl essential oil: chemical composition, and antioxidant and fungitoxic activities and morphological characterization of secretory structures by scanning electron microscopy. Rev Bras Plantas Med.; 13(4):456-466. (2011).
- Avancini CAM, Wiest JM, Mundstock E. Bacteriostatic and bactericidal activity of the Baccharis trimera (Less.) D.C. – Compositae decocto, as disinfectant or antisseptic. Arq Bras Med Vet Zootec.; 52(3):230-234. (2000).