Ana Paula 2018 - Antifungal activity study of the monoterpene

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Natural Product Research
Formerly Natural Product Letters
ISSN: 1478-6419 (Print) 1478-6427 (Online) Journal homepage: https://www.tandfonline.com/loi/gnpl20
Antifungal activity study of the monoterpene
thymol against Cryptococcus neoformans
Anna Paula de Castro Teixeira, Rafaela de Oliveira Nóbrega, Edeltrudes de
Oliveira Lima, Wylly de Oliveira Araújo & Igara de Oliveira Lima
To cite this article: Anna Paula de Castro Teixeira, Rafaela de Oliveira Nóbrega, Edeltrudes de
Oliveira Lima, Wylly de Oliveira Araújo & Igara de Oliveira Lima (2018): Antifungal activity study
of the monoterpene thymol against Cryptococcus�neoformans, Natural Product Research, DOI:
10.1080/14786419.2018.1547296
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Published online: 25 Dec 2018.
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SHORT COMMUNICATION
Antifungal activity study of the monoterpene thymol
against Cryptococcus neoformans
Anna Paula de Castro Teixeiraa, Rafaela de Oliveira N�obregaa,
Edeltrudes de Oliveira Limab, Wylly de Oliveira Ara�ujoa and
Igara de Oliveira Limaa
aDepartament of Pharmacy of Health Academic Unit, Federal University of Campina Grande, Cuit�e,
Brazil; bDepartment of Pharmaceutical Sciences, Federal University of Para�ıba, Jo~ao Pessoa, Brazil
ABSTRACT
Cryptococcus neoformans is a yeast fungus, which causes cryptococ-
cosis, triggered by basidiospore inhalation and consequent dissem-
ination to the central nervous system. In this study, we analyzed
the antifungal action of thymol against 10 clinical strains of C. neo-
formans and analyzed the interaction of this monoterpene with
sterols. The MICs of thymol ranged from 20 to 51lg/ml, while the
MFC values varied between 40 and 101lg/ml. For the strains
ICB-2601 and LM-39, in the presence of ergosterol, the MIC of thy-
mol was 64lg/ml, and in the presence of cholesterol, its MIC was
32lg/ml. Based on the results, thymol presents antifungal action
and seems to interact with ergosterol, but not with cholesterol.
Complementary studies are needed to analyze its full effects.
ARTICLE HISTORY
Received 26 September 2018
Accepted 2 November 2018
KEYWORDS
Cryptococcus neoformans;
microdilution; phytocon-
stituent; terpene; thymol
1. Introduction
Cryptococcus neoformans is a cosmopolitan and opportunistic yeast, present in pigeon
excrement (for being rich in urea and creatine), and remaining viable for many years
CONTACT Anna Paula de Castro Teixeira annapaula.1993@gmail.com
Supplemental data for this article can be accessed at https://doi.org/10.1080/14786419.2018.1547296
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NATURAL PRODUCT RESEARCH
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(El-Fane et al. 2015). It is an etiological agent of cryptococcosis, a systemic infection
that manifests clinically as subacute to chronic meningoencephalitis in immunocom-
promised patients, and in domestic mammals such as dogs and cats (Barnett 2010).
The treatment of choice is administration of amphotericin B in combination with
flucytosine (Chastain, Henao-Mart�ınez, Franco-Paredes 2017). However, the high inci-
dence of the disease, antifungal resistance, and the side effects caused by these drugs
has encouraged researchers to analyze new molecules seeking alternatives for treat-
ment of the disease (Perfect et al. 2010; Belato et al. 2018). In this context, researchers
analyze molecules isolated from essential plant oils such as the terpenes. Thymol, pre-
sent in the essential oil of Thymus vulgaris (Laminaceae) has been noted. Thymol is a
small hydrophilic molecule with a neutral pH and is soluble in alcohol and other
organic solvents (S�anchez et al. 2009). In a study conducted by Lima et al. (2017), thy-
mol demonstrated bacteriostatic action against Escherichia coli. There are also reports
in the literature of thymol’s biological action as an antifungal, an antioxidant and an
antiseptic; making the phytoconstituent a promising molecule (Belato et al. 2018).
Facing the need to search for new antifungal drugs as an alternative treatment for
cryptococcosis, our objective was to investigate the antifungal activity of thymol
against C. neoformans.
2. Results and discussion
Table S1 presents the minimum inhibitory concentration (MIC) results for thymol and
a standard antifungal, amphotericin B, against strains of C. neoformans using serial
microdilution technique in broth (CLSI 2002; Cleeland and Squires 1991; Eloff 1998).
Belato et al. (2018) determined thymol’s MIC against C. albicans, S. aureus and S.
mutans using the microdilution technique, in which the MIC was 10lg/ml for C. albi-
cans, and 160lg/ml for S. aureus and S. mutans; presenting satisfactory results. Dantas
et al. (2015) analyzed thymol against strains of Penicillium using microdilution tech-
nique and obtained a discreet superiority of activity when compared to Amphotericin
B, since the MIC values were the same for both substances (160 lg/mL).
Minimum fungicide concentration (MFC) analyses were also performed; understood as
the lowest concentration sufficient to reduce colony forming units (CFU) by 99.9% (Ernst
et al. 1996; Klepser et al. 1998; Ernst et al. 1999; Cant�on et al. 2003). The MFC of thymol
was measured at twice its respective MIC values, except for the LM-120 and LM-310
strains which were measured at 4x their respective MIC values, as shown in Table S1.
Comparing thymol in this study to Carvacrol against strains of C. neoformans, (MFCs) in
a study by Nobrega et al. (2016), the results were similar (thymol varied between 40
and 102lg/ml and Carvacrol from 25 to 101lg/ml) possibly due to isomerism of these
molecules regarding the position of the hydroxyl group on the phenolic ring.
In accordance with the methodology of Escalante et al. (2008) and Lima et al.
(2013), respectively in Table S2, the next stage of the study explored the mode of
action of the phytoconstituent thymol, against C. neoformans LM-39 and ICB-2601
(clinical strains) to verify interactions of thymol with exogenous ergosterol (which
constitutes the fungal membrane), or with exogenous cholesterol (signaling for pos-
sible toxicity).
2 A. P. DE CASTRO TEIXEIRA ET AL.
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The presence of exogenous ergosterol doubled the MIC of thymol, indicating the dis-
crete affinity of this phytoconstituent to ergosterol. Corroborating this result, a study
carried out by Mota et al. (2012) against Rhizopus oryzae strains; thymol in the presence
of exogenous ergosterol presented augmented MIC values (8x), when compared to the
absence of ergosterol. The addition of cholesterol to the culture medium did not inter-
fere with thymol’s MIC in this study (Table S2). Thymol probably does not interact withcholesterol, suggesting that thymol toxicity is not be related to this interaction.
The antifungal activity of thymol may be associated with increased cellular perme-
ability due to membrane phospholipid attacks which cause cellular lysis (Emiroglu
et al. 2010). The exogenous ergosterol and cholesterol tests were carried out with the
antifungal amphotericin B, a standard control (Table S2). Amphotericin B acts on the
cell due to sterol affinity, having an affinity for fungal membrane, and interacting with
ergosterol (Cuenca-Estrella 2010), and cholesterol, thus forming transmembrane pores
and leaking intracellular content necessary for cellular maintenance, thus causing
apoptosis (Carraro et al. 2014).
Although cholesterol and ergosterol are similar molecules, ergosterol has an add-
itional methyl group, with a double bond in the lateral chain, and in the steroid
nucleus; the very short amphotericin B molecules (monomers) present little interaction
with cholesterol. Administration of this drug in lipid and liposome formulations in
patients maintained under prolonged treatment, minimizes interactions with choles-
terol and consequent toxicity (Huang et al. 2002; Denning and Hope 2010).
4. Conclusion
According to our results, thymol inhibits the growth of Cryptococcus neoformans and
under the evaluated conditions may be involved in complexation with ergosterol, yet
more studies must be carried out to characterize the complete effects of this drug.
Disclosure statement
No conflict of interest was reported by the authors.
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4 A. P. DE CASTRO TEIXEIRA ET AL.
	Abstract
	Introduction
	Results and discussion
	Conclusion
	Disclosure statement
	References