CHARACTERIZATION AND ANTIFUNGAL ACTIVITY OF THE ESSENTIAL OIL OF TAGETES MINUTA FRONT OF THE CRYPTOCOCCUS SPP. ISOLATES FROM THE ENVIRONMENT

Jessyka A da Cunhaa*, Leandro P Bolzanc, Viviane P Faustob, Graciele Scheidc, Cecília A Scheerend, Rodrigo G Madalossob, Berta M Heinzmanna, JANICE LGIONGOe, Rodrigo de-A Vaucherb,c*

aPrograma de Pós-Graduação em Farmacologia, Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000, Prédio 21, BR-97105-900, Santa Maria, RS, Brazil, bPrograma de Pós Graduação em Nanociências, Centro Universitário Franciscano, Rua Dos Andradas, 1614, BR-97010-032, Santa Maria, RS, Brazil, cCurso de Biomedicina, Centro Universitário Franciscano, Rua Dos Andradas, 1614, BR-97010-032, Santa Maria, RS, Brazil, dCurso de Farmácia, Universidade Federal de Santa Maria (UFSM), Av. Roraima, 1000, Prédio 21, BR-97105-900, Santa Maria, RS, Brazil, eLaboratório de Tecnologia Farmacêutica, Universidade Regional Integrada do Alto Uruguai e Das Missões/URI, Av. Batista Bonoto Sobrinho, 733, BR-97700-000, Santiago, RS, Brazil
Email: rodvaucher@hotmail.com  

Received: 26 Oct 2015 Revised and Accepted: 25 Jan 2016

ABSTRACT

Objective: This study evaluated the chemical composition and antifungal activity of the essential oil of inflorescences of Tagetes minuta (EOTM) belonging to the Asteraceae family against Cryptococcus spp. This microorganism is the encapsulated yeast-like and is recognized as an opportunistic fungal pathogen of great clinical importance.

Methods: The inflorescences of T. minuta were collected in Itaara/RS, Brazil, in April 2013, and identification of the components was performed by GC-MS. The species of fungi are environmental isolates of Cryptococcus spp. identified by direct examination with India ink, urease test, culture and agar Niger medium canavanine glycine bromothymol blue, and all fungi isolates were confirmed by the use of automated panel MicroScan® Rapid Yeast ID (SIEMENS®). ATCC strains of C. gattii, C. neoformans and C. grubii belonging to the Microbiology Laboratory of the Centro Universitário Franciscano of Santa Maria/RS, Brazil were also used. The antifungal activity of the EOTM was evaluated by microdilution.

Results: Most strains of Cryptococcus spp. were sensitive to EOTM even at low concentrations, except when the microorganism in question was Cryptococcus grubii which the essential oil showed a weak antifungal action.

Conclusion: The EOTM appears as promising in prospecting for new drugs for the treatment of cryptococcosis.

Keywords: Cryptococcosis, Natural products, Antifungal,Marigold

© 2016 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

Cryptococcus spp is an encapsulated yeast fungus widely spread and worldwide recognized as opportunistic pathogenic fungus. Cryptococcus genus includes more than 30 species, among which there are the Cryptococcus neoformans and Cryptococcus gattii, causing more severe clinical manifestations of cryptococcosis [1]. The infection is acquired through inhalation of viable proposals in the environment, affecting the lungs as the primary site of infection, and the spread can lead to meningoencephalitis, a clinical manifestation diagnosed in humans [2]. The detection of the fungus in environmental samples and the antifungal activity of constituents are important towards of decreased resistance of microorganisms over the drugs that are already used for the treatment of cryptococcosis [3]. The therapeutic use of the plant species has been described since the dawn of humanity, even without scientific basis [4]. Among the various plant species of medicinal interest, are included plants of the Asteraceae family, one of the families with the highest number of species between dicotyledonous and being also known as compositae [5]. Tagetes minuta L. belongs to the Asteraceae family and is an aromatic species native of the fields and mountainous regions of South America, although it has spread around the world. This plant species popularly known as marigold are often found growing in areas with loose soil, blooming between October and November. A better quality of the essential oil is obtained when it is produced in temperate regions, where the nights are humid and it's very cold during the growth and flowering plant [6]. Morphologically is an upright plant, sparsely branched, measuring about 1-2 m tall with reproduction by seed only [7]. The plant shows photosensitizers and antiviral activities due to thiophenes. The antimicrobial activity is related to the presence of acyclic monoterpenes and ketones in the constitution. One of the constituents of the plant, E-ocimenone, is responsible for the larvicidal activity of the oil against Aedes aegypti [8]. The decoction is obtained from the whole plant and this vegetal species has antibacterial activity against Gram-positive bacteria such as Staphylococcus aureus and Enterococcus faecium and in the more moderate way on the Gram-negative bacterium Salmonella gallinarum [4]. The essential oil from the aerial parts of T. minuta presented antibacterial activity against Staphylococcus [9]. This study aims to evaluate the chemical composition and antifungal activity of essential oil of T. minuta (EOTM) against the Cryptococcus spp.

The inflorescences of T. minuta were collected in Itaara (The Rio Grande do Sul, Brazil), in april 2013. The species were deposited in the herbarium of the Department of Biology, UFSM as vouchers specimens (N ° HDCF 6400). After that, the inflorescences were subjected to hydro distillation for 3 h using a Clevenger apparatus for essential oil extraction [10]. The identification of the components of EOTM was performed by gas chromatography system coupled to mass spectrometry detector Agilent 7890A and 5975C masses. Analysis parameters: the split mode (1: 100), carrier gas: Helium (1 ml/min); HP5MS capillary column (Hewlett-Packard, 5% phenyl methyl siloxane, 30 m x 0.25 mm x 0.25 mM), temperature program: 40 °C for 4 min, 40-320 °C, 4 °C/min, injector temperature: 250 °C, the interface temperature: 250 °C, the ionization energy: 70 eV.

The OE components were identified based on retention indices determined by the use of an n-alkane calibration curve injected under the same conditions and the samples based on mass spectrum [11, 12]. The quantification of the components was performed using Agilent 7890A with a flame ionization detector in accordance with the parameters mentioned above, except for the following: the splitless mode; injector and detector temperature: 300 °C. The species of fungi come from environmental isolates of Cryptococcus spp. (14.1, 29.1, 15.1, 13.1, 7.1, 15, 44, 45.4, 13.2) and strains of C. gattii (ATCC 56990), C. grubii (ATCC 208821) and C. neoformans (ATCC 28957) stored at the Microbiology Laboratory of the Centro Universitário Franciscano of Santa Maria/RS, Brazil. These samples were subcultured on Sabouraud dextrose agar for 24 h at 37 °C for the identification and determination of minimum inhibitory concentration through the serial microdilution technique. Direct examination of the colonies was performed using a blade containing a heave of the colony, an India ink drop and overlapping a coverslip, and observed by microscopy in a 40x objective. The presence of the yeast with regular shapes, globular, round, with or without encapsulated bud and the presence of Cryptococcus spp, were evaluated [1]. Identification of the isolates was obtained by using physiological methods and biochemical test. The suggestive colonies of Cryptococcus spp. were picked in Agar urea and Agar Niger, respectively, to evaluate the action of urease and phenol oxidase, and incubated at 30 °C for 48 h. For biochemical tests the automated MicroScan® Rapid Yeast ID panel (SIEMENS®) was used. The system displays a list of microorganisms and their relative probability of identification based on a number of biotypes formed by nine digits. All the methodology used in this system, with regard to technical and interpretation of results, was made systematically according to the manufacturer's recommendations (SIEMENS®). The microdilution technique was performed as recommended by the CLSI M27-A3 protocol [12]. In 96-well plate were inoculated into each well 100 µl of RPMI-1640 supplemented with MOPS. Then, 100 µl EOTM was pipetted in the first well and a serial dilution to the proportion 1:64 was performed. After dilution, 10 µl of the standardized inoculum over the range of 0.5 McFarland (1.5 X 108 CFU/ml) was added to all wells. This test was performed in triplicate. Plates were incubated for 48 h and the reading was made microscopically for the presence or absence of pits in yeast. However, due to the viscosity of the EOTM it was seeded all proportions (1:2 to 1:64) on Sabouraud agar, and the plates incubated at 37 °C for 48 h to confirm the result shown in microscopy. Positive and negative controls were performed in the test.In the chemical composition of the EOTM were qualified and quantified ten constituents, being identified about 91% of the sample as shown in table 1.


Table 1: Chemical identification the essential oil of inflorescences of Tagetes minuta

Constituent

Retention time

Calculated kovats index

Tabulated kovats index

Source

Percentage (%)

E-Tagetone

21.67

1159

1152

Adams

40.37

Z-Tagetone

20.91

1138

1138

NIST

29.50

Dihydro Tagetone

17.70

1053

1053

NIST

7.57

Ocimene

17.093

1037

1044

NIST

6.11

Limonene

16.68

1026

1027

NIST

3.99

E-caryophyllene

30.47

1419

1421

Adams

0.86

Octanal

15.65

999

1001

NIST

0.38

a-pinene

13.15

937

939

NIST

0.32

a-humulene

31.59

1455

1451

NIST

0.40

According to the literature, this plant species is rich in essential oil and has with main components: ß-phellandrene, limonene, ß-ocimene, dihydro tagetone, tagetone and tagetenone [13, 14] as evidenced on the study results by GC-MS. There are a lot of reports in the literature about in vitro evaluation of antimicrobial, acaricide, antioxidant, antiseptic, insect repellent and nematicides activity of T. minuta, still showing antimicrobial activity with marked selectivity for Gram-positive bacteria such as Staphylococcus aureus and Enterococcus faecium, and more moderate way on the Gram-negative bacterium Salmonella gallinarum [4]. In addition to these aforementioned properties, the EOTM is used in the perfumery and food industry market, including alcoholic beverages, dairy frozen, sweets, cakes, cookies, jellies, puddings, and condiments. In direct examination performed with India ink, all the environmental isolates showed positive results for Cryptococcus spp. For urease activity, urea was hydrolyzed after 48 h incubation in urea agar. The pH change was observed when the color of the medium changed from orange to pink. The species differentiation tests observed that in Niger agar the samples showed beige, light colored colonies, and 4% samples showed an activity of phenoloxidase, visualized by the formation of brown pigmentation in the colonies. Environmental isolates 29.1 and 45.4 were identified as C. neoformans. In chemical characterization comprising the use of L-canavanine medium glycine bromothymol blue, the results were analyzed according to the change of pH of the yellow to blue, showing that all other analyzed environmental isolates were identified as C. gattii.

The results obtained for biochemical tests in automated ID Rapid Yeast Micro Scan® panel (SIEMENS®) confirmed the presence of two strains of C. neoformans and seven strains characterized as C. gattii. In this study, environmental isolates Cryptococcus spp. mostly presented susceptible to EOTM, with MIC less than or equal to 3.22±0.18 µg/ml of the value shown by the negative control, Amphotericin B (a widely used drug for the treatment of various fungal infections). Among the nine strains of Cryptococcus spp., only the strain ATCC Cryptococcus grubii (ATCC 208821) was resistant compared to the other strains and the negative control, with MIC of 51.4±1.89 µg/ml. However, all MIC essential oil compared to the microorganisms tested showed lower results than the MIC presented by the positive control that was carried out with the medium itself (RPMI-1640) in which was inoculated only the fungus as shown in table 2.


Table 2: Minimum inhibitory concentration (MIC) of the essential oil of inflorescences of Tagetes minuta against 12 pathogenic fungi estimated by the microdilution method

Microorganism

MIC (µg/ml)*

C. neoformans

6.43±0.08

C. grubii

51.4±1.89

C. gattii

6.43±0.67

15.1 (C. gattii)

3.22±0.03

15 (C. gattii)

3.22±0.01

44(C. gattii)

6.43±0.05

14.1(C. gattii)

3.22±0.05

7.1 (C. gattii)

1.28±0.06

29.2 (C. neoformans)

1.28±0.08

13.1(C. gattii)

3.22±0.08

13.2 (C. gattii)

3.22±0.07

45.4 (C. neoformans)

3.22±0.05

Amphotericin B

3.22±0.18

Positive Control

411.5±0.14

*The results were expressed as mean±SEM; n=3.


The data obtained in this study indicate that EOTM is effective against Cryptococcus spp. Essential oils can be a good option to treating since they are renewable and less harmful to humans. Information regarding the interaction and to address the effect of the EOTM in strains of Cryptococcus spp. were not found in the literature reviewed. However, a recent study has associated an excellent antifungal property to the EO of the vegetal species Satureja calamintha nepeta against the fungi Fusarium spp. and Aspergillus spp [15], showing that EO obtained from native plants are a promising alternative to combat pathogenic fungi. In this study, it was possible to conclude that most of the environmentally isolated strains of Cryptococcus spp. were sensitive to the EOTM even at low concentrations, except against C. grubii, where the OE showed a weaker action compared to other strains. Thus, the EOTMis presented as promising in prospecting of new drugs for the treatment of cryptococcosis.

Acknowledgement

The authors acknowledge the financial support of CNPq and CAPES, Brazil.

CONFLICTS OF INTERESTS

Declared none

References

  1. Cotin JT, Quaresma GS, Silva EF, Linardi VR. Ocorrência de Cryptococcus neofomans em fezes de pombos na cidade de caratinga, MG-Brasil. Rev Assoc Med Minas Gerais 2010;21:24-19.
  2. Gomes FS, Sarmento DN, Santo EPTE, Silvia HMS. Quimiotipagem e caracterização fenotípica de Cryptococcus isolados em Belém, Estado do Pará, Brasil. Rev Pan-Amaz Saúde 2010;1:49-43.
  3. Lacaz CS, Porto E, Martins JEC. Tratamento de micologia médica. São Paulo; 2002.
  4. Souza CAS, Avancini CAM, Wiest JM. Antimicrobial activity of Tagetes minuta L.–Compositae (Chinchilho) against gram-positive and gram-negative bacteria. Brazilian J Veterinary Res Animal Sci 2000;37.
  5. Boldrini II, Eggers L. Vegetação campestre do sul do Brasil: dinâmica de espécies à exclusão do gado. Acta Botanica Brasilica 1996;10:50-37.
  6. Letizia CS, Api AM. Plantas medicinais: cura segura? química nova (Impresso). Toxicologist 2000;54:397.
  7. Beserra EB, Ferdandes CRM, Queiroga MFC, Castro FPJ. Resistance of Aedes aegypti (L.) (Diptera: Culicidae) populations to organophosphates temephos in the paraíba state, Brazil. Neotrop Entomol 2007;6:307-303. 
  8. Maradufu AR, Lubeya DF. Isolation of (5E) ocimenone, a mosquito larvicide from Tagetes minuta. Lloydia 1998;41:183-1.
  9. Farmacopéia Brasileira. 4th Ed. Parte I. São Paulo: Atheneu; 1988.
  10. Adams RP. Identification of essential oil component by chromatography/mass spectroscopy. Allured Publishing Co., Carol Stream; 2007.
  11. NIST. Special Publication Technical Guide to Information Security Testing and Assessment. Gaithersburg, MD; 2008.
  12. Clinical and laboratory standards institute. Method for broth dilution antifungal susceptibility testing of yeasts: approved standard. 3rd. Edition. M27-A3. CLSI: Wayne, PA, USA; 2008.
  13. Andreotti R, Guerrero FD, Soares MA, Barros JC, Miller RJ, León AP. Acaricide resistance of Rhipicephalus (Boophilus) microplus in State of mato grosso does sul, Brazil. Rev Bras Parasitol Vet 2011;20:133-27.
  14. Veiga VF, Pinto A, Maciel MAM. Plantas medicinais: cura segura? Quim Nova (Impresso) 2005;28:528-19. 
  15. Labiod R, Aouadi S, Bouhaddouda N. Chemical composition and antifungal activity of essential oil from Satureja calamintha nepeta against phytopathogens fungi. Int J Pharm Pharm Sci 2015;7:208-11.