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Acta Odontológica Latinoamericana

versión On-line ISSN 1852-4834

Acta odontol. latinoam. vol.25 no.3 Buenos Aires dic. 2012

 

ARTÍCULOS ORIGINALES

In vitro activity of Schinus terebinthifolius (Brazilian pepper tree) on Candida tropicalis growth and cell wall formation

 

Lívia A. Alves1, Irlan de A. Freires2, Tricia M. P. A. de Souza3, Ricardo D. de Castro4

1 Department of Oral Diagnosis, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil.
2 Department of Physiological Sciences, Piracicaba Dental School, State University of Campinas, Piracicaba, SP, Brazil.
3 Department of Comprehensive Dentistry, Federal University of Pernambuco, Recife, PE, Brazil.
4 Department of Clinics and Social Dentistry, School of Dentistry, Federal University of Paraiba, PB, Brazil.

CORRESPONDENCE Irlan de Almeida Freires 475 Cajazeiras St., Apt. 102, Coral Gables, Manaira, Joao Pessoa, Paraiba, 58038-040, Brazil e-mail: irlan.almeida@gmail.com


ABSTRACT

The aim of this study was to evaluate the in vitro antifungal activity of Schinus terebinthifolius (Brazilian pepper tree) tincture on planktonic Candida tropicalis (ATCC 40042), which is a microorganism associated to oral cavity infections. Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) were determined through the microdilution technique. Possible action of the tincture on fungal cell wall formation was also studied by adding an osmotic protector (0.8M sorbitol) to the microplates. Nystatin was used as standard control and tests were performed in triplicate. S. terebinthifolius was found to have MIC and MFC values of 625 μg/mL on the strain assayed, whereas nystatin showed MIC and MFC of 6.25 μg/mL. Results suggest that S. terebinthifolius tincture acts on fungal cell walls, since the sorbitol test indicated a MIC of 1.250 μg/mL. It may be concluded that S. terebinthifolius has potential in vitro antifungal activity against C. tropicalis strains, and probably acts by inhibiting fungal cell wall formation.

Key-words: Candida tropicalis; Anacardiaceae; Natural products.

RESUMO

Atividade in vitro de Schinus terebinthifolius (aroeira) sobre o crescimento e formação da parede celular de Candida tropicalis

O objetivo deste estudo foi avaliar in vitro a atividade antifungica da tintura de Schinus terebinthifolius (aroeira) sobre Candida tropicalis (ATCC 40042) planctonica, que e um microrganismo associado a infeccoes na cavidade oral. A Concentracao Inibitoria Minima (MIC) e a Concentracao Fungicida Minima (CFM) foram determinadas por meio da tecnica de microdiluicao. Tambem foi investigada uma possivel acao da tintura na formacao da parede celular de fungos, adicionando-se um protetor osmotico (sorbitol 0,8M) nas microplacas. Nistatina foi utilizada como controle e os testes foram realizados em triplicata. A tintura de S. terebinthifolius apresentou MIC e MFC de 625 μg/mL sobre a cepa ensaiada, enquanto que a nistatina mostrou MIC e MFC de 6,25 μg/mL. Os resultados sugerem que S. terebinthifolius atua sobre a parede celular fungica, uma vez que o teste com sorbitol indicou uma MIC de 1.250 μg/mL. Pode-se concluir que S. terebinthifolius apresentou, in vitro, atividade antifungica potencial contra a cepa de C. tropicalis, e provavelmente atua inibindo a formacao da parede celular fungica.

Palavras-chave: Candida tropicalis; Anacardiaceae; Produtos biologicos.


 

INTRODUCTION

Candida species are commonly found in the oral cavity of healthy individuals. These yeasts usually inhabit the host as saprophytic microorganisms, establishing an ecological balance in the oral microbiota. Changes in such balance and the presence of predisposing factors result in the disruption of organic integrity and the modification of the yeastlike conformation into a fusiform shape, which is pathogenic and causes candidiasis1-3. Oral candidiasis may be caused by different species of the genus Candida, including C. albicans, C. tropicalis, C. glabrata, C. krusei, C. parapsilosis, and C. guilliermondii4.
Mycotic studies have drawn attention to the increasing importance of non-albicans species, especially C. tropicalis, as a pathogenic microorganism that is responsible for several diseases in human beings. C. tropicalis has been described as the major cause of candidiasis induced by non-albicans species, and has frequently been found in samples from the oral cavity of HIV+ patients, showing oropharyngeal candidiasis in 18% of patients wearing prosthesis, but present in healthy individuals as well5. Considering the group of non-albicans species, C. tropicalis has become an emerging worldwide pathogen. The major factors responsible for such emergence are: (1) increasing use of antifungal agents; (2) increasing number of immunocompromised individuals; (3) prolonged use of catheters; (4) use of broad-spectrum antibiotics; and (5) complications in the treatment of subclinical underlying conditions along with intolerance to antifungal drugs. In addition, drug resistance in immunocompromised patients who survive longer has been increasing together with mortality rate6. Taking into account the resistance of Candida genus yeasts to current antifungal agents, it may be inferred that studies searching for new plant antifungal compounds are increasingly relevant. It is possible to observe the antibiotic potential of plant species, and consequently, the feasibility of applying them to the prevention and treatment of fungal diseases7.
Schinus terebinthifolius
is a small to medium-sized tree widely present in many continents, including America, Africa and Oceania8. It is native to South America, especially Brazil, Paraguay and Argentina, and commonly known as beach-, white- or redpepper tree, among other designations. It belongs to Anacardiaceae family and is well known for its astringent, antiseptic and anti-inflammatory activities according to folk medicine and use in cosmetic formulations9. The Brazilian pepper tree has also shown an antiulcer effect, and is used as an antiseptic and in the treatment of stomatitis. Those properties are owed to its stabilizing effect on the membrane and anticholinergic and anti-histaminic action10. The aim of this study is to evaluate the antifungal activity of S. terebinthifolius tincture (Brazilian pepper tree) on Candida tropicalis, establishing a possible mechanism of antimicrobial action.

MATERIALS AND METHODS

Research Center
Microbiological assays were performed at the Oral Microbiology Laboratory of the Nucleus of Tropical Medicine, Center for Health Sciences, Federal University of Paraiba, Joao Pessoa, Brazil.

Fungal strain and preparation of inoculum
The Candida tropicalis strain (ATCC 40042) was provided by the National Institute of Quality Control in Health at Oswaldo Cruz Foundation (NIQCH). The yeast was routinely grown in Sabouraud dextrose agar plates incubated at 35oC for 24h from stock cultures in SDB-glycerol and stored at 4oC during the procedures. In the antimicrobial trial, fungal inoculum was standardized to approximately 5.0 x 106 CFU/ml according to the 0.5 McFarland turbidity range (CLSI Clinical and Laboratory Standards Institute - NCCLS).

Study product
The study product was a tincture from the stem bark of Schinus terebinthifolius, which was provided by a compounding pharmacy in Joao Pessoa, Paraiba, Brazil. The product, detailed in Table 1, met all required specifications concerning quality control according to the technical survey provided by the supplier.

Table 1: Characterization of the experimental product.

Determination of the Minimum Inhibitory Concentration (MIC)
The MIC determination of the S. terebinthifolius tincture was performed by the microdilution technique proposed by Ellof (1998)11, using 96-well Ubottom microtiter plates (ALAMARR, Diadema, Sao Paulo, Brazil). Initially, 100 μL of Sabouraud Dextrose Broth (SDB, HIMEDIAR, Sao Paulo, Brazil) doubly concentrated were placed in each well on the plates. Then 100 μL of the S. terebinthifolius tincture were added at an initial concentration of 5,000 μg/mL. Serial dilutions were prepared from these concentrations by taking 100 μL from the most concentrated well and placing it in the following well. Concentrations then ranged from
5,000 μg/mL to 39 μg/mL. Finally, 10 μL aliquots of inoculum were placed into the wells of each column. At the same time, yeast viability was checked and yeast susceptibility verified by using powdered nystatin at an initial concentration of 100 μg/mL, ranging up to 0.78 μL/mL.
Tests were performed in triplicate, and the plates were incubated at 35°C for 48 hours. The MIC of the tincture on the yeast strain was determined visually. The formation or non-formation of cell clusters ("buttons") at the bottoms of the wells was considered. MIC was taken as the lowest concentration of the study product capable of producing visible inhibition of the growth of the yeast strain used in the microbiological assay11.

Determining Minimum Fungicidal Concentration (MFC)
After the MIC determination, the inhibitory and two following higher concentrations as well as the positive controls were sub-cultured on plates containing Sabouraud Dextrose Agar, in triplicate. The MFC readings were based on the growth of the controls after 24 hours incubation at 35°C. MFC was taken as the lowest drug concentration that hindered visible growth of the sub-culture.

Effect of S. terebinthifolius on fungal cell wall
MIC of S. terebinthifolius tincture in presence of sorbitol (0.8M) was determined using the microdilution technique11, in triplicate. To each well, we added 100 μL SDB previously supplemented with sorbitol, with molecular weight 132.17 g (Compounding pharmacy, Joao Pessoa, Paraiba, Brazil), both doubly concentrated. Subsequently, 100 μL of the tincture were also placed to the wells in the first row of the plate. Through a serial dilution at a ratio of two, concentrations were obtained ranging from 5,000 μL/mL to 39 μL/mL of the S. terebinthifolius tincture and, in relation to sorbitol, a final concentration of 0.8 M was obtained in each well. Finally, 10 μL of inoculum were added to each well. Microorganisms were controlled by placing 100 μL of doubly concentrated DSB plus sorbitol (0.8M) and 10 μL of inoculum in the wells. Sterility was controlled by using the same culture medium and sorbitol without fungal suspension. Plates were incubated at 35o C for 48h, after which they were read12. Data were tabulated and analyzed using descriptive statistics.

RESULTS

Table 2 shows the MIC and MFC of S. terebinthifolius tincture on Candida tropicalis. MIC was considered as the lowest product concentration capable of inhibiting fungal growth, whereas MFC was the lowest concentration capable of eliminating the fungus.

Table 2: Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC) in μg/mL of the Schinus terebinthifolius (Brazilian pepper tree) tincture on Candida tropicalis. Values are expressed in μg/mL.

Table 3 shows the MIC values of the S. terebinthifolius tincture in presence or absence of sorbitol (osmotic protector/O.8M) on C. tropicalis. This study shows S. terebinthifolius might act upon fungal cell wall synthesis.

Table 3: Action of Schinus terebinthifolius tincture on C. tropicalis cell wall. Values are expressed in μg/mL.

DISCUSSION

Plants represent a substantial source of biologically active natural products, many of which have been used as models for the synthesis of a number of drugs. In Brazil, there are about 100,000 plant species cataloged, but only 8% have been studied chemically, and it is estimated that only 1,100 species have been assessed as regards therapeutic properties13. Traditional, folk or popular usages are not enough for ethical validation of medicinal plants as safe and effective drugs, i.e., medicinal plants do not differ from any other synthetic xenobiotic. Their recommendation or the official authorization for use must be based on experimental evidence, which attests that the risks to which users are exposed are outweighed by the expected benefits. Their use must be previously validated, i.e., their expected action must be proven and their potential toxicity to humans must be evaluated, as required for any drug14.
Brazil has been involved in establishing guidelines for using medicinal plants or drugs, meeting safety requirements and considering the policy established for the rational use of drugs15. The findings of our research are of great importance because the Candida species evaluated has been indicated as pathogenic in cases of oral candidiasis, especially in immunocompromised patients16,17. The susceptibility of strains of C. albicans, C. tropicalis and C. krusei to nystatin was evaluated in the study by Alves and Cury (1992)18. They found MIC and MFC values ranging from 0.5 to 8.0 μg/mL and 8 to 64.0 μg/mL, respectively. These data were similar to what was found in this study, wherein nystatin had MIC and MFC values of 6.25 μg/mL against C. tropicalis. Freires et al. (2011)19 studied the action of 10% S. terebinthifolius tincture on C. tropicalis by means of the disc diffusion test. They found mean zones of inhibition measuring 25.32 mm promoted by the tincture, compared to 33.32 mm corresponding to the control (nystatin). A study by Alves et al. (2009)20 using the agar diffusion method found in vitro antifungal activity of S. terebinthifolius hydroalcoholic extract against C. albicans, with MIC of 1:8. On C. tropicalis and C. krusei, MIC was 1:16. Nevertheless, in the abovementioned studies19,20 antifungal activity was assayed by solid medium diffusion method, which differs from this study, in which microdilution was used, hampering comparisons between studies. The hydroalcoholic extract of Brazilian pepper was found to have a healing effect on cystotomies in rats21. Soares et al. (2007)22 demonstrated effective in vitro action of the tincture at 20% in the decontamination of toothbrushes contaminated with Streptococcus mutans. These findings highlight the anti-inflammatory and antimicrobial properties of S. terebinthifolius established in the literature.
A recent study23 evaluated the anti-inflammatory and healing effects of the hydroalcoholic extract of S. terebinthifolius at 30% orabase. To do so, 60 Wistar rats were submitted to electrically produced sores on the skin and back. By means of macroscopic and microscopic examination, the authors showed that if the product was used daily, it had both anti-inflammatory and healing activity. MIC values of the S. terebinthifolius tincture in presence or absence of sorbitol against C. tropicalis were also recorded, suggesting that sorbitol protects cells from the tincture's inhibitory effects, once changes occurred in the MIC of the product under evaluation. The control using sorbitol guaranteed the reliability of the results and methods employed since the strain was capable of growing in presence of sorbitol and absence of the experimental product. These outcomes suggest that the antifungal activity of S. terebinthifolius tincture somehow involves its direct interaction with the fungal cell wall. The test with sorbitol performed in this study, is based on the extent and capacity of the damage that substances with antifungal activity have on the components of the fungal cell wall. If the product acts somehow on fungal cell wall, it will cause cellular lysis in absence of an osmotic stabilizer. This assay therefore compares the MIC of antifungal agents in absence and presence of 0.8 M sorbitol, which is, as mentioned above, an osmotic protector used to stabilize fungal protoplasts24.
According to findings of this research, S. terebinthifolius may have a mechanism of action on fungal cell wall, since tincture MIC was 625 μg/mL in absence of sorbitol, and 1,250 μg/mL in its presence, proving that S. terebinthifolius activity was reduced in presence of sorbitol, which hampered the effect of the test product against fungal cell wall. Protection with sorbitol is a test that has a wide range of possibilities, detecting not only agents which interfere with the synthesis of cell wall polymers and surrounding areas, but also regulatory mechanisms involved in these processes, which are complementary to microscopic observation of malformations detected in fungal strains analyzed previously25. This study used stem bark because it is the part used in most studies testing antimicrobial and antiinflammatory activities19,22,26. S. terebinthifolius bark contains numerous chemical compounds, such as flavonoids, tannins, and triterpenes. These constituents are considered to be responsible for the antibacterial, antifungal and anti-inflammatory action, making the plant widely used in the treatment of infectious and inflammatory diseases27. Tannins have the ability to precipitate proteins from surface cells of tissues and mucous membranes, creating a protective layer (tannin complex / protein) on the damaged skin or mucosa, in addition to having high antimicrobial activity28. Flavonoids have proven anti-inflammatory and antioxidant activity29, and triterpenes have an anti-inflammatory property as they act as phospholipase A2 competitive inhibitors30.
The clinical efficacy of S. terebinthifolius tincture was tested to treat denture stomatitis. Authors selected 18 users of removable prostheses diagnosed with denture stomatitis type II and presence of candidiasis associated with prosthesis, confirmed by clinical and mycological examinations. All patients were instructed to sanitize the prosthesis using a brush and dentifrice, and then apply the product on the palatal mucosa and prosthesis surface 3 times daily for 15 consecutive days. They concluded that the tincture was effective in the treatment of denture stomatitis, providing remission of the inflammatory process and infection by Candida spp. Their results were similar to the traditional therapy for this disease. Nevertheless, this clinical trial refers to the need for further studies researching the long-term success of the therapy with this plant26.
It is worth noting that this study is an initial assessment of the determination of the antifungal activity of S. terebinthifolius tincture, which points to the need for other pre-clinical trials, including toxicological evaluation. The findings of this study allow the continuity of research on S. terebinthifolius, in view of its potential antifungal activity against C. tropicalis and thus applicability in the medical/dental fields. Accordingly, it is shown to be an alternative for the treatment of oral candidiasis that may overcome the shortcomings of the synthetic agent (i.e. Nystatin) regarding cost, substantivity, treatment extension, adverse effects and above all, microbial resistance. Clinical studies are suggested to further research the advantageous effects of S. terebinthifolius on the treatment of superficial candidiasis. Given the above, Schinus terebinthifolius (Brazilian pepper tree) has in vitro antifungal activity (fungistatic and fungicidal) against Candida tropicalis, and probably acts by inhibiting fungal cell wall formation.

REFERENCES

1. Repentigny L, Lewandowski D, Jolicoeur P. Immunopathogenesis of Oropharyngeal Candidiasis in Human Immunodeficiency Virus Infection. Clin Microbiol Rev. 2004;17: 729-759.         [ Links ]

2. Ramos INC, Vasconcelos LCS, Lima MGGC, Figueiredo LQ. Oral candidosis in positive HIV patients. Braz J Clin Dent. 1999;3:59-61.         [ Links ]

3. Alves PM, Leite PHAS, Pereira JV, Pereira LF, Pereira MSV, Higino JS, et al. Antifungal activity of the extract of Psidium guajava Linn. ("goiabeira") upon leavens of Candida of the oral cavity: an in vitro evaluation. Braz J Pharmacog. 2006;16:192-196.         [ Links ]

4. Candido RC, Azevedo RVP, Komesu MC. Enzymotiping of species of the genus Candida isolated from the oral cavity. J Braz Soc Trop Med. 2000;33:437-442.         [ Links ]

5. Dorko E, Pilipcinec E, Bracokova I, Jenca A, Svicky E, Danko J, et al. Relative Pathogenicity of Candida tropicalis in rat tongue mucosa. Folia microbial. 2000;45:561-565.         [ Links ]

6. Kothavade JR, Kura MM, Valand AG, Panthaki MH. Candida tropicalis: its prevalence, pathogenicity and increasing resistance to fluconazole. J Med Microbiol. 2010; 59:873-880.         [ Links ]

7. Lima IO, Oliveira RAG, Lima EO, Farias NMP, Souza EL. Antifungal activity from essential oils on Candida species. Braz J Pharmacog. 2006;16:197-201.         [ Links ]

8. Carlini, EA, Duarte-Almeida JM, Rodrigues E, Tabach R. Antiulcer effect of the pepper trees Schinus terebinthifolius Raddi (aroeira-da-praia) and Myracrodruon urundeuva Allemao, Anacardiaceae (aroeira-do-sertao). Braz J Pharmacog. 2010;20:140-146.         [ Links ]

9. Paulo PTC, Diniz MFFM, Medeiros IA, Morais LCSL., Andrade FB, Santos HB. Phase I clinical toxicological assays of a complex herbal medicine (Schinus terebinthifolius Raddi, Plectranthus amboinicus Lour and Eucaliptus globulus Labill). Braz J Pharmacog. 2009;19:68-76.         [ Links ]

10. Paulo EMM. Evaluation of the anti-inflammatory and healing effects of the natural extract of aroeira-do-sertao at 30% in wounds in the dermis of the dorse of rats by electro-surgery: in vivo study. Monograph (Graduation)Available in: Biblioteca Brasileira de Odontologia.– Pernambuco School of Dentistry, University of Pernambuco, 1988.

11. Ellof, JN. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Medica. 1998;64:711-713.         [ Links ]

12. Frost DJ, Brandt KD, Cugier D, Goldman R. A whole-cell Candida albicans assay for the detection of inhibitors towards fungal cell wall synthesis and assembly. Journal of Antibiotics. 1995;28:306-309.         [ Links ]

12. Varanda EA. Mutagenic Activity of Medicinal Plants. Journal of Basic and Applied Pharmaceutical Sciences. 2006; 27:1-7.         [ Links ]

13. Simoes CMO. Pharmacognosy: from the plant to the drug. 6nd edition. Florianopolis: Ed. Universidade Federal de Santa Catarina, 2011.         [ Links ]

14. Brazil, Ministry of Health. Health Surveillance National Agency. Ordinance n. 3,916 10/30/1998. Approves the National Drug Policy. Official Diary of Brazil, Brasilia/DF, Sec. N. 215-E, p. 18, 1998.         [ Links ]

15. Marcos-Arias C, Vicente JL, Sahand IH, Equia A, De-Juan A, Madariaga L, et al. Isolation of Candida dubliniensis in denture stomatitis. Archives of Oral Biology. 2009;54:127-131.         [ Links ]

16. Samaranayake L. Commensal Oral Candida in Asian Cohorts. Int J Oral Sci. 2009;1:2-5.         [ Links ]

17. Alves SH, Cury AE. Candida from cancer patients: susceptibility in vitro to polyene antifungal agents. J Trop Med Inst Sao Paulo. 1992;34:251-254.         [ Links ]

18. Freires IA, Alves LA, Jovito VC, Castro RD. Antifungal activity of Schinus terebinthifolius (Aroeira) on Candida strains. ROBRAC. 2011;20:68-72.         [ Links ]

19. Alves PM, Queiroz LMG, Pereira JV, Pereira MSV. In vitro antimicrobial, antiadherent and antifungal activity of Brazilian medicinal plants on oral biofilm microorganisms and strains of the genus Candida. J Braz Soc Trop Med. 2009;42:222-224.         [ Links ]

20. Lucena PLH, Ribas Filho JM, Mazza M, Czeczko NG, Dietz UA, Correa Neto MA et al., Evaluation of the aroeira (Schinus terebinthifolius Raddi) in the healing process of surgical incision in the bladder of rats. Acta Cirurgica Brasileira. 2006;21:16-51.         [ Links ]

21. Soares DGS, Oliveira CB, Leal C, Drumond MRS, Padilha WWN. Antibacterial activity in vitro of the tincture of aroeira (Schinus terebinthifolius) in the decontamination of dental brushes contamined by S. mutans. Braz Res Ped Dent Int Clin.. 2007; 7:253-257.         [ Links ]

22. Martorelli SBF, Pinheiro ALB, Souza IA, Higino JS, Bravo F. The anti-inflammatory and healing effects of an 30% hydroalcoholic solution of Schinus terebinthifolius Raddi (Aroeira) - an oral base in "vivo" study. Int J Dent. 2011; 10:80-90.         [ Links ]

23. Pereira FO. Antifungal Activity of the Essential Oil of Cymbopogon winterianus Jowitt ex Bor on dermatophytes of Trichophyton genus. [Doctorate Thesis]. Joao Pessoa: Department of Pharmaceutical Sciences, Federal University of Paraiba, Brazil, 2009.         [ Links ]

24. Lesage G, Bussey H. Cell wall assembly in Saccharomyces cerevisiae. Microbiol Molec Biol Rev. 2006; 70:317-343.         [ Links ]

25. Soares DGS, Oliveira CB, Paulo MQ, Carvalho MFFP, Padilha WWN. Clinical and Microbiological Evaluation of the Treatment of Denture Stomatitis with Schinus terebinthifolius Raddi (Aroeira) Tincture. Braz Res Ped Dent Int Clin. 2010;10:365-370.         [ Links ]

26. Lima MRF, Luna JS, Santos AF, Andrade MCC, Sant'Ana AEG, Genet JP, et al. Antibacterial activity of some brazilian medicinal plants. J Ethnopharmacol. 2006;105:137-147.         [ Links ]

27. Haslam E. Natural polyphenols (vegetable tannins) as drugs and medicine: possible modes of action. J Nat Prod. 1996; 59:205-215.         [ Links ]

28. Velazquez E, Tournier HA, Mordujovich de Buschiazzo P, Saavedra G, Schinella GR. Anti-oxidant activity of Paraguayan plant extracts. Fitoterapia. 2003;74:91-97.         [ Links ]

29. Jain MK, Yu BZ, Rogers JM, Smith AE, Boger ET, Ostrander RL, et al. Specific competitive inhibitor of secreted phospholipase A2 from berries of Schinus terebinthifolius. Phytochemistry. 1995;39:537-547.         [ Links ]

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