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Antifungal activity of Cymbopogon winterianus jowitt ex bor against Candida albicans

Candida albicans is an opportunistic yeast and a member of the normal human flora that commonly causes infections in patients with any type of deficiency of the immune system. The essential oils have been tested for antimycotic activity and pose much potential as antifungal agents. This work investigated the activity of the essential oil of Cymbopogon winterianus against C. albicans by MIC, MFC and time-kill methods. The essential oil (EO) was obtained by hydrodistillation using a Clevenger-type apparatus. It was tested fifteen strains of C. albicans. The MIC was determined by the microdilution method and the MFC was determined when an aliquot of the broth microdilution was cultivated in SDA medium. The phytochemical analysis of EO showed presence of citronellal (23,59%), geraniol (18,81%) and citronellol (11,74%). The EO showed antifungal activity, and the concentrations 625 µg/mL and 1250 µg/mL inhibited the growth of all strains tested and it was fungicidal, respectively. The antimicrobial activity of various concentrations of EO was analyzed over time, it was found concentration-dependent antifungal activity, whose behavior was similar to amphotericin B and nystatin.


INTRODUCTION
Candida albicans is an opportunistic yeast and a member of the normal human flora. The individual is colonized at birth or near birth and transmission is primarily through physical contact. This yeast normally colonizes the skin and mucosal epithelium in healthy individuals (25). In patients hospitalized with cancer, immunocompromised or who use immunosuppressive drugs colonization may progress to invasion, resulting in severe systemic disease (26,38). Antifungal activity of C. winterianus Systemic infection by C. albicans is often associated with catheters, surgery, parenteral nutrition and damage the skin, mucous membranes and the digestive tract (39). Invasive fungal infections are associated with high mortality rates and over 40% in bloodstream infections caused by C. albicans (8).
The common sites for superficial candidiasis include under the breasts, in the groin, in skin folds of obese persons and the perineal area (18). The major antifungal agents used in the therapy of these infections are azoles such as fluconazole and itraconazole, and the polyene compounds such as amphotericin B and nystatin (6,30).
The increasing resistance of fungi to azole derivatives as a result of long-term therapies may limit the use of these drugs in the future (10,17,30). Due to limitations of current antifungal therapy and the emergence of resistant strains, it is necessary the search for new antifungal drugs through the testing of substances from plants (3,34). Among the candidates, the essential oils are known to possess several biological activities, such as antibacterial and antifungal action (2,7).

Essential oil analysis
The essential oil was analyzed using a gas chromatograph

Time-kill
The time-kill of C. albicans in presence of the essential oil was performed according to Klepser et al. (22), with some modifications. Before testing, the microrgamism was cultivated in SDA. Colonies derived from culture were suspended in 0.85% NaCl and turbidity adjusted to the range of 0.5 McFarland (1-5 x 10 6 CFU/mL). One milliliter of fungal suspension was added to 9 mL of broth Sabouraud with or without the essential oil in various appropriate concentrations.
The initial inoculum contains 1-5 x 10 5 CFU/mL. The log 10 CFU/mL was plotted on a graph as function of time and used to compare the rate and extent of antifungal activity in various concentrations of essential oil. It was considered fungicidal activity when there was a decrease greater than or equal to 3 log 10 CFU/mL of the initial inoculum, resulting in reduction of 99.9% or more CFU/mL in 24 hours compared with the initial inoculum. Activity lower than that described was considered fungistatic (16,24,35).

Chemical characterization of oil constituents
Essential oil of C. winterianus was subjected to GC and GC-MS analysis. The phytochemicals and their respective percentage in the essential oil composition and retention times are shown in Table 1. The majority constituents were citronellal (23,59%), geraniol (18,81%) and citronellol (11,74%).

Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC)
By the broth microdilution technique was determined the MIC and the MFC, which are shown in Table 2. Antifungal activity of C. winterianus As seen in Table 2, the MIC of the essential oil tested ranged between 78 and 625 g/mL. The concentration of 625 g/mL inhibited the growth of all strains, while 312 g/mL was able to inhibit 60% of the strains tested. MFC of microorganisms ranged between 312 and 1250 g/mL, being the latter fungicidal for all strains tested.

Time-kill
The microorganism growth was analyzed over time when it was subjected to various concentrations of essential oil of C.
winterianus. Two strains of C. albicans (ICB 12 and ATCC 13803) were submitted to the experiment of microbial time-kill (Figures 1 and 2).
The graphs show the log 10 of CFU/mL versus time for several multiples of the MIC (625 g/mL) and the control without essential oil. The analysis of the graph to point out that at concentrations less than or equal to 1xMIC, the essential oil has fungistatic activity (reduction of less than 99.9% or 3log 10 the number of CFU/ml of initial inoculum), and at concentrations greater than or equal to 2xMIC it has fungicidal activity (reduction greater than or equal to 99.9% or 3log 10 the number of CFU/mL of initial inoculum), therefore, the essential oil of C. winterianus has concentration-dependent fungicidal activity.
Although the rate and extent of antifungal activity has varied slightly between the two strains tested, their behavior in front of the essential oil was similar. The antifungal activity improved with increasing concentration of essential oil, like this, when the higher concentration of essential oil, the lower time is required for fungicidal activity. Pauli and Schilcher (32) suggest that oral administration of essential oil compounds is not suitable to cure severe infections in children. Topical application or inhalation of selected compounds for the treatment or additional treatment of mild infections is reasonable. Dorman and Deans (12) reported that administered orally, volatile oils may be able to control a wide range of microbes, but there is also the possibility that they may cause an imbalance in the gut microflora, allowing opportunistic pathogens coliforms to become established in the gastrointestinal tract with resultant deleterious effects. Other possible side effects of essential oils are irritation, allergic contact dermatitis and spasmolytic or spasmogenic properties (7). The essential can to be used for treatment some diseases like dermatomycoses and superficial candidiasis mainly in topical applications (18). The essential oils can be used alone or in combination with other drugs which can exhibit synergism (19,20).
The increased resistance of C. albicans and the nature of the fungistatic azoles have valued the search for new antifungal agents, once the activity against this type of pathogen is essential (16). The antifungal activity demonstrated by the essential oil of C. winterianus makes it a potential candidate as a major agent in controlling fungi growth that cause infections or are present in the environment.