Antimycotic effect of Melaleuca alternifolia on cutaneous mycosis

Cutaneous fungi in human infections are serious medical threat. Recently, based on reports, numerous fungal species were regarded as natural contaminants. Generally, researchers and other clinicians in the field have deemed it fit and worthy that new potent, effective and safe antifungal drugs are needed. This study reports the antifungal activity of the aqueous extract of Melaleuca alternifolia (tea tree) leaves, using the standard drugs, ketoconazole as the positive control. Antifungal assay was done using Clinical and Laboratory Standard Institutes (CLSI) procedures against isolates such as yeast; Rhodoturular and moulds; Aspergillus flavus, Aspergillus niger and Aspergillus fumigatus. The results showed significant zone of inhibitions (>12 mm and <25 mm diameter) for Melaleuca alternifolia compared to ketoconazole (>5 mm and <19 mm). Cytotoxic test of the crude extract using tadpoles produced mortality of 18 ± 2.50 % at the lowest concentration of 21.9 μg/ml, and 55 ± 3.40 % at the highest concentration of 175 μg/ml over a 24hour period. The phytochemical screening of the extract showed the presence of tannins, saponins, flavonoids, alkaloids, cardiac glycosides and terpenes. The in vitro antifungal activities gave remarkable effect on the selected fungi isolates. The remarkable antifungal activity shown by Melaleuca alternifolia methanol extract, against yeast and moulds, suggested its usefulness as a natural antifungal agent for the treatment of some cutaneous mycoses with no cytotoxic effect, and also highlights its significance use as potential healthcare product in pharmaceutical formulations when adopted.


Introduction
Cutaneous fungi infection poses serious medical issues and more than a hundred thousand fungal species have been considered as natural contaminants [1]. Therefore, it is quite logical that any recent search for new prototype antifungal products should also include a variety of plant extracts [2]. Also, to design a search for novel prototype antifungal agent, it seems reasonable to assume that if new agents are found to have different structures and different activities from those in current use, higher plants are a logical choice, chiefly because of their seemingly infinite variety of novel molecules, which are often referred to as secondary metabolite [2]. Antifungal agents are widely distributed among higher plants [3], but only a few have been evaluated for their activity against human pathogenic fungi. In the past few decades, a worldwide increase in the incidence of fungal infections has been observed as well as a rise in the resistance of some species of fungi to conventional antifungal drugs used in medical practice. Therefore, new prototype antimicrobial agents are needed to address this situation [4].
Fungi are one of the most neglected pathogens as demonstrated by the fact that the amphotericin B, a polyene antibiotic discovered as long as 1956, is still used as a gold standard for antifungal therapy [5]. Exploitation of naturally occurring compounds from plants and microbes have been suggested [6]. The majority of clinically used antifungal agents have various draw backs in terms of toxicity, efficacy as well as cost and their frequent use has led to emergence of resistant strains. Hence there is a great demand for novel antifungal drugs belonging to a wide range of structural classes, selectively acting on new targets with least side effects. Natural products, either as pure compounds or as standardized plant extracts, provide unlimited opportunities for new drug leads. In addition, edible plants have been proven to be harmless and are economical [7], in which one of them is the tea tree known as Melaleuca alternifolia.
Comprehensive investigation of the susceptibility of fungi to the oil of Melaleuca alternifolia has only recently been completed [8,9]. Prior to this, data were somewhat piecemeal and fragmentary. Early data were also largely limited to Candida albicans, which was a commonly chosen model test organism. Recently, data now shows that a range of yeasts, dermatophytes, and other filamentous fungi are susceptible to TTO [10]. Although test methods differ, minimum inhibitory concentration (MICs) generally range between 0.03 and 0.5%, and fungicidal concentrations generally range from 0.12 to 2%. The notable exception is Aspergillus niger, with minimal fungicidal concentrations (MFCs) of which are as high as 8% reported for this organism [11]. However, these assays were performed with fungal conidia, which are known to be relatively impervious to chemical agents. Subsequent assays have shown that germinated conidia are significantly more susceptible to TTO than non-germinated conidia [10], suggesting that the intact conidial wall confers considerable protection. TTO vapors have also been demonstrated to inhibit fungal growth and affect sporulation [12]. Studies investigating the mechanisms of antifungal action have focused almost exclusively on C. albicans. Similar to results found for bacteria, TTO also alters the permeability of C. albican cells. The treatment of C. albicans with 0.25% TTO resulted in the uptake of propidium iodide after 30 min [13], and after 6 hours significant staining with methylene blue and loss of 260 nm light-absorbing materials had occurred [14]. TTO also inhibits respiration in C. albicans in a dose-dependent manner [15]. Additional studies have shown that when cells of C. albicans are treated with diethyl stilbestrol to inhibit the plasma membrane ATPase, they had a much greater susceptibility to TTO than control cells [9], suggesting that the plasma membrane ATPase has a role in protecting cells against the destabilizing or lethal effects of TTO. Tea Tree Oil inhibits the formation of germ tubes, or mycelial conversion, in C. albicans [16]. Two studies have shown that germ tube formation was completely inhibited in the presence of 0.25 and 0.125% TTO, and it was further observed that treatment with 0.125% TTO resulted in a trend of blastospores changing from single or singly budding morphologies to multiply budding morphologies over the 4-hour test period [17]. These cells were actively growing but were not forming germ tubes, implying that morphogenesis is specifically inhibited, rather than all growth being inhibited. Interestingly, the inhibition of germ tube formation was shown to be reversible, since cells were able to form germ tubes after the removal of the TTO [17]. However, there was a delay in germ tube formation, indicating that TTO causes a post antifungal effect.
Tea tree oil possessed antifungal activity against filamentous fungi associated with invasive fungal wound infections (IFIs); and it indicated a potential use as topical treatment of IFIs [18]. The study of in-vitro susceptibilities of candida and Aspergillus species to melaleuca alternifolia (tea tree) oil [19] showed that Melaleuca alternifolia inhibited the candida species. Thus, this study investigates the effects of the crude aqueous extract of the tea tree Melaleuca alternifolia on cutaneous mycosis.

Extraction of the plant
The leaves of Melaleuca alternifolia were air-dried under shade, at room temperature (20-25 °C ), for 3 weeks. The dried leaves were grinded into powder (340 g), using laboratory milling machine. The dried powder was cold-macerated using distilled water for 48 hours. The liquid extract was filtered and then concentrated using rotary evaporator at 40 °C , and to complete dryness inside fume cupboard. The concentrated and dried extract which weighed 7.7 g was stored in a sample bottle and refrigerated prior to bioassay work.

Phytochemical screening
The plant extract was assessed for the presence of phytochemicals using method described by [20].

Preparation of fungal strains
Viability tests were carried out by picking the organism from the stock using sterile loop and inoculated into sabouraud dextrose agar (SDA) media and then left at room temperature. The test fungi were confirmed using morphological and microscopic characteristics, using direct plate culture method and slide culture techniques respectively.

Morphological / microscopic characterization of fungi isolates
The identification of the isolated fungi organism was done by slide culture technique. 10 ml of glycerol was added into a beaker containing 25 ml of sterile distilled water. A folded whatman filter paper was soaked into the beaker and placed in a sterile plate. A slide was placed on the whatman paper as a base. Using a sterile blade, sabouraud dextrose agar (SDA) was cut into squares (block) small enough to fit under a cover slip. A square of the agar was placed in the middle of the slide in the plate and the fungal isolates were inoculated on the cut agar block on the slide. Then a cover slip was placed on the agar block on the slide, which was placed on the whatman filter paper in the sterile petri dish and left under room temperature for 2 days until growth and sporulation has occurred.
After visible growth, the cover slip was removed and 2 drops of lactophenol cotton blue was placed on the agar block and then the cover slip was placed back onto the wet agar block. The slide containing the wet agar block was examined under the microscope at x40. This procedure was repeated for each fungus isolate in other to view their conidia, hyphae and micro conidia.

Molecular identification of test organism
Fungal isolates were obtained from skin scraping from skin of infected cows from abattoirs' in Owerri, Imo state, Nigeria. The exudates from the skin scrapings were cultured using SDA and incubated for 2-5 days and their Molecular Identification were verified using the 16S RNA primer and gene sequencing. The isolates include, Aspergillus niger, aspergillus fumigatus, aspergillus flavus and rhodoturular specie.

Preparation of Mcfarland standard
Mcfarland standard is used as a reference to adjust the turbidity of fungal suspension so that fungal organisms will be within a given range. Accurately 0.5 Mcfarland equivalent turbidity was prepared by adding 0.6 ml of 1% Barium chloride solution (BaCl2. 2H2O) to 99.4 ml of 1% Tetraoxosulphate acid (H2SO4) and mixed thoroughly. Then, a small of the turbid solution was transferred to a cap tube of the same type that was used to prepare the test organism and control inoculum. It was then stored in the dark at room temperature (25°C). Exactly 0.5 Mcfarland gives an equivalent approximate density of fungi 1.5 x 10 8 colony forming units per ml (CFU) mL -1 [21].

Preparation of extract
2.5 g of crude extract was weighed into 25 ml sterile distilled water (100 mg/ml) and was mixed for 30 minutes in an electric shaker, to obtain a homogenous solution. Then, the extract was filtered using whatman No.1 filter paper to exclude the granules. The extract solution was transferred into a sterile screw cap bottle and labeled stock solution, then stored in the refrigerator (4°C).

Disc diffusion method
The use of agar disc diffusion method to screen for antifungal activities of the crude tea aqueous extract was done accordingly to the National Committee of Clinical and Laboratory Standards [22] now Clinical and Laboratory Standard Institutes (CLSI). The fungal inoculums for susceptibility test were standardized using barium sulphate standard equivalent to McFarland No 0.5, giving cell density of 1.5 x 10 8 Colony Forming Units per ml (CFU/ml). Circular paper discs (6mm diameter) made of whatman No.1 filter paper were prepared with the aid of an office paper perforator. The discs were placed in a petri-dish and sterilized in an autoclave. Dilutions of several concentrations of the crude extracts were made. A Positive control was made using azole group of compound (ketoconazole), and the negative control were prepared using sterile distilled water. Blank sterile papers disc measuring 6 mm were impregnated with 20 L of test concentration of crude extract. The discs were then aseptically transferred into respective inoculated plates. Briefly, approximately 1.5 x 10 8 cells of freshly grown fungal suspension were uniformly streaked in the sterile malt extract agar dishes using wire loop. The discs with respective crude extract solution concentrations were placed on a Malt extract agar plates containing chloramphenicol to which the test fungi have been inoculated. The inoculated plates were left for 48 hours before the activity was determined. The activities of the crude extract were established by the presence of zones of inhibition which were measured in millimeter. ketoconazole discs were used as antifungal reference standards for positive control. Similarly, the sterile distilled water was set as negative controls.

Well in agar diffusion method
2 drops of diluted inoculum with different concentration was placed into different sterile cap bottle containing 15 ml of sterilized malt extract agar and chloramphenicol. They were mixed and transferred into sterile plates respectively and allowed to solidify. Using a sterile cock borer, holes were made in the agar plate and labeled according to concentrations. Different concentration was poured into the holes respectively. This procedure was done for the antifungal drugs ketoconazole and the extracts with different dilutions of the drug. These plates were left for 48 hours before observation for the zone of inhibition.

Cytotoxic assay of crude extract using tadpoles (Ranicepsranninus)
Tadpoles (5-7 days old) were scooped from fresh water in the crown estate, Okada, Edo State, Nigeria; and were properly identified in the Department of Animal and Environmental Biology, Faculty of Life Science, University of Benin, Edo State, Nigeria. 10 tadpoles of same sizes together with 30 mL of their habitat water were transferred into different beakers. Distilled water was added to make contents up to 49 mL, after which 1 mL of each concentration (175, 87.5, 43.7, 21.9 g/ml) of the extract in 5% dimethyl sulfoxide (DMSO) was added to make the mixtures up to 50 mL [23]. The experiments were incubated for 24 hours after which mortality was determined. Control assay was done with 50 mL of habitat water containing 1 mL of 5 % DMSO in distilled water. All tests were done in triplicate.

Results and discussion
The phytochemical analysis of the plant extracts which yielded 2.26% was shown in table 1. Table 2 showed the Cutaneous fungi isolates of the macroscopic and microscopic identification of the test organism A, B, C and D with their diagrams. Figure 1 shows a chart presentation of the in vitro antifungal activity of crude aqueous extract of melaleuca alternifolia using agar well diffusion method.    Concentration of 100mg/mL exhibited more zone of inhibition for organism A with 25mm, followed by organism D (23 mm), organism B (22 mm), organism C (19 mm) respectively.
For concentration 50 mg/mL, organism B and D exhibited more zone of inhibition with 20 mm each followed by organism A with 19 mm and organism C with 14 mm.
For concentration of 25 mg/mL, organism A with 18 mm showed more zone followed by organism B with 15 mm, organism D with 13 mm and organism C with 12 mm respectively.
The antifungal activity of ketoconazole (Standard Drug), using well diffusion method at concentration of 20 mg/mL, 10 mg/mL and 5 mg/mL to the test organisms are presented in fig. 2. For organism A, the concentration of 5 mg/mL showed greater zone of inhibition with 10 mm, then the concentrations 20mg/mL and 10 mg/mL had no zone of inhibition, thus, there was resistance (5mm diameter is estimated (considered) as no zone of inhibition).
The organism B is resistant to the antifungal drug (ketoconazole) at all concentrations (5mm is considered as no zone of inhibition).
For organism C, the concentration 5 mg/mL had greater zone of inhibition with 19 mm, then concentration 20 mg/mL had 17 mm; but concentration 10 mg/mL is resistant with no zone (5mm is considered as no zone of inhibition).
For organism D, concentration 20 mg/mL showed zone of inhibition with 8 mm and 10 mg/mL and 5 mg/mL showed no zone of inhibition.
The antifungal activity of ketoconazole with various concentrations of 20 mg/mL, 10 mg/mL, 5 mg/mL, 2.5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, 0.3125 mg/mL using a disc of 6 mm in disc diffusion method are presented in fig. 3. The inhibitory effect against the four organisms; A, B, C, D (dermatophytes) were taken by their zones of inhibition (mm).
Organism A, at concentration of 2.5 mg/mL exhibited more zone of inhibition with diameter 22 mm followed by concentration 0.625 mg/mL at 20 mm, then 5 mg/mL at 16 mm, 1.25 mg/mL with 14 mm and 20 mg/mL, 10 mg/mL and 0.325 mg/mL with no zone of inhibition (5mm diameter is estimated (considered) as no zone of inhibition).
For organism B, concentration at 2.5 mg/mL exhibited the highest zone of inhibition with 15 mm diameter, then concentrations at 20 mg/mL, 10 mg/mL, 5 mg/mL, 1.25 mg/mL, 0.625 mg/mL, and 0.3125 mg/mL showed no zone of inhibition.
A=Rhodoturula (yeast), B=Aspergillus fumigatu, C= Aspergillus flavus, D= Aspergillus niger  Shift in the treatment of infectious diseases is necessary where appropriate alternatives to antibiotics ought to be considered, and also to prevent antibiotics from becoming obsolete. There are already several non-antibiotic approaches to the treatment and prevention of infection, including probiotics, phages, and phytomedicines. Melaleuca alternifolia is an old, over the counter remedy that possesses in vitro antifungal activity against a broad spectrum of candida species [25]. It has antibacterial and antifungal activity properties that have secured it a place in the commercial pharmaceutical market. Melaleuca compounds may be a valuable addition to the management of fungal infections in the future [10].
For all the organism tested in this study, organism A (Rodotorula), a yeast was the most sensitive fungus to the crude extract, organism B (Aspergillus flavus), D and C also showed great inhibitions sequentially, using well in agar method. For other assay method used, such as disc diffusion, the entire test organisms used were resistant. Therefore, assay method could also affect the result of any activity of drugs on organism, especially fungi.
Our results indicate a considerable difference in antifungal (antimycotic) of Melaleuca alternifolia and that of the standard antifungal drug (ketoconazole), which confirms that the crude aqueous extract has antimicrobial effect against the test organism (organism A,B,C,D) using pour plate method/well in agar. Organism A was the most sensitive fungus to the crude extract of Melaleuca alternifolia. For the antimicrobial activity of ketoconazole, organism D showed greater sensitivity than the other organisms used. The result of the comparative analysis of the extract and ketoconazole using pour plate method/well in agar method showed that, the extract of the plant showed more antifungal activity against the test organisms than ketoconazole. Extract activity with the test organism A (Rhodotorula) which is a yeast, showed greater zone of inhibition diameter of >18 mm, while organism D (Aspergillus niger) was moderately sensitive with inhibition zone diameter of >13 mm, followed by organism B with zone of inhibition >15 mm, and then organism C having >12 mm zone of inhibition. While for ketoconazole activities, organism D showed greater inhibition zone < 19 mm, and organism B was resistant with inhibition of zone diameter of 5 mm. The results in this study were in line with the report of [26], where it was stated that ketoconazole is not useful in treating aspergillosis, zygomycosis or sporotrichosis. Due to the concentration of ketoconazole used, the organisms exhibited more resistant than sensitivity to the antifungal drug.
Tea tree oil, as a direct contact solution, was the most effective at inhibiting fungal growth of both test species among all the agents assessed in the current study. This result is in agreement with previously published data on the in vitro antimicrobial efficacy of TTO from a clinical setting [17], where it was found that TTO had both an inhibitory effect and a fungicidal effect on filamentous fungi. The authors found both germinated conidia and non-germinated conidia of the fungal isolates demonstrated susceptibility to TTO. The mechanism of TTO's antifungal action is believed to be by the alteration of the cell membrane structure, causing it to become permeable, which leads to the leakage of cellular material and disruption to cellular functions [17].
The phytochemical study revealed the presence of tannins, saponins, alkaloids, cardiac glycosides, terpenes, flavonoids and phenols (Table 1). These are significant components that are capable of causing varied physiochemical and pharmacological effects [27]. The results were in line with similar studies carried out [28], who also reported the presence of saponins, flavonoids, phytosterols, triterpenoids, phenolics and tannins in the leaves extracts of Melaleuca alternifolia. Phytochemical screening, apart from revealing the metabolites responsible for activities, can also help to disclose new sources for vital economical secondary metabolite materials, as a precursor for the synthesis of complex chemical substances [29].
In addition, comparison of our data with those previously published, demonstrate very similar susceptibility results.
Unfortunately, it is difficult to compare data from different investigators [17] since the chemical composition may differ or other environmental factors could also affect the components of the drugs used.

Conclusion
Melaleuca alternifolia crude aqueous extract had greater zone of inhibition to the test organisms than the test antifungal drug, ketoconazole, which showed that it is more active in the treatment of the test organisms used in this study than ketoconazole. This plant can therefore be utilised as an alternative source of useful antifungal drugs.