In vitro Antifungal Efficacies of Ethyl Acetate Fractions of Mitracarpus villosus from Abuja, Nigeria

Introduction: The use of medicinal plants in the treatment of diseases is as old as man. The development of synthetic (orthodox) drugs led to a decline in the use of herbs however in the past few decades, there has been an increase in the use of medicinal plants, especially in developing countries. Several reports have shown that herbal medicines are well tolerated when compared with synthetic drugs. Over eighty percent of the population in Africa, most especially West Africa, has been reported to depend on medicinal plants for the treatment of infections and diseases. Aims: The main objective of this study is to assess the anti-fungal potentials of the ethyl acetate extract and fractions of the aerial parts of Mitracarpus villosus (Sw.) DC from Abuja, Nigeria. Methods: The powdered plant was extracted successively and exhaustively with hexane, ethyl acetate, ethanol and water. Thirty fractions were obtained from the extract using the bioassay-Original guided fractionation by means of column chromathographic technique. Antifungal activities of the ethyl acetate extract and fractions of M. villosus against clinical isolates of Candida albicans, Candida krusei , Trichophyton mentagrophytes, Trichophyton verrucosum, Aspergillus fumigatus and Aspergillus niger were investigated using agar diffusion and micro broth dilution methods. Results: All the fractions showed good antifungal activity against test fungi. The minimum inhibitory concentration of the extract and fractions ranged between 250 - 4000 µg / ml while the minimum fungicidal concentration of the ethyl acetate extract and fractions against the test organisms were found to fall between 500 – 16000 µg / ml. Conclusion: The plant promises to hold good potentials for development of novel antifungal drug.


INTRODUCTION
Medicinal plants have been a source of wide variety of biologically active compounds for many centuries and used extensively as crude material or as pure compounds for treating various disease conditions [1]. More than 80% of the population in developing countries has been reported to depend on plants for their medical needs [2]. Research have shown that plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties [3].
In recent times, there has been a worldwide increase in the incidence of fungal infections, as well as a rise in the resistance of some species of fungi to different fungicidal agents used in medicinal practice [4]. The incidence of fungal infections is thus increasing at an alarming rate, presenting an enormous challenge to healthcare professionals. This increase is directly related to the growing population of immunocompromised individuals especially children resulting from changes in medical practice such as the use of intensive chemotherapy and immunosuppressive drugs [5].
Mitracarpus villosus (S.W) D.C belongs to the family Rubiaceae. It grows as an erect perennial annual plant with height up to 60 cm. It can be found on old and abandoned farmlands where it grows as a weed with a wide distribution ranging from forest to savanna zones in tropical climates. Phytochemical evaluation of the leaves of this plant has been reported to show the presence of alkaloids, tannins, cardiac glycosides, saponins, soluble carbohydrate, flavonoids, reducing sugar, cyanide, glycoside, steroid and terpenoid [6]. In Tropical Africa, fresh extracts of M. villosus have been employed in traditional medicine for the treatment of sore throat. In West Africa, natives use extract of the plant for management and cure of several disease conditions which include headaches, toothaches, amenorrhoea, hepatic diseases, gastrointestinal disturbance (like dyspepsia), sexually transmitted diseases, leprosy [7]. The juice from the fresh plant is used to treat skin infections like ringworm and eczema, stoppage of bleeding and as first aid treatment for fresh cuts, wounds and ulcer. The aerial parts of M. villosus have also been formulated into lotions and skin ointments for the treatment of skin infections [8]. In Nigeria, the extracts of the juice from the fresh aerial parts of the plant is applied on the skin surface to treat skin diseases and heal wounds [9]. There is limited documentation on the antifungal potential of the ethyl acetate extract of Mitracarpus villosus. The main objective of this study is to evaluate the anti-fungal activities of the ethyl acetate extract of the aerial parts of Mitracarpus villosus (Sw.) DC from Abuja, Nigeria.

Drying of Plant Material
The aerial parts fresh plant was air dried at 25ºC for 10 days. The completely dried aerial parts were crushed to coarse powder by grinding with wooden mortar and pestle.

Extraction of Plant Material
Successive extraction was employed for the extraction of active component of the plant. Simply, the dried powdered aerial parts of the plant was placed in a Soxhlet extractor (Quick Fit-England) and the plant material extracted successively and exhaustively each time with each of the various solvents in the order of increasing polarity i.e. hexane, ethyl acetate and ethanol respectively. Lastly, the marc was macerated in water and all the extract concentrated, dried and weighed [10].

Fractionation of Ethyl Acetate Extract
The fractionation of ethyl acetate extract was done using the bioassay-guided fractionation by employing the accelerated gradient chromatographic (AGC) technique [11]. Silica gel G (E-Merck, Germany) was used as an absorbent. Gradient elution was effected using hexane and ethyl acetate sequentially with increasing polarity. The column was successively eluted with hexane (100%), hexane-ethyl acetate

Preparation of antifungal agents
Stock solutions of fluconazole powder (Sigma Aldrich, Cat No. F8929) and ketoconazole powder (Sigma Aldrich, Cat No. K1003) was prepared in dimethyl sulfoxide (DMSO, BDH, Germany). Sabouraud dextrose broth was used to dilute the stock solutions to their required concentrations.

Fungi used
The

Cultivation and standardization of test fungi
Eighteen-hour culture of the test Candida spp. on Sabouraud dextrose agar (SDA) was standardized according to National Committee for Clinical Laboratory Standards [12]. Colonies of the pure culture of the fungi on solid medium was gradually added to normal saline and its turbidity compared to 0.5 McFarland standard of which is approximately 1.5 × 10 8 cfu/ml. This was finally diluted with SDB to a population of 1.5 × 10 6 cfu/ml. Fungal spores Trichophyton spp. and Aspergillus spp., were harvested from SDA slant cultures (7-10 day old) by flooding with 10 ml sterile normal saline containing 3% w/v Tween 80. Sterile glass beads were used to dislodge the spores [13]. Standardization of the fungal spore suspension to 1.0 x 10 6 spores / ml was carried out using a single-beam spectrophotometer (Spectronic 20D; Milton Roy Company, Pacisa, Madrid, Spain) at a wave length of 530 nm (OD530) and adjusted to 80 -85% transmittance (Aspergillus spp.) and 70 -72% (Trichophyton spp.). Quantification was done by spreading 100µL of suspension on Sabouraud dextrose agar plate and incubated at 37ºC for 18 h and 30ºC for 72 hours for yeast and moulds respectively [14]. The cultures were checked for purity based on their morphological characteristics on media, morphology on staining and biochemical tests. The fungi were maintained on Sabouraud dextrose agar (SDA) at 4ºC until required for use [14].

Susceptibility of the organisms to ethyl acetate extract of M. villosus
One milliliter of standardized culture of each test organism was spread on Sabouraud dextrose agar (excess aseptically drained) and the plates allowed to dry at 37ºC temperature in a sterilized incubator. The susceptibility of the organisms to the plant extracts was carried out using the agar diffusion cup plate method [15]. Simply, a sterile cork borer (6 mm) was used to bore holes in the agar plates and the bottoms of the wells sealed with the molten Sabouraud dextrose agar. With the aid of a micropipette, 0.1 ml each of gradient concentrations (mg/ml) of the crude ethyl acetate extracts was dispensed into the holes. Sterile distilled water was used as control. The plates were allowed to stand at room temperature for one hour in order to allow extract diffuse into the agar before incubation at 37ºC for 18 hours (yeast) and 30ºC for 72 hours up to 5 days (dermatophytes and moulds). The zones of inhibition produced by the extract on the test organisms were measured using a wellcalibrated meter ruler to the nearest millimeter.
The experiment was carried out in triplicates.

Determination of minimum inhibitory concentration (MIC) of the extract and fractions
The minimum inhibitory concentration (MIC) of the extract and fractions was tested using the serial broth micro dilution method [16]. Round bottom 96-well microtitre plates was used for the assay. Fifty microliters of the working solutions of extracts and fractions was added into the wells in row 1 and 2 of each column. The last eleven wells from rows 2 to 12 were filled with 50μl of sterilized Saboraud dextrose broth and an identical two-fold serial dilution were made from wells in rows 2 to the rows 11. The last wells in row 12 served as drug-free controls. Sterile SDB was used as negative control while ketoconazole and fluconazole served as positive controls. Lastly, 50μl of standardized fungal inoculum (10 6 cfuml -1 ) were added in all the wells from column A to H and mixed thoroughly to give final concentrations. Tests were done in triplicates. The innoculated microplates were sealed with parafilm and the plates incubated at 37ºC for 18 hours (yeast) and 30ºC for 48 hours and up to 7 days (dermatophytes and moulds). MIC was defined as the first well with no visible growth after 24 hours.

Determination of minimum fungicidal concentration (MFC) of the extract and fractions
The in vitro minimum fungicidal concentration was done by transferring fifty microliters from the wells without any visible growth after MIC determination into fresh wells containing Saboraud dextrose broth and incubated at 37ºC for 18 hours (yeast) and 30ºC for 48 hours and up to 7 days (dermatophytes and moulds). The lowest concentration resulting in no growth on subculture was taken as the minimum fungicidal concentration [17].

Statistical Analysis
The results obtained from the experiment were expressed as mean±standard deviation. One way ANOVA (Smith's Statistical Package version 2.80) at p=0.05 was adopted in the analysis of the findings.

RESULTS AND DISCUSSION
A total of 30 fractions were collected from the elution of the ethyl acetate extract. Identical fractions were combined based on thin layer chromatographic values giving 6 fractions M1 -M6 (Table 1). The result of preliminary antifungal activities showed that the ethyl acetate extract inhibited the growth of the test fungi with varying degree of inhibition which increased with increase in the concentration of the agent ( Table  2). The results of the minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) is shown in Tables 3 and 4 respectively. The effect of ethyl acetate extract and fractions was strongest on T. verrucosum. Trichophyton verrucosum a zoophilic dermatophyte is known to be the predominant causative agent of cattle ringworm infection. It is also known to affect (with lower prevalence), sheep, goats and other ruminants. Trichophyton verrucosum has also been implicated in, several human outbreaks by direct contact with infected animals or indirect contact with infectious propagules in the environment [18]. Plants contain bioactive constituents as protective substances against bacteria, fungi, viruses and pests [19]. The mode of action of ethyl acetate extract could be related to their ability to alter membrane properties leading to cell death [20]. All the test fungi were found to be susceptible to ethyl acetate extracts and fractions of M. villosus (Table 2), which is in line with the works done in the past [21] which reported the potent antifungal activity of M. villosus. However, there were differences in the inhibition of growth among the compounds against different strains of fungi. The ethyl acetate extract and fractions were shown to be fungistatic at lower concentrations and fungicidal at higher concentrations. The inhibitory action of M. villosus against Candida spp. has been linked to the presence of acetophenone derivatives [9].   [22]. With the strong antifungal activity shown by the fractions M. villosus against this organism, this plant shows promising characteristics for the use in the treatment and management of invasive fungal infections. Generally, the anti fungal activity of the ethyl acetate extract and fractions of M. villosus was strong and comparable to that of ketoconazole and fluconazole, which has been linked to the combined action of the secondary metabolites present in the extract [20].

CONCLUSION
The ethyl acetate extract and fractions of M. villosus possess antifungal activity against a broad spectrum of fungi ranging from yeast to dermatophytes. This medicinal plant holds great promise for the development of novel antifungal drug.

CONSENT
It is not applicable.