Introduction  

One of the health problems in Indonesia is fungal infections, such as candidiasis and dermatomycosis [1-4]. During the past years, fungal infections have been increased due to the risen immunocompromised population (e.g., patients with AIDS or cancer, and organ transplant recipients). The prevalence of these diseases has been increased since the 1980s [5]. Candidiasis is caused by Candida albicans (C. albicans), a normal flora in the human body which in certain conditions becomes pathogenic and causes various diseases such as thrush, vulvanginitis, and even dangerous systemic candidiasis diseases. Generally, candidiasis treatment uses topical therapies, while systemic treatment is given to patients who experience resistance or show less effective response to topical therapies. Some antifungal drugs that are used to treat candidiasis include the azole group, nystatin, and amphotericin B [6].  Approximately, 69.5% of dermatomycosis in humans is caused by Trichophyton rubrum (T. rubrum). This fungus usually infects the tissues containing keratin, such as nails, hair, and stratum corneum in epidermidis layer, which can expand into lesions [7]. Some antifungal drugs used to treat dermatomycosis are amphotericin B, azole group, alyl amine group, and griseofulvin [8, 9]. 

Most antifungal drugs can change the life cycle of fungi and growth patterns of fungi, leading to resistance in certain pathogenic fungi [10]. Therefore, the discovery of novel antifungal agents becomes a necessity, to widen the spectrum of antifungal activity against resistant fungi such as C. albicans and T. rubrum. The effort in exploring antifungal agents could be from conventional antifungal drugs, as well as from medicinal plants. Antifungal properties of some medicinal plants have been reported based on folklore data, while others are from studies on the inhibitory activity of the plants against some pathogenic fungi.

Medinilla speciosa (M. speciosa) Blume (Melastomaceae), is a plant originating from Mount Muria, Kudus, Central Java, Indonesia, which has been used by the community to treat diarrhea, inflammation, and bacterial infections [11]. Methanol, ethanol and ethyl acetate extracts of this fruit showed antibacterial activity against Staphylococcus aureus, Escherichia coli, Pseudomonas aureginosa, Bacillus subtilis, Methicillin-Resistant S. aureus (MRSA), and ESBL E. coli [12]. M. speciosa Blume fruit contains various secondary metabolites, including terpenes, tannins, flavonoids, saponins, and glycosides. Antifungal activity study from Melastoma malabathricum, in the same family with M. speciosa, revealed that the extract inhibited the growth of Candida krusei [13].  Hence, the study on the antifungal activity of M. speciosa Blume fruit has not been reported yet. Therefore, this present study was conducted to determine the antifungal activity of M. speciosa fruit extract against C. albicans ATCC 10231 and T. rubrum ATCC 28188. It is expected that the data can be used as evidence of the potential of M. speciosa Blume fruit to be used as an alternative antifungal agent.

Materials and Methods

Materials

M. speciosa Blume fruit was collected from Mount Muria, Kudus, Central Java, Indonesia. C. albicans ATCC 10231 and T. rubrum ATCC 28188 were obtained from Hasan Sadikin Hospital, Bandung, West Java, Indonesia. The culture media were Potato Dextrose Agar/PDA (Oxoid) and Potato Dextrose Broth/PDB (Oxoid). Ketoconazole, as a reference antibiotic, was obtained from PT. Dexa Medica Indonesia.

The other materials used in this study were n-hexane (Merck), ethyl acetate (Merck), methanol (Merck), ammonia (Merck), chloroform (Merck), hydrochloric acid (Merck), Mayer reagent, Dragendorf reagent, Liebermann-Burchard reagent, magnesium powder (Bratachem), amyl alcohol (Merck), potassium hydroxide (Merck), iron (III) chloride (Merck), gelatin (Bratachem), ether (Merck), vanillin (Merck), dimethyl sulfoxide/DMSO (Merck), 96% ethanol (Bratachem), normal saline solution (Otsu-NS), 0.5 McFarland standard solution, TLC Silica Gel 60 F245 (Merck),  paper discs 6 mm (Whatman), filter paper No. 1 (Whatman), and distilled water.

Plant determination and sample preparation

Leaves, fruits, stems, and roots of this plant were determined at Plant Taxonomy Laboratory, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Sumedang, West Java, Indonesia. The healthy fresh fruit was sorted, then washed with running tap water and distilled water to remove any adsorbed contaminant from the fruit surface. The cleaned sample was dried at room temperature and ground to a coarse powder.

Sample extraction

The sample was extracted by a gradual maceration using three types of solvents of polar (methanol), semipolar (ethyl acetate), and nonpolar (n-hexane) solvents. The dried fruit powder (182 g) was macerated with 1 L of n-hexane at room temperature for 24 h. Next, the sample was filtered using filter paper No.1. The extraction process was repeated several times until the last drop of the extract was colorless. The residual sample was dried.  The remaining sample was subjected to extraction by maceration using methanol and ethyl acetate as solvents. The solvents were removed using a rotary evaporator at 45°C, then concentrated in a 60°C water bath until the solid mass of extracts was obtained. The extracts were observed organoleptically and continued by yields determination. 

Phytochemicals screening

Phytochemical compounds of M. speciosa Blume fruit extracts were screened using standard procedures as described by Fansworth (1966) in the former publication [14].

Thin layer chromatography (TLC) profile assay

TLC profile assay of n-hexane, ethyl acetate, and methanol extract from M. speciosa Blume was carried by using the stationary phase of TLC Silica Gel 60 F245, while the mobile phase was determined from the optimization process. The plate was observed under visible light, UV light 254, and 366 nm. Rf value was determined for each spot. 

Antifungal activity test

Antifungal activity of M. speciosa Blume fruit extracts was tested against C. albicans ATCC 10231 and T. rubrum ATCC 28188 using paper disc diffusion assay (Kirby-Bauer method) [15] with some modifications. The extracts stock solutions were prepared at a concentration of 50 % w/v in DMSO 2% v/v. The sample solutions were prepared by diluting the stock solution to give concentrations of 20, 30, 40, and 50 % w/v against C. albicans ATCC 10231 and 5, 10, 15, and 20% w/v against T. rubrum ATCC 28188.

About 20 mL of warm PDA was added to a sterilized petri dish and allowed to solidify at room temperature. Briefly, 50 µL of fungus suspensions were objected to PDA using a sterile drigalski rod. The sterile paper disc containing 20 μL of the extract was placed on the agar. As a comparison, the positive (PDA inoculated with fungal suspension) and negative (PDA) controls were also prepared. All plates were incubated at 30ºC for 1-2 days for C. albicans ATCC 10231 and 3-5 days for T. rubrum ATCC 28188. The inhibition zones were measured using calipers. All measurements were carried out in triplicate.

Determination of minimum inhibitory concentration (MIC) and minimum fungicidal concentration (MFC) values

Determination of MIC and MFC values of the most potential extract of M. speciosa Blume fruit against C. albicans ATCC 10231 and/or T. rubrum ATCC 28188 were performed in 96-well microtiter plates, using two-fold broth microdilution methods in triplicates [16, 17] with some modifications according to procedures of Nejad [18]. The crude extract of M. speciosa Blume was dissolved in 2% v/v DMSO, at a concentration of 10 % w/v or 100,000 ppm served as a stock solution. For the assay, stock extract solutions were diluted twice with sterile distilled water to produce serial decreasing dilutions ranging from columns 3 to 12. The adjusted inoculum suspensions should be diluted in sterile normal saline solution, so that, after inoculation, each well contained approximately 5 × 10² - 2.5 × 10³ CFU/mL for C. albicans ATCC 10231 and 0.4 × 10⁴ - 5 × 10⁴ CFU/mL) for T. rubrum ATCC 28188.

About 50 µL fungus suspension was inoculated against each well containing 50 µL of extract in the dilution series (and a positive control containing only broth) and were mixed thoroughly. Column 1 served as a negative control (PDB), while column 2 as a positive control was also used as a blank control to verify fungal growth. The microplates were then incubated at 30ºC for 1-2 days for C. albicans ATCC 10231 and 3-5 days for T. rubrum ATCC 28188. The MIC value is the lowest concentration of extract to give complete inhibition recorded as visible growth.

The MFC was determined by introducing the solution from each well and cultured on PDA. The plates were incubated at 30ºC for 1-2 days for C. albicans ATCC 10231 or 3-5 days for T. rubrum ATCC 28188. The lowest concentration producing no visible fungal growth after incubation was considered to be MFC value. Each fungus was tested in triplicate with 3 plates.

Comparative study of antifungal activity

The comparative study of antifungal activity was performed to determine the equivalence of antifungal activity of the most potential extract of M. speciosa Blume fruit compared with ketoconazole as a standard antifungal agent against C. albicans ATCC 10231 and/or T. rubrum ATCC 28188. The experiment was performed on the same plate using the same procedure as the antifungal activity test [15]. Ketoconazole was used as a standard due to its commonly used antifungal drug for the treatment of dermatomycoses [8]. 

The extract and Ketoconazole were dissolved and diluted to give various concentrations of samples. The log concentration values as x-axis were plotted against the inhibition diameter (mm) on the y-axis. The linear regression as a mathematical model was obtained and used to calculate comparative antifungal activity value. By comparing the concentration of M. speciosa Blume fruit extracts with the concentration of Ketoconazole producing the same inhibitory diameter, the comparison number can be evaluated.

Results and Discussion

Plant determination and sample preparation

The plant determination results showed that the plant is identic with Medinilla speciosa Blume from Melastomataceae family, Myrtales order, Magnoliopsida class, and Traceophyta division.

Sample extraction

Extraction was performed by maceration to avoid degradation of the thermolabile compound due to the heating process during extraction. The percentage yields of n-hexane, ethyl acetate, and methanol extracts were 3.34, 1.47, and 8.79% w/w, respectively. Characteristics of the extracts were thick, sticky, distinctive smelling with specific colour (light green for n-hexane, dark green for ethyl acetate and red brown for methanol extract).  

It can be concluded that methanol extract gave the highest yield, followed by n-hexane and ethyl acetate extract. The results revealed that the compounds extracted from M. speciosa Blume fruit were dissolved more in the polar solvent than non-polar or semi-polar solvents.

Phytochemicals screening

The phytochemicals contained in M. speciosa Blume fruit extracts are presented in Table 1.

+ : Presence, - : Absence

Considering Table 1, it can be seen that the n-hexane extract of M. speciosa Blume fruit contains polyphenols, steroids, triterpenes, and quinones. Non-polar compounds, such as fat, steroids, coumarin, and some terpenes dissolve in n-hexane [19]. Furthermore, Ethyl acetate extract contains saponins, sesquiterpenes, monoterpenes, flavonoids, tannins, polyphenols, and alkaloids, while methanol extract contains saponins, quinones, flavonoids, tannins, polyphenols, and alkaloids. The compounds contained in the three M. speciosa Blume fruit extracts are polyphenols. Polyphenols, tannins, flavonoids, and saponins compounds can generally be dissolved by polar or semi-polar solvents, such as methanol and ethyl acetate [19]. Flavonoid compounds have polyhydroxy groups, so these compounds are more partitioned in semi-polar and polar solvents. Flavonoids and phenolic acids have been the object of various investigations concerning their antioxidant and antimicrobial activities.

TLC profile assay

Based on the optimization results, the mobile phase was ethyl acetate:n-hexane (3:7) for n-hexane and ethyl acetate extracts, while for methanolic extract was n-hexane: ethyl acetate (1:9). The result of the TLC profile assay of M. speciosa fruit extracts is shown in Table 2.

Concerning Table 2, it can be seen that the methanol and ethyl acetate extracts gave more spots than that from n-hexane extract. There are one or two yellow-greenish spots under visible light and blue fluorescence under UV light 366 nm indicating the presence of flavonoid compounds [20]. 

Antifungal activity test

Antifungal activity of M. speciosa Blume fruit extracts against C. albicans ATCC 10231 and T. rubrum ATCC 28188 are listed in Table 3 and Figure 1.

Paper Discs Diameter = 6 mm