ANTIFUNGAL EFFECT OF ETHANOL PLANT EXTRACT ON CANDIDA SP

In this study, we investigated the in-vitro antimicrobial activity of some medicinal plants in the Arabian peninsula, including Rhamnus globosa, Ocimum basilicum, Tecoma stans and Coleus forskohlii. Our results showed high inhibitory growth in yeast after treatment with R. globosa and O. basilicum. C. tropicalis was shown to be a sensitive strain with an inhibition of 29, 28, 35, 25 and 27 mm after treatment with R. globosa, R. globosa* “leaf with thorns,” O. basilicum, Tecoma stans and Coleus forskohlii, respectively. Thus, our results confirmed the fungicidal effect of O. basilicum and R. globosa with a 20 and 30% reduction in CFU compared with the starting inoculums in the time-kill.


INTRODUCTION
Candida albicans is a common microflora in humans and an opportunistic fungal infection, which is often found in compromised immune systems and the overgrowth of this yeast causes candidiasis. In addition, these fungal pathogens have become increasingly important over the past 20 years; with the success of modern medical practices, this provides hope for the survival of weakened and immunosuppressed patients (Kourkoumpetis et al., 2010;Kothavade et al., 2010;Tanushree et al., 2010;Dalirsani et al., 2011). Treatment of pathogenic fungi involves antifungal medications, which include several groups. However, fungal and human cells are similar at the molecular level and thus it is difficult to identify medicines that target fungi without affecting human cells. Consequently, the side effects of these drugs include allergic reactions, liver damage and altered estrogen levels. Moreover, improper use of antifungals can be life threatening (McMichael and Hordinsky, 2008). Thus, new antifungal drugs that are safer and more effective are needed.
Natural products derived from medicinal plants are among the safest sources of new medications and antifungal drugs. Several studies have investigated natural products and essential oils with antimicrobial and antifungal effects. For example, in a study of alcoholic curry leaves, a maximum zone of inhibition on Candida albicans following treatment with aqueous tea leaves was observed. However, other plant extracts such as alcoholic onion leaves, alcoholic tea leaves, alcoholic onion bulb, alcoholic aloe vera and alcoholic mint leaves inhibited the growth of Candida albicans, but to a lesser extent (Doddanna et al., 2013). Salvia officinalis (L.) demonstrated less inhibitory effects against C. albicans or C. tropicalis and the root extracts of Labisia pumila showed a higher activity in response to Candida sp. compared with leaf extracts (Celi Garcia et al., 2012;Karimi et al., 2013). In a study on Chinese medicinal plants, water, ethanol, acetone and n-hexane extracts for each plant were tested on C. albicans, as well as extracts of Pseudolarix kaempferi Gord., acetone extract of Sophora flavescens Ait., ethanol, acetone and hexane extracts of Pogostemon cablin (Blanco) Benth. and Alpinia officinarum hance, hexane extract of Eugenia caryophyllata Thunb. ethanol and acetone extracts of Melia toosendan Sieb. et Zucc. and Polygonum hydropiper L., which showed an inhibition of more than Science Publications AJABS 50% of C. albicans growth and was comparable to miconazole in some cases (Liu et al., 2012).
In this study, the objective was to investigate the antifungal activity of ethanol extracts of some medicinal plants in the Arabian Peninsula. These medicinal plants included Rhamnus globosa, Ocimum basilicum, Tecoma stans and Coleus forskohlii.

Plant Studies and Extract Preparation
The plants were collected from different locations in the Arabian Peninsula. They were identified in the Botany section at the biology department, faculty of science, KAU as Rhamnus globosa, Ocimum basilicum, Tecoma stans and Coleus forskohlii. The plant leaves, except R. globosa, which consisted of two types of extracts (leaves and leaves with thorns) were washed several times with distilled water, spread onto plates and dried at 40°C. After drying, the samples were grounded and solubilized with ethanol solvent at 10 mg mL −1 . The mixtures were maintained on a shaker at 120 rpm at 30°C for 24 h and then filtered using Whatman No. 1 filter paper. The samples were dried under a reduced pressure at 40°C and the thick deposits obtained were used as crude extracts (Vijayakumar et al., 2013).

Antifungal Assays
The antimicrobial activity of each crude plant extract was determined in vitro in response to the Candida species. The activities were measured using disc diffusion and broth dilution methods, as previously described by the Clinical and Laboratory Standards Institute (CLSI; formerly known as the National Committee for Clinical Laboratory Standards) (NCCLS, 2004;Fothergill, 2011).
Each thick deposit extract was dissolved in Dimethylsulfoxide (DMSO) at 50 µg mL −1 and filtered through a 0.22 µm pore filter (Millipore, Billeria, MA). The antibacterial activities of each extract were investigated by disc diffusion using filter paper discs (1-mm diameter impregnated with 100 µL), which were then placed on the pre-inoculated agar surface. Negative controls were prepared with the same solvent. Plates were then incubated at 35°C for 48 h and the inhibitory zones of each disc were measured. All tests were performed in triplicate.

Minimum Inhibitory Concentration (MIC) and Minimum Fungicidal Concentration (MFC)
Extracts that inhibited the growth of bacteria were investigated to determine the MIC and MFC using a broth-microdilution method. The yeast were cultured overnight on mueller-hinton agar and then resuspended in 1 mL mueller-hinton broth (OXOID CM 405) to obtain a final concentration of 100 cfu mL −1 . Each extract was serially diluted with muellerhinton broth using methods approved by the National Committee for Clinical Laboratory Standards (M27-A) (NCCLS, 1997). After incubation, the MIC was determined as the lowest concentration of extract for which there was no visible growth compared with the control (CLSI, 2008;. The MFC was determined by inoculating 0.1 mL of negative growth in MIC onto sterile SDA (OXOID) plates. The plates were incubated at 35°C for 48 h. The lowest concentration of plant extract that did not demonstrate growth of the tested organisms was considered the MFC; the negative control was a plate grown with media only (Ernst et al., 2002;Wiegand et al., 2008).

Time-Kill Determination
Liquid cultures (1 mL) were diluted to an initial inoculum of 2×10 5 -5×10 5 CFU/mL in Mueller-Hinton broth and R. globosa, O. basilicum, T. stans and C. forskohlii were added at one-half and one and two times the MICs. The cultures were incubated for 0, 2, 4, 8, 12 and 48 h at 30°C. After each time point, 50 µL aliquots were obtained from the cultures, plated onto the SDA and incubated at 35°C for 48 h. Visible colonies were read using an Interscience scan 500 colony counters and each treatment was performed in triplicate (Nostro et al., 2000;Lewis et al., 2002).

Statistical Analysis
The results were analyzed by paired-samples t-test using the IBM SPSS 20 statistical software to compare the mean values of each treatment. The results are expressed as means ± SE. Probability levels of less than 0.01 were considered highly significant.

RESULTS
Investigation of new antifungal agents from natural sources is a major field of research and the results of these studies have identified several new sources of plant medicines and their synthetic compounds. As shown in Table 1, high inhibitory growth of the tested yeast was observed after treatment with R. globosa and O. basilicum and C. tropicalis was shown to be a sensitive strain with an inhibition of 29, 28, 35, 25 and 27 mm after treatment with R. globosa, R. globosa* "leaf with thorns," O. basilicum, Tecoma stans and Coleus forskohlii, respectively. The ethanol extract of the R. globosa leaf and R. globosa leaf with thorns showed that there was no significant difference between these bacteria on yeast growth inhibition and the highest inhibitory effect was observed in C. tropicalis, with an inhibition of 29 mm for R. globosa.
The MIC and MFC values are shown in Table 2 and 3. According to these results, the highest MICs were 8 µ L mL −1 and 4 µ L mL −1 , which were obtained by treatment with O. basilicum on C. albicans and C. tropicalis, respectively. In contrast, the lowest MICs were obtained by treatment with Tecoma stans extracts on the tested yeast. The most sensitive yeast was C. tropicalis with MICs of 4, 8, 4, 16 and 8 µ L mL −1 after treatment with R. globosa, R. globosa* "leaf with thorns," O. basilicum, Tecoma stans and Coleus forskohlii, respectively. The MFCs were approximate in most plant extracts. Most fungicidal concentrations were affected by treatments with O. basilicum on C. albicans and C. tropicalis with values of 32 and 16 µ L mL −1 , respectively and the fungicidal extract concentrations of Tecoma stans increased to 128 and 128 µ L mL −1 , for C. albicans and C. tropicalis, respectively.
The concentration of MICs and MBCs reflected the kill-times of the tested yeast. As shown in Fig. 1 and  2, the kill-time of one-half MIC, MIC and two MIC of each plant extract had no significant differences. The tested yeast had an endpoint of kill-times at a concentration of two MICs within 4 h except T. stans and C. forskohlii, which had an endpoint of kill-time of 12 h and 8 h, respectively. The CFUs of the tested bacteria decreased after 2 h and continued to decrease until they reached the kill-time. The fungicidal endpoint was not reached at a concentration of onehalf MIC and extracts of R. globosa, O. basilicum and C. forskohlii resulted in 20, 30 and 10% reduction in CFU from the starting inoculums, respectively.

DESSCUSION
Previous studies have performed screens on the antimicrobial activity of extracts from plants. On the basis of these studies, it was concluded that diethyl ether extracts were the most efficient antimicrobial compounds and this activity was more pronounced against grampositive bacteria (Nostro et al., 2000). Our results on ethanol extracts were consistent with the results of several studies on alcoholic and non-alcoholic plant extracts. A previous study of alcoholic curry leaves demonstrated the strongest growth inhibition of C. albicans followed by aqueous tea leaves, alcoholic onion, tea, mint leaves extracts and alcoholic onion bulb and aloe vera extracts (Duarte et al., 2005). Moreover, a study of Traditional Chinese Medicine (TCM) and Chinese folk medicine demonstrated antifungal activity against Candida albicans; however, out of 58 extracts examined, two plant extracts, Codonopsis pilosula and Tussilago farfara, showed high inhibitory effects against C. albicans (Karimi et al., 2013;Zhang et al., 2013). In addition, a recent study on six natural commodities and four commercial medicines against C. albicans revealed that Mayaca extracts could act as a potential antifungal agent for oral thrush caused by C. albicans (Reena et al., 2013). Furthermore, extracts of Althaea officinalis and Matricaria recutita and Combretum molle, Piper capense, Solanum aculeastrum, Syzygium cordatum and Zanthoxylum davyi have a fungicidal effect on C. albicans (Steenkamp et al., 2007;Shakib et al., 2013). However, a study on essential oils and ethanolic extracts from the leaves and roots of 35 medicinal plants commonly used in Brazil, were tested for an antifungal effect on C. albicans and essential oils from 13 plants showed antifungal activity, including Aloysia triphylla, Anthemis nobilis, Cymbopogon martini, Cymbopogon winterianus, Cyperus articulatus, Cyperus rotundus, Lippia alba, Mentha arvensis, Mikania glomerata, Mentha piperita, Mentha sp., Stachys byzantina and Solidago chilensis. Moreover, the ethanol extract was not effective at any of the concentrations tested (Duarte et al., 2005).
The high antimicrobial effect of medicinal plant extracts may be due to the compensation between the secondary metabolic compounds in plant tissues. Results of the phytochemical screening on medicinal plants showed that the antimicrobial activity was most likely due to the compounds reduced from plant metabolism, such as flavonoids, terpenes, alkaloids, tannins, the hydroxyl group and phenol; 1,8-cineole, geranial, germacrene-D, limonene, linalool, fatty acids, esters and menthol; and essential oils, such as yarrow, carvacrol, thymol, glycosides, tannins, saponins and steroids (Gregory et al., 2009;Choudhury et al., 2013;Jadha et al., 2013;Joshua and Takudzwa, 2013;Mulyono et al., 2013).

CONCLUSION
In conclusion, we highlighted the antifungal activity of ethanol extracts of Rhamnus globosa, Ocimum basilicum, Tecoma stans and Coleus forskohlii. These results confirmed the traditional uses of R. globosa as medicinal plants. In addition, extracts of O. basilicum primarily produced fungicidal effects, with a limited number of observed growths, which was consistent with the MIC and MBC and kill-time of both plant extracts.