Combining Fluconazole and Leaf Extracts from <em>Vernonia adoensis</em> Enhances the Antifungal Effects on <em><em>Candida</em></em> krusei

Special Article – Mycology

J Bacteriol Mycol. 2018; 5(5): 1077.

Combining Fluconazole and Leaf Extracts from Vernonia adoensis Enhances the Antifungal Effects on Candida krusei

Nyamuriya R, Sithole S and Mukanganyama S*

Department of Biochemistry, University of Zimbabwe, Mt. Pleasant, Harare, Zimbabwe

*Corresponding author: Stanley Mukanganyama, Biomolecular Interactions Analyses Group, Department of Biochemistry, University of Zimbabwe, Pleasant, Harare, Zimbabwe

Received: August 01, 2018; Accepted: September 07, 2018; Published: September 14, 2018


Background: Invasive infections or colonization with Candida krusei have increased due to the prophylactic use of antifungals and widespread use to suppress fungal infections in HIV infected individuals. C. krusei is intrinsically resistant to fluconazole, an azole antifungal which works principally by inhibiting the cytochrome P-450 14a- demethylase and C- 14a- demethylase required for ergosterol synthesis. The aim of the study was to investigate if leaf extracts of Vernonia adoensis can enhance the effect of fluconazole on C. krusei. Eight solvent extracts of the V. adoensis leaves were prepared and these included DCM/Methanol extracts, hexane, DCM, ethyl acetate, ethanol, methanol and water extracts. The extracts were tested for their potency on inhibiting the growth of C. krusei. The MIC were determined from the most potent extract. The effects of combining fluconazole and the most potent extract were also investigated as well as the effect of the drug/plant extract combination on the efflux of rhodamine 6G from C. krusei. The distilled water extract was the most potent in inhibiting growth of C. krusei and was, therefore, combined with fluconazole to determine if there was enhanced effects. The combination lowered the minimum inhibitory concentration of fluconazole on C. krusei from 125 μg/ml to 32 μg/ml. Exposure to fluconazole caused a leakage of 1.03 μg/ ml of cellular proteins but when fluconazole was combined with the distilled water leaf extract, the protein leakage increased to 4.13 μg/ml. The distilled water extract enhanced the inhibition of transported across the membrane shown by increasing R6G concentration of 3.56μM with fluconazole to 4.46μM it was combined with fluconazole. In conclusion, the distilled water leaf extract enhanced the antifungal activity of fluconazole. This action may be due to increased protein leakage and inhibition of transport across the cell membrane. There is need to isolate the active chemical entities from V. adoensis so that they can be used as templates for design of novel antifungals with antifungal potentiation activities.

Keywords: Vernonia adoensis; Candida albicans; Candida krusei; Drug/ Herb Combinations; Candidiasis


Candida species are found globally and are part of the human normal flora [1]. Candida infections are found more in people whose immune has been compromised leading to extensions in hospital treatment and death that is significant [2]. The chief source of bloodstream fungal infections are caused by Candida species mainly C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei, which is the fifth in dominancy [3]. Candidemia is generally linked with an increase in morbidity leading to a rise in healthcare costs [4]. In HIV/AIDS patients, Candida species are the main cause of fungal infections. During the early and late phases of an HIV infection, oesophageal candidiasis and oropharyngeal candidiasis can develop due to Candida species colonisation in the oral mucosa [5]. Compared to the HIV negative women, vulvovaginal candidiasis is experienced more in HIV positive women [6]. Candida species can cause systemic and local infections in patients [7].

C. krusei is a fungal pathogen that has a possibility of being multi-drug resistant fungi due to its reduced susceptibility to amphotericin B and flucytosine as well as its intrinsic resistance to fluconazole [8]. However, in vitro, C. krusei remains susceptible to voriconazole as the drug binds more effectively to the C. krusei cytochrome P-450 isoenzyme [9]. Nosocomial infections are a main cause financial problem on patients, morbidity and death. When immunocompromised patients are suffering from a disease or undergo a surgery, they are at risk of nosocomial infections and are more affected if they are admitted in the intensive care unit [10]. The urinary tract, bloodstream, respiratory tract and surgical positions are the major sites of nosocomial infections [11]. The ability of Candida species to form biofilms are a possible cause of nosocomial infections. Biofilms are microbial adherences on abiotic or biotic surfaces to form a polymer matrix, which becomes the microbes’ growth substrate [12]. Invasive fungal infections are infections where fungi get into deep tissues and establish themselves leading to a prolonged illness [12]. In patients who are immunocompromised, the main cause of illness and death are opportunistic invasive fungal infections [13]. The prophylactic use of antifungals has been linked to invasive infections or C. krusei colonisation [2]. Invasive fungal infections are being increased by medical developments leading to a population that is prone to repressed immunological defenses against fungal infections [14].

Presently, over 25% of all artificial medicines have been developed after the discovery of chemical compounds, from plants, that have pharmacological values [15]. Despite advances observed in modern medicine, plants still contribute largely to health care and many modern pharmaceutical drugs contain plant ingredients [16]. The value of plant medicines is due to their phytochemical constituents [17]. Vernonia adoensis belongs to the Asteraceae family. It has a great anti-plasmodia activity [18]. Sexually transmitted diseases such as gonorrhoea are treated using the plant roots of V. adoensis [19]. V. adoensis leaves, contain phenols, flavonoids, terpenoids, steroids, saponins, alkaloids and tannins [20]. V. adoensis, leaves have a lot of pharmacological value in the treatment of sore throat infections, food poisoning and stomach problems [15].

Fluconazole is efficient in treatment of most Candida species except C. krusei [14,20]. Fluconazole is very active against numerous pathogens which results in systemic mycoses. C. krusei is intrinsically resistant to fluconazole [4]. People infected with fluconazole-resistant strains are at a higher risk of reinfection than those infected with susceptible strains [6].

Combination therapy has possible benefits, which comprise less resistant organisms, increased potency of antifungal effectiveness, and reduced toxicities as lesser doses will be administered. The micro dilution checkerboard is one of the traditional methods used for the evaluation of antifungal synergy with synergy determined as the viable growth of an organism that is reduced as a result of a combination of two drugs or a drug and an extract compared with most effective antifungal when tested alone [22]. Synergy requires a four-fold reduction in the MIC of antifungals in combination compared with each use alone [23]. There have been reports that show enhancement of antifungal activity by natural products when combined with conventional antifungals. One such report by Hirasawa and Takada (2004) showed that the catechins from green tea resulted in enhanced antifungal effect of amphotericin against C. albicans [24]. The aim of the study was to investigate if leaf extracts of V. adoensis can enhance the effect of fluconazole on C. krusei.

Materials and Methods

Chemicals used were obtained from Sigma-Aldrich (Steinheim, Germany). The list included Sabouraud dextrose broth and agar, glucose, reserpine, Rhodamine 6G, NaOH, sodium azide, glycine, HCl, sodium phosphate monobasic dehydrate and potassium phosphate dibasic trihydrate, anhydrous sodium carbonate, potassium sodium tartrate, bovine serum albumin and Folin and Ciocalteu’s phenol reagent. Methanol, ethanol, hexane, ethyl acetate, acetone and dichloromethane were obtained from Labchem (Edernvale, South Africa). C. krusei as well as the reference control fungi C. albicans (ATCC 10231) were obtained from the Department of Microbiology, Medical School, University of Zimbabwe, Harare, Zimbabwe. V. adoensis leaves were collected from Centenary (16.8°S, 31.1167°E, and 1156m above sea level), Mashonaland Central Province, Zimbabwe. The plant samples collected (C1E7) were authenticated by Mr Christopher Chapano, a taxonomist at the National Herbarium located at the Harare Botanical Gardens, Harare, Zimbabwe.

Extraction of phytochemicals

Fifty grams of powdered V. adoensis leaves were weighed. In total extraction, 250 ml of dichloromethane (DCM) and 250 ml of methanol were added to a beaker containing the weighed leaves. The mixture was left to stir overnight using a magnetic stirrer. The extract was then filtered [25]. The filtrate was left to evaporate in a fume hood and a fan was used to speed up the evaporation. When the filtrate had dried, the total extract was weighed, put into a falcon tube then stored at 4°C. The residue was discarded. Serial exhaustive extraction was performed using non-polar solvents first to polar solvents. Fifty grams of powdered V. adoensis leaves were weighed. The first solvent that was used for extraction was 400 ml of hexane. Hexane was added to 50 g of powdered V. adoensis leaves, which were in a beaker, and the mixture was left to stir overnight on a magnetic stirrer. The extract was then filtered from the marc and the filtrate was concentrated by evaporation in the fume hood. The residue was dried and 400 ml of DCM was added to it and left stirring overnight then filtered. After DCM, 400 ml of acetone, ethyl acetate, ethanol, methanol and distilled water were added, each time drying the residue after extraction. The filtered extracts were dried in a fume hood at ambient temperature (25oC) using a fan and the dried extracts were weighed, put in labelled falcon tubes and stored at 4°C.

Cell culturing

The media was prepared from Sabouraud dextrose broth (SDB). Thirty grams of SDB was dissolved in 1000ml of distilled water and autoclaved. Two centrifuge tubes were used and these were labelled control and C. krusei isolate 1 and C. krusei isolate 2. A volume of 20ml of SDB was added to each tube. In the growth tube, 20μl of inoculum from glycerol stock were added and in the control tube, there were no additions [25]. The tubes were incubated at 37oC overnight in an incubator shaking at 120 revolutions/minute. To culture in agar plates, 47g of Sabouraud and 2% glucose agar were dissolved in 1000 ml of boiled distilled water then autoclaved. The agar was poured into petri dishes and allowed to solidify. A volume of 20 μl of inoculum was pipetted at the centre and spread using a glass spreader that has been sterilised in 70% and burnt over the flame to remove the ethanol. The plates were left to incubate at 37°C overnight in an incubator without shaking.

Determination of minimum inhibitory concentrations (MIC)

The assay to determine the least concentration of the extracts and of the antifungal drug fluconazole that would inhibit the growth of C. krusei (both isolate 1 and isolate 2) was carried out according to CLSI reference method [26]. After incubating for 16-24 hrs at 37°C cells were diluted in relation to the McFarland’s standard [27]. The solution of 0.5 McFarland’s must be in a range of 0.08 to 0.10 optical density at 600nm and would contain 1.5x108 CFU/ml of cells. Calculations were done so that the final cell concentrations in the micro titre well was 2×106 CFU/ml of cells.

Cells were exposed to extracts or fluconazole in a 96-well plate. The concentration of the standard antibiotic ranged from 0-1 μg/ml. When assessing the effects of the extracts, the drug was substituted with extracts at concentrations ranging from 100 μg/ml to 0 μg/ml. The plates were sealed with parafilm™ paper and incubated in a container containing wet paper towel over night at 37°C in a non-shaking incubator (Jeio Tech, Korea). The MIC was defined by comparing the amount of growth in the test tubes and the growth control tubes as reported by Santos and Hamdan (2005) and recommended by the Clinical and Laboratory Standards Institute [28].

Determination of the combination of fluconazole and extract on growth of C. krusei

The assay was carried out following the procedure by Ahmad et al., [29] to evaluate if there was enhanced antifungal activity when fluconazole and the leaf extract were combined The final cell concentrations in the 96-well microtitre wells was 2×106 CFU/ml of cells. To each well, 50 μl of the drug was added to 50 μl of the extract then 100μl of the diluted cells were added. The plate was left in a nonshaking incubator (Jeio Tech, Korea) at 37oC.

Protein leakage assay

Determination of protein leakage was carried out according to the method by Ladokhin et al., [30], C. krusei was sub-cultured in 20 μl of Sabouraud Dextrose broth media in a falcon tube and left to incubate overnight at 37°C with shaking at 120 rpm. A volume of 200 μl of the sub-cultured cells were added to 200 ml of SDB media in a 1 litre container and left to incubate overnight at 37°C with shaking at 120 rpm. The cells were then poured into 4 centrifuge tubes, distributing them equally. The tubes were then centrifuged at 3000 rpm for 5 min and the supernatant was discarded. The pellets were diluted with 0.9% saline until their cell density was 1.526 at 600 nm. To 5 centrifuge tubes which were labelled A, B, C, D and E, 6mls of cells were added. To A, a volume of 120 μl of 5 mg/ml stock of the distilled extract and 96 μl of 2 mg/ml fluconazole were added. The combination concentrations were 100μg/ml extract and 32 μg/ ml fluconazole. To B, only 96 μl of 2 mg/ml fluconazole was added. To C, only 120 μl of 5 mg/ml stock of the distilled extract was added. To D, 216 μl of sterile distilled water was added and to E, 600 μl of 1% SDS so that the final concentration was 0.1%. The tubes were left to incubate for 2 hr at 37°C in a shaking incubator SI-300, incubator Lab companion, (Jeio Teicho, Korea) and then centrifuged at 3500 rpm for 4 min. The pellet was discarded the supernatants were used for the protein determination assays. The controls were D (cells only) and E (0.1% SDS). Protein content was measured by the method of Lowry (1951), using BSA as a standard.

The effects of extract/fluconazole combination on drug efflux in C. krusei

The determination of drug efflux assay was carried out according to the method by Maesaki et al [31]. Using rhodamine 6G as a probe substrate for the efflux pumps. C. krusei was first cultured in a sterile 50 ml centrifuge tube and then 200 μL of the cells were inoculated into 2000 μL of Sabouraud dextrose broth and left in an incubator overnight at 37°C shaking at 120 rpm. The cells were divided into 6x50 ml centrifuge tubes and centrifuged at 4000rpm for 10ml using Hettich Rotofix 32 Centrifuge, (Tuttingen, Germany). The supernatant was discarded and the cells were washed twice in phosphate buffered saline (PBS) pH 7.2 by mixing the cells with the buffer, centrifuging at 4000 rpm for 10 min and discarding the supernatant. The pellet was collected in a weighed tube and the mass of the pellet was weighed before being suspended in PBS containing 10mM NaN3 at a density of 40mg/ml. Rhodamine 6G (R6G) was immediately added to the cells to achieve a final concentration of 10 μM. The cells were incubated at 120 rpm for 1 hour. The cells were divided into two tubes A and B in the ratio 1:2. The tubes were centrifuged at 4000 rpm for 5 min. After discarding the supernatant, the pellet in tube A was resuspended in PBS at 40 mg/ml while the pellet in tube B was resuspended in PBS containing 1M glucose at 40 mg/ml. Tube B was further divided into 10 tubes each with 5 ml of the cells suspended in 1M glucose. To tubes B1 and B2, 60 μg/ml of reserpine was added. To tubes B3 and B4, fluconazole was added such that the final concentration of fluconazole was 32 μg/ml. To tubes B5 and B6, the distilled water leaf extract of V. adoensis was added such that the final concentration of the extract was 100 μg/ml. To tubes B7 and B8, a combination of fluconazole and the distilled water extract was added, the same volumes added in B3 and B5. Tubes B9 and B10 were controls with glucose only. The contents of the tubes were mixed and incubated in an incubator at 37°C shaking at 120 rpm for 30 minutes. The tubes were then centrifuged at 4000 rpm for 10min and the supernatants were collected in other clean tubes. The pellets were resuspended in 5 ml of 0.1M glycine HCl, pH 3 to lyse. The cells were incubated for 18 hours at 37°C shaking at 120rpm. After, the tubes were centrifuged at 4000 rpm for 10 minutes and the supernatant was collected. The absorbance of R6G was measured at 527 nm using the Unico 2100 UV spectrophotometer, (United Product and Instruments Inc, USA). The standards were prepared by diluting 10 μM of R6G in glycine HCl.

Reading of MICs and Statistical analyses

The absorbance of the plate was read at 590nm before and after incubation using a Multimode micro plate reader, GENios Pro, (TECAN Austria G.M.B.H, Untersbergstrasse, Austria). The difference in the absorbance values between pre-incubation and post incubation was used to plot a graph using the GraphPad prism 5 for Windows (GraphPad Software Inc., San Diego, California, USA version 5.03). Statistically significant differences between the mean of the controls and the tests were analysed using one-way ANOVA with Dunnett’s multiple comparison post-test. Enzyme kinetics was analysed with nonlinear regression and allosteric sigmoidal using GraphPad Prism 5 (Version 5.03 GraphPad Software Inc. San Diego, California U.S.A).


Extraction of phytochemicals from Vernonia adoensis leaves

The V. adoensis DCM/Methanol (1:1) had the highest yield (10%) followed by distilled water extract (5%), methanol (4%) and acetone (2%) as shown in Table 1. The yields did not depend on the polarity of the solvent used for extraction.