Malva sylvestris inhibits Candida albicans biofilm formation

M. sylvestris inhibits C. albicans biofilm formation Journal of Herbmed Pharmacology, Volume 6, Number 2, April 2017 http://www.herbmedpharmacol.com 63 increased expression of drug efflux pump genes and metabolic plasticity provide protection by preventing the penetration of host immune factors and antifungals agents into the extracellular matrix in comparison to planktonic cells (3,6-10). The ability of C. albicans to form drug-resistant biofilms has important clinical repercussions. The increasing prevalence of drug-resistant strains of Candida species necessitates searches for new targets for new antifungal agents. Among the alternative therapies, the use of natural medicines obtained from the plants has attracted great attention because, according to the World Health Organization (WHO), these would be the best source for obtaining a wide variety of drugs that could benefit a large portion of the patient population (3,9,10). Nature has been a source of medicinal products for centuries, Malva sylvestris, Dorema aucheri, Ferulago angulata and Citrullus colocynthis are important traditional medicinal plants. M. sylvestris (11-13), D. aucheri (14,15), F. angulate (16,17) and C. colocynthis (1821) have inhibitory activities against microorganisms. Moreover, M. sylvestris (11,12), F. angulata (16) and C. colocynthis (19,20) extracts exhibited in vitro antifungal activity against some Candida species. Here we performed the aqueous and ethanol extracts of the different parts of M. sylvestris, D. aucheri, F. angulata and C. colocynthis plants to identify best plant extract that inhibits growth of C. albicans or C. krusei, and conducted a series of follow-up studies to examine the inhibitors of C. albicans biofilm formation of the identified plant extract. Methods Microorganisms Three clinical isolates of C. albicans and Candida krusei from Shahid Beheshti hospital, Yasooj, Iran were obtained. All samples were isolated from patients with systemic candidiasis. Candida albicans ATCC 10231 and C. krusei ATCC 6258 as reference quality-control strains were employed. After all the isolates were identified by conventional methods (22) and using CHROM agar Candida medium (CHROM agar Company, France), all the isolates maintained as sterile 20% (v/v) glycerol stocks and subcultured on sabouraud dextrose agar with chloramphenicol (SDA, Difco Laboratories, Detroit, Michigan) at 35–37°C for 24–48 hours to ensure viability and purity prior to testing. Plants materials Malva sylvestris, D. aucheri, F. angulata and C. colocynthis plants were harvested in Yasooj, Iran. Plant materials were transported to the lab immediately and voucher specimens were verified by Zardband Pharmaceuticals Company, Yasooj. Different parts of the plant were immediately cleaned and further processed according to the method described by Khodavandi et al (23), with slight modifications. Different parts of the plant were washed with sterile distilled water, sliced and dried in the oven for at least 2 days. One gram of tissues powder was mixed with 5 mL of sterile distilled water or ethanol and incubated at 37°C for 72 hours. The tissues extract was undergone a sequential extraction using soxhlet. The tissues extract was filter-sterilized (0.22-μm durapore, Millipore). Effect of Malva sylvestris, Dorema aucheri, Ferulago angulata and Citrullus colocynthis on growth of Candida albicans and Candida krusei The Clinical and Laboratory Standards Institute guidelines (CLSI) (24) were used for the antifungal disk diffusion susceptibility test. The experiments were conducted in triplicate in 2 independent assays. The plant extracts were subjected to broth microdilution antifungal susceptibility test using CLSI reference method for yeast (25), with end points determined at 24 hours post inoculation. Effect of ethanol extract of Malva sylvestris root on Candida albicans biofilm formation Candida albicans ATCC 10231 was used to form biofilm according to the method described by Khodavandi et al (26). Briefly, a suspension of C. albicans cell with a density of 1 × 106 cells/mL was added to 100 μL of plant extract at different concentrations based on minimum inhibitory concentration (MIC) (1/4 MIC, 1/2 MIC, 1 MIC and 2 MIC) using 96-well microplate (Brand 781660, Wertheim, Germany) and incubated at 35°C for 90 minutes. Subsequently, the mixture was incubated at 35°C for 24 hours with gentle shaking. Quantitative and qualitative analysis The cultures were used for quantitative and qualitative analysis using crystal violet (CV) assay and morphological response. The protocol of CV assay by Khodavandi et al (26) was adopted. The biofilm was fixed for 15 minutes by adding 100 μL of 99% methanol to each well and then the supernatants were ejected. Further, the cells were airdried and then stained with CV solution (1:50 from stock solution, Sigma). The extra CV was removed and the wells were filled with 150 μL of acetic acid 33% (Sigma, USA). The absorbance was measured at 630 nm using ELISA microplate reader. We employed light microscopy method as a qualitative assay to investigate the anti-biofim activity of plant extract against C. albicans. The C. albicans biofilms were prepared as previously described (26). Thereafter, the media were discarded and coverslips were washed twice by PBS and viewed with a light microscope (Leica, DMRA II, Germany). qRT-PCR analysis of Candida albicans hypha-specific gene A suspension containing different concentrations of antifungal agents and 1×106 cells/mL of C. albicans ATCC 10231 were prepared as previously described (26). Total RNA was extracted from biofilm sample using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The quantity and purity of the isolated RNA were measured by Nano Drop