Evaluation of Antifungal Potentials and Antioxidant Capacities of Some Foliose Lichen Species

This work is aimed to assess of antioxidant and antifungal potential of the foliose lichen species: Flavoparmelia caperata , Xanthoparmelia stenophylla and Xanthoparmelia conspersa . The antifungal activity of lichens were studied against some pathogenic fungi by disc diffusion method. The acetonitrile extracts of these lichens were obtained with Soxhlet extraction. While F. caperata exhibited maximum antifungal activity (32 mm) against the C. albicans , the minimum antifungal activity (10 mm) was obtained from X. stenophylla lichen against S. cerevisiae . In CUPRAC assay, absorbance values was in order of BHT> X. conspersa > F. caperata > X . stenophylla . As a result of the study, it was concluded that these lichen species may be a potential source for the development of new antifungal and antioxidant compounds.


Introduction
Antioxidant term is prominent in human lifetime.Various biological roles such as anti-mutagenicity, anti-aging and anti-carcinogenicity are thought to be based on this concept.The growth of medicinal plants has been connected with phytochemicals which is in their structure.These phytochemicals provide health maintenance and protection against many diseases and cancers [1].Many studies were revealed on the preventive actions of natural antioxidants against diseases, like AIDS, Parkinson, Alzheimer, diabetes, eye diseases, and cardiovascular diseases [2].As understanding of the function of free radicals in human diseases has enhanced, antioxidants have gained remarkable concern because of their role in inhibiting harmful effects of free radicals and their help in preventing the body against injury by reactive oxygen species [1].
Infectious which was originated from pathogenic fungi is a crucial hazard on public health.Of late years, increase in immunocompromised patients like organ transplant recipients, HIV and cancer patients cause the rise in frequency of fungal infections.Some commensal fungi like Candida species generate infections when their host is immunocomprised.These problem leads to resistance and toxicity to antifungal drugs during prolonged therapy [3].
Nature provide a large source of medicinal agents for many years and many efficient modern drugs has been isolated from plants.Plants are still the first source of pharmaceutical agents utilized in traditional medicine [4].Plants possess various secondary compounds, which most of them have antifungal action [3].
Lichens are symbiotic associations that consist of a mycobiont and photosynthetic partners (a photobiont) often from green algae, and/or cyanobacteria.This complex community is a potential source of brand and efficient medicinal agents [5].Lichens have been utilized in traditional medicines by different cultures in the world commonly for curing wounds, skin illnesses, respiratory, digestive problems and gynecological concerns [6].
Lichens and their metabolites possess plenty of biological activities such as antibiotic, antitumor, ecological roles, antiviral, antiherbivore and enzyme inhibitory [7].

Table 1. Traditional usage of the tested lichens
Lichen Traditional Usage F. caperata F. caperata is used to treat the intestinal worms.Dried powder of thallus of it also used on skin burns [10].Moreover, this lichen has used as decoction to heat in China [11].
X. conspersa X. conspersa is utilized to cure wounds and cuts, therapy of inflammatory gingiva and sore throats [6].
X. stenophylla X. stenophylla is utilized to treat snake bites and venereal diseases like syphilis [12].

Lichen specimens
Lichen specimens of F. caperata (Herbarium no: 6398), X. conspersa (Herbarium no: 6399) and X. stenophylla (Herbarium no: 6400) was collected from Giresun province in 2012.They were kept at the Department of Biology, Faculty of Science, University of Giresun, Turkey.Lichen identification was done with some flora books [8][9].

Fungi strains
Four fungi strains were used to determine antifungal action of the extracts.Saccharomyces cerevisiae was obtained from Giresun Province Control Laboratory; Candida albicans and Candida tropicalis were obtained from Department of Biology, Fırat University and Candida parapsilosis was obtained from Faculty of Education, Giresun University.

Lichen preparation
Dried and finely grounded lichen samples (30 g) were extracted with acetonitrile (300 ml) in a Soxhlet extractor.The extracts were filtered then concentrated in a rotary evaporator.The crude extracts were kept at -18 °C till further antioxidant and antifungal analyses.The extracts were dissolved in dimethyl sulphoxide (DMSO) for further analysis [13].
Tube was vortexed and stored in a dark place for 30 min.Absorbance was measured at 450 nm.Butylated hidroxytoluene (BHT) was utilized as standard antioxidant substance [17].Absorbance values of the extracts were compared with absorbance values of BHT.

Results and Discussion
Lichens synthesize many metabolites which knows as lichen substances or lichen metabolites.These substances are dibenzofurans, amino acid derivatives, aromatic compounds, depsides and depsidones.They possess significant biological action such as antiviral, enzyme inhibitory, antioxidant and antibiotic [18].   2 reveals that the lichen extracts were effective (except for S. cerevisiae) in significantly reducing the growth of fungi as compared with flucanozole.DMSO which was used as negative control had no activity against test fungi.
Antifungal activity of the extracts are presented in Table 2.The acetonitrile extracts of the tested lichens showed maximum and minimum activity against C. albicans (32 mm) and S. cerevisiae (10 mm), respectively.Antifungal action of lichen extracts are increased in the following order: F. caperata>X.conspersa>X.stenophylla.
There are limited studies about antifungal activity of F. caperata lichen.For example, Mitrovic et al. [19] reported that methanol extract of F. caperata had effective against C. albicans.In agreement with this study we also observed that acetonitrile extract of F. caperata is active against C. albicans.On the other hand, in a study which was conducted by Millot et al [5] it was concluded that acetone extract of F. caperata were inactive against C. albicans To the best of our knowledge, there is not any survey about antifungal potential of the extracts of X. stenophylla against C. albicans.
The degree of the antimicrobial activity changes according to the used extract type, extract concentration, tried microorganisms and localities where plant samples collected [20].
Researches also done about antifungal actions of various lichen species.For example, Halama and Haluwin [21] studied acetone extracts of Evernia prunastri, Hypogymnia physodes and Cladonia portentosa against eight plant pathogenic fungi.
Piovani et al. [23] investigated antifungal activity of lichen aromatic metabolites against C. albicans and S. cerevisiae.
Methanol, acetone and chloroform extracts of Usnea florida was analyzed against C. albicans and C. parapsilosis [24].
The CUPRAC test of antioxidant activity is attributed to the absorbance reading of Cu(I)-Nc chelate created as a result of the redox reaction of chain-breaking antioxidants by CUPRAC reagent, Cu(II)-Nc [25].The ferric ion is altered with cupric ion, tripyridyltriazine is replaced by neocupreine and formed color can be observed with a spectrophotometer at 450 nm [26].
The CUPRAC values of the extracts were demonstrated in Table 3.The extract of X. conspersa showed the highest reducing power Cu 2+ to Cu + .The extract of X. stenophylla exhibited the lowest activity.Decreasing CUPRAC values is in order of X. conspersa>F.caperata>X.stenophylla.This is the first report about CUPRAC test of F. caperata, X. stenophylla and X. conspersa lichens.CUPRAC activity was also studied by Zlatanovic et al. [29] in the acetone extract of Umbilicaria crustulosa.

Conclusion
Recently, the search for biologically active compounds from plants to improve pharmaceutical, cosmetics and agriculture industry have gained more attraction.The lichen metabolites were studied for their phytochemical features.Lipid peroxidation caused by oxygen leads to deterioration of foods and diseases such as cancer and ageing.Therefore there was an urgent demand for medicines of natural origin for treating many illnesses.As the result of this study we have concluded that tested lichen extracts have antioxidant and antifungal activity.Hereby, F. caperata, X. conspersa and X. stenophylla might be a potential source for improving brand antifungal and antioxidant compounds.

Figure 1 .
Figure 1.Inhibition effects of the tested lichen extracts against a) C. tropicalis b) C. albicans c) S. cerevisiae Antifungal activity of F. caperata, X. conspersa and X. stenophylla was investigated towards to different yeasts namely S. cerevisiae, C. albicans and C. tropicalis and C. parapsilosis.The inhibition zones as demonstrated in Table2reveals that the lichen extracts were effective (except for S. cerevisiae) in significantly reducing the growth of fungi as compared with flucanozole.DMSO which was used as negative control had no activity against test fungi.

Table 2 .
Inhibition zones of the acetonitrile extracts of the lichens (mm)