Antidermatophytic Activity of the Fruticose Lichen Usnea orientalis

In the present study, the new biological sources in the form of lichen Usnea orientalis Motyka was screened for its antidermatophytic potential. Six species of dermatophytes were chosen on the basis of their prevalence for antidermatophytic assays, and the Clinical Laboratory Standard Institute (CLSI)-recommended broth microdilution procedure was used to detect the efficacy of extract against dermatophytes. Thin layer chromatography of lichen extracts reveals the presence of two secondary metabolites viz. salazinic acid and usnic acid. U. orientalis extract exhibited promising antidermatophytic activity against all tested pathogens. Amongst all tested pathogens, Epidermophyton floccosum exhibited most susceptibility towards extract, whereas Trichophyton mentagrophytes exhibited the least susceptibility. Topical application of U. orientalis extract might be helpful in the cure of dermal infections.


Introduction
Lichen thallus (a composite organism) is mainly composed of mycobiont and photobiont in a mutualistic relationship [1]. Lichens are known for their secondary metabolites, which are quite unique to them and have several properties such as photoprotection, allelopathy, and antioxidant, antimicrobial, and antiviral activities [2]. The genus Usnea is well known for the worldwide distribution and for the role of secondary metabolite, i.e., usnic acid, in medicines. More than 300 species of genus Usnea were reported throughout the world, of which 57 are from India [3]. Usnea orientalis Motyka (fruticose and corticolous lichen) was used ethno-medicinally in urinary tract problems, swelling, and edema [4]. Cutaneous infections caused by fungi generally produce boggy nodular swelling called kerion [5]. Cutaneous mycoses (skin infection) caused by filamentous keratinophilic fungi known as dermatophytes, composed of the three genera Trichophyton, Microsporum, and Epidermophyton [6]. In humans, about 30 species of dermatophytes have been identified as pathogens [7]. Dermatophytes are cosmopolitan in distribution. Several reports from different parts of the world have reported the occurrence of dermatophytes. A study involving 16 European countries showed that 35%-40% of the analyzed individuals had infection of the foot (tinea pedis) caused by dermatophytes [8]. A study in the US revealed that between 22% and 55% had hair scalp infection of dermatophytes [9]. Another study conducted in Brazil showed that Trichophyton rubrum, Microsporum canis, and Epidermophyton floccosum were the most prevalent species infecting humans in developing countries [10]. The World Health Organization estimated that dermatophytes affect about 25% of the world population [6]. Apart from wide prevalence, the dermatophytes have exhibited resistance against griseofulvin, terbinafine, and fluconazole [6,[11][12][13][14]. Although the prevalence of drug resistance in dermatophytes is rare, recurrence in patients is common with 60%-80% [5]. Based on the aforementioned literature, the new biological source in the form of U. orientalis was screened for its antidermatophytic property.

Preparation and Percent Yield of Extract
Lichen thalli were collected from Koti, Chakrata district, Uttarakhand, India and identified with the help of relevant keys [15]. The voucher specimen was deposited in the Botanical Survey of India, Allahabad, India: U. orientalis (Accession No. BSA-8760). Two grams of air-dried thallus (vegetative as well as fruiting) was washed thoroughly using tap water followed by distilled water and pat-dried. Then, thalli were subjected to cold extraction of secondary metabolites in 50 mL of acetone. Subsequently, the solvent was filtered by Whatman No. 1 filter paper after 48 h.
The weight of crude extract obtained was 0.16 g after vacuum drying the filtrate via rotary evaporator. Percent yield of crude extract was calculated according to the equation below: Percent yield (%) = (Dry weight of extract/Dry weight of sample) × 100.
Stock solution (50 mg/mL) of crude extract was prepared in dimethyl sulphoxide (DMSO) for the evaluation of antidermatophytic and free radical scavenging activity.

Determination of Fungistatic Concentration
Antifungal susceptibility test was performed according to the Clinical Laboratory Standard Institute (CLSI)-recommended broth microdilution method in RPMI-1640 medium HEPES modification (Sigma Aldrich, St. Louis, MO, USA) supplemented with MOPS buffer (3-morphollinopropane-1-sulfonic acid) (Qualigens Fine Chemicals, Mumbai, India) [18]. Brief steps involved per plate were as follows: Inocula prepared was diluted 1:50 times in testing media, i.e., RPMI 1640; the test was performed in 96-well flat bottom microtiter plates; Column 1 was named as negative control consisting of 100 µL of RPMI-1640 broth media and 100 µL of inocula prepared in formaldehyde (less than 0.5%); Column 2 was named as broth control consisting of 200 µL of media; Columns 3 and 4, 6 and 7, and 9 and 10 were vertically diluted with extract having a final concentration of 1.25 to 0.009 mg/mL and named as treated; Column 5, 8, and 11 were taken as positive controls and contained only 100 µL of inocula and 100 µL of RPMI-1640 broth media, respectively; Column 12 was named as extract control and contained vertically diluted extract in the aforementioned concentrations. To nullify the effect of extract color, optical density (O.D.) of the extract control was subtracted from treated columns corresponding to extract treated [19]. Percent inhibition was calculated using following equation:  (Figure 1).
The antifungal activity of the chemical drug Sertaconazole nitrateBP (SN) (Glenmark Pharmaceuticals, Nasik, India) was taken as the reference standard. An amount of 50 mg/mL of stock solution of SN was prepared and tested at the same concentrations as the U. orientalis extract.

Determination of Fungicidal Concentration
A portion of 20 μL of U. orientalis-treated columns from the above MIC wells were transferred into 7-mL tubes of a fresh RPMI 1640 medium. Tubes were incubated at 30 ± 2 °C for 4 weeks and checked for the turbidity. The aforementioned procedure was performed with SN, and the concentration at which no turbidity has been achieved was defined as the minimum fungicidal concentration (MFC) [20,21].

Statistical Analysis
An independent sample t-test was performed between the positive control and sertacoazole-treated dermatophytes; and between the positive control and extract-treated dermatophytes for the measure of Levene's test for equality of variances and t-test for equality of means via SPSS v20.

Percent Yield of Extract
Percent yield of extract obtained from U. orientalis thallus was 8%. The antifungal activity of the chemical drug Sertaconazole nitrateBP (SN) (Glenmark Pharmaceuticals, Nasik, India) was taken as the reference standard. An amount of 50 mg/mL of stock solution of SN was prepared and tested at the same concentrations as the U. orientalis extract.

Determination of Fungicidal Concentration
A portion of 20 µL of U. orientalis-treated columns from the above MIC wells were transferred into 7-mL tubes of a fresh RPMI 1640 medium. Tubes were incubated at 30 ± 2 • C for 4 weeks and checked for the turbidity. The aforementioned procedure was performed with SN, and the concentration at which no turbidity has been achieved was defined as the minimum fungicidal concentration (MFC) [20,21].

Statistical Analysis
An independent sample t-test was performed between the positive control and sertacoazole-treated dermatophytes; and between the positive control and extract-treated dermatophytes for the measure of Levene's test for equality of variances and t-test for equality of means via SPSS v20.

Percent Yield of Extract
Percent yield of extract obtained from U. orientalis thallus was 8%.

Thin Layer Chromatography
A light yellow turning into a green-colored spot was observed and confirmed as usnic acid in U. orientalis extract and the other compound was salazinic acid. The chromatographic study confirmed the presence of two compounds viz. usnic acid and salazinic acid in the U. orientalis extract.

Antifungal Test for Opportunistic Filamentous Fungi
U. orientalis, fruticose lichen, contains usnic and salazinic acid and is used ethno-medicinally in swelling. The U. orientalis extract was tested for its efficacy against six dermatophytic species and compared with sertaconazole nitrate. Sertaconazole nitrate is a highly active chemical drug having low fungistatic and fungicidal activities, but it causes inflammation and itching in patients [22]. Due to the side effects of SN and the development of resistance in dermatophytes against first-line clinical drugs, there is a need for new antidermatophytic compounds. The antidermatophytic activity of SN and U. orientalis were represented graphically in the form of IC 50 (50% inhibitory concentration) and MIC (minimum inhibitory concentration) values were represented graphically in Figure 1.
The IC 50 , MIC, and MFC for T. rubrum were achieved at 0.033 mg/mL, 0.064 mg/mL, and 0.078 mg/mL against SN and 0.249 mg/mL, 0.54 mg/mL, and 0.625 mg/mL against U. orientalis extract. T. mentagrophytes was found least susceptible amongst all tested pathogens with IC 50 = 0.037 mg/mL, MIC = 0.09 mg/mL, and MFC = 0.156 mg/mL against SN and IC 50 = 0.204 mg/mL, MIC = 1.04 mg/mL, and MFC = 1.25 mg/mL against U. orientalis extract. The efficacy of U. orientalis extract is equivalent to sertaconazole nitrate against E. floccosum, but was found to be less effective against all other dermatophytes.
In another study, extracts of U. florida exhibited an MIC between 0.050 and 0.100 mg/mL against M. gypseum, T. mentagrophytes, and T. rubrum [23]. In the present study U. orientalis exhibited a MIC between 0.531 and 1.04 mg/mL and was found to be less active than U. florida. The ethno-medicinal use of U. orientalis extract in swelling caused by dermatophyte infection might be cured, but in vivo efficacy and potency of the lichen extract needs to be investigated.

Statistical Analysis
The level of significance was calculated in terms of p-value. Results obtained were statistically significant except between U. orientalis treatments and control T. rubrum (p-value = 0.19) and U. orientalis treatments and control E. floccosum (p-value = 0.19); p-values calculated for SN-treated E. floccosum, M. fulvum, and M. gypseum were less than 0.01, whereas, for M. canis, T. mentagrophytes, and T. rubrum, p-values were less than 0.05. p-value calculated for U. orientalis treated all pathogens except T. rubrum, which showed a level of significance less than 0.01 (Figure 1).

Conclusions
U. orientalis extract exhibited broad-range antidermatophytic activity against all three genera of dermatophytes, and usnic acid (a well-known antifungal compound) was present in the lichen extract. Topical application of lichen extract might be helpful in the cure of cutaneous infections.