Allium sativum Extract Chemical Composition, Antioxidant Activity and Antifungal Effect against Meyerozyma guilliermondii and Rhodotorula mucilaginosa Causing Onychomycosis

Onychomycosis is a major health problem due to its chronicity and resistance to therapy. Because some cases associate paronychia, any therapy must target the fungus and the inflammation. Medicinal plants represent an alternative for onychomycosis control. In the present work the antifungal and antioxidant activities of Alium sativum extract against Meyerozyma guilliermondii (Wick.) Kurtzman & M. Suzuki and Rhodotorula mucilaginosa (A. Jörg.) F.C. Harrison, isolated for the first time from a toenail onychomycosis case, were investigated. The fungal species were confirmed by DNA molecular analysis. A. sativum minimum inhibitory concentration (MIC) and ultrastructural effects were examined. At the MIC concentration (120 mg/mL) the micrographs indicated severe structural alterations with cell death. The antioxidant properties of the A. sativum extract were evaluated is a rat turpentine oil induced inflammation, and compared to an anti-inflammatory drug, diclofenac, and the main compound from the extract, allicin. A. sativum reduced serum total oxidative status, malondialdehyde and nitric oxide production, and increased total thiols. The effects were comparable to those of allicin and diclofenac. In conclusion, the garlic extract had antifungal effects against M. guilliermondii and R. mucilaginosa, and antioxidant effect in turpentine-induced inflammation. Together, the antifungal and antioxidant activities support that A. sativum is a potential alternative treatment in onychomycosis.

LOD -limit of detection, R 2 -coefficient of determination for the calibration curves (at six levels of concentrations). Indicated intervals represents the average ± standard deviations (n = 4).
Regarding phenolics, only gentisic acid, chlorogenic acid, 4-hydroxybenzoic acid, and p-coumaric acid were found to be above the limit of quantification (LOQ) ( Figure S1B, Table S2), but no flavonoid was detected. LOD -limit of detection, R 2 -coefficient of determination for the calibration curves (at six levels of concentrations). Indicated intervals represents the average ± standard deviations (n = 4).
Chromatograms of the analysed extract exhibit other important peaks that were not identified by mass alone. The UV spectra of the unidentified peaks, as registered by DAD detector after HPLC separation, and the scatterplot of the scores for the first two components when PCA was applied on these spectra, are illustrated in Figure S2A and S2B respectively. The data indicate that these main unidentified constituents corresponding to the chromatographic peaks of the A. sativum extract are nonphenolic in nature and rather similar with alliin and allicin in terms of UV-vis spectral features, most probably non-aromatic sulphurcontaining compounds such as peptides and their derivatives. Figure S2. A. Mean UV-vis absorption molecular spectra of A. sativum main chromatographic peaks and of the standards, indicating the two classes, phenolics and non-phenolics compounds. B. Scatterplot of the first two components in the Principal Component Analysis (PCA) applied on the UV-vis spectra. Standards are indicated by name while the main chromatographic peaks are labelled with S1-S14.
Chromatograms of the analysed extract exhibit other important peaks that were not identified by mass alone. The UV spectra of the unidentified peaks, as registered by DAD detector after HPLC separation, and the scatterplot of the scores for the first two components when PCA was applied on these spectra, are illustrated in Figure 2A and 2B respectively. The data indicate that these main unidentified constituents corresponding to the chromatographic peaks of the A. sativum extract are nonphenolic in nature and rather similar with alliin and allicin in terms of UV-vis spectral features, most probably non-aromatic sulphurcontaining compounds such as peptides and their derivatives.

Phytochemical analysis
The separations were performed on an Agilent 1200 HPLC system (Waldbronn, Germany) equipped with an on-line vacuum degasser, quaternary pump, temperature-controlled sample tray, automatic injector, a column thermostat compartment a DAD detector followed by a 6320 Ion Trap MS detector. The chromatographic separations were run on a Zorbax SB-C18 column (250 mm x 4.6 mm, 5 µm particle size) also from Agilent. The injection volume was 15 µL (0.22 µm filtered extract), the column temperature was set to 30 °C and the flow rate was 1 mL/min. Several preliminary tests were employed for method optimization using different experimental conditions. The optimum methods consisted of a multistep gradient elution system using solvent A, 10 mM ammonium formiate pH 2.5 for protocol one and ammonium acetate pH 5.5 for second protocol and as solvent B acetonitrile. For the first protocol, the steps were as follows: 0-5 min isocratic at 0% B, 5-14 min from 0 to 70% B, 14-15 min from 70% to 90% B, 15-18 min from 90% to 100% B, 18-22 min isocratic at 100% B and 22-22.1 min back to 0% B where was kept until 25 min.
For the second protocol, the steps were as follows: 0-2 min isocratic at 5% B, 2-10 min from 5 to 35% B, 10-20 min from 35% to 45% B, 20-25 min from 45% to 95% B, 25-28 min from 95% to 100% B, 28-32 min isocratic at 100% B and 32-32.1 min back to 5% B where was kept until 35 min. The UV-Vis detection of the compounds was performed using the DAD detector that measured the entire spectrum in 210-600 nm region (2 nm resolution), every 2 seconds and the chromatograms were monitored at 210, 220, 230, 240 and 280 nm for first protocol and 242, 260, 280, 320 and 340 for second protocol. As standards there were allicin, alliin, gentisic acid, 3,4-dihydroxybenzoic acid, chlorogenic acid, 4-hydroxybenzoic acid, cafeic acid, syringic acid, rutin, isoquercitrin, p-coumaric acid, quercitrin, ferulic acid, myricetin, morin, luteolin, quercetin, apigenin, kaempferol, galangin, all of analytical grade purity for different commercial available sources, except allicin which was synthesized as described below. Calibration curve was constructed 39, 78, 156, 313, 625, 1250 µg/mL for allicin and alliin determination, in the case of first protocol and 35, 53, 70, 105, 140, 210, 280 µg/mL for phenolic compounds, using the area of the peak by integration employed by the Agilent soft. The limit of detection (LOD) and limit of quantification (LOQ) were determined by formula LOD = (3.3 × standard deviations of intercept)/calibration curve slope and LOQ = (10 × standard deviation of intercept)/calibration curve slope, respectively. The identification of the compounds was employed by both chromatographic retention time and spectral similarities that were done by the built-in soft, as well as using the MS spectrum. The chromatograms and the mean spectra of the main chromatographic peaks were exported and analysed using Excel and Statistica 10 software packages.
In order to investigate the chemical nature of these compounds, a chemometric approach of chemomapping was applied on their UV-vis spectra. For this purpose, the UV-vis spectra of the main chromatographic peaks (with a maximum higher than 50 mAbs at 210 nm, 14 compounds, the four detected phenolic compounds were not among them) and the standards used in the calibrations were exported from the Agilent ChemStation soft and were analysed with Principal Component Analysis (PCA) using Statistica 10.

Allicin synthesis and characterization
Allicin was synthesized and analyzed according to Jansen et al., 1987 [1]. Briefly, 200 µL pure diallyldisulfide (Sigma Aldrich, St. Louis, USA) was dissolved in 14 mL dichloromethane to which 6 mL solution containing 357.7 mg 3-chloroperbenzoic (Sigma-Adrich, St. Louis, USA) was added dropwise under vigorous stirring in a cooling bath at -10 °C during one hour. Following this, the resulting solution was further kept at room temperature for one hour and washed two times with 2.5% sodium bicarbonate and water and then the organic phase was treated with anhydrous sodium sulfate for water removal. The solvent was removed under vacuum at room temperature and the resulting clear, oily and garlic-smelling substance was aliquoted and kept at -80 °C. MS and 1 H, 13 C NMR analysis proved it was allicin at purity higher than 96%. 1