Antifungal and antimycotoxigenic metabolites from native plants of northwest Argentina: isolation, identification and potential for control of Aspergillus species

Extracts from aerial parts of Prosopis ruscifolia , Bidens pilosa , Cercidium praecox and Phoradendron liga were assayed against toxigenic Aspergillus species. They were obtained by sequential extraction of the aerial parts with hexane (fHex), dichloromethane (fDCM), ethyl acetate (fEtOAc) and methanol (fMeOH). The fMeOH from P. ruscifolia showed the highest antifungal spectrum (MIC=750-1500 µg mL -1 ; MID=50-200 µg; DI=1.7-3.0 mm). Indolizidine alkaloids (juliflorine and juliprosine) and tryptamine were identified with strong (MIC=188 µg mL -1 ) and moderate antifungal activities (MIC=750 µg mL -1 ), respectively, towards A. parasiticus and A. flavus . The fMeOH, the indolizidine alkaloids and tryptamine synergized the fungitoxic effect of potassium sorbate and propiconazole. They completely suppressed the biosynthesis of aflatoxins at concentrations of 47, 94 and 375 µg mL -1 , respectively. Our results indicate that fMeOH and its identified alkaloids are promisory additives of commercial antifungals and are antiaflatoxigenic agents at concentrations below of those required for complete suppression of fungal growth. The inhibitory effect of fMeOH, tryptamine, juliflorine and juliprosine accumulation of NRRL2999 ( A. parasiticus) and LABI 216 ( A. flavus ) was tested in YES medium. Aliquots of 8 mL of the medium supplemented with fMeOH, tryptamine, juliflorine or juliprosine were placed in 125 mL Erlenmeyer flasks. Each flask was inoculated with 3.10 4 conidia of A. parasiticus or A. flavus suspended in 1 mL of medium in triplicate. Concentrations assayed ranged from 47 to 1500 µg mL -1 of liquid medium. The flasks were then incubated in the darkness at 30 °C in an orbital shaker at 150 rev min -1 for 7 days. Following incubation, cultures were centrifuged at 3000 g for 10 min. Supernatants were stored at -20°C before quantification of aflatoxins. Fungal biomass was measured by weighing the mycelial pellet after 48 h of freeze-drying. The initial pH of the culture medium was not affected by the supplementation, and the final pH conditions did not vary between treatments. Contents of mycotoxins were measured in the supernatants expressed as µg mg -1 of dry mycelial biomass. The obtained data were subjected to ANOVA and a subsequent post hoc Dunnet T3 test, with P = 0.05.

fractionation of the components of the aerial parts. Each filtered organic fraction was evaporated under reduced pressure at 40°C, the dry residues were weighed and weights were used to calculate the yields of dry matter (DM) as: DM (%) = (mg of dry residue/mg of dry plant material) x 100 The dry residues of the filtered organic fractions were stored at -15 °C in sealed flasks until use.

Antifungal assays
The antifungal activity of the organic fractions obtained from the aerial parts of the plant species was assessed by the dot blot and the broth microdilution assays. In the dot blot assay, thin layer chromatography (TLC) plates (silica gel G60 F254, Merck, 6 x 8 cm x 0.2 mm) were submerged in 96% ethanol and dried in sterile conditions. Then, the organic plant extracts were punctually loaded on the plate at equidistant positions in doses of 200 µg to 2000 µg of dry matter. Organic solvents (10 µL) or propiconazole (20 µg) were also spotwise applied on the plates as negative and positive controls, respectively. Then, each plate was covered with 3 mL of semisolid MPA medium containing 1 x 10 3 spores mL -1 . Plates were incubated at 30 °C for 48-72 h. Then, they were covered with 1.5 mL of a solution of 3-(4,5-dimethylthyazol-2-yl) 2,5diphenyltetrazolium (MTT) and incubated at 30 °C for 24 h. Dehydrogenases of the living fungal cells convert MTT into blue (Bluma et al., 2008). Hence, dead cells were visualized on the plates as yellow zones under a blue background. The minimal dose of an organic plant extract required to suppress fungal growth (MID) was determined. Two perpendicular diameters were measured in the each yellow zone observed at the MID with the software Fiji win 32 (Image J 1.44c, Wayne Rasband, NIH, Bethesda MD).
The organic extracts recovered from the aerial plant parts were also subjected to the broth microdilution assay in 96 flat well microplates. The protocol was developed according to M38-A document from the National Committee for Clinical Laboratory Standards with some modifications (NCCLS, 2002). Fungal colonies were grown in Petri dishes for 7 to 15 days in solid MPA medium in the darkness at 30 °C. Then, the fungal colonies were washed with 2 mL of physiological solution (0.9% of NaCl in distilled water) to obtain microconidial suspensions. The asexual spores were counted in a Neubauer chamber, and the suspension was diluted in semiliquid YES medium (Yeast-malt extract-sucrose: 2% yeast extract, 15% sucrose, 0.05 % magnesium sulphate, 0.125 % agar) to obtain a density of 1 x 10 5 spores mL -1 .The stock solutions of each organic extract were prepared in DMSO and diluted with culture medium to prepare two fold dilution series comprised between 3000-87.5 μg mL -1 , in semiliquid YES medium. The final volume in each well was 200 μL containing 2% DMSO. This volume corresponded to 100 μL of fungal spore suspension and 100 μL of a dilution of an organic plant extract. Controls of growth consisted in 100 μL of YES medium plus 100 μL of spore suspension in each well. Controls of sterility were 200 μL of YES medium per well. Each treatment (organic extracts or controls) had three repetitions per microplate. Each microplate was prepared twice. The microplates were incubated 72 h at 30°C. Then, the minimum concentration of each organic fraction required to inhibit 100% of the microbial growth (MIC) was visually determined. The MIC values presented in tables are means of three replicates obtained from two experiments.

Isolation and identification of the antifungal constituents from the methanolic extract of Prosopis ruscifolia
The constituents of the methanolic extract of P. ruscifolia were separated following a bioassay guided isolation. The antifungal activity was evaluated by the dot blot assays on A. flavus and A. parasiticus at each step of the separation process. The dry residue of the methanolic extract (4 g) of P. ruscifolia, dissolved in a mixture of chloroformmethanol (8:2, v/v), was loaded on a column (123 g of silica gel, pore size 60 Å, 230-400 mesh particle size). Then, the column was eluted with a gradient of chloroformmethanol (200 mL, 8:2; 150 mL, 7:3; 150 mL, 6:4, v/v). The composition of the eluted fractions (20 mL each) was monitored by TLC with ethyl acetate-formic acid-water (65:15:10, v/v/v) as mobile phase. The TLC chromatograms were visualized under UV light at 254 nm and 365 nm, and then revealed with chromogenic reagents (vanillin/sulfuric acid, p-anisaldehyde or dragendorff) according to Wagner and Bladt (1996). Fractions were combined in eight groups (F1-F8). The constituents of F6 were loaded in a Sephadex LH 20 column (250 x 17 mm, Amersham Pharmacia Biotech) and recovered in three pools (P1-P3). The P1 and P2 were subjected to preparative thin layer chromatography (PTLC) on silica gel 60 F 254 (2 mm thickness). PTLC bands 1 (B1) from P2 and 2 (B2) and 3 (B3) from P1 were scrapped from the plate and eluted with methanol. After 24 h, the methanolic suspensions were centrifuged and the supernatants were recovered. Each supernatant was passed through 280 mg of silica gel packaged in a Pasteur pipette. The 1 H NMR spectra of B1, B2 and B3 recovered from the Pasteur pipettes (hereafter referred as Pp1, Pp2 and Pp3, respectively) were recorded using a Bruker Avance-III 400 MHz spectrometer in deuterated methanol. NMR Fourier transform, peak picking and integration were done with Bruker TopSpin software.
These spectra allowed the identification of the compounds based on their matching with those obtained for standard compounds (i. e. tryptamine) or previously published in the literature (Barry et al., 2005).

Brine shrimp assay
The cytotoxicity of the antifungal constituents was evaluated by the brine shrimp assay (Meyer et al., 1982). Eggs of Artemia salina were placed in a plastic cage with seawater (3.8% NaCl). Half of the cage was exposed to light provided by a 60-watt lamp and was incubated for 48 h at 27 °C. The dried residue of the methanolic extract from P. ruscifolia, juliflorine, juliprosine and tryptamine were dissolved in the sea water and tested at concentrations of 3000-87.5 μg mL -1 . Positive controls were K 2 Cr 2 O 7 (González Pérez and Aportela Gilling, 2001) and propiconazole. Brine shrimps placed in sea water served as the viability control. Ten brine shrimp larvae were placed in each well of 24-well microplates and incubated for 18 h at 27 °C. The surviving nauplii were counted under a magnifying glass at a 3x magnification and percentages of inhibition were determined. Percentages of inhibition were calculated and then the concentration required to kill 50% of the nauplii (LC 50 ) was interpolated by probit analysis. Each assay was performed in triplicate. The LC 50 values were interpreted as follows: <1.0 μg mL -1 , highly toxic; 1.0-10.0 μg mL -1 , toxic; 10.0-30.0 μg mL -1 , moderately toxic; 31-100 μg mL -1 , mildly toxic, and >100 μg mL -1 , as non-toxic (Meyer et al., 1982).

Joint action of the methanolic extract of Prosopis ruscifolia and its alkaloids with commercial antifungals
The methanolic extract of P. ruscifolia, juliflorine and juliprosine were assayed in combination with propiconazole and with potassium sorbate on A. parasiticus and A.  (Vitale et al., 2005).

Antiaflatoxigenic activity of fMeOH and its alkaloids
The inhibitory effect of fMeOH, tryptamine, juliflorine and juliprosine on aflatoxin accumulation of NRRL2999 (A. parasiticus) and LABI 216 (A. flavus) was tested in YES medium. Aliquots of 8 mL of the medium supplemented with fMeOH, tryptamine, juliflorine or juliprosine were placed in 125 mL Erlenmeyer flasks. Each flask was inoculated with 3.10 4 conidia of A. parasiticus or A. flavus suspended in 1 mL of medium in triplicate. Concentrations assayed ranged from 47 to 1500 µg mL -1 of liquid medium. The flasks were then incubated in the darkness at 30 °C in an orbital shaker at 150 rev min -1 for 7 days. Following incubation, cultures were centrifuged at 3000 g for 10 min. Supernatants were stored at -20°C before quantification of aflatoxins. Fungal biomass was measured by weighing the mycelial pellet after 48 h of freeze-drying. The initial pH of the culture medium was not affected by the supplementation, and the final pH conditions did not vary between treatments. Contents of mycotoxins were measured in the thawed supernatants with ELISA kits (Ridascreen Fast Aflatoxin, Biopharm,Germany),and expressed as µg mg -1 of dry mycelial biomass. The obtained data were subjected to ANOVA and a subsequent post hoc Dunnet T3 test, with P = 0.05.