Tropolone

Russet potatoes were purchased from local markets. p-Coumaric acid (pCA) (≥98.0%, HPLC), L-tyrosine (reagent grade, ≥98.0%, HPLC), β-cyclodextrin (βCyD) (≥97%), chlorogenic acid (CA; preferred IUPAC numbering 5-O-caffeoylquinic acid; ≥95%), L-ascorbic acid (AA) (reagent grade, crystalline), and tropolone (98%) were purchased from Sigma–Aldrich (USA). chlorogenic acid stock solutions were prepared in dilute acid (10 mM phosphoric acid) to prevent autoxidation [24 (link)].

Tricyclazole (5-methyl-1,2,4-triazole[3,4-b]benzothiazole), pyroquilon (1,2,5,6-tetrahydropyrrolo [3,2,1,-ij]quinolin-4-one), and tropolone (2-Hydroxy-2,4,6-cycloheptatrien-1-one) were purchased from Sigma-Aldrich (St. Louis, MO) and used at 50 ppm to study in vivo the effect on melanin biosynthesis and the mycelium growth effect. Both were dissolved in ethanol (10 mg/ml stock solution) and added to 50 ml of potato dextrose broth (PDB) (Difco Laboratories). To estimate fungal biomass growth, 100 mg of mycelial mat was obtained from 7-day-old cultures that were actively grown in PDB. This was put in a 12-multiwell plate with 2.5 mL of fresh PDB medium for each well and incubated for 7 days at 27°C and 150 r/min. After incubation, the mycelia were filtered through Whatman filter paper in vacuo to separate them from the culture. The material was dried to constant mass at 60°C and weighed. The experiments were performed in triplicate. Results are reported as the mean ± SD.

All cell culture reagents except fetal bovine serum (FBS, Cambrex IEP GmbH (Wiesbaden, Germany)) were obtained from Gibco, Invitrogen (Paisley, UK). [³H]-dopamine (2220 GBq/mmol) was purchased from Perkin Elmer (Hopkinton, MA, USA), an E.Z.N.A.^® Total RNA Kit I from Omega Bio-Tek (Norcross, GA, USA), and a High Capacity cDNA Reverse Transcription Kit, TaqMan Gene Expression Assays and TaqMan^® Universal PCR Master Mix from Applied Biosystems (Carlsbad, CA, USA). Decynium-22 (D22), nortriptyline HCl, apomorphine HCl, haloperidol, L-DOPA, L-deprenyl HCl and tropolone were obtained from Sigma Aldrich (St. Louis, MO, USA), and desipramine HCl was obtained from Sandoz (Cham, Switzerland). GBR12909 was obtained from Tocris (Bristol, UK).