The mycelia were recovered from the liquid medium by filtration, washed with distilled water and dried at 40 °C during 24 h to obtain the yield of biomass. The residual amount of reducing sugars in the filtrates was evaluated by using the dinitrosalicylic method ( Miller, 1959). Fruiting bodies and the mycelia were dried and

milled to a fine powder (40 mesh). The samples (5 g) were extracted by stirring with 100 mL of ethanol 70:30 (in water) at 25 °C and at 130 rpm for 3 h and filtered through Whatman n° 1 paper. Ethanol was chosen because of its abundance and low cost. The extractions were repeated two times. No increases in yield were achieved by further extractions. The combined filtrates were concentrated with a rotary vacuum evaporator at 40 °C to eliminate ethanol, freeze-dried and weighted. The freeze-dried powders www.selleckchem.com/products/SGI-1776.html were stored in freezer until use. The reducing and total carbohydrates of extracts were determined by using the dinitrosalicylic (Miller,

Alpelisib in vivo 1959) and anthrone (Morse, 1947) methods, respectively, and expressed as glucose equivalents. Evaluation of the reducing and non-reducing sugars present in the extracts was also carried out using thin layer chromatography. The samples were spotted onto Silica Gel 60 plates (Merck, Darmstadt, Germany) and developed with butanol–pyridine–water (15:30:20, vol/vol) as the mobile phase. The spots were detected by spraying with 5 g/L KMnO4 in 1 mol equi/L NaOH. Glucose, mannitol and trehalose at 20 g/L were used as standards. Total phenolic compounds were

Selleckchem Fludarabine measured using Lowry reagent (Singleton & Rossi, 1956) and expressed as gallic acid equivalents. The determination of flavonoids was done by means of a colorimetric assay (Zhishen, Mengcheng, & Jianming, 1999) and expressed as catechin equivalents. Compounds bearing free amino groups were measured using the ninhydrin method with l-alanine as standard (Starcher, 2001). Amino acids were evaluated also using paper chromatography (Moffat & Lytle, 1959). The hydroalcoholic extracts from both mycelia and fruiting bodies of A. brasiliensis were fractionated by means of high-performance liquid chromatography (HPLC). The HPLC system (Shimadzu, Japan) consisted of a system controller SCL-10AVP, two pumps, model LC10ADVP, a column oven, model CTO-10AVP, and a UV–Vis detector, model SPD-10AVP. A reversed-phase C18 CLC-ODS column (5 μm; 250 × 6 mm i.d.; Shimadzu) protected with a GHRC-ODS pre-column (5 μm; 10 × 4 mm i.d.; Shimadzu) was used. The absorbance of each sample solution was measured at 280 nm. The mobile phase was distilled water:glacial acetic acid (99.9:0.1) (solvent A) and acetonitrile:glacial acetic acid (99.9:0.1) (solvent B). The gradient was 0 min, 92:8 (A:B); 0–2 min, 90:10 (A:B); 2–27 min, 70:30 (A:B); 27–50 min, 10:90 (A:B); 50–60 min, 0:100 (A:B); 60–63 min, 92:8 (A:B). Run time was 63 min using a flow rate of 1 mL/min ( Kim et al., 2008).