Preparation of Microsomes and (1,3)-β-D-Glucan Synthase Activity

Quantification of (1,3)-β-D-glucan synthase activity was performed by aniline blue assay with microsomal membranes. Microsomal membranes were prepared from exponential phase C. albicans SC5314 cells according to Shedletzky’s method [16] with slight modifications [15]. The protein concentration of the prepared microsomal membranes with apricot color was determined by Bradford microassay according to the manufacturer’s manual (Bio-Rad, USA), and stored at -27°C prior to use. The (1,3)-β-D-glucan synthase activity was measured based on the method of Frost et al. [17] with some modifications [15]. The (1,3)-β-D-glucan synthase activity assay was conducted with or without the A. lappa extract for 40 min at 25°C, and the synthesized glucans were stained specifically with aniline blue solution. Each reaction product was placed into a black, 96-well, flat-bottom microplate in quadruplicate, and fluorescence intensity was measured at 400 nm (20 nm width) excitation and 485 nm emission (20 nm width) wavelengths using a fluorometer. The quantity of (1,3)-β-glucan synthase activity in the presence of the A. lappa extract was represented as a percentage of the DMSO control. The data show the mean of quadruplicate measurements.