Phenolic compounds was measured working with a Folin iocalteu assay described by Sankam et al. [19]. The sample and Folin iocalteu reagent have been mixed and incubated at 45 C for 15 min. The absorbance at 750 nm was measured applying a UV-visible spectrometer. The total phenolic content material was calculated using a gallic acid normal curve and expressed as mg of gallic acid equivalents (GAE) per g of extract. The content material of total carbohydrates was determined using a phenol ulfuric acid assay [20] utilizing glucose as a normal. A variety of red yeast extracts have been incubated with sulfuric acid at 90 C for 30 min, followed by adding phenol option, plus the mixture was additional incubated at room temperature for five min. The carbohydrate content was measured at 490 nm using a UV-visible spectrometer and calculated as mg of glucose per g of extract working with the calibration curve of glucose. The content material of carotenoid derivatives was analyzed utilizing reverse-phase HPLC in line with the process of Shi et al. [8]. HPLC was carried out on a reverse phase C18 column (Agilent 4.6 mm 250 mm, five ). The mobile phase system consisted of a gradient composed of acetonitrile/water/formic acid (86:ten:four v/v/v) as phase A and ethyl acetate: formic acid (96:4 v/v) as phase B with a flow rate of 1 mL/min. The optical density at wavelengths of 338, 426, 452, and 478 nm was detected. The content material of carotenoid derivatives was characterized and calculated making use of typical –RelB supplier carotene and lycopene. 2.four. Mutagenicity and Antimutagenicity of Red Yeast Utilizing Salmonella Mutation Assay The mutagenicity of red yeast powder and its extracts, at concentrations ranging from 40 to 5000 /plate, was assessed working with a Salmonella mutation assay as outlined by the process of Inboot et al. [21]. Salmonella typhimurium tester strains TA98 and TA100 were kindly supplied by Dr. Kei-Ichi Sugiyama, National Institute of Overall health, Tokyo, Japan. AF-2 and 2-AA were used as regular mutagens within the absence (-S9) and presence (+S9) of metabolic activation, respectively. S9 fraction was prepared from 80 week-old male Wistar rat (Rattus norvegicus) injected with phenobarbital and -naphthoflavone. Mutagenicity was expressed making use of the mutagenic index (MI) calculated in the number of revertant colonies divided by the number of spontaneous revertant colonies. The mutagenicity was classified when the MI value was over 2-fold. The antimutagenicity test of red yeast powder and its extracts was modified in the preceding procedure on the mutagenicity test. The concentrations of test compounds, ranging from 40 to 1000 ug/plate, were PKCμ supplier neither cytotoxic nor mutagenic to bacterial tester strains. AFB1 concentrations at 25.0 and 12.5 ng/plate had been applied as a good mutagen in TA98 and TA100, respectively, under metabolic activation conditions. -Carotene and lycopene, the possible constituents in red yeast, have been also assessed for their antimutagenic activities against AFB1 -induced mutagenesis. The percentage of inhibition of each sample was calculated as described by Inboot et al. [21]. two.five. Animals Three-week old male Wistar rats (500 g body weight (bw)) had been bought from Nomura Siam International (Bangkok, Thailand). Rats have been acclimatized for 1 week before beginning the experiment. They were housed in controlled environments using a dark ight cycle of 12:12 h and at a temperature of 25 1 C. Water and basal diet plan have been supplied ad libitum. The protocol was approved by the Animal Ethic Committee of the Faculty of Medicine, Chiang Mai Universit.
