Melanoidins were extracted from filtered decaffeinated coffee by way of dialysis against milli-Q water through a 12- to 14-kDa cutoff membrane for 1 week at 4°C. The nondialyzed fraction was collected and reconstituted to the starting volume with milli-Q water. The product was aliquoted and stored at −20°C until use as a melanoidin-based beverage. Polyphenols were extracted from decaffeinated coffee powder by way of solid/liquid extraction with ethanol (1:4 wt/vol). Filtered ethanol was dried under a vacuum at room temperature and recovered by the container dissolving the dry extract with the same starting volume of a 0.5% ethanol aqueous BYL719 solubility dmso solution.
Aliquots of the polyphenol-based beverage were stored at −20°C until use. The above preparations were diluted daily in 20 mL water to afford rats a daily dosage of 1.5 mL. The final beverages consumed by rats per 100 mL had the following composition: coffee, 280 mg and 150 mg of polyphenols and melanoidins, respectively; polyphenol-based beverage, 280 mg of polyphenols and no melanoidins; and melanoidin-based beverage, 150 mg melanoidins and no polyphenols. Male Wistar rats weighing 180-220 g were randomly housed in wire-bottomed cages. Animals were obtained from Harlan Italy (Udine, Italy) and maintained under controlled
temperature conditions of 22 ± 1°C with a 12-hour light/dark cycle and free access to water. NASH BAY 80-6946 purchase was induced by way of a high-fat diet (HFD) for 3 months as described by Svegliati-Baroni et al.4; the diet composition was: 58% of energy derived from fat, 18% from protein, and 24% from carbohydrates; 5.6 kcal/g (Harlan Italy). Control rats were fed a standard pellet diet (5% of energy derived from fat, 18% from proteins, and 77% from carbohydrates [3.3 kcal/g]) for 3 months. Thirty rats were divided into five groups. Animals in four groups were fed an HFD for 3 months to develop NASH. Over the same period, the fifth group of animals was fed a standard diet (normal chow) and represented the healthy control group. Beginning with the second month, the four HFD-fed these groups were
differentiated for beverages: they drank decaffeinated coffee, a solution of coffee polyphenols or melanoidins, or water. Healthy control rats drank water throughout the study period. The amount of coffee or coffee fractions was administered at a daily dose corresponding to 6 cups of espresso coffee or 2 cups of filtered coffee for a person weighing 70 kg. The experimental protocol was approved by the University’s ethics committee. Food intake was recorded daily, and body weight was measured weekly. All animals were sacrificed 3 months after beginning the diet. All animals received humane care according to the criteria outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication 86-23; revised 1985).