2.1. Test Supplement
reflux
REFLUX
To obtain the unripe black raspberry extract (BRE), 500 kg of dried unripe black raspberries, sourced from Gochang, Jeollabuk-do, Korea, were subjected to extraction using ethanol at a temperature of 80 ± 2 °C for a duration of 2 h. This mixture was then filtered using a 50 µm filter housing to obtain the initial extract. This extract, with a Brix concentration of 10–20%, was further concentrated at a temperature of 80 °C and subsequently lyophilized to yield the final BRE extract. Quantitative analysis of ellagic acid in BRE was performed using HPLC analysis. The average yield of the final BRE was 21.5 ± 0.9%, and the content of ellagic acid, an indicative component of BRE, was found to average 59.7 ± 1.8 mg/g. A standard stock solution was prepared by dissolving approximately 5 mg of ellagic acid (analytical standard, purity 95%, Sigma-Aldrich, MO, United States) in 25 mL of methanol. This solution was then diluted with methanol to create standard solutions with concentrations of 6.25, 12.5, 25, 50, 100, 150, and 200 μg/mL. After preparation, the stock and standard solutions were refrigerated at 4 °C until further use. About 0.1 g of BRE was placed into a 100 mL volumetric flask. To this, 10 mL of a pre-processing solvent (composed of 20 mL HCl, 20 mL tertiary distilled water, and 60 mL ethanol) was added, and the mixture was thoroughly dissolved. This mixture was then hydrolyzed for 3 h in a reflux extraction device at 90 °C, and subsequently cooled to room temperature. The hydrolyzed solution was transferred to a 50 mL volumetric flask, further dissolved with methanol, and then filtered through a 0.45 μm syringe filter before use. The ellagic acid content in BRE was determined using an ACQUITY UPLC H- Class HPLC instrument from Waters. The amount of ellagic acid was calculated from a calibration curve constructed from the peak areas at each concentration of the standard solution. Chromatograms were obtained using a Shiseido CAPCELL PAK CAll extraction procedures were performed by the Berry and Bio Food Research Institute in Gochang, Jeollabuk-do, Republic of Korea, and the test BRE product was provided in the form of yellow-brown powder tablets. The placebo product contained maltodextrin and was identical to the test product in appearance, flavor, and weight (