BIOACTIVE POTENTIAL OF GRAPE SEED FLOUR: PHENOLIC COMPOUNDS AND ANTIOXIDANT ACTIVITY

The main bioactive compounds present in grape seeds include catechins, epicatechins, and proanthocyanidins, which are recognized for their high antioxidant activity. This study aimed to determine the total phenolic content and evaluate the antioxidant capacity of grape seed flour extract. The sample was extracted using 70% ethanol at a 1:40 (w/v) ratio and agitated on an orbital shaker at 200 rpm for 90 minutes at 25 °C. It was then centrifuged at 8,000 rpm for 10 minutes at 10 °C. The supernatant was filtered through qualitative filter paper, placed in amber vials, and stored frozen at -18 °C. Total phenolic content was determined using the Folin-Ciocalteu method, initially employing a 1:10 (v/v) dilution of the extract. Briefly, 0.5 mL of extract, 2.5 mL of Folin-Ciocalteu reagent (10%), and 2 mL of 7.5% Na₂CO₃ were mixed and incubated in a water bath at 50 °C for 6 minutes. Absorbance was measured at 750 nm using a UV/Vis spectrophotometer. Antioxidant activity, evaluated via the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging method, was quantified by adding 100 μL of the diluted sample (1:10, v/v) to 4.0 mL of ethanol and 1.0 mL of a 250 μM ethanolic DPPH• solution. After 30 minutes, absorbance was measured at 517 nm. To determine antioxidant activity via the scavenging of the ABTS+• radical (2,2'-azino-bis(3-ethylbenzothiazoline)-6-sulfonic acid), 30 μL of the diluted sample (1:10, v/v) was added to 3.0 mL of the ABTS+• solution. After homogenization, the tubes were left in the dark for 6 minutes. Absorbance was measured at 734 nm. Reducing power was evaluated using the FRAP (Ferric Reducing Antioxidant Power) assay, with a 90 μL aliquot of the diluted sample (1:10, v/v) to which 270 μL of distilled water and 2.7 mL of the FRAP reagent were added. Following homogenization, the samples were incubated at 37 °C for 30 minutes, and absorbance was measured at 595 nm. The total phenolic content of 22.174 ± 0.533 mg GAE/g indicates a substantial amount of extractable phenolic compounds. All three assays detected antioxidant activity, with values ​​of 145,840 ± 3,074, 151,765 ± 9,825, and 134,995 ± 0.308 µmol TE/g for DPPH, ABTS, and FRAP, respectively. Similar responses across the assays indicate a consistent antioxidant potential, although the higher values ​​for ABTS and DPPH compared to FRAP may reflect differences in the chemical mechanisms of free radical scavenging versus the reduction of ferric ions. Previous studies have identified catechin, epicatechin, and proanthocyanidins as significant phenolic constituents of grape seeds. Therefore, the phenolic content, combined with the results obtained from complementary antioxidant assays, reinforces the potential of grape seed flour as a source of bioactive compounds. These characteristics support the interest in valorizing grape seeds for the development of value-added food ingredients.