Effect of storage conditions on phenolic compounds and antioxidant activity of blueberry extract and the effect of anthocyanins from selected cultivars of Georgia-grown blueberries on apoptosis and phase-ii enzymes
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Blueberry (Vaccinium corymbosum) of the family Ericuceae is reported to have high antioxidant activity compared to other fruits and vegetables. This is highly correlated with the anthocyanins and total polyphenolic content. Blueberries are often converted to extracts such as juice or juice concentrate for subsequent use in beverages, syrups and other food products. Phenolic compounds are highly unstable and may be lost during processing, particularly when heat treatment is involved. Blueberry extract was prepared and stored at different temperatures (-20±1, 6±1, 23±1, and 35±1 oC) in glass bottles. Changes were observed in total polyphenols (TPP), total anthocyanin (TACY), Troloxequivalent antioxidant capacity (TEAC), phenolic acids, individual anthocyanins, and cell proliferation during storage. Two Georgia-grown cultivars, Tifblue and Powderblue were chosen for the study. Recovery of TPP, TACY and TEAC in blueberry extract after pressing and heating were ~25, ~29, and ~69%, respectively, for both cultivars. Recovery of gallic acid, catechin and quercetin was ~25% in final extract. Ferulic acid was not detected in the final extract in both Tifblue and Powderblue cultivars. Recovery of peonidin, malvidin and cyanidin was ~20% in final extract in both the cultivars. These results suggest that most of the phenolic compounds were lost during removal of residue and during heating. Losses due to storage were less when compared with initial loss due to processing. There was no statistically significant loss (P < 0.05) of TPP, TACY and TEAC observed up to 30 days at -20±1 oC. At 6 ºC storage, a significant loss of TPP, TACY and TEAC was observed from 15 to 30 days. Similar results were obtained at 23 ºC and 35 ºC (P < 0.05). A linear relationship was observed between TEAC values and total polyphenols and total anthocyanins. There was retention of more than 40% of ellagic acid and quercetin after 60 days at 35±1 oC. Anthocyanins were not detected after 60 days of storage at 35±1 oC. Significant retention (P < 0.05) was obtained for malvidin (42.8 and 25.8%) and peonidin (74.0 and 79.5%) after 60 days storage at 23±1oC in glass bottles for Tifblue and Powderblue, respectively. Cell viability assay was performed using HT-29 cancer cell line and anthocyanins extracted from 30, 60, and 90 days stored extract at 6±1 and 23±1 oC. Significant cell proliferation inhibition percentage was observed in 30 days, although this was reduced significantly after 30-90 days. These results suggest that initial preparatory steps like washing, removal of residue mainly skin, heating and storage conditions were significantly affecting the phenolic compounds and their biological activity. Anthocyanin fractions from four cultivars of Georgia-grown blueberries namely Tifblue, Powderblue, Brightblue, and Brightwell were used for apoptosis study. Apoptosis was confirmed using two different methods: DNA fragmentation and caspase- 3 activity. The effect of anthocyanins on the activity of detoxifying enzymes glutathioneS- transferase (GST) and quinone reductase (QR) were also determined. Cells were treated with 50, 100, and 150 μg/mL of anthocyanin fraction. Low concentration of anthocyanin from all cultivars showed DNA fragmentation. There was a significant difference in the caspase-3 activity (P < 0.05) between the control cells and the cells treated with anthocyanins from all the cultivars. A positive dose-response relationship was found in all the cultivars. Highest activity (1.4 fold increase over control) was observed in cells treated with 150 μg/mL anthocyanin fraction from the Brightwell cultivar. QR activity was lower in all treated cells than in control cells (0.25 μM/mg protein); A positive dose-response relationship was found in all the cultivars except Brightblue, where activity was the same for all three concentrations. GST activity was statistically higher (P < 0.05) in control cells than in cells treated with anthocyanin fractions from all the cultivars and at all levels of concentration. These results indicated that anthocyanins were not highly active in induction of detoxifying enzymes; however, apoptosis was confirmed in HT-29 cancer cells when treated with anthocyanins consisting predominantly of malvidin.