Effect of anthocyanin fractions from selected cultivars of Georgia-grown blueberries on apoptosis and phase II enzymes.

In recent years, considerable attention has been paid to anthocyanins due to their abilities to inhibit oxidative stress and cell proliferation. The regulations of apoptosis and the phase II enzymes glutathione-S-transferase (GST) and quinone reductase (QR) are other potential mechanisms through which flavonoids such as anthocyanins may prevent cancer. Our study confirmed that anthocyanin fractions from high bush blueberry cultivars increased apoptosis using two different methods: DNA fragmentation and caspase-3 activity. The effect of anthocyanins on the activity of the detoxifying enzymes GST and QR was also determined. Major anthocyanins identified were delphinidin, cyanidin, peonidin, petunidin, and malvidin. In Tifblue and Powderblue cultivars, DNA fragmentation increased at anthocyanin concentrations from 50 to 150 microg/mL, but cells treated with the anthocyanin fraction of Brightblue and Brightwell showed a prominent ladder at 50-100 microg/mL when compared to cells treated with 150 microg/mL. 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 of the cultivars. The response correlated positively with dose. The QR activity was lower in all cells treated with an anthocyanin fraction from Tifblue, Powderblue, Brightblue, and Brightwell cultivars than in control cells (P < 0.05). The activity decreased gradually when treated with increased concentrations of anthocyanin fractions (50-150 microg/mL) in the Tifblue and Powderblue cultivars. The GST activity was lower (P < 0.05) in cells treated with anthocyanin fractions from all of the cultivars and at all concentrations. These results indicated that apoptosis was confirmed in HT-29 cells when treated with anthocyanins from blueberry cultivars at 50-150 microg/mL concentrations, but these same concentrations decrease QR and GST activities rather than induce them.

Effect of storage conditions on the biological activity of phenolic compounds of blueberry extract packed in glass bottles.

Recent research suggests that blueberries are rich in total polyphenols and total anthocyanins. Phenolic compounds are highly unstable and may be lost during processing, particularly when heat treatment is involved. There is no systematic study available providing information on the fate of phenolic compounds during storage and how that affects their biological activity. We provide a systematic evaluation of the changes observed in total polyphenols (TPP), total anthocyanins (TACY), Trolox equivalent antioxidant capacity (TEAC), phenolic acids, and individual anthocyanins of blueberry extract stored in glass bottles and the ability of blueberry extract to inhibit cell proliferation. The extract was stored at different temperatures (-20 +/- 1, 6 +/- 1, 23 +/- 1, and 35 +/- 1 degrees C). Two cultivars, Tifblue and Powderblue, were chosen for the study. The recoveries of TPP, TACY, and TEAC in blueberry extract after pressing and heating were approximately 25, approximately 29, and approximately 69%, respectively, for both cultivars. The recovery of gallic acid, catechin, and quercetin was approximately 25%. Ferulic acid was not detected in the final extract in both Tifblue and Powderblue cultivars. The recovery of peonidin, malvidin, and cyanidin glycosides was approximately 20% in the final extract in both cultivars. Losses due to storage were less when compared with initial losses due to processing. At -20 degrees C, no statistically significant loss of TPP, TACY, and TEAC was observed up to 30 days (P < 0.05). At 6 degrees C storage, there was a significant loss observed from 15 to 30 days. Similar results were obtained at 23 and 35 degrees C (P < 0.05). There was retention of more than 40% of ellagic and quercetin after 60 days at 35 +/- 1 degrees C. Anthocyanins were not detected after 60 days of storage at 35 +/- 1 degrees C. Significant retention (P < 0.05) was obtained for malvidin (42.8 and 25.8%) and peonidin (74.0 and 79.5%) after 60 days of storage at 23 +/- 1 degrees C in glass bottles for Tifblue and Powderblue, respectively, when compared with other individual anthocyanins. A linear relationship was observed between TEAC values and total polyphenols or total anthocyanins. A cell viability assay was performed using HT-29 cancer cell lines and anthocyanins extracted from 30, 60, and 90 days of stored extract at 6 +/- 1 and 23 +/- 1 degrees C. A significant cell proliferation inhibition percentage was observed in 30 days, although this was reduced significantly after 30-90 days. These results suggest that heating and storage conditions significantly affect the phenolic compounds and their biological activities. Frozen and low temperature storage are suggested for blueberry extract in order to retain the bioactive components.

Absorption of anthocyanins from blueberry extracts by caco-2 human intestinal cell monolayers.

Recent studies have shown that dietary polyphenols may contribute to the prevention of cardiovascular disease and cancer. Anthocyanins from different plant sources including blueberries have been shown to possess potential anticancer activities. One of the key factors needed to correctly relate the in vitro study results to human disease outcomes is information about bioavailability. The objectives of the current study were to evaluate the absorption of blueberry anthocyanin extracts using Caco-2 human intestinal cell monolayers and investigate the effects of different aglycones, sugar moieties, and chemical structure on bioavailability of different types of anthocyanins. The results of this study showed that anthocyanins from blueberries could be transported through the Caco-2 cell monolayers although the transport/absorption efficiency was relatively low compared to other aglycone polyphenols. The transport efficiency of anthocyanins averaged approximately 3-4% [less than 1% in delphinidin glucoside (Dp-glc)]. No significant difference in transport/absorption efficiency was observed among three blueberry cultivars. The observed trends among different anthocyanins generally agreed well with some published in vivo results. Dp-glc showed the lowest transport/absorption efficiency, and malvidin glucoside (Mv-glc) showed the highest transport/absorption efficiency. Our result indicates that more free hydroxyl groups and less OCH(3) groups can decrease the bioavailability of anthocyanins. In addition, cyanindin glucoside (Cy-glc) showed significantly higher transport efficiency than cyanidin galactoside (Cy-gal), and peonidin glucoside (Pn-glc) showed significantly higher transport efficiency than peonidin galactoside (Pn-gal), indicating that glucose-based anthocyanins have higher bioavailability than galactose-based anthocyanins.

Phenolic compounds from blueberries can inhibit colon cancer cell proliferation and induce apoptosis.

Research has shown that diets rich in phenolic compounds may be associated with lower risks of several chronic diseases including cancer. This study systematically evaluated the bioactivities of phenolic compounds in rabbiteye blueberries and assessed their potential antiproliferation and apoptosis induction effects using two colon cancer cell lines, HT-29 and Caco-2. Polyphenols in three blueberry cultivars, Briteblue, Tifblue, and Powderblue, were extracted and freeze-dried. The extracts were further separated into phenolic acids, tannins, flavonols, and anthocyanins using an HLB cartridge and LH20 column. Some individual phenolic acids and flavonoids were identified by HPLC with >90% purity in anthocyanin fractions. The dried extracts and fractions were added to the cell culture medium to test for antiproliferation activities and induction of apoptosis. Flavonol and tannin fractions resulted in 50% inhibition of cell proliferation at concentrations of 70-100 and 50-100 microg/mL in HT-29 and Caco-2 cells, respectively. The phenolic acid fraction showed relatively lower bioactivities with 50% inhibition at approximately 1000 microg/mL. The greatest antiproliferation effect among all four fractions was from the anthocyanin fractions. Both HT-29 and Caco-2 cell growth was significantly inhibited by >50% by the anthocyanin fractions at concentrations of 15-50 microg/mL. Anthocyanin fractions also resulted in 2-7 times increases in DNA fragmentation, indicating the induction of apoptosis. The effective dosage levels are close to the reported range of anthocyanin concentrations in rat plasma. These findings suggest that blueberry intake may reduce colon cancer risk.

Phenolic compounds and antioxidant capacity of Georgia-grown blueberries and blackberries.

Blueberries and blackberries grown at various locations in Georgia were collected and analyzed for flavonoids, total anthocyanins, total polyphenols, and Trolox-equivalent antioxidant capacity (TEAC). Each sample was analyzed for phenolic acids (gallic acid, p-hydroxybenzoic acid, caffeic acid, ferulic acid, and ellagic acid) and flavonoids (catechin, epicatechin, myricetin, quercetin, and kaempferol). A high-performance liquid chromatographic (HPLC) method with photodiode array detection was used for analysis. Compounds were analyzed as aglycons after acid hydrolysis with 1.2 M HCl. Identification of each compound was based on retention time and UV spectra by comparison with pure commercial standards. Phenolic acids ranged from 0.19 to 258.90 mg/100 g fresh weight (FW), and flavonoids ranged from 2.50 to 387.48 mg/100 g FW. Total polyphenols ranged from 261.95 to 929.62 mg/100 g FW, and total anthocyanins ranged from 12.70 to 197.34 mg/100 g FW. TEAC values varied from 8.11 to a maximum of 38.29 microM/g FW. A linear relationship was observed between TEAC values and total polyphenols or total anthocyanins. The data indicate that blueberries and blackberries are rich sources of antioxidants.

Phenolic content and antioxidant capacity of muscadine grapes.

Fruits of 10 cultivars of muscadine grapes (five bronze skin and five purple skin) grown in southern Georgia were separated into skin, seed, and pulp. Each fruit part and the leaves from the corresponding varieties were extracted for HPLC analysis of major phenolics. Total phenolics were determined colorimetrically using Folin-Ciocalteu reagent. Total anthocyanins were determined according to a pH-differential method, using a UV-visible spectrophotometer. Antioxidant capacity was determined by the Trolox equivalent antioxidant capacity (TEAC) assay. Gallic acid, (+)-catechin, and epicatechin were the major phenolics in seeds, with average values of 6.9, 558.4, and 1299.4 mg/100 g of fresh weight (FW), respectively. In the skins, ellagic acid, myricetin, quercetin, kaempferol, and trans-resveratrol were the major phenolics, with respective average values of 16.5, 8.4, 1.8, 0.6, and 0.1 mg/100 g of FW. Contrary to previous results, ellagic acid and not resveratrol was the major phenolic in muscadine grapes. The HPLC solvent system used coupled with fluorescence detection allowed separation of ellagic acid from resveratrol and detection of resveratrol. Reported here for the first time are the phenolic content and antioxidant capacity of muscadine leaves. Major phenolics in muscadine leaves were myricetin, ellagic acid, kaempferol, quercetin, and gallic acid, with average concentrations of 157.6, 66.7, 8.9, 9.8, and 8.6, respectively. Average total phenolics were 2178.8, 374.6, 23.8, and 351.6 mg/g gallic acid equivalent in seed, skin, pulp, and leaves, respectively. Total anthocyanin contents were 2.1 and 132.1 mg/100 g of FW in the skins of bronze and purple grapes, respectively, and 4.3 and 4.6 mg/100 g of FW in seeds and pulps, in that order. Antioxidant capacity values were, on average, 2.4, 12.8, 281.3, and 236.1 microM TEAC/g of FW for pulps, skins, seeds, and leaves, respectively.

Effects of blueberry (Vaccinium ashei) on DNA damage, lipid peroxidation, and phase II enzyme activities in rats.

Blueberry extracts have high antioxidant potential and increase phase II enzyme activities in vitro. This study tested the hypothesis that blueberries would reduce DNA damage and lipid peroxidation and increase phase II enzyme activities in vivo. Young, healthy male Sprague-Dawley rats (n = 8 per group) were fed control AIN-93 diets or AIN-93 diets supplemented with blueberries or blueberry extracts for 3 weeks. Diets were supplemented with 10% freeze-dried whole blueberries, blueberry polyphenol extract and sugars to match the 10% blueberry diet, or 1 and 0.2% blueberry flavonoids, which were primarily anthocyanins. Liver and colon mucosa glutathione-S-transferase (GST), quinone reductase, and UDP-glucuronosyltransferase activities in colon mucosa and liver were not significantly increased by freeze-dried whole blueberries or blueberry fractions. Liver GST activity, however, was approximately 25% higher than controls for the freeze-dried whole blueberry, blueberry polyphenol, and 1% flavonoid groups. DNA damage was significantly lower than control only in the liver of animals fed the 1% flavonoid diet. The level of urinary F(2)-isoprostanes, a measure of lipid peroxidation, was unaffected. In summary, in healthy rats, short-term supplementation with freeze-dried whole blueberries, blueberry polyphenols, or blueberry flavonoids did not significantly increase phase II enzyme activities. However, supplementation with 1% blueberry flavonoids did decrease oxidative DNA damage in the liver.