Blueberry Fractions Research Articles

Wild-Blueberry Volatile and Phenolic Fractions Mitigate LPS-Induced Inflammatory Responses in Whole Blood of Children

Abstract Objectives The anti-inflammatory effects of berries have been attributed to their polyphenol content. In vitro models of activated murine macrophages have shown that volatiles in berries exert anti-inflammatory effects as well. However, human data in this area are scarce. To address this limitation we measured the effects of wild-blueberry phenolic (WBB-P) and WBB volatile (WBB-V) fractions in LPS-treated whole blood (WB) of children. Methods Fasting WB was collected (n = 19), and treated with high and low doses of either WBB-P (2 μg/mL or 0.02 μg/mL), WBB-V (2 ng/mL or 0.02 ng/mL), or culture medium (RPMI) as control and incubated for 1 hour at 37°C. To evaluate potential anti-inflammatory effects of WBB molecules, lipopolysaccharide (LPS, 0.2 μg/mL or 1 μg/mL) was added to the wells, and samples incubated for 48 hours. Interleukin (IL)-1β, IL-6, IL-10 and TNF-α supernatant concentrations were measured using enzyme-linked immunosorbent assay. One-factor ANOVA analysis with participant ID as random effect was used to compare cytokine concentrations (ng/ml) of WBB fraction treatments against their respective RPMI + LPS controls. Results Children (12.0 ± 0.6 years old) were predominantly white (79%), and with a BMI percentile of 51 ± 35. At both LPS levels, IL-6 concentrations were lower in the high-dose WBB-P and WBB-V treatments compared to the control group. LPS-High: WBB-P: 22.2 ± 6.7 vs. RPMI: 28.2 ± 8.3 (p = 0.005); WBB-V: 24.4 ± 7.2 vs. RPMI: 28.2 ± 8.3 (p = 0.013). LPS-low: WBB-P: 20.4 ± 5.5 vs. RPMI: 26.6 ± 7.7 (p = 0.001); WBB-V: 20.6 ± 6.5 vs. RPMI: 26.6 ± 7.7 (p < 0.0001). At the lowest LPS level, TNF-α concentration was 23% lower in the high-dose WBB-V treatment compared to the control group (WBB-V: 0.45 ± 0.06 vs. RPMI: 0.55 ± 0.12, p = 0.003). IL-1β, and IL-10 were not modified by WBB fraction treatments. Conclusions These preliminary results indicate that both WBB-P and WBB-V fractions mitigate inflammatory responses in LPS treated WB of children. Blueberry fractions do not impact IL-1β or IL-10 secretion by immune cells in WB. Funding Sources Center for Childhood Obesity Prevention NIH-NIGMS 5P20GM109096. USDA-ARS 6026–51,000-012–06S. The Arkansas Center for Advancing Pediatric Therapeutics NIH 8UG1OD024945.

The effect of Chinese wild blueberry fractions on the growth and membrane integrity of various foodborne pathogens.

The objective of this study was to evaluate the antibacterial effect of Chinese wild blueberry extract and its fractions against Listeria monocytogenes, Staphylococcus aureus, Salmonella Enteritidis, and Vibrio parahaemolyticus. Chinese wild blueberry (Vaccinium uliginosum) crude extract (BBE) was obtained using methanol extraction, and sugars plus organic acids (F1), phenolics fraction (F2), and anthocyanins plus proanthocyanidins (F3) fractions were separated using C-18 Sep-Pak columns. The minimal inhibitory concentration and minimal bactericidal concentration of each fractional component were determined using a two-fold-serial dilution method. Nucleic acid leakage (OD260 nm ) and protein release (Bradford protein assay) were determined by spectrophotometry, to evaluate the permeability of the cell membrane. F3 was found to exhibit the greatest antimicrobial activity against the four tested strains, followed by F2, F1, and BBE. V. parahaemolyticus was the most sensitive to the all fractions, followed by S. Enteritidis, L. monocytogenes, and S. aureus. Survival curve analysis showed that the number of bacteria decreased from six log colony-forming units (CFU) to less than 10 CFU after bacteria were treated with fractions for 12 hr, which demonstrated the bactericidal effect of blueberry fractions. Furthermore, when the pathogens were treated with fractions for 2 hr, the OD260 nm and OD595 nm values increased significantly (P <0.01), which indicated the significant release of nucleic acid and protein. The results from this study indicated that blueberry fractions, especially F3, inhibited the growth of foodborne pathogens by damaging their cell membrane, and may be developed as a natural preservative to prevent and control foodborne pathogens. PRACTICAL APPLICATION: A blueberry crude extract and its sugars plus organic acids, phenolics, and anthocyanins plus proanthocyanidins fractions, inhibited the growth of foodborne pathogens by destroying their cell membrane. Therefore, Chinese wild blueberries have potential as a natural preservative to prevent and control foodborne pathogens.

Blueberry polyphenols prevent cardiomyocyte death by preventing calpain activation and oxidative stress.

The purpose of this study was to examine the efficacy of an aqueous wild blueberry extract and five wild blueberry polyphenol fractions on an in vitro model of heart disease. Adult rat cardiomyocytes were pretreated with extract and fractions, and then exposed to norepinephrine (NE). Cardiomyocyte hypertrophy, cell death, oxidative stress, apoptosis and cardiomyocyte contractile function as well as the activities of calpain, superoxide dismutase (SOD) and catalase (CAT) were measured in cardiomyocytes treated with and without NE and blueberry fraction (BF). Four of five blueberry fractions prevented cell death and cardiomyocyte hypertrophy induced by NE. Total phenolic fraction was used for all further analysis. The NE-induced increase in oxidative stress, nuclear condensation, calpain activity and lowering of SOD and CAT activities were prevented upon pretreatment with BF. Reduced contractile function was also significantly improved with BF pretreatment. Blueberry polyphenols prevent NE-induced adult cardiomyocyte hypertrophy and cell death. The protective effects of BF may be in part attributed to a reduction in calpain activity and oxidative stress.

Antioxidant and skin pores contraction activity of Vaccinium corymbosum

Blueberry has attracted much attention due to their potential health benefits. Blueberry contains a diverse of biological properties such as antioxidant and anti‐inflammatory activities. The aim of this study is to confirm the effect of blueberry extracts on skin pores contraction through antioxidant activity, quantification of tannin contents, protein aggregation assay and inhibition of 5‐alpha‐reductase type 1 mRNA expression. Blueberry was extracted with 70% ethanol and then fractionated with hexane, CHCl3, EtOAc, BuOH and water to get active fractions. Antioxidant activity of each fraction was determined by using the DPPH and FRAP assay. All fractions had high antioxidant activities, tannin contents than crude extract, but blueberry ethyl acetate (BB‐E) fraction was at the top of the list. Protein aggregation of blueberry fraction has an effect as compared with positive control tannic acid. The major factor contributing to excess sebum is 5‐alpha reductase type1 which related to enlarge skin pores. We confirmed that blueberry fractions reduced 5‐alpha reductase type1 expression in HaCaT using RT‐PCR method. In conclusion, we expected the beneficial effect of blueberry on skin pores contraction.Grant Funding Source : Ipeeres Ltd.

A nonpolar blueberry fraction blunts NADPH oxidase activation in neuronal cells exposed to tumor necrosis factor-α.

Inflammation and oxidative stress are key to the progressive neuronal degeneration common to chronic pathologies, traumatic injuries, and aging processes in the CNS. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-α) orchestrates cellular stress by stimulating the production and release of neurotoxic mediators including reactive oxygen species (ROS). NADPH oxidases (NOX), ubiquitously expressed in all cells, have recently emerged as pivotal ROS sources in aging and disease. We demonstrated the presence of potent NOX inhibitors in wild Alaska bog blueberries partitioning discretely into a nonpolar fraction with minimal antioxidant capacity and largely devoid of polyphenols. Incubation of SH-SY5Y human neuroblastoma cells with nonpolar blueberry fractions obstructed the coalescing of lipid rafts into large domains disrupting NOX assembly therein and abolishing ROS production characteristic for TNF-α exposure. These findings illuminate nutrition-derived lipid raft modulation as a novel therapeutic approach to blunt inflammatory and oxidative stress in the aging or diseased CNS.

The antimicrobial properties of the lowbush blueberry (Vaccinium angustifolium) fractional components against foodborne pathogens and the conservation of probiotic Lactobacillus rhamnosus

The antimicrobial properties of lowbush blueberry (Vaccinium angustifolium) were studied against Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and Lactobacillus rhamnosus to determine which fractional components have antimicrobial effects and which microorganisms are most susceptible to these antimicrobial properties. Lowbush blueberry extract (F1) was separated using a C-18 Sep-Pak cartridge into monomeric phenolics (F2) and anthocyanins plus proanthocyanidins (F3). Fraction 3 was further separated into anthocyanins (F4) and proanthocyanidins (F5) using a LH-20 Sephadex column. Each fraction was initially screened for antimicrobial properties using agar diffusion assay. Treatments that demonstrated inhibition were further analyzed for inhibition in liquid culture. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using a two-fold dilution series and viable cell counts taken at 0 and 24 h to examine growth reduction. Fraction 3 demonstrated the lowest MICs/MBCs followed by F1, F2, F4, and F5. L. monocytogenes was the most susceptible to blueberry fraction treatment, followed by E. coli O157:H7, and S. Typhimurium. L. rhamnosus was the least susceptible to each fraction treatment. The results can be applied to the field of preventive medicine, food safety, and enrich the understanding of the health benefits of lowbush blueberries.

Differential Protection among Fractionated Blueberry Polyphenolic Families against DA-, Aβ42- and LPS-Induced Decrements in Ca2+ Buffering in Primary Hippocampal Cells

It has been postulated that at least part of the loss of cognitive function in aging may be the result of deficits in Ca(2+) recovery (CAR) and increased oxidative/inflammatory (OX/INF) stress signaling. However, previous research showed that aged animals supplemented with blueberry (BB) extract showed fewer deficits in CAR, as well as motor and cognitive functional deficits. A recent subsequent experiment has shown that DA- or Abeta(42)-induced deficits in CAR in primary hippocampal neuronal cells (HNC) were antagonized by BB extract, and (OX/INF) signaling was reduced. The present experiments assessed the most effective BB polyphenol fraction that could protect against OX/INF-induced deficits in CAR, ROS generation, or viability. HNCs treated with BB extract, BB fractions (e.g., proanthocyanidin, PAC), or control medium were exposed to dopamine (DA, 0.1 mM), amyloid beta (Abeta(42), 25 muM) or lipopolysaccharide (LPS, 1 microg/mL). The results indicated that the degree of protection against deficits in CAR varied as a function of the stressor and was generally greater against Abeta(42) and LPS than DA. The whole BB, anthocyanin (ANTH), and PRE-C18 fractions offered the greatest protection, whereas chlorogenic acid offered the lowest protection. Protective capabilities of the various fractions against ROS depended upon the stressor, where the BB extract and the combined PAC (high and low molecular weight) fraction offered the best protection against LPS and Abeta(42) but were less effective against DA-induced ROS. The high and low molecular weight PACs and the ANTH fractions enhanced ROS production regardless of the stressor used, and this reflected increased activation of stress signals (e.g., P38 MAPK). The viability data indicated that the whole BB and combined PAC fraction showed greater protective effects against the stressors than the more fractionated polyphenolic components. Thus, these results suggest that, except for a few instances, the lesser the polyphenolic fractionation, the greater the effects, especially with respect to prevention of ROS and stress signal generation and viability.

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.

Effects of blueberry extracts on DNA damage levels and phase II enzyme activities in rats.

Blueberry extracts been shown to reduce oxidative stress and increase phase II enzyme activities in vitro. We examined these effects in vivo. Male Sprague-Dawley rats (n=8 per group) were fed AIN-93 or modified AIN-93 diets for 3 weeks. Modified diets were 10% freeze dried blueberries substituted for sucrose (BLU), blueberry polyphenol extract and sugars to match the 10% blueberry diet (POLY), sugars equivalent to the 10% blueberry diet, 1% blueberry flavonoids (1% FL) and 0.2% blueberry flavonoids. The polyphenol extract was obtained using a 40:40:20:0.1 (v:v:v:v) ratio of acetone/methanol/water/formic acid solution. By further purifying the polyphenol extract with an Oasis HLB cartridge then eluting with acidified methanol, total flavonoids were obtained. Liver and colon mucosa glutathione-S-transferase (GST) and quinone reductase (QR) and liver UDP-glucuronosyltransferase (UGT) activities were measured as were urinary F2-isoprostanes and liver and lymphocyte DNA damage. GST, QR and UGT activities were not significantly increased at p<0.05 by blueberries or blueberry fractions, but some slight increases in activity were seen. For example, compared to controls, the BLU, POLY, and 1% FL groups had 28%, 27% and 24% higher GST activity in liver tissue. DNA damage assessed with the Comet assay was lowest for both liver and colon mucosa in the 1%FL group, but results were not significant. This study shows that in vivo results do not always agree with in vitro findings and that extremely high doses may be required to elicit significant effects. Support: GA FOODPAC, GA Ag Exp Sta., GA Blueberry Growers Assoc.

Blueberry flavonoids inhibit matrix metalloproteinase activity in DU145 human prostate cancer cells

Regulation of the matrix metalloproteinases (MMPs), the major mediators of extracellular matrix (ECM) degradation, is crucial to regulate ECM proteolysis, which is important in metastasis. This study examined the effects of 3 flavonoid-enriched fractions (a crude fraction, an anthocyanin-enriched fraction, and a proanthocyanidin-enriched fraction), which were prepared from lowbush blueberries (Vaccinium angustifolium), on MMP activity in DU145 human prostate cancer cells in vitro. Using gelatin gel electrophoresis, MMP activity was evaluated from cells after 24-hr exposure to blueberry fractions. All fractions elicited an ability to decrease the activity of MMP-2 and MMP-9. Of the fractions tested, the proanthocyanidin-enriched fraction was found to be the most effective at inhibiting MMP activity in these cells. No induction of either necrotic or apoptotic cell death was noted in these cells in response to treatment with the blueberry fractions. These findings indicate that flavonoids from blueberry possess the ability to effectively decrease MMP activity, which may decrease overall ECM degradation. This ability may be important in controlling tumor metastasis formation.

Differential effects of blueberry proanthocyanidins on androgen sensitive and insensitive human prostate cancer cell lines

Blueberries are rich in health-promoting polyphenolic compounds including proanthocyanidins. The purpose of this study was to determine if proanthocyanidin-rich fractions from both wild and cultivated blueberry fruit have the same inhibitory effects on the proliferation of LNCaP, an androgen-sensitive prostate cancer cell line, and DU145, a more aggressive androgen insensitive prostate cancer cell line. When 20 μg/ml of a wild blueberry proanthocyanidin fraction (fraction 5) was added to LNCaP media, growth was inhibited to 11% of control with an IC 50 of 13.3 μg/ml. Two similar proanthocyanidin-rich fractions from cultivated blueberries (fractions 4 and 5) at the same concentration inhibited LNCaP growth to 57 and 26% of control with an IC 50 of 22.7 and 5.8 μg/ml, respectively. In DU145 cells, the only fraction that significantly reduced growth compared to control was fraction 4 from cultivated blueberries with an IC 50 value of 74.4 μg/ml, indicating only minor inhibitory activity. Differences in cell growth inhibition of LNCaP and DU145 cell lines by blueberry fractions rich in proanthocyanidins indicate that blueberry proanthocyanidins have an effect primarily on androgen-dependant growth of prostate cancer cells. Possible molecular mechanisms for growth inhibition are reviewed.