Journal of Berry Research

Studies to advance the potential health benefits of berries continue to increase as was evident at the sixth biennial meeting of the Berry Health Benefits Symposium (BHBS). The two and a half-day symposium was held on October 13–15, 2015, in Madison, Wisconsin, United States. The 2015 BHBS featured new and emerging research further bolstering the positive biological effects of berry consumption on human health, performance, and disease prevention. The papers presented at the 2015 BHBS consisted of invited papers from an international group of leading berry researchers, as well as poster abstracts. Oral sessions were organized around themes including heart health, cancer prevention, gut health/gut microflora, brain aging, metabolism, and berry compositional chemistry. These thematic health areas, while not exhaustive, encompass the more prominent research success stories on berries, the vast majority of which are backed by published animal and human studies. Similar to the past meetings, the research findings at the 2015 BHBS primarily focused on blackberries, blueberries, black raspberries, cranberries, red raspberries, and strawberries. However, research on other berry fruits, including chokeberry (aronia berry), cloudberry, blue honeysuckle berry, bilberry, jamun berry, and elderberry, was also featured as was data on major classes of berry polyphenols/phytochemicals including anthocyanins and other flavonoids and their in vivo derived metabolites. The BHBS continues to be a leading forum for interactions between scientists and berry industry stakeholders. The cluster of papers in this issue represents a snapshot of presentations at the 2015 BHBS which support the positive biological effects of berries on human health and diseases.

BACKGROUND: Many foods rich in antioxidant and anti-inflammatory compounds have been shown to increase health and reduce markers of aging. A number of berry fruits high in polyphenols are known to ameliorate age-related declines in cellular, cognitive and behavioral function in rats.

OBJECTIVES: This study evaluated the effectiveness of a red raspberry-supplemented diet on age-sensitive measures of learning, memory and motor performance in aged (19 mo) F344 rats.

METHODS: Red raspberry extract was prepared from fresh whole fruit and incorporated in standard rodent chow to create a 2% diet. Following ten weeks on a control or 2% raspberry diet, cognitive and motor performance was assessed using the Morris Water Maze (MWM) and a battery of five psychomotor tasks.

RESULTS: The supplemented diet significantly improved performance on three of the five motor tasks, but did not affect MWM performance. Specifically, old rats fed the 2% raspberry diet had significantly better performance on the rod and plank walks, which measure psychomotor coordination and balance, and on the inclined screen, which measures muscle tone, strength, stamina and balance.

CONCLUSIONS: Given that falls are the number one health hazard for otherwise healthy older adults, these results may have important implications for increasing healthy aging.

BACKGROUND: Blueberry (BB) and black raspberry (BRB) have been shown to be chemopreventive against estrogen-mediated breast cancer in pre-clinical studies. However, therapeutic efficacy of these berries against lung cancer is not known.

METHODS: In this study we investigated i) relative efficacy of individual anthocyanidins vs. respective anthocyanins, ii) relative antiproliferative activity of mixture of anthocyanidins compared to individual anthocyanidins, iii) antitumor activity of dietary BB, iv) Tumor inhibitory activity of diet supplemented with BB, alone and in combination with BRB, against lung tumor xenograft using nude mice, and finally, v) the efficacy of select polyphenolics present in BB and BRB against lung tumor xenograft.

RESULTS: Our findings indicated that individual anthocyanidins (aglycones) were significantly more potent (2-3 fold lower IC₅₀) in inhibiting the non-small-cell lung cancer (NSCLC) cell growth vs. respective anthocyanins (glycones). Further, anthocyanidins mixture at equimolar concentrations exhibited synergistic antiproliferative activity vs. individual anthocyanidins. When tested against NSCLC (A549 and H1299) cells in nude mice, dietary BB (7.5%, w/w) showed >40% reduction in tumor volume against H1299 xenografts. The maximal growth inhibition occurred with 5% BB dose, with no additional protection occurring at a higher dose (7.5%). However, somewhat lower protection was found when the BB diet initiated prior to tumor cell inoculation. The mixture of BB (5%, w/w) and BRB (2.5%) resulted in higher inhibition of tumor growth vs. BB alone (71% vs 42%). Likewise, a combination of delphinidin (bioactive of BB) and punicalagins (a bioactive of BRB, which gets converted to ellagic acid in vivo) showed higher tumor growth inhibition compared to delphinidin.

CONCLUSIONS: The therapeutic effects of the berries and berry polyphenolics observed against lung cancer in this study are highly encouraging. Further investigation into the mechanism of action of the combinations of the berry bioactives will be valuable for clinical use of this potent natural product against lung cancer.

BACKGROUND: Populations that consume procyanidin-rich diets are less susceptible to inflammatory disease. Allergic asthma is an inflammatory lung disease perpetuated by a hyperreactive airway epithelium and eosinophil infiltration into the lung. Eotaxin-1 (CCL11) mediates eosinophil migration into tissues and its modulation could represent a means to assist the management of airway inflammation.

OBJECTIVE: Here we evaluated procyanidins as a means of modulating CCL11 production in vitro.

METHODS: We used human lung epithelial cells (A549) and optimized the conditions to induce CCL11 production in vitro. Cells were exposed to procyanidins for 6 h prior to an inflammatory insult of 5 ng/mL IL-4 with 5 ng/mL TNFα for 48 h. An enzyme-linked immunosorbent assay was used to measure CCL11 production.

RESULTS: Cells exposed to 5 μM procyanidin A2 prior to the inflammatory challenge showed significantly inhibited (36%) CCL11 production. Under the same conditions, procyanidins B1 and B2 elicited no effect. Furthermore, combinations of procyanidins A2 and B2 (5 μM total) demonstrated no evidence of a synergistic interaction.

CONCLUSIONS: These data demonstrate that the regulation of CCL11 by lung epithelial cells is not ubiquitous among the three investigated procyanidins. We demonstrate a differential inhibition of CCL11 by A-type and B-type procyanidins. This evidence supports further studies into procyanidins, specifically A-type, for managing inappropriate airway inflammation.

BACKGROUND: We recently reported that a cranberry proanthocyanidin rich extract (C-PAC) induces autophagic cell death in apoptotic resistant esophageal adenocarcinoma (EAC) cells and necrosis in autophagy resistant cells. EAC is characterized by high morbidity and mortality rates supporting development of improved preventive interventions.

OBJECTIVE: The current investigation sought to investigate the role of reactive oxygen species (ROS) in the context of C-PAC induced cell death.

METHODS: A panel of human esophageal cell lines of EAC or BE (Barrett’s esophagus) origin were treated with C-PAC and assessed for ROS modulation using CellROX^® Green reagent and the Amplex Red assay to specifically measure hydrogen peroxide levels.

RESULTS: C-PAC significantly increased ROS levels in EAC cells, but significantly reduced ROS levels in CP-C BE cells. Increased hydrogen peroxide levels were also detected in C-PAC treated EAC cells and supernatant; however, hydrogen peroxide levels were significantly increased in medium alone, without cells, suggesting that C-PAC interferes or directly acts on the substrate. Hydrogen peroxide levels did not change in C-PAC treated CP-C BE cells.

CONCLUSION: These experiments provide additional mechanistic insight regarding C-PAC induced cancer cell death through modulation of ROS. Additional research is warranted to identify specific ROS species associated with C-PAC exposure.

BACKGROUND: Data from human intervention studies have highlighted potential cardiovascular benefits of blueberry (poly)phenols. However, such biological effects are dependent on their bioavailability and, as such, information on the absorption, metabolism and excretion of such compounds is necessary.

OBJECTIVE: To investigate whether the bioavailability of blueberry (poly)phenols is intake-dependent, a group of nine healthy volunteers consumed three wild blueberry drinks containing 766 mg (lower), 1278 mg (medium) and 1791 mg (higher) total (poly)phenols, corresponding to 34, 56 and 80 g, respectively, freeze-dried blueberry powder.

METHODS: Plasma levels of (poly)phenol metabolites were assessed at baseline and at 1, 2, 4 and 6 hours post-consumption, using UPLC-Q-TOF mass spectrometry.

RESULTS: Twenty-three phenolic acid metabolites were quantified in plasma after blueberry consumption. Increases in plasma (poly)phenol metabolites were observed in all the interventions tested. The area under the curve of the concentration over time (AUC) significantly increased when comparing the lower and higher (poly)phenol interventions. Linear dose-response regressions were obtained for 11 metabolites, while the plasma concentration of the remaining 12 metabolites was not affected by increasing amounts of (poly)phenols in the blueberry interventions.

CONCLUSION: Absorption and metabolism of blueberry (poly)phenols are not exclusively intake-dependent at the amounts tested, evidencing a complex metabolic fate of these compounds.

BACKGROUND: The formation and accumulation of advanced glycation end-products (AGEs) are implicated in several chronic human illnesses including type-2 diabetes, renal failure, and neurodegenerative diseases. The cranberry (Vaccinium macrocarpon) fruit has been previously reported to show anti-AGEs effects, attributed primarily to its phenolic constituents. However, there is lack of similar data on the non-phenolic constituents found in the cranberry fruit, in particular, its carbohydrate constituents. Herein, a chemically characterized oligosaccharide-enriched fraction purified from the cranberry fruit was evaluated for its potential anti-AGEs and free radical scavenging effects.

OBJECTIVE: The aim of this study was to evaluate the in vitro anti-AGEs and free radical scavenging effects of a chemically characterized oligosaccharide-enriched fraction purified from the North American cranberry (Vaccinium macrocarpon) fruit.

METHOD: The cranberry oligosaccharide-enriched fraction was purified from cranberry hull powder and characterized based on spectroscopic and spectrometric (NMR, MALDI-TOF-MS, and HPAEC-PAD) data. The oligosaccharide-enriched fraction was evaluated for its anti-AGEs and free radical scavenging effects by the bovine serum albumin-fructose, and DPPH assays, respectively.

RESULTS: Fractionation of cranberry hull material yielded an oligosaccharide-enriched fraction named Cranf1b-CL. The ¹H NMR and MALDI-TOF-MS revealed that Cranf1b-CL consists of oligosaccharides ranging primarily from 6-mers to 9-mers. The monosaccharide composition of Cranf1b-CL was arabinose (25%), galactose (5%), glucose (47%) and xylose (23%). In the bovine serum albumin-fructose assay, Cranf1b-CL inhibited AGEs formation in a concentration-dependent manner with comparable activity to the synthetic antiglycating agent, aminoguanidine, used as the positive control (57 vs. 75%; both at 500μg/mL). In the DPPH free radical scavenging assay, Cranf1b-CL showed superior activity to the synthetic commercial antioxidant, butylated hydroxytoluene, used as the positive control (IC₅₀ = 680 vs. 2200μg/mL, respectively).

CONCLUSION: The in vitro anti-AGEs and free radical scavenging effects of cranberry oligosaccharides support previous data suggesting that these constituents may also contribute to biological effects of the whole fruit beyond its phenolic constituents alone. Also, this is the first study to evaluate a chemically characterized oligosaccharide fraction purified from the North American cranberry fruit for anti-AGEs and free radical scavenging properties.

BACKGROUND: Fruits and berries are known to contain relatively high amounts of antioxidant/bioactive compounds. Several methods have been used for measurement of antioxidant capacity (AC), but not all methods have direct relevance to in vivo antioxidant status.

OBJECTIVE: Determine AC in berry/fruit samples, processed berry products, and purified compounds by utilizing 5 different biologically relevant free radical/oxidant sources.

METHODS: Samples were assayed for AC capacity using 5 different free radical/oxidant sources: peroxyl radical (ORAC), hydroxyl radical (HORAC), peroxynitrite (NORAC), superoxide anion (SORAC) and singlet oxygen (SOAC)]. Total AC (sum of AC with 5 individual radicals) was expressed as Oxygen Radical Absorption Capacity using Multiple Radicals (ORAC_MR5).

RESULTS: SOAC contributed more than 60% of ORAC_MR5 in blackberries, sweet and tart cherries; and no detectable levels of SOAC were found in strawberries, black currants and raspberries. Whole fruit purees of mango, wild blueberry and cherry contained 95, 85 and 67% respectively of total ORAC_MR5 from SOAC. However, freeze dried wild blueberry powder from same production season had only 28% as SOAC and 42 and 22% as ORAC and HORAC. Blueberry/Pomegranate and Mango/Pineapple smoothies had 67% and 77% of ORAC_MR5 as SOAC.

CONCLUSIONS: The antioxidant quenching potential using 5 different radical/oxidant sources of different berries and fruits varied widely and understanding this variation may be helpful in understanding health benefits of different berries and foods.

BACKGROUND: Many brightly colored fruits and vegetables owe their pigmentation and beneficial health effects to anthocyanins. Unfortunately, anthocyanins in the presence of ascorbic acid are readily degraded over juice processing and storage, which adversely affects color stability and potential health benefits.

OBJECTIVE: This project focused on the effect of ascorbic acid as a catalyst in anthocyanin degradation.

METHODS: The project involved searching for novel pigmented compounds in a simple model system composed of the most common anthocyanin cyanidin-3-O-β-glucoside and ascorbic acid, and a second model system consisting of blackberry extract supplemented with ascorbic acid. Degradation products were identified by HPLC-PDA and HPLC-MS. ESR was used to monitor hydroxyl radical formation in the model systems.

RESULTS: Over 72 hours at ambient temperature, 67% of cyanidin-3-O-β-glucoside was lost in the model system during which time an unknown pigmented compound was formed. The unknown compound was also formed in a more complex model system consisting of blackberry extract and ascorbic acid. HPLC with PDA monitoring at 510 nm was used to detect a novel compound and HPLC-ESI-MS³ allowed a proposed structure to be built based on the fragmentation patterns. The unknown structure formed via oxidation of cyanidin 3-O-β-glucoside by ascorbic acid was identified as 6-hydroxy-cyanidin-3-O-β-glucoside. The mechanism was substantiated with malvidin-3-O-β-glucoside and ascorbic acid, which produced a hydroxylated malvidin-3-O-β- glucoside. Production of hydroxyl radical in the base and blackberry model systems was confirmed by ESR.

CONCLUSIONS: We propose that the pigmented compound is formed from hydroxyl radicals via the Haber-Weiss reaction. The addition of food grade hydroxyl radical scavengers to juices may be a viable treatment to prevent ascorbic acid-catalyzed degradation of anthocyanins.

BACKGROUND: Blueberry anthocyanins are susceptible to degradation during juice processing and storage of juice at ambient temperature. Methods are needed to stabilize the health-promoting anthocyanins in blueberry and other anthocyanin-rich berry juices.

OBJECTIVE: In this study we determined the effect of acidification (pH 2.1, 2.5 and 2.9) of blueberry juice on changes in anthocyanins and percent polymeric color in response to juice processing and during eight months of storage at ambient and refrigerated temperatures.

METHODS: Three subsamples of non-pasteurized blueberry juice were adjusted to three pH levels: 2.9 (control, no pH adjustment), 2.5, and 2.1. After pH adjustment, juices were pasteurized and placed in storage at 4 and 25°C. Samples were analyzed before (non-pasteurized) and after pasteurization, and after 2, 4, 6, and 8 months of storage at each temperature (4 and 25°C) for anthocyanin composition by HPLC and percent polymeric color.

RESULTS: Blueberry juice acidified to pH 2.1 retained higher levels of total anthocyanins and had lower percent polymeric color values than juice acidified to pH 2.5 and control juice (pH 2.9) following pasteurization. Anthocyanin arabinosides were more susceptible to thermal degradation than glucosides, galactosides and acetylated derivatives. Levels of total anthocyanins declined markedly over 8 months of storage, but juices stored at 4°C had on average 56% higher levels of total anthocyanins than juices stored at 25°C. Juice acidified to pH 2.1 had on average 12% and 26% higher levels of total anthocyanins than pH 2.5 and control juices, respectively. After 8 months of storage, juice acidified to pH 2.1 had 11 and 22% higher levels of total anthocyanins than pH 2.5 and control juices stored at 4°C, and 26% and 59% higher levels of total anthocyanins than pH 2.5 and control juices stored at 25°C. Acetylated derivatives were more prone to losses during storage than glycosides, especially in acidified juices.

CONCLUSIONS: Acidification of blueberry juice coupled with refrigerated storage are effective treatments to retain health-promoting anthocyanins.

BACKGROUND: Products derived from black chokeberry are claimed to be beneficial in treating chronic diseases, such as obesity and diabetes.

OBJECTIVE: The objective of this study was to determine if supplementation with Aronia melanocarpa (black chokeberry) juice concentrate (AJC) has anti-obesity properties in mice fed obesogenic diets.

METHODS: Male C57BL/6J mice (n = 10/dietary treatment) were placed on either a low-fat, high-sucrose (LFHS; 5% fat), LFHS+AJC (1.44 g AJC/kg diet), high-fat (HF; 30% fat), or HF+AJC for 12-weeks.

RESULTS: Final body weight was lower in LFHS+AJC compared to LFHS, HF and HF+AJC (∼14, 20% and ∼16%, respectively; P < 0.05). Mice receiving LFHS and LFHS+AJC had significantly higher (P = 0.001) energy intake than HF and HF+AJC. LFHS-fed mice had less (–30%) epididymal fat (p < 0.05) than HF-fed mice, however mice on the LFHS+AJC had less epididymal fat per gram body weight than LFHS controls. There was no effect of diet or AJC on adipose tissue gene expression. There was no difference in plasma insulin, glucose or triglycerides between groups, however there was a positive effect of AJC on adiponectin (P = 0.059). There was also a significant effect of diet (LFHS versus HF) on HOMA-IR (P = 0.004) and HOMA-BCF (P = 0.002).

CONCLUSIONS: The results from this study demonstrate that AJC supplementation has the potential to prevent weight gain and markers of obesity. Further research is needed to determine mechanisms of action.

BACKGROUND: A significant body of research demonstrates that intake of berries and berry phytochemicals favorably influence metabolism via a number of different biological mechanisms. However, raspberries and select phytochemicals present in raspberries have been less studied than several other berry fruits.

OBJECTIVE: The current study tested the relative effect of whole raspberry products or raspberry phytochemicals using the C57BL/6J mouse fed a high-fat, high-sucrose diet as a model for the obesigenic Western diet. The development of obesity and related metabolic complications were measured.

METHODS: C57BL/6J mice were fed experimental diets for ten weeks. Diets were formulated to include a typical dietary level of whole raspberry food products, or for the case of ellagic acid (EA) and raspberry ketone (RK), levels that could be reasonably achieved with typical dietary supplement use. In week nine, a glucose tolerance test was conducted. After ten weeks, animals were sacrificed, serum collected, and liver tissue saved for histology and lipid accumulation measurements.

RESULTS: Addition of raspberry food products and phytochemicals to high-fat diet reduced body weight gain significantly when raspberry juice and puree concentrates (RJC and RPC), and the EA+RK combination were added. Food efficiency values reflected these weight changes, with the same three groups having food efficiencies similar to normal-weighted low-fat fed mice. Serum insulin and resistin levels were measured and resistin levels were reduced in the two groups fed high fat diets with RPC or RJC added. A histological evaluation of liver tissue was completed and some differences were impacted by diet.

CONCLUSIONS: When added to a high-fat diet, raspberry juice and puree concentrates and the combination of EA+RK decreased weight gain and food efficiency obesity associated with consumption of the obesigenic high-fat, high-calorie diet. Future studies are required to determine mechanisms that may be responsible for these changes.

The ability of a wild blueberry-enriched diet to improve risk factors related to Metabolic Syndrome (MetS) such as endothelial dysfunction and chronic inflammation in the Obese Zucker Rat (OZR), a model of the MetS, was studied.

Obese Zucker Rats (OZRs) and their lean controls (LZR) were placed either on a Wild Blueberry-enriched (WB) or a control (C) diet for 8 weeks. Obese Zucker rats exhibited reduced vasoconstrictor response to phenylephrine (Phe) and exaggerated vasorelaxant response to acetylcholine (Ach). WB diet partially restored Phe-induced constrictor responses and attenuated Ach-induced relaxant responses in OZR. Plasma nitric oxide (NO) was significantly attenuated and aortic effluent prostaglandin I₂ PGI₂ concentration significantly increased in the WB diet. Downregulation of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2) expression in the OZR aorta was observed in the WB diet.

WB consumption decreased plasma concentrations of tumor necrosis factor (TNF)-α (–25.6%, P < 0.05), interleukin (IL)-6 (–14.9%, P < 0.05), C-reactive protein (CRP) (–13.1%, P < 0.05) and increased adiponectin concentration (+21.8%, P < 0.05). Expression of IL-6, TNF-α and nuclear factor (NF)-kB was downregulated in both the liver (–65%, –59% and –25%, respectively) and the abdominal adipose tissue (–64%, –52% and –65%), while CRP expression was downregulated only in the liver (–25%). Thus, WB consumption improved endothelial function and exerted an anti-inflammatory effect in the OZR.

The incidence of type 2 diabetes mellitus (T2DM) has reached near-epidemic proportions in the Western world with other parts of the world following close behind. Glycemic control is paramount in type 1 diabetes mellitus (T1DM) and T2DM. Berries may beneficially influence glycemic control through the action of their polyphenolic components. While clinical studies on the anti-diabetic effects of berry polyphenols are limited, epidemiological data have indicated favorable effects of berry/anthocyanins intake on development and/or management of T2DM. Furthermore, data derived from in vivo animal studies and in vitro cell culture models are promising. Various molecular targets and modulation of cell signaling pathways in pancreatic β-cells, hepatocytes, adipocytes, and skeletal muscle cells are among the proposed mechanisms for berry polyphenols and their metabolites’ action. Berry polyphenols may exert anti-diabetic effects by (i) enhancing insulin production and reducing apoptosis and promoting proliferation of pancreatic β-cells, (ii) regulating glucose metabolism by interfering with absorption or by increasing peripheral tissue glucose uptake through insulin receptor-dependent or independent mechanisms via modification of oxidative stress, inflammation or perceived energy status of cell. This mini review discusses recent findings from our laboratory and other studies on the anti-diabetic effects of berry polyphenolic compounds with special emphasis on the cellular and molecular mechanisms involved in the beneficial effects of berry polyphenol compounds.

BACKGROUND: Black raspberries (BRB) inhibit a broad range of cancers in preclinical models, including in vivo models of oral, esophageal, colon, breast and skin cancer. Promising preclinical results have led to clinical evaluations in cancer patients or patients at increased risk for cancer development.

OBJECTIVE: To summarize clinical investigations targeting cancer or precancerous lesions with BRB and discuss future directions.

METHODS: A thorough literature search was conducted through December 1, 2015 to identify all published studies evaluating BRB in cancer focused clinical trials.

RESULTS: Research investigating BRB in clinical settings report positive effects on preneoplastic lesions or cancers of the oral cavity, esophagus and colon. BRB treatment resulted in: histologic regression of oral intraepithelial neoplasia associated with improved histologic grade and significantly reduced loss of heterozygosity at tumor suppressor gene loci, modulated genes linked to RNA processing and growth factor recycling; in the colon, BRB inhibited FAP-associated polyp progression, demethylated tumor suppressor genes and improved plasma cytokine profiles; in Barrett’s patients, BRB consumption increased tissue levels of GST-pi and decreased 8-isoprostane, a marker of lipid peroxidation/oxidative stress.

CONCLUSIONS: The precise dose, duration and optimum mode of BRB delivery for cancer inhibition remains to be fully elucidated. Common themes across studies support that BRB are anti-proliferative, anti- inflammatory, reduce oxidative stress and restore tumor suppressive activity. Future directions are included in the conclusions section.