Berry polyphenols metabolism and impact on human gut microbiota and health.

Berries are rich in phenolic compounds such as phenolic acids, flavonols and anthocyanins. These molecules are often reported as being responsible for the health effects attributed to berries. However, their poor bioavailability, mostly influenced by their complex chemical structures, raises the question of their actual direct impact on health. The products of their metabolization, however, may be the most bioactive compounds due to their ability to enter the blood circulation and reach the organs. The main site of metabolization of the complex polyphenols to smaller phenolic compounds is the gut through the action of microorganisms, and reciprocally polyphenols and their metabolites can also modulate the microbial populations. In healthy subjects, these modulations generally lead to an increase in Bifidobacterium, Lactobacillus and Akkermansia, therefore suggesting a prebiotic-like effect of the berries or their compounds. Finally, berries have been demonstrated to alleviate symptoms of gut inflammation through the modulation of pro-inflammatory cytokines and have chemopreventive effects towards colon cancer through the regulation of apoptosis, cell proliferation and angiogenesis. This review recapitulates the knowledge available on the interactions between berries polyphenols, gut microbiota and gut health and identifies knowledge gaps for future research.

Children's liking and wanting of foods vary over multiple bites/sips of consumption: A case study of foods containing wild blueberry powder in the amounts targeted to deliver bioactive phytonutrients for children.

To encourage children to frequently consume wild blueberries as part of their dietary patterns, incorporating blueberry powder into specific foods may be an effective strategy. Thus, it is important to determine appropriate types of food products that both minimize food processing-induced loss of beneficial components of blueberries (e.g., anthocyanins) and satisfy at the same time both nutritional and sensory aspects. For clinical studies assessing the health benefits of blueberry-containing food samples, it would be important to ensure children consume the entire portion of the test samples. This study, therefore, aimed at determining how sensory acceptability (liking) and psychological reward (wanting) of wild blueberry-based foods vary over multiple steps of ad libitum consumption: appearance, first bite/sip, half bite/sip, and full consumption. Five different types of foods containing the targeted amount of wild blueberry powder were prepared for sensory testing (oatmeal bar, beverage, ice pop, gummy, and cookie), and the residual amounts of total anthocyanin and chlorogenic acid were measured to confirm levels targeted to deliver bioactive amounts of these phytochemicals. Results showed that while overall liking and desire to eat did not differ among the five samples at the appearance and first bite/sip steps, they differed significantly at the end of consumption. Although children liked and wanted to consume the cookies more when compared to beverages, ice pops, and/or gummies, total anthocyanin contents were lower in cookies and gummies than in wild blueberry powder, beverage, and ice pop samples. Notably, the oatmeal bars with significant amounts of total anthocyanin and chlorogenic acid did not significantly differ from the cookies with respect to overall liking, desire to eat, and the amount consumed. In conclusion, this study shows that sensory evaluation using multiple bites/sips of ad libitum food consumption, along with a measurement of beneficial compounds, is efficient in determining appropriate vehicles for clinical studies of wild blueberry-containing foods.

Recent Research on the Health Benefits of Blueberries and Their Anthocyanins.

Awareness of the human health benefits of blueberries is underpinned by a growing body of positive scientific evidence from human observational and clinical research, plus mechanistic research using animal and in vitro models. Blueberries contain a large number of phytochemicals, including abundant anthocyanin pigments. Of their various phytochemicals, anthocyanins probably make the greatest impact on blueberry health functionality. Epidemiological studies associate regular, moderate intake of blueberries and/or anthocyanins with reduced risk of cardiovascular disease, death, and type 2 diabetes, and with improved weight maintenance and neuroprotection. These findings are supported by biomarker-based evidence from human clinical studies. Among the more important healthful aspects of blueberries are their anti-inflammatory and antioxidant actions and their beneficial effects on vascular and glucoregulatory function. Blueberry phytochemicals may affect gastrointestinal microflora and contribute to host health. These aspects have implications in degenerative diseases and conditions as well as the aging process. More evidence, and particularly human clinical evidence, is needed to better understand the potential for anthocyanin-rich blueberries to benefit public health. However, it is widely agreed that the regular consumption of tasty, ripe blueberries can be unconditionally recommended.

Gene expression microarray analysis of the effects of grape anthocyanins in mice: a test of a hypothesis-generating paradigm.

The mechanism(s) through which fruits, vegetables, and whole grains favorably affect health is not well established. Using an anthocyanin-rich grape as a model, we examined the ability of an agnostic analytical approach using gene expression microarrays to generate novel testable hypotheses regarding the mechanisms of action of potentially healthful foods and food components. C57BL/6 mice were divided into 2 groups and fed a proatherogenic diet with or without a semipurified anthocyanin extract (70% anthocyanins) incorporated at a level of 0.1 mg/mL into the drinking water. After 6 weeks, compared with control mice, mice supplemented with anthocyanins tended to gain more weight and have increased adipose tissue mass, although these effects did not achieve statistical significance. Anthocyanin-supplemented mice had significantly reduced relative liver weights and heart weights. Serum lipids and inflammatory cytokines were not different between the groups. Gene expression microarray analysis of the liver and skeletal muscle identified a number of molecular pathways significantly affected by anthocyanin treatment. Two distinct clusters emerged. The first cluster included down-regulated pathways in both muscle and liver involving cellular defense, whereas the second included hepatic genes involved in energy metabolism. From these data, 3 hypotheses were developed for future investigation.

Processing and storage effects on monomeric anthocyanins, percent polymeric color, and antioxidant capacity of processed blackberry products.

Blackberries are a rich source of polyphenolics, particularly anthocyanins, that may contribute to the reduced risk of chronic disease; however, as with most berries, the fresh fruit are only seasonally available. With most of the blackberries consumed as frozen or in thermally processed forms after long-term storage, the purpose of this study was to evaluate the effects of processing and 6 months of storage on the anthocyanins and antioxidant capacity of blackberries that were individually quick-frozen (IQF), canned-in-syrup, canned-in-water, pureed, and juiced (clarified and nonclarified). Monomeric anthocyanins, percent polymeric color, and antioxidant capacity by oxygen radical absorbance capacity (ORAC FL) and photochemiluminescence (PCL) were determined postprocessing (1 day) and after 1, 3, and 6 months of storage. Processing resulted in increases in polymeric color values (up to 7%) and losses in monomeric anthocyanins (up to 65%). For most products, processing also resulted in losses in antioxidant capacity (by ORAC FL and PCL). Storage at 25 degrees C of all processed products resulted in dramatic losses in monomeric anthocyanins with as much as 75% losses of anthocyanins throughout storage, which coincided with marked increases of percent polymeric color values of these products over 6 months of storage. There were no changes in ORAC FL or PCL for processed products throughout long-term storage. No significant changes in antioxidant capacity or anthocyanin content were observed in IQF fruit during long-term storage at -20 degrees C.

Whole berries versus berry anthocyanins: interactions with dietary fat levels in the C57BL/6J mouse model of obesity.

Male C57BL/6J mice received diets with either 10% of calories from fat (LF) or a high-fat diet [45% (HF45) or 60% (HF60) calories from fat] for 92 days (expt 1) or 70 days (expt 2). These were given with or without freeze-dried powders from whole blueberries (BB) or strawberries (SB) (expt 1) or purified anthocyanin extracts from BB or SB (expt 2). Body composition was determined utilizing Echo MRI. Berries added to the LF diet did not alter weight gain, final body weights, body fat, or protein (percent body weight) or diet (grams) or energy (kilocalories) intake. However, in both HF45- and HF60-fed mice, weight gain, final weights, body fat (percent), and epididymal fat weights increased and body protein decreased ( p < 0.01) compared to LF mice. In mice fed HF45 diet plus BB, body weight gains, body fat (percent of BW), and epididymal fat weights were significantly greater than those in the HF45-fed controls, whereas weights of mice fed SB HF were similar to those of HF controls. SB or BB feeding did not alter glucose tolerance, although glucose tolerance decreased with age and in HF45 versus LF mice. Baseline plasma glucose was lower in SB- versus HF45-fed mice. After 8 weeks, mice fed the HF60 diet plus purified anthocyanins from BB in the drinking water had lower body weight gains and body fat than the HF60-fed controls. Anthocyanins fed as the whole blueberry did not prevent and may have actually increased obesity. However, feeding purified anthocyanins from blueberries or strawberries reduced obesity.

Ellagitannin composition of blackberry as determined by HPLC-ESI-MS and MALDI-TOF-MS.

Blackberries ( Rubus sp.) were evaluated by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF-MS) to identify the ellagitannins present in flesh, torus (receptacle tissue), and seeds. Most ellagitannins were present (or detectable) only in seed tissues. Ellagitannins identified by HPLC-ESI-MS in the seeds included pedunculagin, casuarictin/potentillin, castalagin/vescalagin, lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D. For several of the ellagitannins, isomeric separation was also obtained. The MALDI-TOF-MS analysis was primarily utilized to evaluate and identify high molecular mass (>1000 Da) ellagitannins. The MALDI analysis verified the presence of the ellagitannins identified by HPLC-ESI-MS including lambertianin A/sanguiin H-6, lambertianin C, and lambertianin D, but the analysis also indicated the presence of several other compounds that were most likely ellagitannins based on the patterns observed in the masses (i.e., loss or addition of a gallic acid moiety to a known ellagitannin). This study determined the presence of several possible isomeric forms of ellagitannins previously unidentified in fruit and presents a possible analytical HPLC method for the analysis of the major ellagitannins present in the fruit.

Identification and quantification of glycoside flavonoids in the energy crop Albizia julibrissin.

Oxygen radical absorbance capacity (ORAC) values showed that methanolic extracts of Albizia julibrissin foliage displayed antioxidant activity. High performance liquid chromatography (HPLC) and mass spectrometry (MS) techniques were utilized in the identification of the compounds. The analysis confirmed the presence of three compounds in A. julibrissin foliage methanolic extract: an unknown quercetin derivative with mass of 610 Da, hyperoside (quercetin-3-O-galactoside), and quercitrin (quercetin-3-O-rhamnoside). Fast performance liquid chromatography (FPLC) was employed to fractionate the crude A. julibrissin foliage methanolic extract into its individual flavonoid components. The flavonoids were quantified in terms of mass and their respective contribution to the overall ORAC value. Quercetin glycosides accounted for 2.0% of total foliage.

Stabilization of anthocyanins in blackberry juice by glutathione fortification.

Blackberry anthocyanins provide attractive color and antioxidant activity. However, anthocyanins degrade during juice processing and storage, so maintaining high anthocyanin concentrations in berry juices may lead to greater antioxidant and health benefits for the consumer. This study evaluated potential additives to stabilize anthocyanins during blackberry juice storage. The anthocyanin stabilizing agents used were: glutathione, galacturonic acid, diethylenetriaminepentaacetic acid and tannic acid, which were added at a level of 500 mg L-1. Juice anthocyanin, flavonol, and ellagitannin content and percent polymeric color were measured over five weeks of accelerated storage at 30 °C. Glutathione had the greatest protective effect on total anthocyanins and polymeric color. Therefore a second study was performed with glutathione in combination with lipoic and ascorbic acids in an effort to use antioxidant recycling to achieve a synergistic effect. However, the antioxidant recycling system had no protective effect relative to glutathione alone. Glutathione appears to be a promising blackberry juice additive to protect against anthocyanin degradation during storage.

Changes in polyphenolics during maturation of Java plum (Syzygium cumini Lam.).

Java plum (Syzygium cumini Lam.) is a rich source of polyphenolics with many purported health benefits, but the effect of maturation on polyphenolic content is unknown. Freeze-dried samples of Java plum from seven different maturity stages were analyzed for anthocyanin, flavonol, flavanonol and hydrolysable tannin composition by HPLC. Anthocyanins were first detected at the green-pink stage of maturity and increased throughout maturation with the largest increase occurring from the dark purple to black stages of maturation. Levels of gallotannins, ellagitannins, flavonols, gallic acid and ellagic acid were highest at early stages of maturation and decreased as the fruit ripened. For production of antioxidant-rich nutraceutical ingredients, fruit should be harvested immature to obtain extracts rich in hydrolysable tannins and flavonols. The exceptional anthocyanin content of black fruit may prove useful as a source of a natural colorant.

A glycoside flavonoid in Kudzu (Pueraria lobata): identification, quantification, and determination of antioxidant activity.

Kudzu (Pueraria lobata) foliage has been touted as a possible energy crop. High-performance liquid chromatography and mass spectrometry analysis of the methanolic kudzu foliage extracts confirmed the presence of robinin (kaempferol-3-O-robinoside-7-O-rhamnoside). Robinin accounted for 0.65 +/- 0.16% (dry basis) of kudzu biomass. Fast performance liquid chromatography (FPLC) was employed to fractionate robinin from the crude extract. The antioxidant capacity of robinin was evaluated by an oxygen radical absorbance capacity (ORAC) assay. The ORAC values of pure standard were compared with those of the extract fractions. One milligram of the FPLC-fractionated robinin generated an ORAC value of 5.15 +/- 2.00 micromol/mg of Trolox, whereas 1 mg of pure robinin generated an ORAC value of 12.34 +/- 0.45 micromol/mg of Trolox. Because of its antioxidant properties, robinin may be a flavonoid worth extracting prior to energy production.

Effects of solvent and temperature on pressurized liquid extraction of anthocyanins and total phenolics from dried red grape skin.

Pressurized liquid extraction (PLE) was used to extract anthocyanins from the freeze-dried skin of a highly pigmented red wine grape with six solvents at 50 degrees C, 10.1 MPa, and 3 x 5 min extraction cycles. Temperature (from 20 to 140 degrees C in 20 degrees C increments) effects on anthocyanin recovery by acidified water and acidified 60% methanol were also studied. Acidified methanol extracted the highest levels of total monoglucosides and total anthocyanins, whereas the solvent mixture (40:40:20:0.1 methanol/acetone/water HCl) extracted the highest levels of total phenolics and total acylated anthocyanins. Acidified water extracts obtained by PLE at 80-100 degrees C had the highest levels of total monoglucosides, total acylated anthocyanins, total anthocyanins, total phenolics, and ORAC values. Acidified methanol extracts obtained by PLE at 60 degrees C had the highest levels of total monoglucosides and total anthocyanins, whereas extracts obtained at 120 degrees C had the highest levels of total phenolics. High-temperature PLE (80-100 degrees C) using acidified water, an environmentally friendly solvent, was as effective as acidified 60% methanol in extracting anthocyanins from grape skins.

Extraction of antioxidant compounds from energy crops.

Energy crops offer enormous opportunities for increasing the sustain ability of agriculture and energy production in the United States. Nevertheless, opportunities for sustaining biomass energy production may well hinge on producing energy and extracting high-value products from the same crop. Seven potential energy crops (mimosa, sericea, kudzu, arunzo, switchgrass, velvet bean, and castor) were extracted and assayed for the presence of potentially high-value antioxidant compounds. Of these crops, mimosa and sericea had the highest antioxidant potential and were selected for further study. High-performance liquid chromatography (ultraviolet) and liquid chromatography/mass spectrometry techniques were then utilized to help identify the compounds with high antioxidant potential using extract fractionation, and total phenolics and oxygen radical absorbance capability assays as a guide. These analyses indicate that methanol extracts of mimosa foliage most likely contain quercetin, a flavonol that has been associated with cardio- protection. Future work will concentrate on quantifying the quercetin content of mimosa (likely parts-per-million levels), as well as identifying and quantifying other antioxidants found in energy crops.

Urinary excretion of phenolic acids in rats fed cranberry, blueberry, or black raspberry powder.

Dietary polyphenolics can be converted into smaller phenolic acids (PA) by microorganisms in the colon and may contribute to health benefits associated with the parent polyphenolics. Urinary excretion of 18 PA and their conjugates was studied, using HPLC-MS/MS, in rats fed AIN93G-based diets containing 5% (dry weight basis) of either cranberry (CB), blueberry (BB), or black raspberry (BRB). Hippuric, 4-hydroxyphenylacetic, 3-methoxy-4-hydroxyphenylacetic, and 4-hydroxybenzoic acids were excreted in greatest quantity in the urine over a 24 h period in all diets. Primary PA excreted in the berry diets were 4-hydroxycinnamic acid for CB; chlorogenic, ferulic, and 3,4-dihydroxycinnamic acids for BB; and 3-hydroxyphenylpropionic, 3-hydroxybenzoic, and 3-hydroxycinnamic acids for BRB. PA were present in conjugated form with cinnamic acid derivatives being 50-70% and phenylacetic acid derivatives conjugated <10%. Conjugated, and not just the free, PA are significant contributors to total urinary excretion.

Changes in chokeberry (Aronia melanocarpa L.) polyphenols during juice processing and storage.

Chokeberries are an excellent source of polyphenols, but their fate during juice processing and storage is unknown. The stability of anthocyanins, total proanthocyanidins, hydroxycinnamic acids, and flavonols at various stages of juice processing and over 6 months of storage at 25 °C was determined. Flavonols, total proanthocyanidins, and hydroxycinnamic acids were retained in the juice to a greater extent than anthocyanins, with losses mostly due to removal of seeds and skins following pressing. Anthocyanins were extensively degraded by thermal treatments during which time levels of protocatechuic acid and phloroglucinaldehyde increased, and additional losses occurred following pressing. Flavonols, total proanthocyanidins, and hydroxycinnamic acids were well retained in juices stored for 6 months at 25 °C, whereas anthocyanins declined linearly. Anthocyanin losses during storage were paralleled by increased polymeric color values, indicating that the small amounts of anthocyanins remaining were present in large part in polymeric forms.

Improved stability of chokeberry juice anthocyanins by ß-cyclodextrin addition and refrigeration.

Chokeberry anthocyanins are susceptible to degradation during processing and storage of processed products. This study determined the effects of three pH levels (2.8, 3.2, and 3.6) and four ß-cyclodextrin (BCD) concentrations (0, 0.5, 1, and 3%) alone and in combination on the stability of chokeberry juice anthocyanins before and after pasteurization and over 8 months of storage at 4 and 25 °C. Lowering the pH from 3.6 to 2.8 in the absence of BCD provided marginal protection against anthocyanin losses during processing and storage. Addition of 3% BCD at the natural chokeberry pH of 3.6 resulted in excellent protection of anthocyanins, with 81 and 95% retentions after 8 months of storage at 25 and 4 °C, respectively. The protective effect of BCD was lessened with concentrations <3% and reduction in pH, indicating changes in anthocyanin structure play an important role in BCD stabilization of anthocyanins.

The blackberry fruit: a review on its composition and chemistry, metabolism and bioavailability, and health benefits.

Blackberry (Rubus sp.) fruit contains high levels of anthocyanins and other phenolic compounds, mainly flavonols and ellagitannins, which contribute to its high antioxidant capacity and other biological activities. Blackberry phenolic composition and concentrations are known to be influenced by genetics, growing conditions, and maturation. Despite the current knowledge of their chemistry, research specific to blackberry phenolic compounds' health benefits, metabolism, bioavailability, and mechanism by which they confer health benefits is scarce. Blackberry phenolic compounds have protective effects on age-related neurodegenerative diseases and bone loss in vivo and can inhibit low-density lipoprotein and liposomal oxidation in vitro. Blackberry extracts have also exerted antimutagenic effects in vitro and in vivo by modifying cell signaling pathways and suppressing tumor promotion factors. However, the antiobesity, antidiabetic, antimicrobial, and anti-inflammatory properties of blackberry phenolic compounds need investigation. Similarly, studies that elucidate the in vivo physiologically effective concentrations of blackberry phenolic compounds are necessary.

Processing and storage effect on berry polyphenols: challenges and implications for bioactive properties.

Anthocyanins and tannins in blueberries, blackberries and black raspberries are susceptible to degradation during processing, with juices showing the greatest losses due to physical removal of skins and seeds. Anthocyanins and procyanidins are also degraded in processed products stored at ambient temperature with losses accompanied by increased polymeric pigments (PPs). Using chokeberry as a model, formation of PPs occurred in both pasteurized and aged juices and pasteurized juice contained a greater proportion of low molecular weight PPs than aged juice, while aged juice contained a greater proportion of higher molecular PPs. Formation of PP accounts for some of the losses of anthocyanins and procyanidins during processing and storage, but the complete fate of anthocyanins remains unclear. In this review we highlight the steps in processing where significant losses of polyphenols occur, and discuss potential mechanisms responsible for losses, methods to mitigate losses, and implications on bioactive properties.

Design and optimization of a semicontinuous hot-cold extraction of polyphenols from grape pomace.

Grape pomace contains appreciable amounts of polyphenolic compounds such as anthocyanins and procyanidins which can be recovered for use as food supplements. The extraction of these polyphenols from the pomace is usually accomplished at slightly elevated temperatures, frequently employing hydroethanolic solvents. Due to governmental regulations and the cost involved in using ethanol as a solvent, as well as the loss in polyphenolics due to thermal degradation, improved extraction techniques are required. In this study, a semicontinuous extraction apparatus employing only water was developed to maximize the recovery of anthocyanins and procyanidins from red grape pomace (Vitis vinifera). Water is preheated prior to its entry to the extraction cell containing the grape pomace sample, where it is allowed to then flow continuously through the unheated extraction vessel prior to its collection at ambient conditions. Extraction variables that impacted the polyphenolic recovery included pomace moisture content (crude or dried), sample mass, water flow rate, and extraction temperature. A response surface method was used to analyze the results from the extraction, and the optimal conditions were found to be 140 °C and 9 mL/min water flow rate. These conditions can produce an extract containing 130 mg/100 g DW of anthocyanins and 2077 mg/100 g DW of procyanidins. Higher yields of polyphenolics were observed using crude (wet) rather than dried pomace, hence avoiding the need to dry the pomace prior to extraction. The described semicontinuous extraction method using only water as the extraction solvent under subcritical conditions allowed the efficient extraction of polyphenols from red grape pomace without the attendant loss of polyphenolic content due to having to heat the extraction vessel prior to commencement of extraction.