Anthocyanins and Their C6-C3-C6 Metabolites in Humans and Animals.

Research on the bioavailability of anthocyanins has focused, historically, on the non-flavonoid (C6-Cn) products that arise from anthocyanins in vivo. However, this review focuses on the products of anthocyanins that still possess the flavonoid structure (C6-C3-C6). Described herein are aspects of the in vivo pool of C6-C3-C6 anthocyanin-derived intermediates. Properties related to molecular size, shape, and polarity conveyed by six major anthocyanidin structures are discussed. The presence of a glycoside or not, and a variety of possible phase 2 conjugates, gives rise to a chemically diverse pool of C6-C3-C6 intermediates. Chemical properties influence the in vivo stability of anthocyanin-derived products, as well as their suitability as a substrate for xenobiotic conjugation and transport, and their association with the biomatrix. The flavonoid structure is associated with bioactivity and the particular properties of these C6-C3-C6 products of anthocyanins determines their deposition in the body, which may influence in vivo processes and ultimately health outcomes.

Enhanced neural activation with blueberry supplementation in mild cognitive impairment.

OBJECTIVES: Preclinical studies have shown that blueberry supplementation can improve cognitive performance and neural function in aged animals and have identified associations between anthocyanins and such benefits. Preliminary human trials also suggest cognitive improvement in older adults, although direct evidence of enhancement of brain function has not been demonstrated. In this study, we investigated the effect of blueberry supplementation on regional brain activation in older adults at risk for dementia. METHODS: In a randomized, double-blind, placebo-controlled trial we performed pre- and post-intervention functional magnetic resonance imaging during a working memory (WM) task to assess the effect of blueberry supplementation on blood oxygen level-dependent (BOLD) signal in older adults with mild cognitive impairment, a risk condition for dementia. RESULTS: Following daily supplementation for 16 weeks, blueberry-treated participants exhibited increased BOLD activation in the left pre-central gyrus, left middle frontal gyrus, and left inferior parietal lobe during WM load conditions (corrected P < 0.01). There was no clear indication of WM enhancement associated with blueberry supplementation. Diet records indicated no between-group difference in anthocyanin consumption external to the intervention. DISCUSSION: These data demonstrate, for the first time, enhanced neural response during WM challenge in blueberry-treated older adults with cognitive decline and are consistent with prior trials showing neurocognitive benefit with blueberry supplementation in this at-risk population.

Phenolic compounds isolated from fermented blueberry juice decrease hepatocellular glucose output and enhance muscle glucose uptake in cultured murine and human cells.

BACKGROUND: We recently reported that blueberry juice fermented (FJ) with Serratia vaccinii bacterium has antidiabetic activities both in vivo and in vitro. The purpose of this project was to elucidate the effect of FJ on glucose homeostasis in liver and skeletal muscle cells and to identify active fractions/compounds responsible for this effect. METHODS: FJ was fractionated using standard chromatography procedures. Hepatic (H4IIE, HepG2) and skeletal muscle cells (C2C12) were treated with maximum non-toxic concentrations of FJ, fractions and isolated compounds thereof. Glucose-6-phosphatase (G6Pase) activity was measured using glucose oxidase method. To measure glucose uptake and glycogen synthase (GS) activity, radioactive assays were used. RESULTS: Fractionation of FJ yielded seven fractions. FJ and its phenolic fractions F2, F3-1 and F3-2 respectively inhibited G-6Pase by 31, 45, 51 and 26%; activated GS by 2.3-, 2.3-, 2.2- and 2-fold; and stimulated glucose uptake by 19, 25, 18 and 15%, as compared to DMSO vehicle control. Subfractionation of the active fractions yielded 4 compounds (catechol, chlorogenic, gallic and protocatechuic acid). Catechol, yielding the greatest bioactivity in G6Pase and glucose uptake assays, decreased G6Pase activity by 54%, increased GS by 2-fold and stimulated glucose uptake by 44% at 45.5 µM. CONCLUSIONS: This study identifies novel potential antidiabetic compounds that can help standardize FJ.

Fermented blueberry juice extract and its specific fractions have an anti-adipogenic effect in 3 T3-L1 cells.

BACKGROUND: Obesity and Type 2 diabetes have reached epidemic status worldwide. Wild lowbush blueberry (Vaccinium angustifolium Aiton) is a plant of the North American Aboriginal traditional pharmacopeia with antidiabetic potential, especially when it is fermented with Serratia vaccinii. METHODS: A phytochemical fractionation scheme was used to identify potential bioactive compounds as confirmed by HPLC retention times and UV-Vis spectra. 3 T3-L1 cells were differentiated for 7 days with either Normal Blueberry Extract (NBE), Fermented Blueberry Extract (FBE/F1), seven fractions and four pure compounds. Triglyceride content was measured. Examination of selected intracellular signalling components (p-Akt, p-AMPK) and transcriptional factors (SREBP-1c and PPARγ) was carried out by Western blot analysis. RESULTS: The inhibitory effect of FBE/F1 on adipocyte triglyceride accumulation was attributed to total phenolic (F2) and chlorogenic acid enriched (F3-2) fractions that both inhibited by 75%. Pure compounds catechol (CAT) and chlorogenic acid (CA) also inhibited adipogenesis by 70%. Treatment with NBE, F1, F3-2, CAT and CA decreased p-AKT, whereas p-AMPK tended to increase with F1. The expression of SREBP1-c was not significantly modulated. In contrast, PPARγ decreased in all experimental groups that inhibited adipogenesis. CONCLUSIONS: These results demonstrate that fermented blueberry extract contains compounds with anti-adipogenic activity, which can serve to standardize nutraceutical preparations from fermented blueberry juice and to develop novel compounds with anti-obesity properties.

Effects of blueberry polyphenolic extract on vascular remodeling in spontaneously hypertensive rats.

Blueberry is considered a functional food due to various beneficial health effects associated with its consumption. Therefore, we examined the cardiovascular benefits of a blueberry polyphenolic extract in spontaneously hypertensive rats (SHR). Male SHR and Wistar-Kyoto (WKY) rats were administered with blueberry polyphenolic extract for 15 weeks. SHR showed significant augmented media-to-lumen ratio compared to WKY rats and blueberry polyphenolic extract significantly improved media-to-lumen ratio. SHR also had high blood pressure (BP), cardiac remodeling, and diastolic dysfunction and treatment did not affect BP or cardiac structure and function. SHR showed significantly increased the levels of malondialdehyde (MDA) and blueberry polyphenolic extract did not lower MDA. The levels of interleukin 6 and nitrate/nitrite ratio were unaltered in SHR. SHR showed a significant increase in the pro-apoptotic marker, Bax. Blueberry polyphenolic extract significantly lowered Bax. Our study shows that blueberry polyphenolic extract is beneficial in preventing vascular remodeling and cardiac apoptosis. PRACTICAL APPLICATIONS: Similar to many other berries, blueberries are repertoire of many phytochemicals including polyphenols. Along with its considerably well-established role as a sought after berry, blueberries have been at the forefront of approaches to hharnessing health benefits from plant food sources. Several studies have attempted to unravel the role of blueberry and their major phytochemicals in reducing the risk of cardiovascular diseases and reported their beneficial effects. Our pre-clinical study found that blueberry polyphenolic extract can reduce vascular remodeling in the setting of hypertension. This new finding further suggests the potential of blueberry-based phytochemicals. Further exploration of blueberries and their phytochemicals and positive outcomes from such studies can lead to substantial benefits for consumers and economy as a whole.

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.

Identification of anthocyanins in the liver, eye, and brain of blueberry-fed pigs.

Dietary intervention with anthocyanins may confer benefits in brain function, including vision. Research to date indicates that animals have only a limited capacity to absorb anthocyanins, compared to other types of flavonoids. Pigs, which are a suitable model for human digestive absorption, were used to examine the deposition of anthocyanins in tissues including the liver, eye, and brain tissue. Pigs were fed diets supplemented with 0, 1, 2, or 4% w/w blueberries ( Vaccinium corymbosum L. 'Jersey') for 4 weeks. Prior to euthanasia, pigs were fasted for 18-21 h. Although no anthocyanins were detected in the plasma or urine of the fasted animals, intact anthocyanins were detected in all tissues where they were sought. LC-MS/MS results are presented for the relative concentration of 11 intact anthocyanins in the liver, eye, cortex, and cerebellum. The results suggest that anthocyanins can accumulate in tissues, including tissues beyond the blood-brain barrier.

Cognitive response to fish oil, blueberry, and combined supplementation in older adults with subjective cognitive impairment.

Given evidence that eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and anthocyanin-rich blueberries provide neurocognitive benefit, we investigated long-term supplementation in older adults with cognitive complaints. In a 24-week randomized, double-blind, placebo-controlled trial, elderly men and women received daily fish oil (FO) or blueberry (BB) or both. Diet records confirmed that participants reduced background consumption of EPA, DHA, and anthocyanins as prescribed. Erythrocyte EPA + DHA composition increased in the FO groups (p = 0.0001). Total urinary anthocyanins did not differ between the groups after supplementation but glycoside and native (food) forms increased only in the BB-supplemented groups. The FO (p = 0.03) and BB (p = 0.05) groups reported fewer cognitive symptoms, and the BB group showed improved memory discrimination (p = 0.04), indicating that supplementation improved cognition. Cognitive benefit in the BB group was associated with the presence of urinary anthocyanins reflecting recent BB intake but not with anthocyanin metabolites. However, combined FO + BB treatment was not associated with cognitive enhancement as expected.

Blueberry polyphenols increase lifespan and thermotolerance in Caenorhabditis elegans.

The beneficial effects of polyphenol compounds in fruits and vegetables are mainly extrapolated from in vitro studies or short-term dietary supplementation studies. Due to cost and duration, relatively little is known about whether dietary polyphenols are beneficial in whole animals, particularly with respect to aging. To address this question, we examined the effects of blueberry polyphenols on lifespan and aging of the nematode, Caenorhabditis elegans, a useful organism for such a study. We report that a complex mixture of blueberry polyphenols increased lifespan and slowed aging-related declines in C. elegans. We also found that these benefits did not just reflect antioxidant activity in these compounds. For instance, blueberry treatment increased survival during acute heat stress, but was not protective against acute oxidative stress. The blueberry extract consists of three major fractions that all contain antioxidant activity. However, only one fraction, enriched in proanthocyanidin compounds, increased C. elegans lifespan and thermotolerance. To further determine how polyphenols prolonged C. elegans lifespan, we analyzed the genetic requirements for these effects. Prolonged lifespan from this treatment required the presence of a CaMKII pathway that mediates osmotic stress resistance, though not other pathways that affect stress resistance and longevity. In conclusion, polyphenolic compounds in blueberries had robust and reproducible benefits during aging that were separable from antioxidant effects.

Flavonoid Metabolites in Human Urine during Blueberry Anthocyanin Intake.

The human health benefits of anthocyanins (Anc) and other flavonoids are widely recognized. However, the flavonoid-based urinary metabolites arising in vivo after Anc intake are not well described. Human (n = 17) urine was collected while blueberry juice (BJ) was consumed daily for 28 days and once after a 7 day washout. MS/MS scanning of 664 urine samples for 18 parent Anc (PAnc) and 42 predicted Anc metabolites (AncM) yielded 371 products (i.e., MS/MS × retention time (RT)). Flavonoid-based AncM, which were likely underestimated, were almost 20 times more abundant than PAnc. Together, PAnc and AncM accounted for about 1% of the daily Anc dose. Aglycone forms were >94% of the total. Cluster analysis of the 371 Anc identified about 55 major Anc that contributed about 80% to the total Anc. The abundance of flavonoid-based Anc-derived products in the gastrointestinal tract could contribute to the health benefits of Anc-rich berries.

Human anthocyanin bioavailability: effect of intake duration and dosing.

While in vitro and animal evidence supports a role for anthocyanins in human health, future opportunities in berry health benefits will rest upon evidence from clinical intervention trials. Because little is known about the behaviour of anthocyanins during long term intake in humans, several clinical design factors were examined. Urine from volunteers (n = 17) who consumed blueberry juice daily was analysed using LC-MS/MS for predicted flavonoid-based products of anthocyanins in relation to a 5-day anthocyanin-free run-in, 28 days of blueberry juice intake, a 7-day washout and two dosing regimens. Total and parent anthocyanin content in urine varied 10-fold among the 17 participants. A high 24-0 h total anthocyanin excretion was associated with high anthocyanin retention (i.e. 0 h, before blueberry juice intake). Total anthocyanin excretion was not different before and after up to 7 days of washout indicative of a slow release of anthocyanins. Urinary excretion of anthocyanins declined during the 36-day study. The 24-0 h excretion was greater for total anthocyanins but not for parent anthocyanins when daily blueberry juice was taken all at once rather than as ⅓ doses taken thrice daily. However parent anthocyanins were retained better (higher 0 h) with 1× dosing. These findings could aid in the design of clinical research on anthocyanins and health.

Gastroretentive systems - a proposed strategy to modulate anthocyanin release and absorption for the management of diabetes.

Several reports have indicated a positive correlation between the consumption of anthocyanins (ACN) and biomarkers relating to the improvement of type 2 diabetes (T2D). However, the results from in vitro studies often do not translate into clinical evidence. Potential causes of these discrepancies are experimental conditions that lack physiological relevancy; extensive degradation of these compounds in vivo due to changes in pH and metabolism; and a short residence time in the absorption window in relation to the absorption rate. Here, gastroretentive systems (GRS) are proposed as a strategy to overcome the limitations in ACN delivery and to reduce the existing bench-to-subject gap. This review summarizes recent literature on the use of ACN for the management and control of T2D, followed by GRS platforms to promote a sustained release of ACN for increased health benefits.

Unraveling Anthocyanin Bioavailability for Human Health.

This review considers the bioavailability of health-protective anthocyanin pigments from foods, in light of the multiple molecular structures and complicated traffic patterns taken by anthocyanins both as flavonoid metabolites and as phenolic acid metabolites within the body. Anthocyanins have generally been considered to have notoriously poor bioavailability, based on the very low levels typically detected in routine human blood draws after ingestion. Although some investigations have assessed anthocyanin bioavailability solely based on the measurement of parent anthocyanins or phenolic acid breakdown products, more recent research has increasingly revealed the presence, qualitative diversity, relatively high concentrations, and tenacity of molecular intermediates of anthocyanins that retain the unique flavonoid C6-C3-C6 backbone structure. We argue that the persistence of anthocyanin metabolites suggests enterohepatic recycling, leading to prolonged residence time, and supports the notion that anthocyanins are far more bioavailable than previously suggested.

Quantitative changes in proteins responsible for flavonoid and anthocyanin biosynthesis in strawberry fruit at different ripening stages: A targeted quantitative proteomic investigation employing multiple reaction monitoring.

To better understand the regulation of flavonoid and anthocyanin biosynthesis, a targeted quantitative proteomic investigation employing LC-MS with multiple reaction monitoring was conducted on two strawberry cultivars at three ripening stages. This quantitative proteomic workflow was improved through an OFFGEL electrophoresis to fractionate peptides from total protein digests. A total of 154 peptide transitions from 47 peptides covering 21 proteins and isoforms related to anthocyanin biosynthesis were investigated. The normalized protein abundance, which was measured using isotopically-labeled standards, was significantly changed concurrently with increased anthocyanin content and advanced fruit maturity. The protein abundance of phenylalanine ammonia-lyase; anthocyanidin synthase, chalcone isomerase; flavanone 3-hydroxylase; dihydroflavonol 4-reductase, UDP-glucose:flavonoid-3-O-glucosyltransferase, cytochrome c and cytochrome C oxidase subunit 2, was all significantly increased in fruit of more advanced ripeness. An interaction between cultivar and maturity was also shown with respect to chalcone isomerase. The good correlation between protein abundance and anthocyanin content suggested that a metabolic control point may exist for anthocyanin biosynthesis. This research provides insights into the process of anthocyanin formation in strawberry fruit at the level of protein concentration and reveals possible candidates in the regulation of anthocyanin formation during fruit ripening. BIOLOGICAL SIGNIFICANCE: To gain insight into the molecular mechanisms contributing to flavonoids and anthocyanin biosynthesis and regulation of strawberry fruit during ripening is challenging due to limited molecular biology tools and established hypothesis. Our targeted proteomic approach employing LC-MS/MS analysis and MRM technique to quantify proteins in relation to flavonoids and anthocyanin biosynthesis and regulation in strawberry fruit during fruit ripening is novel. The identification of peptides and proteins provided reliable design and validation of quantitative approaches using SRM on targeted proteins proposed involved in strawberry fruit. Our data revealed the identifying candidate proteins and their quantitative changes in relation to fruit ripening and flavonoids and anthocyanin biosynthesis and regulation. More importantly, this quantitative proteomic data is also compared with chemical analysis to reveal possible control levels of this important quality trait. Although, MRM approach is not new in plant biology research, the application has been very rare. This is the first systematic multi-targeted interrogation of the possible regulation of entire pathway of flavonoids and anthocyanin biosynthesis in strawberry fruit at different ripening stages using quantitative MRM technique on mass spectrometry. Our results demonstrate the power of targeted quantitative mass spectrometry data for analysis of proteins in biological regulation. These results indicate that distinct and diverse control of flavonoids and anthocyanin biosynthesis mechanisms at metabolism and proteins levels. This important and complementary knowledge will be useful for systematically characterizing the flavonoids and anthocyanin biosynthesis pathway of any fruit/plant species.

Resveratrol, pterostilbene, and piceatannol in vaccinium berries.

A study was conducted to determine the presence of resveratrol, pterostilbene, and piceatannol in Vaccinium berries. Samples representing selections and cultivars of 10 species from Mississippi, North Carolina, Oregon, and Canada were analyzed by gas chromatography/mass spectrometry. Resveratrol was found in Vaccinium angustifolium (lowbush blueberry), Vaccinium arboretum (sparkleberry), Vaccinium ashei (rabbiteye blueberry), Vaccinium corymbosum (highbush blueberry), Vaccinium elliottii (Elliott's blueberry), Vaccinium macrocarpon (cranberry), Vaccinium myrtillus (bilberry), Vaccinium stamineum (deerberry), Vaccinium vitis-ideae var. vitis-ideae (lingonberry), and Vaccinium vitis-ideae var. minor (partridgeberry) at levels between 7 and 5884 ng/g dry sample. Lingonberry was found to have the highest content, 5884 ng/g dry sample, comparable to that found in grapes, 6471 ng/g dry sample. Pterostilbene was found in two cultivars of V. ashei and in V. stamineum at levels of 99-520 ng/g dry sample. Piceatannol was found in V. corymbosum and V. stamineum at levels of 138-422 ng/g dry sample. These naturally occurring stilbenes, known to be strong antioxidants and to have cancer chemopreventive activities, will add to the purported health benefits derived from the consumption of these small fruits.

Blueberry effects on dark vision and recovery after photobleaching: placebo-controlled crossover studies.

Clinical evidence for anthocyanin benefits in night vision is controversial. This paper presents two human trials investigating blueberry anthocyanin effects on dark adaptation, functional night vision, and vision recovery after retinal photobleaching. One trial, S2 (n = 72), employed a 3 week intervention and a 3 week washout, two anthocyanin doses (271 and 7.11 mg cyanidin 3-glucoside equivalents (C3g eq)), and placebo. The other trial, L1 (n = 59), employed a 12 week intervention and an 8 week washout and tested one dose (346 mg C3g eq) and placebo. In both S2 and L1 neither dark adaptation nor night vision was improved by anthocyanin intake. However, in both trials anthocyanin consumption hastened the recovery of visual acuity after photobleaching. In S2 both anthocyanin doses were effective (P = 0.014), and in L1 recovery was improved at 8 weeks (P = 0.027) and 12 weeks (P = 0.030). Although photobleaching recovery was hastened by anthocyanins, it is not known whether this improvement would have an impact on everyday vision.

Anthocyanin metabolites are abundant and persistent in human urine.

LC-MS/MS revealed that metabolites of anthocyanins (Acn) were abundant in human urine (n = 17) even after 5 days with no dietary Acn. After intake of 250 mL of blueberry juice, parent Acn were 4% and Acn metabolites were 96% of the total urinary Acn for the following 24 h. Multiple reaction monitoring revealed 226 combinations of mass transition × retention times for known Acn and predicted Acn metabolites. These were dominated by aglycones, especially aglycone glucuronides. The diversity of Acn metabolites could include positional isomers of Acn conjugates and chalcones. The persistence of Acn metabolites suggested enterohepatic recycling leading to prolonged residence time. The prevalence of Acn metabolites based on pelargonidin, which is not present in blueberry juice, may reflect ongoing dehydroxylation and demethylation of other Acn via xenobiotic and colonic bacterial action. The results suggest that exposure to Acn-based flavonoid moieties is substantially greater than suggested by earlier research.

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.

Prophylactic neuroprotection by blueberry-enriched diet in a rat model of light-induced retinopathy.

The role of anthocyanins is controversial in vision health. This study investigates the impact of a blueberry-enriched diet as neuroprotectant in a rat model of light-induced retinopathy. Thirty-eight albino Wistar rats and 25 pigmented Brown-Norway rats were fed by gavage with long (7 weeks) and short (2 weeks) intervention with fortified blueberry juice (1 ml; 2.8 mg cyanidin 3-glucoside equivalents) or with a placebo solution (7 weeks) that contained the abundant nonanthocyanin blueberry phenolic, namely, chlorogenic acid, before being submitted to 2 hours of intense light regimen (1.8×10(4) lux). Retinal health was measured by fitting electroretinogram responses with the Naka-Rushton equation. The light-induced retinal damage was severe in the placebo groups, with the maximum amplitude of the electroretinogram being significantly reduced in both Wistar and Brown-Norway rats. The maximum amplitude of the electroretinogram was significantly protected from the light insult in the Wistar rats supplemented with blueberry juice for 7 or 2 weeks, and there was no significant difference between these two groups. The same dietary intervention in the Brown-Norway groups failed to protect the retina. Histological examination of retinal section confirmed the electroretinography results, showing protection of the outer nuclear layer of the retina in the Wistar rats fed with blueberries, while all placebo-fed rats and blueberry-fed Brown-Norway rats showed evidence of retinal damage concentrated in the superior hemiretina. The neuroprotective potential of anthocyanins in this particular model is discussed in terms of interaction with rhodopsin/phototransduction and in terms of antioxidative capacity.

Quantitative proteomic investigation employing stable isotope labeling by peptide dimethylation on proteins of strawberry fruit at different ripening stages.

A quantitative proteomic investigation of strawberry fruit ripening employing stable isotope labeling by peptide dimethylation was conducted on 'Mira' and 'Honeoye' strawberry fruit. Postharvest physiological quality indices, including volatile production, total phenolics, total anthocyanins, antioxidant capacity, soluble solids and titratable acidity, were also characterized in white, pink and red fruit. More than 892 and 848 proteins were identified and quantified in the 'Mira' and 'Honeoye' fruit, respectively, using at least two peptides for each protein identification. Using the normalized ratio of protein abundance changes, proteins that changed two-fold or more were identified as proteins that are up- or down-regulated during fruit ripening. Among the quantified proteins, 111 proteins were common to both cultivars and represented five significant clusters based on quantitative changes. Among the up-regulated proteins were proteins involved in metabolic pathways including flavonoid/anthocyanin biosynthesis, volatile biosynthesis, antioxidant metabolism, stress responses and allergen formation. Proteins that decreased during fruit ripening were found to be responsible for methionine metabolism, antioxidant-redox, energy metabolism and protein synthesis. Our results show that strawberry ripening is a highly complex system involving multi-physiological processes made possible through changes in protein expression. This study provides new insights on the regulation of proteins during strawberry fruit ripening that lay the foundation for further targeted studies. BIOLOGICAL SIGNIFICANCE: Research on the postharvest physiology and biochemistry of strawberry fruit as a model of non-climacteric fruit ripening has been conducted for many years. However, the mechanism(s) for the initiation and metabolic regulation of non-climacteric fruit ripening remains unknown. Little information on strawberry fruit ripening is available at the proteome level. This paper is the first report of a quantitative proteomic investigation of strawberry fruit ripening employing stable isotope labeling by peptide dimethylation. Postharvest physiological quality indices, including volatile production, total phenolics, total anthocyanins, antioxidant capacity, soluble solids and titratable acidity, were also characterized in ripening fruit. Significant biological changes associated with ripening were revealed and proteins that change significantly under these conditions were identified. Therefore, our study links the biological events of strawberry fruit ripening with proteomic information and provides insights into possible mechanisms of regulation. Proteins that changed during ripening were analyzed through function analysis, which provides new insights into metabolic changes occurring during ripening. Findings from this paper not only provide proteome information on fruit ripening, but also pave the way for further quantitative studies using SMR to investigate certain proteins and pathways involved in fruit ripening.