Application of quantitative proteomics to investigate fruit ripening and eating quality.

The consumption of fruit and vegetables play an important role in human nutrition, dietary diversity and health. Fruit and vegetable industries impart significant impact on our society, economy, and environment, contributing towards sustainable development in both developing and developed countries. The eating quality of fruit is determined by its appearance, color, firmness, flavor, nutritional components, and the absence of defects from physiological disorders. However, all of these components are affected by many pre- and postharvest factors that influence fruit ripening and senescence. Significant efforts have been made to maintain and improve fruit eating quality by expanding our knowledge of fruit ripening and senescence, as well as by controlling and reducing losses. Innovative approaches are required to gain better understanding of the management of eating quality. With completion of the genome sequence for many horticultural products in recent years and development of the proteomic research technique, quantitative proteomic research on fruit is changing rapidly and represents a complementary research platform to address how genetics and environment influence the quality attributes of various produce. Quantiative proteomic research on fruit is advancing from protein abundance and protein quantitation to gene-protein interactions and post-translational modifications of proteins that occur during fruit development, ripening and in response to environmental influences. All of these techniques help to provide a comprehensive understanding of eating quality. This review focuses on current developments in the field as well as limitations and challenges, both in broad term and with specific examples. These examples include our own research experience in applying quantitative proteomic techniques to identify and quantify the protein changes in association with fruit ripening, quality and development of disorders, as well as possible control mechanisms.

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.

Genome-wide association studies in apple reveal loci of large effect controlling apple polyphenols.

Apples are a nutritious food source with significant amounts of polyphenols that contribute to human health and wellbeing, primarily as dietary antioxidants. Although numerous pre- and post-harvest factors can affect the composition of polyphenols in apples, genetics is presumed to play a major role because polyphenol concentration varies dramatically among apple cultivars. Here we investigated the genetic architecture of apple polyphenols by combining high performance liquid chromatography (HPLC) data with ~100,000 single nucleotide polymorphisms (SNPs) from two diverse apple populations. We found that polyphenols can vary in concentration by up to two orders of magnitude across cultivars, and that this dramatic variation was often predictable using genetic markers and frequently controlled by a small number of large effect genetic loci. Using GWAS, we identified candidate genes for the production of quercitrin, epicatechin, catechin, chlorogenic acid, 4-O-caffeoylquinic acid and procyanidins B1, B2, and C1. Our observation that a relatively simple genetic architecture underlies the dramatic variation of key polyphenols in apples suggests that breeders may be able to improve the nutritional value of apples through marker-assisted breeding or gene editing.

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.

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.

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.

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 supplementation improves memory in older adults.

The prevalence of dementia is increasing with expansion of the older adult population. In the absence of effective therapy, preventive approaches are essential to address this public health problem. Blueberries contain polyphenolic compounds, most prominently anthocyanins, which have antioxidant and anti-inflammatory effects. In addition, anthocyanins have been associated with increased neuronal signaling in brain centers, mediating memory function as well as improved glucose disposal, benefits that would be expected to mitigate neurodegeneration. This study investigated the effects of daily consumption of wild blueberry juice in a sample of nine older adults with early memory changes. At 12 weeks, improved paired associate learning (p = 0.009) and word list recall (p = 0.04) were observed. In addition, there were trends suggesting reduced depressive symptoms (p = 0.08) and lower glucose levels (p = 0.10). We also compared the memory performances of the blueberry subjects with a demographically matched sample who consumed a berry placebo beverage in a companion trial of identical design and observed comparable results for paired associate learning. The findings of this preliminary study suggest that moderate-term blueberry supplementation can confer neurocognitive benefit and establish a basis for more comprehensive human trials to study preventive potential and neuronal mechanisms.

Plum juice, but not dried plum powder, is effective in mitigating cognitive deficits in aged rats.

OBJECTIVE: Normal aging in animals and humans is accompanied by a decline in cognitive performance that is thought to be due to the long-term effects of oxidative stress and inflammation on neurologic processes. Previous findings have suggested that protection against age-related cognitive declines may be achieved by increasing the dietary intake of fruits and vegetables, especially those that are high in antioxidant activity, such as blueberries and strawberries. The objective of this study was to investigate supplementation with Prunus domestica L. in mitigating age-related deficits in cognitive function. METHODS: We investigated the effects of supplementation with P. domestica L., consumed as a 2% dried plum (i.e., prune) powder or 100% plum juice for 8 wk, in mitigating age-related deficits in cognitive function in aged Fischer 344 rats. RESULTS: Rats that drank plum juice from 19 to 21 mo of age had improved working memory in the Morris water maze, whereas rats fed dried plum powder were not different from the control group, possibly due to the smaller quantity of phenolics consumed in the powder group compared with the juice group. CONCLUSION: These results are discussed in relation to the amount and type of phenolics present in the plum products and in relation to other dietary intervention studies in which cognitive benefits have been reported.

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.