Application of HPLC-PDA-MS metabolite profiling to investigate the effect of growth temperature and day length on blackcurrant fruit.

INTRODUCTION: Blackcurrant (Ribes nigrum L.) is an excellent example of a "super fruit" with potential health benefits. Both genotype and cultivation environment are known to affect the chemical composition of blackcurrant, especially ascorbic acid and various phenolic compounds. Environmental conditions, like temperature, solar radiation and precipitation can also have significant impact on fruit chemical composition. The relevance of the study is further accentuated by the predicted and ongoing changes in global climate. OBJECTIVES: The aim of the present study was to provide new knowledge and a deeper understanding of the effects of post flowering environmental conditions, namely temperature and day length, on fruit quality and chemical composition of blackcurrant using an untargeted high performance liquid chromatography-photo diode array-mass spectrometry (HPLC-PDA-MS) metabolomics approach. METHODS: A phytotron experiment with cultivation of single-stemmed potted plants of blackcurrant cv. Narve Viking was conducted using constant temperatures of 12, 18 or 24 °C and three different photoperiods (short day, short day with night interruption, and natural summer daylight conditions). Plants were also grown under ambient outdoor conditions. Ripe berries were analysed using an untargeted HPLC-PDA-MS metabolomics approach to detect the presence and concentration of molecules as affected by controlled climatic factors. RESULTS: The untargeted metabolomics dataset contained a total of 7274 deconvolved retention time-m/z pairs across both electrospray ionisation (ESI) positive and negative polarities, from which 549 metabolites were identified or minimally annotated based upon accurate mass MS. Conventional principal component analysis (PCA) in combination with the Friedman significance test were applied to first identify which metabolites responded to temperature in a linear fashion. Multi-block hierarchical PCA in combination with the Friedman significance test was secondly applied to identify metabolites that were responsive to different day length conditions. Temperature had significant effect on a total of 365 metabolites representing a diverse range of chemical classes. It was observed that ripening of the blackcurrant berries under ambient conditions, compared to controlled conditions, resulted in an increased accumulation of 34 annotated metabolites, mainly anthocyanins and flavonoids. 18 metabolites were found to be regulated differentially under the different daylength conditions. Moreover, based upon the most abundant anthocyanins, a comparison between targeted and untargeted analyses, revealed a close convergence of the two analytical methods. Therefore, the study not just illustrates the value of non-targeted metabolomics approaches with respect to the huge diversity and numbers of significantly changed metabolites detected (and which would be missed by conventional targeted analyses), but also shows the validity of the non-targeted approach with respect to its precision compared to targeted analyses. CONCLUSIONS: Blackcurrant maturation under controlled ambient conditions revealed a number of insightful relationships between environment and chemical composition of the fruit. A prominent reduction of the most abundant anthocyanins under the highest temperature treatments indicated that blackcurrant berries in general may accumulate lower total anthocyanins in years with extreme hot summer conditions. HPLC-PDA-MS metabolomics is an excellent method for broad analysis of chemical composition of berries rich in phenolic compounds. Moreover, the experiment in controlled phytotron conditions provided additional knowledge concerning plant interactions with the environment.

Ascorbate pool, sugars and organic acids in black currant (Ribes nigrum L.) berries are strongly influenced by genotype and post-flowering temperature.

BACKGROUND: Marked effects of the climatic environment on fruit chemical composition have often been demonstrated in field experiments. However, complex covariations of several climatic factors in the natural environment complicate the interpretation of such experiments and the identification of the causal factors. This can be better achieved in a phytotron where the various climatic factors can be varied systematically. Therefore, we grew four black currant cultivars of contrasting origin in a phytotron under controlled post-flowering temperature and photoperiod conditions and analysed the berries for their ascorbic acid, sugar and organic acid contents. RESULTS: The analyses revealed significant effects of genotype on all investigated compounds. Particularly large cultivar differences were observed in the concentrations of l-ascorbic acid (AA) and sucrose. The concentrations of both AA and dehydroascorbic acid (DHAA), as well as the concentrations of all major sugars, decreased consistently with an increasing temperature over the temperature range 12-24 °C. Fructose and glucose were the predominant sugars with concentrations several fold higher than that for sucrose. AA was the main contributor to the total ascorbate pool in black currant berries. The AA/DHAA ratio varied from 5.6 to 10.3 among the studied cultivars. The concentration of citric acid, which was the predominant organic acid in black currant berries, increased with an increasing temperature, whereas the opposite trend was observed for malic and shikimic acid. Quninic acid was always present at relatively low concentrations. By contrast, photoperiod had no significant effect on berry content of any of the investigated compounds. CONCLUSION: It is concluded that the post-flowering temperature has marked effects on the concentration of important chemical compounds responsible for taste and nutritional value of black currant berries, whereas photoperiod has no such effect in the studied cultivars. © 2016 Society of Chemical Industry.

Polyphenol-rich juices reduce blood pressure measures in a randomised controlled trial in high normal and hypertensive volunteers.

Intake of fruits and berries may lower blood pressure (BP), most probably due to the high content of polyphenols. In the present study, we tested whether consumption of two polyphenol-rich juices could lower BP. In a randomised, double-blinded, placebo-controlled trial of 12 weeks, 134 healthy individuals, aged 50-70 years, with high normal range BP (130/85-139/89 mmHg, seventy-two subjects) or stage 1-2 hypertension (140/90-179/109 mmHg, sixty-two subjects), were included. They consumed 500 ml/d of one of either (1) a commercially available polyphenol-rich juice based on red grapes, cherries, chokeberries and bilberries; (2) a juice similar to (1) but enriched with polyphenol-rich extracts from blackcurrant press-residue or (3) a placebo juice (polyphenol contents 245·5, 305·2 and 76 mg/100 g, respectively). Resting BP was measured three times, with a 1 min interval, at baseline and after 6 and 12 weeks of intervention. Systolic BP significantly reduced over time (6 and 12 weeks, respectively) in the pooled juice group compared with the placebo group in the first of the three measurements, both for the whole study group (6·9 and 3·4 mmHg; P= 0·01) and even more pronounced in the hypertensive subjects when analysed separately (7·3 and 6·8 mmHg; P= 0·04). The variation in the BP measurements was significantly reduced in the pooled juice group compared with the placebo group (1·4 and 1·7 mmHg; P= 0·03). In conclusion, the present findings suggest that polyphenol-rich berry juice may contribute to a BP- and BP variability lowering effect, being more pronounced in hypertensive than in normotensive subjects.

Inter seasonal validation of non-contact NIR spectroscopy for measurement of total soluble solids in high tunnel strawberries.

Autonomous field robots are being developed for picking of fruit, where each fruit needs to be individually graded and handled. There is therefore a need for rapid and non-destructive sensing to measure critical fruit quality parameters. In this article we report how total soluble solids (TSS), a measure for total sugar content, can be measured in strawberries in the field by non-contact near-infrared (NIR) interaction spectroscopy. A specially designed prototype system working in the wavelength range 760-1080 nm was tested for this purpose. This novel instrument was compared with a commercial handheld NIR reflection instrument working in the range 900-1600 nm. The instruments were calibrated in the lab using data collected from 200 strawberries of two varieties and tested in a strawberry field on 50 berries in 2022 and 100 berries in 2023. Both systems performed well during calibration with root mean square errors of cross validation for TSS around 0.49 % and 0.57 %, for interaction and reflection, respectively. For prediction of TSS in new berries in 2023, the interaction system was superior, with a prediction error of 1.0 % versus 8.1 % for the reflection system, most likely because interaction probes deeper into the berries. The results suggest that interaction measurements of average TSS are more robust and would most likely require less calibration maintenance compared to reflection measurements. The non-contact feature is important since it reduces the spread of diseases and physical damage to the berries.

The stability of phenolic compounds and the colour of lingonberry juice with the addition of different sweeteners during thermal treatment and storage.

Lingonberries (Vaccinium vitis-idaea L.) are rich in phenolic compounds associated with several health benefits. The berries are also astringent, sour, and bitter and the addition of a sweetener is necessary to increase the palatability of lingonberry products. The addition of a sweetener may, however, affect the stability of phenolic compounds in the product. The aim of this study was thus to determine the effects of the addition of sweeteners (sucrose, acesulfame K or sucralose) and temperature on the stability of anthocyanins, flavonols, flavan-3-ols, hydroxycinnamic acids and the colour of lingonberry juice during thermal treatment and storage. The addition of sweeteners did not affect the stability of phenolic compounds or the colour of lingonberry juice during thermal treatment or storage. The stability of the phenolic compounds was significantly affected by temperature. Anthocyanins were the least stable of the phenolic compounds. The half-lives of total anthocyanins were 3.8, 2.0 and 0.8 h at 75, 85 and 95 °C, respectively. The half-lives during storage were 12.8 and 2.7 weeks at 6 and 22 °C, respectively. Cyanidin-3-galactoside, the major anthocyanin in lingonberries, was extensively degraded during storage, probably due to galactoside side-activities of the enzyme preparation used in juice production. After thermal treatment, the juices were darker and bluer, with lower chromaticity, while after storage, the juices were lighter, more yellow, and had higher chromaticity.

Composition of Sugars, Organic Acids, Phenolic Compounds, and Volatile Organic Compounds in Lingonberries (Vaccinium vitis-idaea L.) at Five Ripening Stages.

Wild lingonberries are a traditional source of food in the Nordic countries and an important contributor to economic activity of non-wood forest products in the region. Lingonberries are a rich source of bioactive compounds and can be a valuable contributor to a healthy diet. However, there are few studies available on how the bioactive compounds in lingonberries develop as they ripen. In this investigation, we examined the content of 27 phenolic compounds, three sugars, four organic acids, and 71 volatile organic compounds at five ripening stages. The study showed that, while the highest content of phenolic compounds was found early in the development, the organoleptic quality of the fruits improved as they ripened. From the first to the last stage of development, anthocyanins went from being nearly absent to 100 mg/100 g fw, and there was an increased content of sugars from 2.7 to 7.2 g/100 g fw, whereas the content of organic acids decreased from 4.9 to 2.7 g/100 g fw, and there were several changes in the profile of volatiles. The contents of flavonols, cinnamic acid derivatives, flavan-3-ols, and the total concentration of phenolic compounds were significantly lower in the fully ripe berries compared to berries in the early green stage. In addition to the changes occurring due to ripening, there was observed variation in the profile of both phenolic compounds and volatiles, depending on the growth location of the berries. The present data are useful for the assessment of harvest time to obtain the desired quality of lingonberries.

Antioxidative protection of dietary bilberry, chokeberry and Lactobacillus plantarum HEAL19 in mice subjected to intestinal oxidative stress by ischemia-reperfusion.

BACKGROUND: Ischemia-reperfusion (I/R) in the intestines is an inflammatory condition which activates leukocytes and reactive oxygen species (ROS) and leads to lipid peroxidation and DNA damage. Bilberry and chokeberry fruits are rich sources of polyphenols which may act as antioxidants and prevent lipid peroxidation. Lactic acid bacteria (LAB) may improve microbial status in the intestines and increase the metabolic activity towards polyphenolic degradation. The aim of the study was to clarify antioxidative effects of bilberry and chokeberry fruits alone and with addition of a LAB-strain, Lactobacillus plantarum HEAL19, in an I/R-model in mice. METHODS: Male BALB/cJ mice were fed the experimental diets for 10 days. Diets consisted of standard chow supplemented with either bilberry (Vaccinium myrtillus) or chokeberry (Aronia × prunifolia) powder alone or in combination with the LAB-strain Lactobacillus plantarum HEAL19. I/R-injury was induced by holding superior mesenteric artery clamped for 30 minutes followed by reperfusion for 240 minutes. Thereafter, colonic and caecal tissues and contents were collected. Malondialdehyde (MDA) was used as indicator of lipid peroxidation and was measured by a calorimetric assay, lactobacilli were cultured on Rogosa agar plates and Enterobacteriaceae on VRBG agar plates, anthocyanins and phenolic acids were analysed by HPLC-DAD-ESI-MSn. RESULTS: MDA was significantly decreased in the colon of groups fed bilberry alone (p = 0.030) and in combination with L. plantarum HEAL19 (p = 0.021) compared to the IR-control but not in chokeberry-fed groups. Supplementation with bilberry or chokeberry alone reduced the total number of lactobacilli on the mucosa. Higher concentrations of anthocyanins were found in the colon than in the caecum content of mice. A more varied composition of different anthocyanins was also observed in the colon content compared to the caecum of bilberry-fed mice. Phenolic acids formed by microbial degradation of the dietary polyphenols in the gut could be detected. More phenolic metabolites were found in the intestines of bilberry-fed mice than in the chokeberry-fed ones. CONCLUSIONS: Bilberry alone and in combination with L. plantarum HEAL19 exerts a better protection against lipid peroxidation than chokeberry. These dietary supplements may be used to prevent or suppress oxidative stress.

Effect of temperature on stability of anthocyanins, ascorbic acid and color in strawberry and raspberry jams.

Strawberry (cv. Senga Sengana) and raspberry (cv. Veten) were processed into jams at 60, 85 or 93 °C and stored at 4 or 23 °C for 8 and 16 weeks. High processing temperature reduced ascorbic acid, total monomeric anthocyanins (TMA) and total phenolics (TP) in strawberries (p<0.05), but not in raspberries. Processing temperature had minor effect on bioactive compounds in the jams during storage (<10% explained variance), but influenced color (L*, °Hue, Chroma), especially L* of the strawberry jams (73.3%). Storage period explained most of the variance in ascorbic acid (>90%), TMA (>42%) and TP (>69%). Storage temperature affected stability of anthocyanins, but had minor effect on ascorbic acid, which declined rapidly independent of storage temperature. Storage temperature also explained most of the variance (>40%) in Chroma of the jams and L* of raspberry jams (53%). Bioactive compounds and color were more stable in raspberry jams than in strawberry jams.

Ellagic acid and urolithin A modulate the immune response in LPS-stimulated U937 monocytic cells and THP-1 differentiated macrophages.

Dietary polyphenols are subjected, following ingestion, to an extensive metabolism, and the molecules that act at the cellular and tissue level will be, most likely, metabolites rather than native polyphenols. The mechanisms behind the positive effects exerted by polyphenols are not yet completely elucidated, since most in vitro studies use unmetabolised polyphenols rather than the metabolites present in the body. The aim of this study was to investigate and compare the potential effect of phenolic metabolites on the immune response using U937 monocyte and THP-1 macrophage cell cultures. Of the 16 metabolites tested, urolithins (Uro), and Uro A, in particular were the most potent, showing a modest increase in basal NF-κB activity and a reduction in lipopolysaccaride (LPS)-induced NF-κB activity, gene expression and secretion of pro-inflammatory cytokines. Protocatechuic acid and its sulfate/glucuronide metabolites reduced LPS-induced NF-κB activity, but not IL-6 and TNF-α cytokine secretion. Interestingly, both ellagic acid and its metabolite Uro A had immunomodulating effects, although they regulated the immune response differently, and both reduced LPS-induced NF-κB activity in U937 cells. However, while Uro A dramatically reduced IL-6 and IL-10 mRNA expression, no effect could be observed with ellagic acid. In THP-1 cells, treatment with ellagic acid dramatically reduced the expression of Toll-like receptor 4, while Uro A had no effect. The dual role observed for Uro A, showing both a modest increase in basal NF-κB activity and a reduction in LPS-induced NF-κB activity, as well as a reduction in LPS-induced pro-inflammatory cytokine secretion, makes this metabolite particularly interesting for further studies in animals and humans.

Major-effect candidate genes identified in cultivated strawberry (Fragaria × ananassa Duch.) for ellagic acid deoxyhexoside and pelargonidin-3-O-malonylglucoside biosynthesis, key polyphenolic compounds.

Strawberries are rich in polyphenols which impart health benefits when metabolized by the gut microbiome, including anti-inflammatory, neuroprotective, and antiproliferative effects. In addition, polyphenolic anthocyanins contribute to the attractive color of strawberry fruits. However, the genetic basis of polyphenol biosynthesis has not been extensively studied in strawberry. In this investigation, ripe fruits from three cultivated strawberry populations were characterized for polyphenol content using HPLC-DAD-MSn and genotyped using the iStraw35k array. GWAS and QTL analyses identified genetic loci controlling polyphenol biosynthesis. QTL were identified on four chromosomes for pelargonidin-3-O-malonylglucoside, pelargonidin-3-O-acetylglucoside, cinnamoyl glucose, and ellagic acid deoxyhexoside biosynthesis. Presence/absence of ellagic acid deoxyhexoside and pelargonidin-3-O-malonylglucoside was found to be under the control of major gene loci on LG1X2 and LG6b, respectively, on the F. × ananassa linkage maps. Interrogation of gene predictions in the F. vesca reference genome sequence identified a single candidate gene for ellagic acid deoxyhexoside biosynthesis, while seven malonyltransferase genes were identified as candidates for pelargonidin-3-O-malonylglucoside biosynthesis. Homologous malonyltransferase genes were identified in the F. × ananassa 'Camarosa' genome sequence but the candidate for ellagic acid deoxyhexoside biosynthesis was absent from the 'Camarosa' sequence. This study demonstrated that polyphenol biosynthesis in strawberry is, in some cases, under simple genetic control, supporting previous observations of the presence or absence of these compounds in strawberry fruits. It has also shed light on the mechanisms controlling polyphenol biosynthesis and enhanced the knowledge of these biosynthesis pathways in strawberry. The above findings will facilitate breeding for strawberries enriched in compounds with beneficial health effects.

A Review of Factors Affecting Anthocyanin Bioavailability: Possible Implications for the Inter-Individual Variability.

Anthocyanins are dietary bioactive compounds showing a range of beneficial effects against cardiovascular, neurological, and eye conditions. However, there is, as for other bioactive compounds in food, a high inter and intra-individual variation in the response to anthocyanin intake that in many cases leads to contradictory results in human trials. This variability could be caused at two levels, one at the bioavailability level and the other at the effect and mechanisms of action. In this context, we have thoroughly reviewed the scientific literature on anthocyanins variability caused by variation in bioavailability. Based on the literature reviewed, we have concluded that the variability in anthocyanins bioavailability might be produced by the lack of homogeneity introduced at three different levels: food matrix and food processing, enzymes involved in anthocyanin metabolism and transport, and anthocyanin metabolizing gut microbiota. However, it should be noted that the literature on anthocyanins bioavailability considering inter or intra-individual variability is still very scarce, which makes it difficult to reach any firm conclusion on the main metabolizing enzymes or bacteria that would be responsible for the variability in anthocyanin bioavailability.

Effect of high pressure and thermal processing on shelf life and quality of strawberry purée and juice.

The purpose of the study was to determine the effects of high pressure processing (HPP; 400-600 MPa, 20 °C, 1.5 or 3 min) and heat treatment (HT; 85 °C for 2 min) of strawberry purée and juice made from the same raw material. Microbiological and enzymatic inactivation, Brix, pH, anthocyanins, vitamin C, colour and sensory properties were analysed after processing and cold storage. The microbiological shelf life of the products was at least 49 days when processed at 500 or 600 MPa. Anthocyanins, vitamin C and colour were well preserved after HPP and HT. During storage, anthocyanins, vitamin C and sensory quality were better conserved in HT than in HPP purées, while there were minor differences between HT and HPP juices. This was probably due to higher enzyme activity in HPP purées, and indicates that raw materials with lower initial enzyme activity, like juices, are more suited for HPP than e.g. purées.

Characterization of phenolic compounds in strawberry (Fragaria x ananassa) fruits by different HPLC detectors and contribution of individual compounds to total antioxidant capacity.

Phenolic compounds in strawberry (Fragaria x ananassa) fruits were identified and characterized by using the complementary information from different high-performance liquid chromatography detectors: diode array, mass spectrometer in positive and negative mode, and coulometric array. Electrochemical profiles obtained from the coulometric array detector contributed to the structural elucidation suggested from the UV-vis and mass spectra. About 40 phenolic compounds including glycosides of quercetin, kaempferol, cyanidin, pelargonidin, and ellagic acid, together with flavanols, derivatives of p-coumaric acid, and ellagitannins, were described, providing a more complete identification of phenolic compounds in strawberry fruits. Quercetin-3-malonylhexoside and a deoxyhexoside of ellagic acid were reported for the first time. Antioxidative properties of individual components in strawberries were estimated by their electrochemical responses. Ascorbic acid was the single most important contributor to electrochemical response in strawberries (24%), whereas the ellagitannins and the anthocyanins were the groups of polyphenols with the highest contributions, 19 and 13% at 400 mV, respectively.

Polyphenol composition and antioxidant activity in strawberry purees; impact of achene level and storage.

In this study the impact of achenes on polyphenolic compounds, ascorbic acids, and antioxidant activities in strawberry purees at production and after storage at 6 and 22 degrees C for 8 and 16 weeks was investigated. Strawberry purees were made from flesh, berry, and achene-enriched homogenate and contained 0, 1.2, and 2.9% achenes, respectively. At production, strawberry purees made from flesh contained more anthocyanins, p-coumaroyl glycosides, and ascorbic acids, whereas increasing achene levels caused increasing levels of ellagic acid derivatives, proanthocyanidins, flavonols, total phenolics (TP), and antioxidant activities. In addition, the anthocyanins, TP, and ferric reducing ability power (FRAP) in purees with more achenes were better retained during storage. Ascorbic acids and anthocyanins declined rapidly during storage, whereas other polyphenols and antioxidant activities were more stable; that is, the contributions from anthocyanins and ascorbic acids to TP and antioxidant activities decreased. The findings that achenes contributed significantly to polyphenol content and stability of strawberry purees may be interesting in a nutritional and, thus, commercial, perspective.

Molar absorptivities and reducing capacity of pyranoanthocyanins and other anthocyanins.

To improve accuracy in the determination of anthocyanin purity and succeeding antioxidant capacity, 1H and 13C nuclear magnetic resonance spectroscopy have been combined with high-performance liquid chromatography (HPLC) equipped with a diode array detector and UV-vis spectroscopy in the analysis of anthocyanidin 3-glycosides and 5-carboxypyranoanthocyanidin 3-glycosides. The molar absoptivity (epsilon) values were found to be relatively similar, in contrast to previously reported literature values, and the average epsilon values for both anthocyanidin 3-monoglycosides and 5-carboxypyranoanthocyanidin 3-glycosides were proposed to be 22,000 and 23,000 in acidified aqueous and methanolic solutions, respectively. To assess the influence of structure on the potential antioxidant capacity of anthocyanins, the 3-glucosides of pelargonidin (1), cyanidin (2), peonidin (3), delphinidin (4), petunidin (5), malvidin (6), 5-carboxypyranopelargonidin (8), 5-carboxypyranocyanidin (9), 5-carboxypyranodelphinidin (11), 5-carboxypyranopetunidin (12), and 5-carboxypyranomalvidin (13) and the 3-galactosides of cyanidin (7) and 5-carboxypyranocyanidin (14) were examined by a ferric ion reducing antioxidant power (FRAP) assay. The reducing capacities of the individual anthocyanins were in the range of 0.9-5.2 micromol of Trolox equivalents/micromol. The two 5-carboxypyranoanthocyanins 11 and 9 and the four common anthocyanins 2, 4, 7, and 14, all possessing pyrogallol or catechol type of B rings, showed the highest antioxidant capacity measured by FRAP. However, the inclusion of the 5-hydroxyl in the D ring and just one oxygen substituent on the B ring in 8 diminished the reducing capacity considerably. Correspondingly, electrochemical behavior of 5-carboxypyranoanthocyanidin 3-glucosides and anthocyanidin 3-glucosides was derived using HPLC coupled to a coulometric array detector set from 100 to 800 mV in increments of 100 mV. The relative order of the reducing capacity of the various 5-carboxypyranoanthocyanidin 3-glucosides and anthocyanidin 3-glucosides were nearly alike, whether determined by coulometric array detection or FRAP.

Development, validation and evaluation of an analytical method for the determination of monomeric and oligomeric procyanidins in apple extracts.

There is a lack of data for individual oligomeric procyanidins in apples and apple extracts. Our aim was to develop, validate and evaluate an analytical method for the separation, identification and quantification of monomeric and oligomeric flavanols in apple extracts. To achieve this, we prepared two types of flavanol extracts from freeze-dried apples; one was an epicatechin-rich extract containing ∼30% (w/w) monomeric (-)-epicatechin which also contained oligomeric procyanidins (Extract A), the second was an oligomeric procyanidin-rich extract depleted of epicatechin (Extract B). The parameters considered for method optimisation were HPLC columns and conditions, sample heating, mass of extract and dilution volumes. The performance characteristics considered for method validation included standard linearity, method sensitivity, precision and trueness. Eight laboratories participated in the method evaluation. Chromatographic separation of the analytes was best achieved utilizing a Hilic column with a binary mobile phase consisting of acidic acetonitrile and acidic aqueous methanol. The final method showed linearity for epicatechin in the range 5-100µg/mL with a correlation co-efficient >0.999. Intra-day and inter-day precision of the analytes ranged from 2 to 6% and 2 to 13% respectively. Up to dp3, trueness of the method was >95% but decreased with increasing dp. Within laboratory precision showed RSD values <5 and 10% for monomers and oligomers, respectively. Between laboratory precision was 4 and 15% (Extract A) and 7 and 30% (Extract B) for monomers and oligomers, respectively. An analytical method for the separation, identification and quantification of procyanidins in an apple extract was developed, validated and assessed. The results of the inter-laboratory evaluation indicate that the method is reliable and reproducible.

Influence of Controlled Postflowering Temperature and Daylength on Individual Phenolic Compounds in Four Black Currant Cultivars.

The effects of postflowering temperature and daylength on the concentration of individual phenolic compounds were studied in black currant (Ribes nigrum L.) berries under controlled phytotron conditions. The four cultivars studied varied greatly in their concentrations of individual phenolic compounds and temperature stability for accumulation. The concentrations of a wide range of identified phenolic compounds were strongly influenced by temperature over the 12-24 °C range, often with opposite temperature gradient patterns for compounds within the same subclass. Accumulation of anthocyanins and flavonols increased under natural long day conditions, which provided an increased daily light integral, while under identical light energy conditions, photoperiod had little or no effect on the concentration of phenolic compounds. Furthermore, with the exception of members of the hydroxycinnamic acid subclass, the concentration of most phenolic compounds was higher in berries ripened outdoors than in the phytotron, apparently due to screening of UV-B radiation by the glass cover.

Phenolic composition and antioxidant activities in flesh and achenes of strawberries (Fragaria ananassa).

High performance liquid chromatography coupled with diode array and coulometric array detectors were used to characterize and quantify phenolic compounds in achenes and flesh of ripe strawberries (cv Totem and Puget Reliance). Total phenolics and total monomeric anthocyanins were measured and antioxidant activities were evaluated by the FRAP and the ORAC assays. Strawberries contained 1% achenes on a fresh weight basis; however, they contributed to about 11% of total phenolics and 14% of antioxidant activities in strawberries. Ellagic acid, ellagic acid glycosides, and ellagitannins were the main contributors to the antioxidant activities of achenes. The major anthocyanin in flesh was pelargonidin-3-glucoside, whereas achenes consisted of nearly equal amounts of cyanidin-3-glucoside and pelargonidin-3-glucoside. Phenolic content and antioxidant activity of strawberry achenes were reduced by industrial processing. However, the levels were still high and strawberry waste byproduct could thus be a possible source of nutraceuticals or natural antioxidants.

Validated Method for the Characterization and Quantification of Extractable and Nonextractable Ellagitannins after Acid Hydrolysis in Pomegranate Fruits, Juices, and Extracts.

Pomegranates are one of the main highly valuable sources of ellagitannins. Despite the potential health benefits of these compounds, reliable data on their content in pomegranates and derived extracts and food products is lacking, as it is usually underestimated due to their complexity, diversity, and lack of commercially available standards. This study describes a new method for the analysis of the extractable and nonextractable ellagitannins based on the quantification of the acid hydrolysis products that include ellagic acid, gallic acid, sanguisorbic acid dilactone, valoneic acid dilactone, and gallagic acid dilactone in pomegranate samples. The study also shows the occurrence of ellagitannin C-glycosides in pomegranates. The method was optimized using a pomegranate peel extract. To quantify nonextractable ellagitannins, freeze-dried pomegranate fruit samples were directly hydrolyzed with 4 M HCl in water at 90 °C for 24 h followed by extraction of the pellet with dimethyl sulfoxide/methanol (50:50, v/v). The method was validated and reproducibility was assessed by means of an interlaboratory trial, showing high reproducibility across six laboratories with relative standard deviations below 15%. Their applicability was demonstrated in several pomegranate extracts, different parts of pomegranate fruit (husk, peels, and mesocarp), and commercial juices. A large variability has been found in the ellagitannin content (150-750 mg of hydrolysis products/g) and type (gallagic acid/ellagic acid ratios between 4 and 0.15) of the 11 pomegranate extracts studied.

Analysis of flavonoids and other phenolic compounds using high-performance liquid chromatography with coulometric array detection: relationship to antioxidant activity.

High-performance liquid chromatography coupled with a coulometric array detector was used to characterize the electrochemical behavior of 17 flavonoids and three cinnamic acid derivatives. The antioxidant activity of these phenolic compounds was evaluated by the ferric reducing activity power (FRAP), the oxygen radical absorbance capacity (ORAC), and the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical assays. All flavonoids, except kaempferol-3-rutinoside, malvidin-3-glucoside, and peonidin-3-glucoside, had two oxidation potentials (100-300 and 700-800 mV). Quercetin and myricetin had an additional oxidation wave at 400 mV. The electrochemical responses at a relatively low oxidation potential (300 mV) and the cumulative responses at medium oxidation potentials (400 and 500 mV) had the highest correlations with antioxidant activities. The highest correlations between electrochemical characteristics and antioxidant activities were found between electrochemical responses and antioxidant activities obtained in the FRAP assay and in the DPPH assay after short reaction periods. Lower correlations were revealed between electrochemical responses and antioxidant activities obtained in the ORAC assay.