Analysis of phenolic compounds in two blackberry species (Rubus glaucus and Rubus adenotrichus) by high-performance liquid chromatography with diode array detection and electrospray ion trap mass spectrometry

High-performance liquid chromatography with diode array (LC-DAD) and electrospray ionization mass spectrometric detection (ESI-MS) was used to analyze phenolic compounds of two blackberry species ( Rubus glaucus Benth. and Rubus adenotrichus Schlech.) growing in South America. UV-visible spectrophotometry was a valuable tool for identifying the class of phenolic compound, whereas MS and MS ( n ) fragmentation data were useful for their structural characterization. Ellagitannins were the major compounds, with sanguiin H-6 and lambertianin C being the predominant ones. The anthocyanin composition as well as the presence or absence of kaempferol glycosides can be used to distinguish the Rubus species studied. Flavonol hexoside-malonates were identified in both berries. Hydroxycinnamic acids were minor compounds and found as ferulic, caffeic, and p-coumaric acid esters. Similar contents were obtained by analysis of soluble ellagitannins and ellagic acid glycosides as ellagic acid equivalents and by analysis of ellagic acid equivalents released after acid hydrolysis.     

Phenolic acid profiles in some small berries

The composition of phenolic acids in several small berries grown in Northeastern Poland, namely, low-bush blueberries, black mulberries, European juneberries, black currants, fruits of blue-berried honeysuckle, and blackberries, was determined by gas chromatography (GC) and mass spectrometry (MS). The total content of phenolic acids, identified by GC-MS, ranged from 2845.8 +/- 141.0 (black mulberries) to 5418.2 +/- 228.0 (blue-berried honeysuckle). Twenty phenolic acids were identified in the berries. Of these, hydroxycaffeic, m- and p-coumaric, and 3,4-dimethoxycinnamic acids were the major phenolic acids in blackberries and blueberries, m-coumaric acid was the major phenolic acid in blue-berried honeysuckle and black currant fruits, while salicylic, caffeic, and m- and p-coumaric acids were the predominant phenolic acids in European juneberries. Syringic and veratric acids were detected only in blueberries, while p-hydroxybenzoic and sinapic acids were present only in black currants and o-coumaric acid was present in blueberries and black mulberries. The phenolic acids liberated from esters and glycosidic bonds were the major fractions of phenolic acids in the berries.     

The influence of early yield on the accumulation of major taste and health-related compounds in black and red currant cultivars (Ribes spp.)

The focus of our study was to investigate the effect of crop load on the accumulation and composition of primary metabolites (sugars and organic acids), selected groups of flavonoids (anthocyanins and flavonols), and total phenolics in two subsequent years in four black currant cultivars ('Titania', 'Triton', 'Tsema', and 'Cacanska crna') and three red currant cultivars ('Junifer', 'Rolan', and 'Stanza'). For the determination and quantification of compounds, high-performance liquid chromatography-photodiode array with a mass spectrometer was used. Significant differences among cultivars were detected in all analyzed compounds. Anthocyanins were the predominant phenolic group and were more abundant in black currant cultivars as compared to red ones. Similar amounts of sugars and organic acids were measured in both Ribes species; however, vitamin C was 3-fold higher in black currants. A larger crop load in the second year had a negative effect on the sugar content of berries and promoted a higher degree of acids, with the exception of vitamin C, which was higher in the year with a lower crop load. On the other hand, the content of anthocyanins and flavonols was higher in the year with a larger crop load, while there were no differences in total phenolic content.     

Identification and quantification of phenolic compounds in berries of Fragaria and Rubus species (family Rosaceae)

High-performance liquid chromatography combined with diode array and electrospray ionization mass spectrometric detection was used to study soluble and insoluble forms of phenolic compounds in strawberries, raspberries (red and yellow cultivated and red wild), arctic bramble, and cloudberries. Hydroxycinnamic acids were present as free forms in cloudberries and mainly as sugar esters in the other berries. Quercetin 3-glucuronide was the typical flavonol glycoside in all of the berries studied. The composition of the predominant anthocyanins can be used to distinguish the studied red Rubus species from each other since cyanidin was glycosylated typically with 3-sophorose (56%) in cultivated red raspberry, with 3-sophorose (30%) and 3-glucose (27%) in wild red raspberry, and with 3-rutinose (80%) in arctic bramble. Ellagic acid was present as free and glycosylated forms and as ellagitannins of varying degrees of polymerization. Comparable levels of ellagitannins were obtained by the analysis of soluble ellagitannins as gallic acid equivalents and by the analysis of ellagic acid equivalents released by acid hydrolysis of the extracts.     

Separation, characterization and quantification of phenolic compounds in blueberries and red and black currants by HPLC-DAD-ESI-MSn

The phenolic profile of four blueberry varieties (Vaccinium corymbosum L., cv. Toro, Legacy, Duke and Bluecrop) and two varieties (Rosenthal and Rovada) of red currants (Ribes rubrum L.) and black currants (Ribes nigrum L.) cultivated in Macedonia have been analyzed using HPLC coupled to diode-array detection and tandem mass spectrometry with electrospray ionization. A complex profile of anthocyanins, flavonols, flavan-3-ols and hydroxycinnamic acid derivatives has been assayed in acetone-acetic acid (99:1, v/v) extracts. Anthocyanins comprised the highest content of total phenolic compounds in currants (>85%) and lower and variety dependent in blueberries (35-74%). Hydroxycinnamic acid derivatives comprised 23-56% of total phenolics in blueberries and 1-6% in currants. Chlorogenic acid was the major hydroxycinnamic acid in blueberries, only in the Legacy variety, two malonyl-caffeoylquinic acid isomers were major components. Flavonols, mainly quercetin and myricetin glycosides, were a minor group, but glucosides of laricitrin and syringetin were also detected in the blueberry varieties counting for 10-34% of total flavonols. From flavan-3-ols, catechin was detected in most samples; the dimer B2 was specific for blueberries whereas epigallocatechin was detected in currants.     

Compositional differences of phenolic compounds between black currant (Ribes nigrum L.) cultivars and their response to latitude and weather conditions

Phenolic compounds in black currants of three Finnish cultivars and their response to growth latitude and weather conditions were analyzed over a six-year period. 'Melalahti' had lower contents of total phenolic compounds (31.4% and 29.2% lower than 'Mortti' and 'Ola', respectively), total anthocyanins (32.6% and 30.5%), and total hydroxycinnamic acid derivatives (23.1% and 23.8%) (p < 0.05) and was less affected by growth latitude and weather conditions than 'Mortti' and 'Ola'. However, all the cultivars grown at higher latitude (66°34' N) had lower contents of total flavonols, total anthocyanins, and total phenolic compounds than those grown at lower latitude (60°23' N) (p < 0.05). The content of total hydroxycinnamic acid conjugates did not vary in 'Melalahti' (p > 0.05) but increased as the latitude increased in 'Mortti' and 'Ola' (p < 0.05). Temperature and radiation were the major weather variables influencing the composition of phenolic compounds. Delphinidin-3-O-glucoside, delphinidin-3-O-rutinoside, and myricetin-3-O-glucoside content showed positive correlations with temperature and radiation in all three cultivars. The study gives important guidelines for the selection of raw materials and growth sites as well as for the berry cultivation for commercial exploitation of black currant berries.     

Identification and quantification of phenolic compounds in grapes by HPLC-PDA-ESI-MS on a semimicro separation scale

Reversed phase high performance liquid chromatography (RP-HPLC) on a semimicro separation scale was employed to develop a straightforward method for the simultaneous separation, identification, and quantification of phenolic compounds occurring in whole berries of Vitis vinifera, which comprise phenolic acids, flavonols, catechins, stilbenes, and anthocyanins. A C-18 narrow bore column of 150 x 2.0 mm I.D. and a semimicro photodiode array detector (PDA) cell of 2.5 microL, in conjunction with a mass spectrometry detector equipped with an electrospray ionization source (ESI-MS) to confirm peak identification, were employed. The C-18 narrow bore column was eluted by a multisegment gradient of increasing concentration of acetonitrile in water-formic acid solution that was optimized on the basis of the results of a study carried out to evaluate the influence of mobile phase composition and gradient shape on separation performance and detection sensitivity by ESI-MS. The identification of individual phenolic compounds was performed on the basis of their retention times and both UV-visible and mass spectra, acquired by a mass spectrometer (MS) equipped with an electrospray ionization (ESI) source, employed in conjunction with the PDA detector. Libraries comprising retention times, UV-visible, and mass spectra for major phenolic compounds expected in grape berries were made by subjecting solutions of each phenolic standard to the optimized RP-HPLC method. Quantification of individual compounds was performed by the external standard method using a six point regression graph of the UV-visible absorption data collected at the wavelength of maximum absorbance of each analyte determined by the PDA spectra. The RP-HPLC method was validated in terms of linearity of calibration graphs, limits of detection, limits of quantification, repeatability, and accuracy, which was evaluated by a recovery study. The developed method was successfully applied to identify the phenolic compounds occurring in the whole berries of nine red and one white grape of different varieties of Vitis vinifera, comprising some autochthonous varieties of south Italy such as Aglianico, Malvasia Nera, Uva di Troia, Negroamaro, Primitivo, and Susumaniello. Large differences in the content of phenolic compounds was found in the investigated grape varieties. As expected, only glycosilated flavonols were quantified, and the total amount of these compounds was higher in the whole berries of red grapes than in the white Moscato, where the most abundant phenolic compound was quercetin 3-O-glucoside. In almost all samples, the most and least abundant anthocyanins were malvidin 3-O-glucoside and cyanidin 3-O-glucoside, respectively, with the exception of Uva di Troia where the least abundant anthocyanin was delphinidin 3-O-glucoside.     

Characterization of phenolic profiles of Northern European berries by capillary electrophoresis and determination of their antioxidant activity

Berries are known to contain phenolic substances (i.e., flavonoids and phenolic acids), which comprise two large and heterogeneous groups of biologically active nonnutrients. This investigation evaluated the content and profile of the phenolic compounds present in six different berries found in Northern Europe. The latter included bilberry (Vaccinium myrtillus), cowberry (Vaccinium vitis-idaea), cranberry (Vaccinium oxycoccus), strawberry (Fragaria ananassa), black currant (Ribes nigrum), and red currant (Ribes rubrum). The study was focused on two areas. The first involved the extraction and analysis of berries for total phenolic content and determination of their antioxidant activity. The total phenolic level of berries was correlated with their antioxidant activity. Second, the berry extracts were analyzed by capillary electrophoresis to determine the content and profile of selected bioactive compounds. The analytes of interest included trans-resveratrol, cinnamic acid, ferulic acid, p-coumaric acid, quercetin, and morin.     

Phenolic profile of quince fruit (Cydonia oblonga Miller) (pulp and peel)

Qualitative and quantitative analyses of phenolic compounds were carried out on quince fruit samples from seven different geographical origins in Portugal. For each origin, both pulp and peel were analyzed by reversed-phase HPLC-DAD and HPLC-DAD/MS.The results revealed differences between the phenolic profiles of pulps and peels in all studied cases. The pulps contained mainly caffeoylquinic acids (3-, 4-, and 5-O-caffeoylquinic acids and 3,5-dicaffeoylquinic acid) and one quercetin glycoside, rutin (in low amount). The peels presented the same caffeoylquinic acids and several flavonol glycosides: quercetin 3-galactoside, kaempferol 3-glucoside, kaempferol 3-rutinoside, and several unidentified compounds (probably kaempferol glycoside and quercetin and kaempferol glycosides acylated with p-coumaric acid). The highest content of phenolics was found in peels.     

Berry phenolics and their antioxidant activity

Phenolic profiles of a total of 26 berry samples, together with 2 apple samples, were analyzed without hydrolysis of glycosides with HPLC. The phenolic contents among different berry genera varied considerably. Anthocyanins were the main phenolic constituents in bilberry, bog-whortleberry, and cranberry, but in cowberries, belonging also to the family Ericaceae genus Vaccinium, flavanols and procyanidins predominated. In the family Rosaceae genus Rubus (cloudberry and red raspberry), the main phenolics found were ellagitannins, and in genus Fragaria (strawberry), ellagitannins were the second largest group after anthocyanins. However, phenolic acids were dominant in rowanberries (genus Sorbus) and anthocyanins in chokeberry (genus Aronia). In the family Grossulariaceae genus Ribes (currants and gooseberry), anthocyanins predominated, as well as in crowberries (family Empetraceae genus Empetrum). In apples, hydroxycinnamic acids were the main phenolic subgroup. Extraction methods for berries and apples were studied to produce phenolic extracts with high antioxidant activity. Evaluation of antioxidant activity was performed by autoxidazing methyl linoleate (40 degrees C, in the dark). The extraction method affected remarkably both the phenolic composition and the antioxidant activity, but with statistical analysis the observed activity could not be well explained with the contents of individual phenolic subgroups.     

Bioactive polyphenols in leaves, stems, and berries of Saskatoon (Amelanchier alnifolia Nutt.) cultivars

The Saskatoon berry is currently cultivated in many parts of the world for its suitability for various food products and due to its high content of nutrients and polyphenols. To determine the phytochemical profile of a Saskatoon plant, polyphenols from leaves, stems, and berries were screened from four cultivars grown in Finland using HPLC-DAD and HPLC-ESI/MS. The phenolic composition and concentrations varied among plant parts and cultivars. The main berry components were cyanidin-based anthocyanins (63% of the phenols), quercetin-derived flavonol glycosides, and hydroxycinnamic acids. The total anthocyanin content varied between 258.7 and 517.9 mg/100 fresh weight among cultivars. Protocatechuic acid was found for the first time in Saskatoon berries. The leaves consisted of quercetin- and kaempferol-derived glycosides (41% of the phenols), hydroxycinnamic acids (36%), catechins, and some neolignans. Quercetin 3-galactoside and 3-glucoside, (-)-epicatechin, and chlorogenic acid were the main phenolics in the leaves of all cultivars. The stem components were flavanone and flavonol glycosides (55% of the phenols), catechins (38%), and hydroxybenzoic acids. Concentrations of the main compound, eriodictyol 7-glucoside, varied among cultivars from 3.3 to 6.5 mg/g of stem dry weight. Very high proanthocyanidin contents were found in stems and leaves (10-14% of dry biomass), whereas berries contained a low amount of proanthocyanidins (3% of dry biomass). The findings reveal that leaves and stems of Saskatoon cultivars possess high amounts of various phenolic compounds that may offer new functional raw materials for a wide range of food and health products.     

Isolation, structure determination, and sensory activity of mouth-drying and astringent nitrogen-containing phytochemicals isolated from red currants (Ribes rubrum)

Application of chromatographic separation and taste dilution analyses recently revealed, besides a series of flavon-3-ol glycosides and (E)/(Z)-aconitic acid, four nitrogen-containing phytochemicals as the key astringent and mouth-drying compounds in red currants (Ribes rubrum). The isolation and structure determination of the astringent indoles 3-carboxymethyl-indole-1-N-beta-D-glucopyranoside (1) and 3-methylcarboxymethyl-indole-1-N-beta-D-glucopyranoside (2), as well as the astringent, noncyanogenic nitriles 2-(4-hydroxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile (3) and 2-(4-hydroxy-3-methoxybenzoyloxymethyl)-4-beta-D-glucopyranosyloxy-2(E)-butenenitrile (4) by means of 1D/2D NMR, LC-MS/MS, and UV-vis spectroscopy are reported. The structures of compounds 1 and 2 were confirmed by synthesis. Using the recently developed half-tongue test, human recognition thresholds for the astringent and mouth-drying nitrogen compounds were determined to be between 0.0003 and 5.9 micromol/L (water). In particular, the extraordinarily low threshold of 0.0003 micromol/L evaluated for the indole 1 represents the lowest recognition threshold of any astringent phytochemical reported to date.     

Influence of growing season on phenolic compounds and antioxidant properties of grape berries from vines grown in subtropical climate

The influence of growing season (winter vs summer) on the synthesis and accumulation of phenolic compounds and antioxidant properties was studied in five grape cultivars for three consecutive years. Four phenolic compound parameters (total phenols, flavonoids, flavan-3-ols, and anthocyanins) and three antioxidant property parameters [2,2-diphenyl-1-picrylhydrazyl radical scavenging, 2,2-azinobis(3-ethylbenzothiazolinesulfonic acid) radical scavenging, and ferric reducing antioxidant power] were investigated. Results showed that both phenolic compounds and antioxidant properties in the seed and skin of winter berries were significantly (p < 0.05) higher than those of summer berries for all of the cultivars investigated. The anthocyanin profiles of berry skins appeared to be extremely consistent in different years for the same crop, whereas they varied greatly between the two crops within the same year (winter vs summer). Winter berries contained richer glucosides of delphinidin, cyanidin, peonidin, and malvidin than summer berries. These seasonal variations of phenolic compounds and antioxidant properties on grape berries were largely contributed by climatic factors such as temperature, solar radiation, rainfall, and hydrothermic coefficient between different growing seasons.     

Influence of domestic processing and storage on flavonol contents in berries

Effects of domestic processing and storage on the flavonols quercetin, myricetin, and kaempferol in five berries were studied using an optimized RP-HPLC method with UV and diode array detection after an acid hydrolysis of the corresponding glycosides. In fresh berries, the total content of flavonols was highest in lingonberry (169 mg/kg) and black currant (157 mg/kg), intermediate in bilberry (41 mg/kg) and strawberry (17 mg/kg), and lowest in red raspberry (9.5 mg/kg). Cooking strawberries with sugar to make jam resulted in minor losses (quercetin 15%, kaempferol 18%). During cooking of bilberries with water and sugar to make soup, 40% of quercetin was lost. Traditional preservation of crushed lingonberries in their own juice caused a considerable (40%) loss of quercetin. Only 15% of quercetin and 30% of myricetin present in unprocessed berries were retained in juices made by common domestic methods (steam-extracted black currant juice, unpasteurized lingonberry juice). Cold-pressing was superior to steam-extraction in extracting flavonols from black currants. During 9 months of storage at 20 C, quercetin content decreased markedly (40%) in bilberries and lingonberries, but not in black currants or red raspberries. Myricetin and kaempferol were more susceptible than quercetin to losses during storage.     

A versatile targeted metabolomics method for the rapid quantification of multiple classes of phenolics in fruits and beverages

Compelling evidence of the health benefits of phenolic compounds and their impact on food quality have stimulated the development of analytical methods for the identification and quantification of these compounds in different matrices in recent years. A targeted metabolomics method has been developed for the quantification of 135 phenolics, such as benzoates, phenylpropanoids, coumarins, stilbenes, dihydrochalcones, and flavonoids, in fruit and tea extracts and wine using UPLC/QqQ-MS/MS. Chromatography was optimized to achieve separation of the compounds over a period of 15 min, and MRM transitions were selected for accurate quantification. The method was validated by studying the detection and quantification limits, the linearity ranges, and the intraday and interday repeatability of the analysis. The validated method was applied to the analysis of apples, berries, green tea, and red wine, providing a valuable tool for food quality evaluation and breeding studies.     

Phenolic composition of grape stems

Grape stems contain significant amounts of polyphenolic compounds, especially phenolic acids, flavonols, and flavanonols such as astilbin. The tannin content was characterized after the depolymerization reaction thiolysis. Tannins consisted of polymeric proanthocyanidins (up to 27 units) mainly consisting of (-)-epicatechin units along with smaller amounts of (+)-catechin, (-)-epicatechin gallate, and (-)-epigallocatechin. Flavanonols (astilbin) have been identified for the first time in stem and characterized by LC/MS and NMR. All phenolic compounds in grape stems were quantified by HPLC: quercetin 3-glucuronide was the most important, followed by catechin, caffeoyltartaric acid, and dihydroquercetin 3-rhamnoside (astilbin). Comparison was made of proanthocyanidin characteristics in different white and red grape varieties and also among parts of the cluster (skin, seed, and stem). Stem-condensed tannins were qualitatively intermediate between seed and skin but could not be differentiated between red and white varieties.     

Anthocyanin-flavanol condensation products from black currant (Ribes nigrum L.)

Putative flavanol-anthocyanin condensation products were detected in a polyphenol-rich concentrate from black currant (Ribes nigrum L.). These compounds had UV-vis spectra similar to those of delphinidin-3-O-rutinoside and cyanidin-3-O-rutinoside, but eluted before all previously described anthocyanins on reversed phase HPLC. Mass spectrometric data indicated that they were rutinoside derivatives of novel aglycons 304 amu greater than delphinidin and cyanidin, respectively. The compounds were partly purified by semipreparative HPLC and gave MS and MS2 spectra consistent with anthocyanin rutinosides covalently linked to epigallocatechin or gallocatechin. These compounds are similar in structure to compounds thought to influence color and quality in red wines and strawberry juice products. There was also evidence for the presence of a range of other flavanol-anthocyanin condensation products. The compounds were present at differing levels in juices of 10 black currant varieties, which were roughly correlated to the content of the parent anthocyanins. The flavanol-anthocyanin products were present in polyphenol-enriched concentrates obtained by solid phase extraction, in commercially produced concentrates, and in fresh extracts of black currants. This suggests that the compounds were not artifacts formed during concentration or purification. However, differences in their comparative contents may be related to the lability of the parent anthocyanins during processing. Although present at low levels, the flavanol-anthocyanin products may influence color or quality parameters of black currant juices, and they may confer enhanced stability to the biological activities reported for their anthocyanin parents.     

Ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from Ribes nigrum

Small fruits such as berries have low energy contents, but high contents of vitamins, micronutrients, and dietary fibers and constitute a good source of natural antioxidant compounds that are important constituents of the human diet. This study identified a large number of compounds in an extract of black currant showing high antioxidant activity and compared their profile in various parts of the plants (leaves, buds, and berries). If it was known that berries contained very high levels of natural phenolic compounds, this study showed that leaves and buds could also be considered good sources of natural antioxidants. Indeed, they contained high amounts of phenolic acids, flavonoids, and carotenoids. An acetone mixture can extract several classes of phenolic compounds with a good yield of flavonols, flavan-3-ols, and anthocyanins.     

UHPLC-PDA-ESI/HRMS/MS(n) analysis of anthocyanins, flavonol glycosides, and hydroxycinnamic acid derivatives in red mustard greens (Brassica juncea Coss variety)

An UHPLC-PDA-ESI/HRMS/MS(n) profiling method was used for a comprehensive study of the phenolic components of red mustard greens ( Brassica juncea Coss variety) and identified 67 anthocyanins, 102 flavonol glycosides, and 40 hydroxycinnamic acid derivatives. The glycosylation patterns of the flavonoids were assigned on the basis of direct comparison of the parent flavonoid glycosides with reference compounds. The putative identifications were obtained from tandem mass data analysis and confirmed by the retention time, elution order, and UV-vis and high-resolution mass spectra. Further identifications were made by comparing the UHPLC-PDA-ESI/HRMS/MS(n) data with those of reference compounds in the polyphenol database and in the literature. Twenty-seven acylated cyanidin 3-sophoroside-5-diglucosides, 24 acylated cyanidin 3-sophoroside-5-glucosides, 3 acylated cyanidin triglucoside-5-glucosides, 37 flavonol glycosides, and 10 hydroxycinnamic acid derivatives were detected for the first time in brassica vegetables. At least 50 of them are reported for the first time in any plant materials.     

High-performance liquid chromatography analysis of black currant (Ribes nigrum L.) fruit phenolics grown either conventionally or organically

Black currants (Ribes nigrum L.) contain a diverse range of phenolics and possess a high antioxidant activity, which makes them an interesting target for the functional food industry. In this study, phenolic profiles of organically and conventionally grown black currant fruits, collected from commercial farms within a climatically similar area, were compared. Compounds were identified using UV/vis and mass spectroscopy techniques and quantified with high-performance liquid chromatography equipped with UV/vis detection. Several different conjugates of hydroxycinnamic acids, flavonols, and anthocyanins were quantified. Statistically significant differences between farms were found for almost all compounds. Differences between the highest and the lowest measured values of major phenolic compounds of different phenolic classes ranged from 24 to 77%. Principal component analysis quite effectively separated farms from each other but did not cluster them according to cultivation technique. Thus, it was concluded that the biochemical quality of organically grown black currant fruits does not differ from those grown conventionally.