HPLC‐MS/MS analysis of anthocyanins in human plasma and urine using protein precipitation and dilute‐and‐shoot sample preparation methods,respectively

A high‐performance liquid chromatography tandem–mass spectrometry (HPLC‐MS/MS) method has been developed to analyze anthocyanins in urine and plasma to further understand their absorption, distribution, metabolism and excretion. The method employed a Synergi RP‐Max column (250 × 4.6 mm, 4 μm) and an API 4000 mass spectrometer. A gradient elution system consisted of mobile phase A (water–1% formic acid) and mobile phase B (acetonitrile) with a flow rate of 0.60 mL/min. The gradient was initiated at 5% B, increased to 21% B at 20 min, and then increased to 40% B at 35 min. The analysis of anthocyanins presents a challenge because of the poor stability of anthocyanins during sample preparation, especially during solvent evaporation. In this method, the degradation of anthocyanins was minimized using protein precipitation and dilute‐and‐shoot and sample preparation methods for plasma and urine, respectively. No interferences were observed from endogenous compounds. The method has been used to analyze anthocyanin concentrations in urine and plasma samples from volunteers administered saskatoon berries. Cyanidin‐3‐galactoside, cyanidin‐3‐glucoside, cyanidin‐3‐arabinoside, cyanidin‐3‐xyloside and quercetin‐3‐galactoside, the five major flavonoid components in saskatoon berries, were identified in plasma and urine samples.     

Polyphenolic Compounds of Crataegus Berry,Leaf, and Flower Extracts Affect Viability and Invasive Potential of Human Glioblastoma Cells

Crataegus contains numerous health-promoting compounds that are also proposed to have anti-cancer properties. Herein, we aimed at a contemporaneous evaluation of the effects of polyphenol-rich extracts of berries, leaves, and flowers of six Crataegus species on the viability and invasive potential on the highly aggressive human glioblastoma U87MG cell line. The treatment with the extracts evoked cytotoxic effects, with the strongest in the berry extracts. All extracts not only promoted the apoptosis-related cleavage of poly (ADP-ribose) polymerase 1 (PARP1) but also substantially inhibited the activity of pro-survival kinases, focal adhesion kinase (FAK), and protein kinase B (PKB; also known as Akt), thus indicating the suppression of proliferative and invasive potentials of the examined glioblastoma cells. The qualitative and quantitative characterization of the extracts’ content was also performed and revealed that amongst 37 polyphenolic compounds identified in the examined Crataegus extracts, the majority (29) was detected in berries; the leaf and flower extracts, exerting milder cytotoxic effects, contained only 14 and 13 compounds, respectively. The highest polyphenol content was found in the berries of C. laevigata x rhipidophylla x monogyna, in which flavan-3-ols and phenolic acids predominated. Our results demonstrated that a high content of polyphenolic compounds correlated with the extract cytotoxicity, and especially berries were a valuable source of compounds with anti-cancer potential. This might be a promising option for the development of an effective therapeutic strategy against highly malignant glioblastomas in the future.     

Anthocyanins as antimicrobial agents of natural plant origin

Anthocyanins are particularly abundant in different fruits, especially in berries. The beneficial effects of these compounds for human health have been known from at least the 16th century. Despite the great number of papers devoted to the different biological effects exerted by anthocyanins only a limited number of studies is focused on the antimicrobial activity of these compounds. Anthocyanin content of berry fruits varies from 7.5 mg/100 mg fresh fruit in redcurrant (Ribes rubum) up to 460 mg/100 g fresh fruit in chokeberry (Aronia melanocarpa). After consumption, anthocyanins are intensively metabolized, mainly in the intestines and liver. Glucorination, methylation and sulfation are the most typical metabolic reactions. Antimicrobial activity of crude extracts of plant phenolic compounds against human pathogens has been intensively studied to characterize and develop new healthy food ingredients as well as medical and pharmaceutical products. However, there is very little information available about the antimicrobial activity of the pure anthocyanins. In the last part of this review we present the collection of papers describing the anthocyanin profiles of different fruits (mainly berries) and the antimicrobial properties of the identified compounds. Generally, anthocyanins are active against different microbes, however Gram-positive bacteria usually are more susceptible to the anthocyanin action than Gram-negative ones. Mechanisms underlying anthocyanin activity include both membrane and intracellular interactions of these compounds. Antimicrobial activity of berries and other anthocyanin-containing fruits is likely to be caused by multiple mechanisms and synergies because they contain various compounds including anthocyanins, weak organic acids, phenolic acids, and their mixtures of different chemical forms. Therefore, the antimicrobial effect of chemically complex compounds has to be critically analyzed.     

Probiotic Yoghurts with Sea Buckthorn,Elderberry, and Sloe Fruit Purees

Elderberries, sea buckthorn, and sloe berries are fruits of wild-grown bushes, valued in folk medicine for their health-promoting properties but still rarely applied in food. The aim of the present study was to produce probiotic yoghurts with a 10% addition of sweetened purees prepared from elderberries (EPY), sea buckthorn (SBPY), and sloe berries (SPY) and to assess their chemical composition, acidity, content of polyphenols and anthocyanins, ferric reducing antioxidant power (FRAP) and antiradical power (ARP), level of starter microbiota, concentration of acetaldehyde and diacetyl, syneresis, instrumentally measured color and texture parameters, and sensory acceptance. The results were compared to those obtained for plain probiotic yoghurt (PPY) and the changes tracked during 1 month of cold storage at 2 week intervals. The addition of elderberry and sloe berries significantly increased the antioxidant capacity of probiotic yoghurts, probably due to a high content of polyphenols, especially anthocyanins. However, anthocyanins were more stable in the EPY when compared to the SPY. All yoghurt treatments were characterized by good sensory quality and viability of starter microorganisms, including probiotic strains during cold storage. Elderberries promoted the evolution of diacetyl in yoghurts during storage and, together with sloe berries, produced increased syneresis and the greatest changes in color profile compared to PPY.     

HPLC determination of the composition and stability of blackcurrant anthocyanins

High-performance liquid chromatography with UV and mass spectrometry detectors are used to monitor the composition and stability of anthocyanins in blackcurrants harvested from different Ribes nigrum breeds at various ripeness phases. The highest amounts of pigments are found in overripe berries. The concentration of anthocyanins is higher in the berries of late blackcurrant breeds (Vakariai and Ben Alder). Delphinidin- 3-rutinoside is the dominant component in the reddish color berries (onset of ripening), and cyanidin-3-rutinoside is a major pigment in the black ones (ripe berries). Studies of the effect of temperature and light on the stability of the main pigments in blackcurrants show that aqueous solution prepared from a dry colorant is more stable when compared with the liquid water and ethanol extracts of coloring substances. Cyanidin-3-rutinoside is found to be the most thermally stable anthocyanin.     

Anthocyanins HPLC-DAD and MS Characterization,Total Phenolics,and Antioxidant Activity of Some Berries Extracts

Extracts of indigenous wild blackberries, mulberries, bilberries, and blackthorns were analyzed for anthocyanin composition, anthocyanin content, total phenolics, and antioxidant capacity. Anthocyanins extraction with acidified methanol in ultrasonic condition (59 kHz, 60 min., 25°C) was carried out. The extracts were analyzed by high-performance liquid chromatography (HPLC) using a Dionex Ultimate 3000 apparatus equipped with photodiode array detector for qualitative characterization of the anthocyanins. The chromatograms revealed the presence of a large number of anthocyanins in fruits extracts: blackberries, 4 compounds; mulberries, 3 compounds; bilberries, 18 compounds; and blackthorns, 5 compounds. The most abundant anthocyanins were cyanidin-3-glucoside in blackberry, mulberry, and bilberry, and cyanidin-3-rutinoside in blackthorn extract. Structural information about anthocyanins was obtained by using a mass spectrometric method based on fully automated chip-nanoelectrospray ionization (nanoESI) high capacity ion trap (HCT). Anthocyanin content was quantified by the pH differential method and total phenolics were determined by Folin-Ciocalteu method. A Jasco V 530 UV-VIS spectrophotometer was used for absorbance measurements. The free radical scavenging activity of the berries extracts was performed by using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The reduction of DPPH was followed by a spectrophotometric method. Also, a correlation of the antioxidant capacities of the extracts with their anthocyanin content and total phenolics was attempted.     

Spectrum–effect relationship study between HPLC fingerprints and antioxidant of honeysuckle extract

Honeysuckle (Lonicera japonica flos) is a well‐known agent of edible and medicinal value in China and its antioxidative activity makes a major contribution to its dual use. However, the compounds responsible for its antioxidative activity are still unknown. In this study, 10 batches of honeysuckle were collected from different origins in China. The fingerprints were established by HPLC technique to investigate the compounds and a 1,1‐diphenyl‐2‐picrylhydrazyl (DPPH) radical scavenging activity assay was carried out to evaluate their antioxidant activity. partial least squares regression analysis was applied to set up the regression equation between DPPH radical scavenging activity and average peak area of common peaks of fingerprints. The results showed that peaks 10 (isochlorogenic acid B), 12 (isochlorogenic acid C), 11 (isochlorogenic acid A) and 9 (cynaroside) in the fingerprints were closely related to the antioxidant activity of 50% methanol extracts of honeysuckle. This study successfully established the spectrum–effect relationship between HPLC fingerprints and DPPH radical scavenging activity and provided a general model for exploring active components with a combination of chromatography and efficacy.     

Grapevine as a Rich Source of Polyphenolic Compounds

Grapes are rich in primary and secondary metabolites. Among the secondary metabolites, polyphenolic compounds are the most abundant in grape berries. Besides their important impacts on grape and wine quality, this class of compounds has beneficial effects on human health. Due to their antioxidant activity, polyphenols and phenolic acids can act as anti-inflammatory and anticancerogenic agents, and can modulate the immune system. In grape berries, polyphenols and phenolic acids can be located in the pericarp and seeds, but distribution differs considerably among these tissues. Although some classes of polyphenols and phenolic acids are under strict genetic control, the final content is highly influenced by environmental factors, such as climate, soil, vineyard, and management. This review aims to present the main classes of polyphenolic compounds and phenolic acids in different berry tissues and grape varieties and special emphasis on their beneficial effect on human health.     

Polyphenolic Constituents of Fruit Pulp of Euterpe oleracea Mart. (Açai palm)

The polyphenolic composition of Euterpe oleracea Mart. (Açai palm) fruit was investigated by HPLC-DAD-UV-Vis and HPLC-MS. Açai palm is widely diffused and cultivated in Amazon regions and especially in the Pará state (Brazil), because the pulp of the fruit is largely consumed as food. This study confirms the presence of cyanidin 3-O-glucoside and cyanidin 3-O-rutinoside as major anthocyanic compounds. Moreover, four main compounds were also identified for the first time, i.e. homoorientin, orientin, taxifolin deoxyhexose, and isovitexin. Traces of a methyl-derivative of homoorientin were also detected. The amount of total anthocyanins was 0.5 mg g^?1 of the dried pulp weight and the amount of the other flavonoids was 3.5 mg g^?1 of the dried pulp weight. No other reports on the presence of non-anthocyanic flavonoids in Euterpe oleracea Mart. have been found so far.     

Quality Characteristics and Anthocyanin Profiles of Different Vitis amurensis Grape Cultivars and Hybrids from Chinese Germplasm

To evaluate the important Vitis amurensis germplasm, the quality characteristics and anthocyanin profiles of the ripe berries of 20 V. amurensis grapes and 11 interspecific hybrids in two consecutive years were analysed. Compared with the V. vinifera grapes, V. amurensis grapes had small berries with low total soluble solids and high titratable acids, and were richer in phenolic compounds except for flanan-3-ols in their skins but had lower phenolic contents in their seeds and showed lower antioxidant activities. An outstanding feature of the V. amurensis grapes was their abundant anthocyanin contents, which was 8.18-fold higher than the three wine grapes of V. vinifera. The anthocyanin composition of V. amurensis was characterized by an extremely high proportion of diglucoside anthocyanins (91.71%) and low acylated anthocyanins (0.04%). Interestingly, a new type of speculated 3,5,7-O-triglucoside anthocyanins was first identified and only detected in V. amurensis grapes and hybrids. Based on the total phenolic and anthocyanin characteristics, V. amurensis grapes were set apart from V. vinifera cultivars and the interspecific hybrids, for the same qualities, fell between them, as assessed by principal component analysis.     

Monitoring of HPLC profiles of selected polyphenolic compounds in sea buckthorn (Hippophaë rhamnoides L.) plant parts during annual growth cycle and estimation of their antioxidant potential

Presented work summarizes the data about polyphenolic profiles in various plant parts (leaves, shoots, berries) of sea buckthorn (Hippophaë rhamnoides L.) during the annual growth cycle. A reversed-phase high performance liquid chromatography method (RP-HPLC) coupled with diode-array detection (DAD) was optimized for determination of catechin, epicatechin, gallic acid, p-coumaric acid, caffeic acid, ferulic acid, rutin (quercetin 3-rutinoside) and quercitrin (quercetin 3-rhamnoside). The content of these polyphenolic compounds was monitored in extracts of sea buckthorn plant samples from April to October. The total antioxidant activity was determined using scavenging of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonate) cation radical (ABTS^·+) and 1,1-diphenyl-2-picrylhydrazyl radical (DPPH^·). The total content of polyphenols was estimated by conventional spectrophotometric method using Folin-Ciocalteu reagent. The monitoring of temporal changes of selected polyphenolic compounds by RP-HPLC showed that catechin, epicatechin and gallic acid were the most abundant analytes in annual green shoots and leaves, and their content varied significantly during the studied period.      

Evaluation of polyphenolic profile and nutritional value of non-traditional fruit species in the czech republic - a comparative study

Dry matter, organic acids, ascorbic acid, minerals (nitrogen, phosphorus, potassium, calcium, magnesium, sodium) and polyphenolic profile of a number of non-traditional fruit species and their genotypes, namely blue honeysuckle (Lonicera spp.), Saskatoon berry (Amelanchier alnifolia), black mulberry (Morus nigra), Tomentosa cherry (Prunus tomentosa Thunb.) and jostaberry (Ribes nigrum x Grossularia uva-crispa) were investigated. The results showed that Lonicera genotypes displayed high levels of ascorbic acid and they were rich in minerals, with the cultivar 'Amfora' achieving the leading position in nitrogen, phosphorus and potassium content among all lesser known fruit species. Amelanchier cultivars represented a valuable source of ascorbic acid and calcium, 'Ti?ňovsky' and 'Smoky' together with Morus nigra 'Jugoslavska' accumulated the highest level of examined polyphenolic compounds. Regular consumption of studied less common fruit species can bring health benefits so they can represent a high potential value for fruit growers and in addition they can be utilised as functional foods.     

Variability of polyphenol compounds in Myrtus communis L. (Myrtaceae) berries from Corsica

Polyphenol compounds were extracted from Myrtus communis L. berries (Myrtaceae) by maceration in 70% ethanol and analysed by HPLC-DAD and electrospray mass spectrometry. The Myrtus berries were collected at maturity from seven localities on the island of Corsica (France) and the sampling was carried out during three years. The polyphenol composition of Corsican Myrtus berries was characterized by two phenolic acids, four flavanols, three flavonols and five flavonol glycosides. The major compounds were myricetin-3-O-arabinoside and myricetin-3-O-galactoside. Principal components analysis (PCA) is applied to study the chemical composition and variability of myrtle berries alcoholic extracts from the seven localities. Canonical analysis and PCA data distinguishes two groups of myrtle berries characterized by different concentrations of polyphenols according to soil and years of harvest. The variations in the polyphenol concentration were due to biotic and abiotic factors.     

Optimization of a new mobile phase to know the complex and real polyphenolic composition: towards a total phenolic index using high-performance liquid chromatography

An HPLC method is reported for the separation and quantification of five major polyphenolic groups found in fruits and related products: single ring phenolic acids (hydroxybenzoic acid and hydroxycinnamic acid derivatives), flavan-3-ols, flavonols, anthocyanins, and dihydrochalcones. A binary mobile phase consisting of 6% acetic acid in 2 mM sodium acetate aqueous solution (v/v, final pH 2.55) (solvent A) and acetonitrile (solvent B) was used. The use of sodium acetate was new and key to the near baseline separation of 25 phenolics commonly found in fruits. A photodiode array detector was used and data were collected at four wavelengths (280, 320, 360, and 520 nm). This method was sensitive and gave good separation of polyphenolics in apple, cherry, strawberry, blackberry, grape, apple juice, and a processing by-product. The improved separation has led to better understanding of the polyphenolic profiles of these fruits. Individual as well as total phenolic content was obtained, and the latter was close to and correlated well with that obtained by the Folin-Ciocalteu method (FC). The HPLC data can be used as a total phenolic index (TPI) for quantification of fruit phenolics, which is advantageous over the FC because it has more information on individual compounds.     

Anthocyanins profile,total phenolics and antioxidant activity of two Romanian red grape varieties: Fetească neagră and Băbească neagră (<Emphasis Type="Italic">Vitis vinifera</Emphasis>)

The phenolic composition of Feteasc? neagr?and B?beasc? neagr? grapes from Dealul Bujorului vineyard (south-east Romania) was studied using the spectrophotometric and high-performance liquid chromatographic (HPLC) methods. The results revealed significant differences between these cultivars. Total anthocyanins ranged from 0.22–5.98 mg g^?1 DW berries in B?beasc? neagr? grapes to 0.54–18.54 mg g^?1 DW berries in Feteasc? neagr?. Both cultivars were characterised by an interesting anthocyanin profile for winemaking with a prevalence of malvidin-3-glucoside. The skins and seeds both had small amounts of flavonoids. In contrast with B?beasc? neagr?, Feteasc? neagr? had more flavonoids in the seeds (69 %) than in the skins. Statistically significant correlations were observed between antioxidant activity and total anthocyanin content in both varieties. The antioxidant activity was also found to be highly correlated to the total phenolic compounds content.     

Differentiation of young red wines based on cultivar and geographical origin with application of chemometrics of principal polyphenolic constituents

Nineteen major polyphenolic phytochemicals including hydroxycinnamate derivatives, flavanols, flavonols, and anthocyanins, were determined in 40 experimental red wines employing HPLC-DAD. All wines analysed were young (non-aged), produced, and stored under identical conditions, in an effort to minimize the effect of oak wood and vinification technology. The data obtained from this examination composed the matrix for the implementation of chemometrics, which aimed at differentiating the wine samples on the basis of cultivar and geographical region of origin. Discriminant analysis performed at a 95% significance level revealed a very satisfactory categorization of the samples both in terms of cultivar and region of origin, thus illustrating the validity of major phenolic phytochemicals for studies pertaining to wine quality and authenticity.     

Ultrasound-Assisted Extraction of Anthocyanins Using Natural Deep Eutectic Solvents and Their Incorporation in Edible Films

Extracts rich in bioactive compounds added to edible films have allowed the development of active packaging that increases the shelf life of food. However, it is necessary to search for solvents that are nontoxic and not harmful to the environment, with natural deep eutectic solvents (NADES) being an attractive and easily synthesized alternative. This research aimed to design NADES by lyophilization to be used in the extraction of anthocyanins from the Chilean Luma chequen (Molina) A. Gray berry, and subsequently adding them to the matrix of edible ƙ-carrageenan films. For this purpose, ultrasound-assisted extraction (UAE) was used and the anthocyanin content was evaluated with the pH differential method. The antioxidant capacity of extracts was determined by DPPH assay and the antibacterial capacity by diffusion agar tests. The results obtained indicate that the designed NADES are efficient at extracting anthocyanins, reaching concentrations between 81.1 and 327.6 mg eq cyanidin 3-glucoside/100 g dw of L. chequen (Molina) A. Gray. The extracts reached inhibition diameters between 5 and 34 mm against Escherichia coli, Staphylococcus aureus, and Salmonella typhi strains. Once the extracts were incorporated into ƙ-carrageenan films, active edible films with antioxidant and antibacterial capacities were obtained.     

Polyphenolic profile by FIA/ESI/IT/MSn and antioxidant capacity of the ethanolic extract from the barks of Maytenus cajalbanica (Borhidi & O. Muñiz) Borhidi & O. Muñiz

Abstract

Medium and high polarity extracts from Maytenus species are known to contain polyphenolic compounds such as proanthocyanidins. The high polarity and structural complexity of these compounds make very difficult their isolation even by modern chromatographic techniques. Maytenus cajalbanica (Borhidi & O. Muñiz) Borhidi & O. Muñiz is endemic from Cuba. So far, there are reports neither of phytochemical work nor of biological evaluation of extracts from this subspecies. The goal of this work is to determine the polyphenolic profile and the antioxidant capacity of the ethanolic extract from the barks of Maytenus cajalbanica. FIA/ESI/IT/MSⁿ analysis allowed the identification of 5 flavan-3-ol monomers, 33 proanthocyanidins, 2 free flavonoids and their respective glycosides as major compounds of the ethanolic extract, which showed a strong radical scavenging capacity and a significant ferric reduction power. FIA/ESI/IT/MSⁿ technique led the rapid, effective and sensitive determination of the polyphenolic profile of Maytenus cajalbanica without previous separation.     

Improved high-performance liquid chromatography-diode array detection method for the determination of phenolic compounds in leaves and peels from different apple varieties

Hydroxycinnamic acids derivatives, monomeric and oligomeric flavan-3-ols, flavonols, and dihydrocalcones are four of the major polyphenolic groups found in apples leaves and peels. A simple extraction with minimal pre-treatment and a high-performance liquid chromatography-diode array detection determination are optimized and validated, in order to identify and quantitate the polyphenolic profile of leaves and peels of four apples varieties (Gala, Topaz, Golden Delicious, and Florina). The improved chromatographic method has led to better separation of some known polyphenols in a single course, and diode-array detection has been used for the previsional identification of some polyphenolic compounds not available as standards. Because the mobile phase and the chromatographic column are compatible with a mass spectrometer, this method could investigate the unknown flavanols, flavonols, hydrocinnamic acid derivatives, and chalcone-related compounds found in apple leaves and peel extracts analyzed.