Evaluation of phenolic compounds in commercial fruit juices and fruit drinks

The total phenolic content of 13 commercially available fruit juices and juice drinks, selected to represent the most popular juice flavors in the United Kingdom, were analyzed using the Folin-Ciocalteu assay. Individual phenolic compounds were identified and quantified using HPLC-PDA-MS2. The catechin content and degree of polymerization of proanthocyanidins were also analyzed. Purple grape juice contained the largest number of individual phenolic compounds and also the highest concentration of total phenolics. The main components were flavan-3-ols, anthocyanins, and hydroxycinnamates, which accounted for 93% of the total phenolic content. In contrast, white grape juice, which contained principally hydroxycinnamates, had the lowest total phenolic content. Antioxidant activity was measured using the ORAC and FRAP assays, and the data obtained were in broad agreement with total phenol content. In view of the recent findings of the Kame project indicating that long-term fruit juice consumption can provide protection against Alzheimer's disease (Dai et al. Am. J. Med. 2006, 379, 464-475), it is suggested that the protective effects may be enhanced by consumption of a combination of juices rich in phenolics and containing a diverse variety of individual phenolic compounds, namely, juices derived from purple grapes, grapefruit, cranberries, and apples.     

Phenolic profiles of raw apricots, pumpkins, and their purees in the evaluation of apricot nectar and jam authenticity

The possibility of proving the undeclared addition of pumpkin puree in apricot nectars and jams has been investigated by using the phenol compound fingerprint and sensory evaluation. The cheaper pumpkin admixtures in apricot nectars and jams could not be detected by the sensory evaluation, particularly if present in quantities of <15%. The lower admixtures of pumpkin puree in apricot nectars and jams could be detected by the presence of syringic acid, a phenolic compound characteristic of the investigated pumpkins (Cucurbita pepo cv. Gleisdorff and Table Gold, Cucurbita maxima cv. Turkinja, and Cucurbita moschata cv. Argenta). Syringic acid was isolated from pumpkin puree and determined by using HPLC with diode array detection. By using the phenolic profile, undeclared pumpkin admixture (> or =5%) in the apricot nectars and jams could be proven.     

Study of the organic acids composition of quince (Cydonia oblonga Miller) fruit and jam

The organic acids present in several samples of quince fruit (pulp and peel) and quince jam (homemade and industrially manufactured) were analyzed by HPLC. The sample preparation was simple, involving only extraction with methanol (40 degrees C) and filtration through a Sep-pack C18 cartridge. The chromatographic separation was achieved using an ion exclusion column, Nucleogel Ion 300 OA (300 x 7.7 mm), in conjunction with a column heating device at 30 degrees C. An isocratic elution with H(2)SO(4) 0.01 N as the mobile phase, with a flow rate of 0.1 mL/min, and UV detection at 214 nm were used. These analyses showed that all samples presented a similar profile composed of at least six identified organic acids: citric, ascorbic, malic, quinic, shikimic, and fumaric acids. Several samples also contained oxalic acid. This study suggests that the organic acids levels and ratios may be useful for the determination of percent fruit content of quince jams. The citric acid value can also be used in the differentiation of the type of manufacture of the commercial quince jams (homemade or industrially manufactured).     

Composition of phenolic compounds and antioxidant activity of commercial aqueous smoke flavorings

The antioxidant activity of 12 aqueous commercial smoke flavorings used in the food industry was determined by two methods: bleaching of the carotenoid crocin and scavenging of the DPPH radical. The reaction with the DPPH radical was evaluated by calculating the effective concentration (EC50) and the antiradical efficiency (AE). A gas chromatography-mass spectrometry method was, moreover, used for the determination of 2-methoxyphenols, 2,6-dimethoxyphenols, and dihydroxybenzenes. The methoxyphenols were extracted from the aqueous smoke by dichloromethane, and also the residue aqueous phase was analyzed to determine the more water-soluble dihydroxybenzenes. The recovery and the repeatability of the method are reported. The total phenolic concentrations of the smoke flavorings showed a wide range, from about 1000 to 25000 mg/kg. Considering the three classes of compounds, the concentrations were about 300-3000 mg/kg for the 2-methoxyphenols, 200-11000 mg/kg for the 2,6-dimethoxyphenols, and 140-10000 mg/kg for the dihydroxybenzenes. The range of the antioxidant activities of the smoke flavorings was wide, reflecting the wide range of the phenolic concentrations. Good correlations were obtained between the total phenolic concentration and the antioxidant activities determined by both the DPPH and crocin assays.     

Influence of jam processing on the radical scavenging activity and phenolic content in berries

Six selected phenolic aglycons (caffeic and ellagic acids, kaempferol, quercetin, myricetin, and morin) in nine types of berries, and their changes as influenced by jam processing, have been evaluated using optimized HPLC with diode-array detection. The berry samples, fresh and after jam processing, were analyzed, and the total amounts of selected phenolics as aglycons were identified and determined by acid hydrolysis. Their contents in fresh and jam samples did not indicate appreciable changes; therefore, the influence of jam processing on these selected phenolics in berries was suggested to be small, and was mostly present in berries as several conjugated forms that were glycosylated, esterified, etc., in the samples. The total phenolic content of each sample was also determined by the Folin-Ciocalteu method. The three samples of each berry, namely fresh, jam, and acid hydrolysate of the berry, had similar total phenolic contents. On the other hand, the scavenging effect on the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was measured, and acid hydrolysates showed stronger activity than that of the fresh and jam-processed samples for all of the berry types.     

Analysis of nonextractable phenolic compounds in foods: the current state of the art

More than 500 phenolic compounds have been reported as present in foodstuffs, and their intake has been related to the prevention of several chronic diseases. Most of the literature on phenolic compounds focuses on those present in the supernatant of aqueous-organic extractions: extractable phenolics. Nevertheless, significant amounts of phenolic compounds remain in the solid residues after such extractions. These nonextractable phenolics are mostly proanthocyanidins, phenolic acids, and hydrolyzable tannins that are closely associated with the food matrix. Studies of this fraction of dietary phenolic compounds are scarce, and the few there are usually refer to particular types of phenolics rather than to the fraction as a whole. The present review reports the state-of-the-art methods that currently exist for analyzing nonextractable phenolic compounds in foods.     

Free amino acid composition of quince (Cydonia oblonga Miller) fruit (pulp and peel) and jam

Twenty-one free amino acids present in several samples of quince fruit (pulp and peel) and quince jam (homemade and industrially manufactured) were analyzed by GC/FID. The analyses showed some differences between quince pulps and peels. Generally, the highest content in total free amino acids and in glycine was found in peels. As a general rule, the three major free amino acids detected in pulps were aspartic acid, asparagine, and hydroxyproline. For quince peels, usually, the three most abundant amino acids were glycine, aspartic acid, and asparagine. Similarly, for quince jams the most important free amino acids were aspartic acid, asparagine, and glycine or hydroxyproline. This study suggests that the free amino acid analysis can be useful for the evaluation of quince jam authenticity. It seems that glycine percentage can be used for the detection of quince peel addition while high alanine content can be related to pear addition.     

Analysis of antioxidative phenolic compounds in artichoke (Cynara scolymus L.)

Artichoke leaf is an herbal medicine known for a long time. A systematic antioxidant activity-directed fractionation procedure was used to purify antioxidative components from the aqueous methanol extractions of artichoke heads and leaves in this study. Seven active polyphenolic compounds were purified from artichoke, and structural elucidation of each was achieved using MS and NMR. Two of these compounds, apigenin-7-rutinoside and narirutin, were found to be unique to artichoke heads, this represents the first report of these compounds in the edible portion of this plant. The contents of these antioxidants and total phenols in dried artichoke samples from leaves and immature and mature heads of three varieties, Imperial Star, Green Globe, and Violet, were then analyzed and compared by colorimetric and validated HPLC methods. Significant differences by variety and plant organ were observed.     

Characterization of phenolic compounds in rooibos tea

Polyphenols present in rooibos, a popular herbal tea from Aspalathus linearis, were isolated in two steps. First, phenolic ingredients were separated by multilayer countercurrent chromatography (MLCCC). Preparative high-performance liquid chromatography (HPLC) was then applied to obtain pure flavonoids. The purity and identity of isolated compounds was confirmed by different NMR experiments, HPLC-diode array detector (DAD), or gas chromatography-mass spectrometry (GC-MS) analysis. This strategy proved to be valid to isolate material in up to gram quantities and to verify known and previously not published polyphenol structures. In addition the chemistry of dihydrochalcones and related intermediates was studied. The dihydrochalcone aspalathin was oxidized to the corresponding flavanone- C-glycosides (( R)/( S)-eriodictyol-6- C-beta- D-glucopyranoside and ( R)/( S)-eriodictyol-8- C-beta- D-glucopyranoside). Flavanone-6- C-beta- D-glucopyranosides were further degraded to flavones isoorientin and orientin.     

Correlations of the phenolic compounds and the phenolic content in some Spanish and French olive oils

The total content of phenolic compounds (TAP) in 29 different monocultivar olive oil samples from France (Aglandau and Tanche) and Spain (Cornicabra, Picual, and Verdial) was assessed by the colorimetric Folin-Ciocalteu method. Also, individual phenolic compounds were determined and quantified by liquid chromatography coupled to mass spectrometry (LC-MS). The French olive oil samples had a lower TAP compared to Spanish samples. The quantity of individual phenolics was similar except for pinoresinol, which was lower in the French olive oil samples. TAP moderately correlated to the sum of quantified compounds (r = 0.64 and p < 0.01) Partial least-squares (PLS) regression analysis emphasized the importance of hydroxytyrosol and the total amount of quantified phenolic compounds by LC-MS in the prediction of the total amount of phenolic compounds as determined by the Folin-Ciocalteu method. The amount of alpha-tocopherol was generally different among the cultivars (Tanche > Picual > Verdial > Aglandau > Cornicabra). Of all quantified phenolic compounds in French olive oil samples, only luteolin correlated well to the altitude of the olive orchards (r = 0.76, p < 0.01).     

Influence of the acetification process on phenolic compounds

Little is known about the change of phenolic compounds and total phenolic content by the acetification process. The aim of this study was to assess the contents of selected phenolic compounds of cider and red and white wines in comparison to phenolic profiles in corresponding vinegars by using a new HPLC method for the simultaneous separation and quantification of polar phenolic acids and less polar flavonoids. Identifications were made by retention times and by means of mass spectra. Additionally, total phenolic contents of wines and vinegars were determined photometrically. The decrease in total phenol content by the acetification process was highest for cider vinegars (40%) and lower for red and white wine vinegars (13 and 8%, respectively). A decrease in the contents of individual phenolic compounds of vinegars from white white and ciders was not observed. In contrast, the contents of individual phenolic compounds in red wine vinegar decreased approximately 50%.     

Biological activity of acetylated phenolic compounds

In recent years an effort has been made to isolate and identify biologically active compounds that are included in the Mediterranean diet. The existence of naturally occurring acetylated phenolics, as well as studies with synthetic ones, provide evidence that acetyl groups could be correlated with their biological activity. Platelet activating factor (PAF) is implicated in atherosclerosis, whereas its inhibitors seem to play a protective role against cardiovascular disease. The aim of this study was to examine the biological activity of resveratrol and tyrosol and their acetylated derivatives as inhibitors of PAF-induced washed rabbit platelet aggregation. Acetylation of resveratrol and tyrosol was performed, and separation was achieved by HPLC. Acetylated derivatives were identified by negative mass spectrometry. The data showed that tyrosol and its monoacetylated derivatives act as PAF inhibitors, whereas diacetylated derivatives induce platelet aggregation. Resveratrol and its mono- and triacetylated derivatives exert similar inhibitory activity, whereas the diacetylated ones are more potent inhibitors. In conclusion, acetylated phenolics exert the same or even higher antithrombotic activity compared to the biological activity of the initial one.     

Binding of selected phenolic compounds to proteins

In the context of this study, the noncovalent binding of selected phenolic compounds (chlorogenic, ferulic, and gallic acids, quercetin, rutin, and isoquercetin) to different proteins (human serum albumin, bovine serum albumin, soy glycinin, and lysozyme) was studied with direct (Hummel-Dreyer/size exclusion chromatography) and/or indirect methods (fluorescence absorbance properties of the binding components). In the latter case, the measurement of the phenol binding was achieved by exploiting the intrinsic fluorescence emission properties of quercetin as a probe. From the data obtained, the binding constants and the number of binding sites were calculated. The binding parameters were influenced by different factors, where, e.g., increasing temperature and ionic strength as well as decreasing pH cause a diminished binding. The structures of the proteins as determined by circular dichroism indicate changes in the tertiary structure with the secondary structure remaining intact.     

High-performance liquid chromatography-mass spectrometry profiling of phenolic compounds for evaluation of olive oil bitterness and pungency

Bitterness and pungency are important parameters for olive oil quality. Therefore, two instrumental methods for evaluation of these taste attributes were developed. The first one is based on the photometric measurement of total phenolic compounds content, whereas the second one is based on the semiquantitative evaluation of hydrophilic compounds by high-performance liquid chromatography-mass spectrometry (HPLC-MS). Evaluation of total phenolic compounds content was performed by a modified method for the determination of the K(225) value using a more specific detection based on the pH value dependency of absorbance coefficients of phenols at λ = 274 nm. The latter method was not suitable for correct prediction, because no significant correlation between bitterness/pungency and total phenolic compounds content could be found. For the second method, areas of 25 peaks detected in 54 olive oil samples by a HPLC-MS profiling method were correlated with the bitterness and pungency by partial least-squares regression. Six compounds (oleuropein aglycon, ligstroside aglycon, decarboxymethyl oleuropein aglycon, decarboxymethyl ligstroside aglycon, elenolic acid, and elenolic acid methyl ester) show high correlations to bitterness and pungency. The computed model using these six compounds was able to predict bitterness and pungency of olive oil in the error margin of the sensory evaluation (±0.5) for most of the samples.     

Effect of pH on the stability of plant phenolic compounds

It is not uncommon to treat plant-derived foods and feeds with alkali. Such exposure to high pH is being used to recover proteins from cereals and legumes, to induce the formation of fiber-forming meat analogue vegetable protein, for preparing peeled fruits and vegetables, and for destroying microorganisms. In addition to their profound effects on functional and nutritional properties in such foods, such treatments may also cause other side reactions, including the destruction of natural polyphenolic compounds. Because plants contain a large number of structurally different antioxidant, anticarcinogenic, and antimicrobial polyphenolic compounds, it is of interest to know whether such compounds are stable to heat and to high pH. In this model study, the stability of the following natural polyphenols to pH in the range 3-11 was studied with the aid of ultraviolet spectroscopy: caffeic acid, (-)-catechin, chlorogenic acid, ferulic acid, gallic acid, (-)-epigallocatechin, rutin, and the nonphenolic compound trans-cinnamic acid. This study demonstrates that caffeic, chlorogenic, and gallic acids are not stable to high pH and that the pH- and time-dependent spectral transformations are not reversible. By contrast, chlorogenic acid is stable to acid pH, to heat, and to storage when added to apple juice. (-)-Catechin, (-)-epigallocatechin, ferulic acid, rutin, and trans-cinnamic acid resisted major pH-induced degradation. The results are rationalized in terms of relative resonance stabilization of phenoxide ions and quinone oxidation intermediates. The possible significance of these findings to food chemistry and microbiology is discussed.     

Analysis of phenolic compounds in white rice, brown rice, and germinated brown rice

Two hydroxycinnamate sucrose esters, 6'-O-(E)-feruloylsucrose and 6'-O-(E)-sinapoylsucrose, were isolated from methanol extracts of rice bran. Soluble and insoluble phenolic compounds as well as 6'-O-(E)-feruloylsucrose and 6'-O-(E)-sinapoylsucrose from white rice, brown rice, and germinated brown rice were analyzed using HPLC. The results demonstrated that the content of insoluble phenolic compounds was significantly higher than that of soluble phenolics in rice, whereas almost all compounds identified in germinated brown rice and brown rice were more abundant than those in white rice. 6'-O-(E)-Feruloylsucrose (1.09 mg/100 g of flour) and 6'-O-(E)-sinapoylsucrose (0.41 mg/100 g of flour) were found to be the major soluble phenolic compounds in brown rice. During germination, an approximately 70% decrease was observed in the content of the two hydroxycinnamate sucrose esters, whereas free phenolic acid content increased significantly; the ferulic acid content of brown rice (0.32 mg/100 g of flour) increased to 0.48 mg/100 g of flour and became the most abundant phenolic compound in germinated brown rice. The content of sinapinic acid increased to 0.21 mg/100 g of flour, which is nearly 10 times as much as that in brown rice (0.02 mg/100 g of flour). In addition, the total content of insoluble phenolic compounds increased from 18.47 mg/100 g of flour in brown rice to 24.78 mg/100 g of flour in germinated brown rice. These data suggest that appropriate germination of brown rice may be a method to improve health-related benefits.     

Assessing antioxidant and prooxidant activities of phenolic compounds

Methods for determining primary antioxidant activity were evaluated. A beta-carotene bleaching method and a free radical method using 2, 2-diphenyl-1-picrylhydrazyl (DPPH(*)) were modified to rapidly test samples for potential antioxidant activity. Malonaldehyde production in a linoleic acid emulsion system assayed by an HPLC method was also used to determine antioxidant and prooxidant activities initiated by a metal catalyst (Cu(2+)). All methods were used to assess activity of selected phenolic compounds including several anthocyanidins/anthocyanins and selected berry extracts. Most phenolic compounds had prooxidant activity at low concentrations, unlike synthetic antioxidants (BHA and BHT). Compounds with similar structures exhibited comparable trends in antioxidant activity. Antioxidant activity usually increased with an increase in the number of hydroxyl groups and a decrease in glycosylation. The antioxidant activity of many phenolic compounds and extracts was comparable to those of synthetic antioxidants using the beta-carotene bleaching and HPLC methods.     

Nitrosation of phenolic compounds: inhibition and enhancement

The nitrosation of phenol, m-, o-, and p-cresol, 2,3-, 3,5-, and 2, 6-dimethylphenol, 3,5-di-tert-butylphenol, 2,4,6-trimethylphenol, o-chlorophenol, and o-bromophenol was studied. Kinetic monitoring of the reactions was accomplished by spectrophotometric analysis of the products at 345 nm. At pH > 3, the dominant reaction was C-nitrosation through a mechanism that appears to consist of an attack on the nitrosatable substrate by NO(+)/NO(2)H(2)(+), followed by a slow proton transfer. The finding of an isokinetic relationship supports the idea that the same mechanism operates throughout the series. The observed sequence of nitrosatable substrate reactivities is explained by (i) the preferred para-orientation of the hydroxyl group for the electrophilic attack of nitrosating agents, (ii) steric hindrance of alkyl substituents, which reduces or prevents attack by nitrosating agents, and (iii) the hyperconjugative effect of the methyl substituent, which causes electronic charge to flow into the aromatic nucleus, as well as the opposite electronic withdrawing effect induced by halogen substituents. The results show that potential nitrosation of widespread environmental species such as chlorophenols is negligible, but more attention should be paid to polyphenols with strongly nucleophilic carbon atoms.     

Antioxidant activity and phenolic compounds in selected herbs.

The antioxidant capacities (oxygen radical absorbance capacity, ORAC) and total phenolic contents in extracts of 27 culinary herbs and 12 medicinal herbs were determined. The ORAC values and total phenolic contents for the medicinal herbs ranged from 1.88 to 22.30 micromol of Trolox equivalents (TE)/g of fresh weight and 0.23 to 2.85 mg of gallic acid equivalents (GAE)/g of fresh weight, respectively. Origanum x majoricum, O. vulgare ssp. hirtum, and Poliomintha longiflora have higher ORAC and phenolic contents as compared to other culinary herbs. The ORAC values and total phenolic content for the culinary herbs ranged from 2.35 to 92.18 micromol of TE/g of fresh weight and 0.26 to 17.51 mg of GAE/g of fresh weight, respectively. These also were much higher than values found in the medicinal herbs. The medicinal herbs with the highest ORAC values were Catharanthus roseus, Thymus vulgaris, Hypericum perforatum, and Artemisia annua. A linear relationship existed between ORAC values and total phenolic contents of the medicinal herbs (R = 0.919) and culinary herbs (R = 0.986). High-performance liquid chromatography (HPLC) coupled with diode-array detection was used to identify and quantify the phenolic compounds in selected herbs. Among the identified phenolic compounds, rosmarinic acid was the predominant phenolic compound in Salvia officinalis, Thymus vulgaris, Origanum x majoricum, and P. longiflora, whereas quercetin-3-O-rhamnosyl-(1 --> 2)-rhamnosyl-(1 --> 6)-glucoside and kaempferol-3-O-rhamnosyl-(1 --> 2)-rhamnosyl-(1 --> 6)-glucoside were predominant phenolic compounds in Ginkgo biloba leaves.     

Antioxidant properties of phenolic compounds from Pelargonium reniforme

Flavonoids and hydrolyzable tannins isolated from Pelargonium reniforme were evaluated for their antioxidant ability using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical generating system and a luminol-dependent chemiluminescence assay. In both assays, the polyphenols tested showed higher radical scavenging activities than the reference antioxidant, ascorbic acid (IC50 2.6-32.9 microM vs 40.9 microM in the DPPH test, and 2-25 times stronger effects in the chemiluminescence assay). A comparison of the flavonoids and the tannins showed that the latter have more potential than the former. Structural requirements for marked antioxidant activities of hydrolyzable tannins were the presence of galloyl and hexahydroxydiphenoyl groups, and apparently carbonyl (ester) functionalities in oxidatively modified dehydrohexa-hydroxydiphenoyl moieties. For flavonoids, it appeared that a catechol (3',4'-dihydroxy) element in the B-ring were important determinants and that O-glycosides were more effective than flavone-based C-glucosyls. Conspicuously, introduction of a galloyl group significantly enhanced their potentials. The demonstrated marked antioxidant effects of the polyphenols provide a clue for beneficial effects of P. reniforme in the treatment of liver disorders among several ethnic groups in areas of southern Africa.