Distribution of flavonoids and furocoumarins in juices from cultivars of Citrus bergamia Risso

HPLC separation of flavonoids and furocoumarins in the crude juices of three cultivars of Citrus bergamia Risso ("Castagnaro", "Fantastico", and "Femminello") was carried out on a C18 reversed phase column. The analysis was performed in a single run using a DAD detector coupled with an ESI-MS-MS source. Two furocoumarins (bergapten and bergamottin) were detected and quantified simultaneously with the sixteen flavonoid components previously found in industrial bergamot juice. Full characterization of the furocoumarins was performed by (1)H NMR analysis on samples separated by means of preparative HPLC. The free-radical scavenging ability of cultivar juices was assessed by using DPPH radical. The data presented show that the "Femminello" cultivar, even though it is the least common of the three, is by far the richest in health-promoting bioactive compounds (both flavonoids and furocoumarins). Given the range of applications of furocoumarins, the preparative separation described herein is proposed as a simple and rapid method to obtain this class of compounds in good yield from crude juice.     

Purification and characterization of a beta-glucosidase from Citrus sinensis var. Valencia fruit tissue

A preliminary survey demonstrated activity for alpha-D-glucosidase, alpha-D-mannosidase, alpha-L-arabinosidase, beta-D-glucosidase, beta-D-xylosidase, and beta-D-galactosidase in orange fruit flavedo and albedo tissue. alpha-L-Rhamnosidase was not detected. Subsequently, a beta-glucosidase was purified from mature fruit rag tissue (composed of intersegmental septa, squeezed juice sacs, and fruit core tissue) of Citrus sinensis var. Valencia. The beta-glucosidase exhibited low levels of activity against p-nitrophenyl-beta-D-fucopyranoside (13.5%) and p-nitrophenyl-alpha-D-glucopyranoside (7.0%), compared to its activity against p-nitrophenyl-beta-D-glucopyranoside (pNPG, 100%). The enzyme was purified by a combination of ion exchange (anion and cation) and gel filtration (Superdex and Toyopearl HW-55S) chromatography. It has an apparent molecular mass of 64 kDa by denaturing electrophoresis or 55 kDa by gel filtration chromatography (BioGel P-100). Hydrolysis of pNPG demonstrated a pH optimum between 4.5 and 5.5. At pH 5.0 the temperature optimum was 40 degrees C. At pH 5.0 and 40 degrees C the K(m) for pNPG was 0.1146 mM and it had a V(max) of 5.2792 nkatal x mg(-1) protein (katal = 0.06 International Units = the amount of enzyme that produces, under standard conditions, one micromol of product per min). Of the substrates tested, the enzyme was most active against the disaccharide cellobiose (1-->4), but was not active against p-nitrophenyl-beta-D-cellobioside. High levels of activity also were observed with the disaccharides laminaribiose (1-->3), gentiobiose (1-->6), and sophorose (1-->2). Activity greater than that observed with pNPG was obtained with the flavonoids hesperetin-7-glucoside and prunin (naringenin-7-glucoside), salicin, mandelonitrile-beta-D-glucoside (a cyanogenic substrate), and sinigrin (a glucosinolate). The enzyme was not active against amygdalin, coniferin, or limonin glucoside.     

Separation and characterization of a salt-dependent pectin methylesterase from Citrus sinensis var. Valencia fruit tissue

A pectin methylesterase (PME) from sweet orange fruit rag tissue, which does not destabilize citrus juice cloud, has been characterized. It is a salt-dependent PME (type II) and exhibits optimal activity between 0.1 and 0.2 M NaCl at pH 7.5. The pH optimum shifted to a more alkaline range as the salt molarity decreased (pH 8.5-9.5 at 50 mM NaCl). It has an apparent molecular mass of 32.4 kDa as determined by gel filtration chromatography, an apparent molecular mass of 33.5 kDa as determined by denaturing electrophoresis, and a pI of 10.1 and exhibits a single activity band after isoelectric focusing (IEF). It has a K(m) of 0.0487 mg/mL and a V(max) of 4.2378 nkat/mg of protein on 59% DE citrus pectin. Deblocking the N-terminus revealed a partial peptide composed of SVTPNV. De-esterification of non-calcium-sensitive pectin by 6.5% increased the calcium-sensitive pectin ratio (CSPR) from 0.045 +/- 0.011 to 0.829 +/- 0.033 but had little, if any, effect on pectin molecular weight. These properties indicate this enzyme will be useful for studying the PME mode of action as it relates to juice cloud destabilization.     

Catechin contents of foods commonly consumed in The Netherlands. 2. Tea, wine, fruit juices, and chocolate milk

Catechins, compounds that belong to the flavonoid class, are potentially beneficial to human health. To enable an epidemiological evaluation of catechins, data on their contents in foods are required. HPLC with UV and fluorescence detection was used to determine the levels of (+)-catechin, (-)-epicatechin, (+)-gallocatechin (GC), (-)-epigallocatechin (EGC), (-)-epicatechin gallate (ECg), and (-)-epigallocatechin gallate (EGCg) in 8 types of black tea, 18 types of red and white wines, apple juice, grape juice, iced tea, beer, chocolate milk, and coffee. Tea infusions contained high levels of catechins (102-418 mg of total catechins/L), and tea was the only beverage that contained GC, EGC, ECg, and EGCg in addition to (+)-catechin and (-)-epicatechin. Catechin concentrations were still substantial in red wine (27-96 mg/L), but low to negligible amounts were found in white wine, commercially available fruit juices, iced tea, and chocolate milk. Catechins were absent from beer and coffee. The data reported here provide a base for the epidemiological evaluation of the effect of catechins on the risk for chronic diseases.     

Multielement analysis of argentinean lemon juices by instrumental neutronic activation analysis and their classification according to geographical origin

Multielement analysis of lemon juices from different Argentinean regions was carried out by instrumental neutronic activation analysis (INAA) with the aim at developing a reliable method in the traceability of the origin of lemon juices. This work presents a characterization of 44 lemon juice samples selected from three different geographical origins in the Northwest region of Argentina. Multivariate chemometric techniques such principal component analysis and lineal discriminant analysis (LDA) were used with the aim of classifying the juices and identifying the most significant parameters. Eleven elements were determined (Br, As, Na, Rb, La, Cr, Sc, Fe, Co, Zn, and Sb). The analytical method was validated by analyzing the standard reference material IAEA V-10 (hay powder); the results are within +/-10% of the reported values for the majority of the elements. Biplots of LDA scores for the INAA data illustrate clear separation between each sample.     

Technological classification of basque cider apple cultivars according to their polyphenolic profiles by pattern recognition analysis

The polyphenolic compositions of 31 Basque cider apple cultivars were determined in pulp, peel, and juice by high-performance liquid chromatography--diode array detection analysis of crude extracts and after thiolysis. Data sets, consisting of individual polyphenol concentrations, total procyanidin content, and the average degree of polymerization of procyanidins, were evaluated by multivariate chemometric techniques, to develop decision rules for classifying apple cultivars technologically into bitter and nonbitter categories. A preliminary study of the data structure was performed by cluster analysis and principal component analysis in each apple material. Bitter apple varieties presented higher contents of flavan-3-ols and/or dihydrochalcones than nonbitter cultivars. Different classification systems for the two categories on the basis of the chemical data were obtained applying several supervised pattern recognition procedures, such as linear discriminant analysis, K-nearest neighbors, soft independent modeling of class analogy, partial least-squares, and multilayer feed forward artificial neural networks. Excellent performance in terms of recognition and prediction abilities for both categories (100% of hits) was achieved in every case (pulp, peel, or juice). Polyphenolic profiles of apple pulp, peel, or juice provide enough information to develop classification criteria for establishing the technological group of apple cultivars (bitter or nonbitter).     

Isolation and characterization of a new compound from Prunus mume fruit that inhibits cancer cells

An active compound that inhibits cancer cells was isolated from the fruit of Prunus mume, and its structure and in vitro activities were characterized. The n-hexane fraction obtained from methanol extracts exhibited the strongest inhibitory effect on the growth of cancer cells. From the n-hexane fraction, a new compound named B-1 was purified through preparative thin-layer chromatography, ODS column chromatography, and reverse phase high-performance liquid chromatography and its structure was analyzed by fast atom bombardment mass spectrometry and 1H and 13C NMR. The molecular formula of B-1 was C19H22O6 {2-hydroxy-1-[(7-hydroxy-2-oxo-2H-chromen-6-yl)methyl]-2-methylpropyl-(2Z)-3-methyl-but-e-enoate:prunate}, and the IC50 value was in the range of 39-58 microg/mL in descending order of the cancer cell lines Hep-2, SW-156, HEC-1-B, and SK-OV-3. B-1 exhibited 81-96% inhibition at a concentration level of 100 microg/mL against all cells, based on an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. However, B-1 showed little effect against normal cells with only 23% or less growth inhibition at 100 microg/mL. Thus, B-1 has a highly specific inhibitory effect against cancer cells but little effect against normal cells. When the cancer cell lines Hep-2 and SK-OV-3 were incubated with B-1 for 72 h, most of the tested cells suffered strong growth inhibition. The compound has the potential to be developed as a nutraceutical.     

Modulation of the biosynthesis of some phenolic compounds in Olea europaea L. fruits: their influence on olive oil quality

The phenolic composition of olive fruits (Olea europaea L.) (cv. Picual, Villalonga, Alfafarenca, and Cornicabra) grown in different areas of Spain was studied by high performance liquid chromatography-mass spectrometry. Different levels of tyrosol, catechin, p-coumaric acid, rutin, luteolin, and oleuropein were observed in the different varieties analyzed. Treating the fruit with 0.3% Brotomax 50 days after anthesis had a beneficial effect on fruit size, oil content, levels of polyphenolic compounds, and Trolox-equivalent antioxidant activity (TEAC) in all the varieties analyzed.     

Cell wall polysaccharides from chalkumra (Benincasa hispida) fruit. Part I. Isolation and characterization of pectins

Pectic polysaccharides were obtained from chalkumra (Benincasa hispida) fruit by sequential extraction with ammonium oxalate (fraction BOX), dilute acid (fraction BHCl), and cold dilute alkali (fraction BOH). The highest yield of polysaccharides was obtained with oxalate and HCl. BOX was enriched in partly methyl-esterified galacturonic acid, whereas BHCl and BOH contained mostly galactose. All of the extracts showed similar elution patterns in size exclusion chromatography although the intrinsic viscosities (eta) were different (132 +/- 6, 100 +/- 5, and 285 + 10 mL/g for BOX, BHCl, and BOH, respectively). From fractionation by anion exchange chromatography, homogalacturonan (as seen from sugar analysis and Fourier transform infrared spectrum) accounted for more than half of BOX and 11% of BHCl. Methylation analyses and hydrolysis of BHCl with endo-beta-(1-->4)-d-galactanase showed the presence of beta-(1-->4)-d-galactan. The neutral galactan represented more than 76% of BHCl and approximately 40% of BOH. The other polysaccharides were complex galactans in BOH and an acidic arabinan (<1%) in BOX and BHCl.     

Antioxidant capacities,phenolic compounds,carotenoids, and vitamin C contents of nectarine,peach, and plum cultivars from California

Genotypic variation in composition and antioxidant activity was evaluated using 25 cultivars, 5 each of white-flesh nectarines, yellow-flesh nectarines, white-flesh peaches, yellow-flesh peaches, and plums, at the ripe (ready-to-eat) stage. The ranges of total ascorbic acid (vitamin C) (in mg/100 g of fresh weight) were 5-14 (white-flesh nectarines), 6-8 (yellow-flesh nectarines), 6-9 (white-flesh peaches), 4-13 (yellow-flesh peaches), and 3-10 (plums). Total carotenoids concentrations (in microg/100 g of fresh weight) were 7-14 (white-flesh nectarines), 80-186 (yellow-flesh nectarines), 7-20 (white-flesh peaches), 71-210 (yellow-flesh peaches), and 70-260 (plums). Total phenolics (in mg/100 g of fresh weight) were 14-102 (white-flesh nectarines), 18-54 (yellow-flesh nectarines), 28-111 (white-flesh peaches), 21-61 (yellow-flesh peaches), and 42-109 (plums). The contributions of phenolic compounds to antioxidant activity were much greater than those of vitamin C and carotenoids. There was a strong correlation (0.93-0.96) between total phenolics and antioxidant activity of nectarines, peaches, and plums.     

Differential response of two taro cultivars to aluminum: I. Plant growth

Abstract

Aluminum (Al) toxicity is one of the major factors limiting plant growth in acid soils. To determine the response of taro [Colocasia esculenta (L.) Schott] to Al‐toxicity, cultivars (cv.) Lehua maoli and Bun long were grown in hydroponic solution at six initial levels of Al (0, 110, 220, 440, 890, and 1330 uM Al). Increasing Al levels significantly depressed fresh and dry weights of taro leaf blades, petioles, and roots, as well as leaf areas and root lengths. No significant cultivar differences were found for plant dry weights. However, significant cultivar differences were found for expansion growth parameters, with cv. Lehua maoli exhibiting greater leaf fresh weights and root lengths in the presence of Al, compared to cv. Bun long. Apparently, differential response of taro cultivars to Al is related to the ability of the Al‐tolerant cultivar to maintain water uptake and cell expansion in the presence of Al. The initial solution Al level that resulted in the greatest separation of growth differences between taro cultivars in their response to Al was 890 μM Al.     

Resonance Raman quantification of nutritionally important carotenoids in fruits, vegetables, and their juices in comparison to high-pressure liquid chromatography analysis

A rapid nondestructive estimation of carotenoid levels in intact fruits and vegetables and their juices could have great value when selecting nutritionally valuable crops for further propagation and commercial use. Carotenoid levels of a variety of agricultural products and juices were measured using resonance Raman spectroscopy and compared to levels determined by extraction and high-pressure liquid chromatography. A strong correlation was observed between the two methods when evaluating juices and when comparing different strains of intact tomatoes at the same stage of ripening.     

Effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars

The purpose of the work was to investigate the effect of the maturation process of the olive fruit on the phenolic fraction of drupes and oils from Arbequina, Farga, and Morrut cultivars. The level in the phenolic content of olive drupes declines rapidly during the black maturation phase. A general decreasing trend was observed too in the phenolic content of olive oils during the ripening process in the three varieties studied. Important differences in the high-performance liquid chromatography profile between varieties were observed. These included the presence of very low amounts of lignans in olive oils proceeding from the Morrut cultivar, and the presence of three peaks after elution of 3,4-DHPEA-EDA in the Farga and Morrut cultivars, which could be used as differentiating parameters. Sensory profile differences were observed between olive cultivars and due to the ripening process.     

Quantitative fractionation of grape proanthocyanidins according to their degree of polymerization.

A method was developed for the fractionation of grape (seed or skin) proanthocyanidins according to their degree of polymerization. After precipitation in chloroform/methanol (75:25, v/v), the grape proanthocyanidins were deposited onto an inert glass powder column and sequentially dissolved in several fractions by increasing proportions of methanol in the solvent. Each fraction from each proanthocyanidin source was quantified and characterized after acidic degradation with phenylmethanethiol (i.e., thiolysis). The comparison of data from total extract and successive fractions showed that a quantitative separation was achieved so that estimation of polymer size distribution in relation to other compositional characteristics (proportions of prodelphinidin units, galloylation rate) was thus possible. Mean degree of polymerization of separated proanthocyanidins ranged increasingly from 4.7 to 17.4 in seed (8.1 for total extract) and from 9.3 to 73.8 in skin (34.9 for total extract). The method proposed is very interesting for the study of grape proanthocyanidins according to their degree of polymerization because it gives both qualitative and quantitative information especially on the highly polymerized forms, which were not fractionated by previous techniques.     

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.     

Chemical characterization of oak heartwood from Spanish forests of Quercus pyrenaica (Wild.). Ellagitannins, low molecular weight phenolic, and volatile compounds

The need for new sources of quality wood supply for cooperage has led to looking into the possibility of utilizing Quercus pyrenaica Wild. oak, a species native to the Iberian peninsula, as an alternative to other European (Quercus robur and Qurecus petraea) and American (Quercus alba) oaks. The low molecular weight phenolic composition, ellagitannins, and volatile compounds (including a wide range of compound families such as volatile phenols, furanic compounds, lactones, phenyl ketones, other lignin-derived compounds, and volatile compounds related to off-flavors) of green heartwood from Spanish forest regions were studied by HPLC and GC, in order to know its enological characteristics. The chemical composition of Q. pyrenaica is similar to that of other species commonly used in cooperage to make barrels, showing only quantitative differences that were more significant with respect to American than to French species. The four provenance regions studied showed similar chemical composition, with high variability among individuals, often higher than the variability among regions of provenance, but in line with that described in other European and American oak woods. Therefore, this species must be considered to be suitable for aging wine.     

Effect of gamma-irradiation on phenolic compounds and phenylalanine ammonia-lyase activity during storage in relation to peel injury from peel of Citrus clementina hort. Ex. tanaka

The influence of gamma-irradiation on the content of phenolic compounds was evaluated on Moroccan Citrus fruits (Citrus clementina Hort. ex. Tanaka) treated at a mean dose of 0.3 kGy and stored for 49 days at 3 degrees C. The results show that irradiation has enhanced the synthesis of total phenolic compounds and is correlated with phenylalanine ammonia-lyase activity (PAL) during storage. Accumulation of phenolic compounds in cells is demonstrated and may be explained by the enhancement of PAL activity. HPLC/UV (diode array detector) analysis demonstrated that hesperidin was the major flavanone and nobiletin and heptamethoxyflavone were the major polymethoxylated flavones. Hesperidin is also the major phenolic compound in clementines. Irradiation stimulates the biosynthesis of hesperidin after 14 days of storage, corresponding to the maximum of PAL activity. p-Coumaric acid was also identified, and its content was particularly high in irradiated fruits after 49 days of storage. Accumulation of flavonoids and p-coumaric acid could be related to a better resistance. The percentage of losses due to peel injury "pitting" during storage was between 1 and 5% after 49 days of storage. The connections between irradiation, enzyme activity, phenolic content, and peel injury are briefly discussed.     

Radical scavenging activities of peels and pulps from cv. Golden Delicious apples as related to their phenolic composition

The relationship between phenolic composition and radical scavenging activity of apple peel and pulp was investigated in fruit produced according to both organic and integrated agricultural methods. Apple tissue extracts were subjected to high-performance liquid chromatography separation, which showed that as compared with pulps, peels are richer in almost all of the quantified phenolics. Flavonols, flavanols, procyanidins, dihydrochalcones, and hydroxycinnamates were the identified phenolic classes in peel tissue, and the most abundant compounds were epicatechin, procyanidin B2, and phloridzin. Pulps were poorer in phytochemicals. Their major phenolics were procyanidins and hydroxycinnamates. Flavonols in amounts <20 mg kg(-1) fresh weight (fw) were also found. In both peels and pulps, integrated production samples were richer in polyphenols. Among the 14 compounds identified, only phloridzin had a tendency to appear higher in organic peels. The total antioxidant capacities (TAC) of extracts were evaluated using the 1,1-diphenyl-2-picrylhydrazyl radical assay and were expressed as Trolox equivalents. Integrated peels gave the highest TAC (18.56 mM kg(-1) fw), followed by organic peels (TAC = 14.96), integrated pulps (TAC = 7.12), and organic pulps (TAC = 6.28). In peels, the top contributors to the antioxidant activity were found to be flavonols, flavanols, and procyanidins, which accounted for about 90% of the total calculated activity whereas in pulps, the TAC was primarily derived from flavanols (monomers and polymers) together with hydroxycinnamates. A good correlation between the sum of polyphenols and the radical scavenging activities was found. Among the single classes of compounds, procyanidins (in peels and pulps) and flavonols (in peels) were statistically correlated to the TAC.     

Virgin olive oil volatile compounds from lipoxygenase pathway and characterization of some italian cultivars.

Fruits from seven different varieties of Olea europaea L., grown in the same environmental conditions, were harvested in two succeeding years at the same ripening degree and immediately processed. The oils obtained were submitted to gas chromatographic determination of the volatile compounds extracted by dynamic headspace technique. The results demonstrated that the accumulation of the different metabolites in the oils obtained from the various cultivars were strictly connected with the varietal parameter because of the enzyme differences genetically determined. This feature made possible the differentiation of the examined cultivars on the basis of the percent of each metabolite from the enzymatic transformation of 13-hydroperoxides of linolenic acid. Oils from Picual and Koroneiki varieties cultivated in Spain and Greece, respectively, showed contents of volatiles very similar to those detected in the oils of the same varieties cultivated in Italy, proving that they were not significantly influenced by the environmental conditions.