Flavonoids in pigmented orange juice and second-pressure extracts

Flavanone glycosides (FGs) and polymethoxylated flavones (PMFs) have been studied in pigmented orange (Citrus sinensis) juices and second-pressure extracts (SPEs) by high-performance liquid chromatography and diode array detector. Detection was performed simultaneously at two different wavelengths: 278 nm (for determination of FGs) and 325 nm (for determination of PMFs). Qualitative distribution patterns of FGs and PMFs in juices and SPEs were similar, although the quantitative results are quite different. An increased narirutin/hesperidin ratio after centrifugation and the presence of high amounts of PMFs in SPEs, which remain unchanged after centrifugation, were observed. Therefore, a simple and affordable procedure to distinguish an orange juice from SPEs was proposed.     

Flavonoids detection by HPLC-DAD-MS-MS in lemon juices from Sicilian cultivars

High-performance liquid chromatography-diode array detector (HPLC-DAD) coupled with electronspray mass spectrometry (ESI-MS) was used to detect the flavonoid profile in lemon juices obtained from the main Sicilian cultivars (Femminello comune, Monachello, and Interdonato). Significant amounts of an unusual constituent were found in the lemon juice of the above-mentioned cultivars together with eriocitrin, hesperidin, and diosmin. Following purification by preparative HPLC, the structure was assigned as 6,8-di-C-glucopyranosyldiosmetin by means of DAD-UV, ESI-MS-MS, and (1)H NMR analysis. Three other minor components were also detected. One of these presented a flavone nature, and spectral data and literature references both suggested a 6,8-di-C-glucopyranosylapigenin structure. The different contents of eriocitrin, hesperidin, diosmin, and above minor components in the cultivars allow juices to be readily differentiated.     

Phenolic compounds and antioxidant activity of extracts from ultrasonic treatment of Satsuma Mandarin (Citrus unshiu Marc.) peels

Ultrasound-assisted extraction (UAE) was used to extract phenolic compounds from Satsuma mandarin ( Citrus unshiu Marc.) peels (SMP), and maceration extraction (ME) was used as a control. The effects of ultrasonic time (10, 20, 30, 40, 50, and 60 min), temperature (15, 30, and 40 degrees C), and ultrasonic power (3.2, 8, 30, and 56 W) on phenolic compounds were investigated. High-performance liquid chromatography (HPLC) coupled with a photodiode array (PDA) detector was used for the analysis of phenolic acids after alkaline hydrolysis (bound phenolic acids) and flavanone glycosides. The contents of seven phenolic acids (caffeic acid, p-coumaric acid, ferulic acid, sinapic acid, protocatechuic acid, p-hydroxybenzoic acid, and vanillic acid) and two flavanone glycosides (narirutin and hesperidin) in extracts obtained by ultrasonic treatment were significantly higher than in extracts obtained by the maceration method. Moreover, the contents of extracts increased as both treatment time and temperature increased. Ultrasonic power had a positive effect on the contents of extracts. However, the phenolic acids may be degraded by ultrasound at higher temperature for a long time. For example, after ultrasonic treatment at 40 degrees C for 20 min, the contents of caffeic acid, p-coumaric acid, ferulic acid, and p-hydroxybenzoic acid decreased by 48.90, 44.20, 48.23, and 35.33%, respectively. The interaction of ultrasonic parameters probably has a complex effect on the extracts. A linear relationship was observed between Trolox equivalent antioxidant capacity (TEAC) values and total phenolic contents (TPC); the correlation coefficient, R(2), is 0.8288 at 15 degrees C, 0.7706 at 30 degrees C, and 0.8626 at 40 degrees C, respectively. The data indicated that SMPs were rich sources of antioxidants. Furthermore, UAE techniques should be carefully used to enhance the yields of phenolic acids from SMPs.     

Flavonoid distribution during the development of leaves, flowers, stems, and roots of Rosmarinus officinalis. postulation of a biosynthetic pathway

The distribution of seven flavonoids, eriocitrin, luteolin 3'-O-beta-d-glucuronide, hesperidin, diosmin, isoscutellarein 7-O-glucoside, hispidulin 7-O-glucoside, and genkwanin, has been studied in Rosmarinus officinalis leaves, flowers, stems, and roots during plant growth. The maximum level reached by luteolin 3'-O-beta-d-glucuronide in leaves during June-August suggests the existence of a delay between the activation of the enzymes involved in the flavanone and flavone biosynthesis. The presence of hesperidin and diosmin in the vascular system is significant, and hesperidin shows even higher levels than the phenolic diterpenes and rosmarinic acid. The distribution of flavonoids observed in R. officinalis suggests a functional and structural relationship between phytoregulators and flavonoids, where flavonoids would be "protectors" of the activity of phytoregulators. A hypothesis for the general pathway of biosynthesis of these compounds in plants of the family Labiatae is proposed.     

Role of the cultivar in choosing Clementine fruits with a high level of health-promoting compounds

Thirteen cultivars and two hybrids of Clementine fruits (Citrus clementina Hort. Ex. Tan) cultivated in Italy were characterized according to pH, titratable acidity, total soluble solids, total polyphenols, carotenoids, vitamin C, hesperidin, rutin, narirutin and naringin and radical scavenging activity. The presence of rutin in Clementine fruit juice is reported for the first time here. The results indicated that all chemical parameters statistically differentiated each cultivar (P < 0.001). In particular, principal component analysis showed a clear discrimination of five cultivars from all the other varieties based on vitamin C and total polyphenols for the Caffin cultivar, which showed also the highest antioxidant activity; narirutin for the Etna hybrid cultivar; hesperidin, rutin and total soluble solids for the SRA 89 cultivar; and naringin, hesperidin and rutin for the Esbal cultivar. Moreover, the Mandalate hybrid cultivar showed the lowest antioxidant activity as well as vitamin C and total polyphenols content, while titratable acidity and naringin level were the highest. The antioxidant activity assessed in all the fruits was closely correlated with vitamin C and total polyphenols content, rather than with the flavonoid compounds.     

Measurement of food flavonoids by high-performance liquid chromatography: A review

The flavonoids are plant polyphenols found frequently in fruits, vegetables, and grains. Divided into several subclasses, they include the anthocyanidins, pigments chiefly responsible for the red and blue colors in fruits, fruit juices, wines, and flowers; the catechins, concentrated in tea; the flavanones and flavanone glycosides, found in citrus and honey; and the flavones, flavonols, and flavonol glycosides, found in tea, fruits, vegetables, and honey. Known for their hydrogen-donating antioxidant activity as well as their ability to complex divalent transition metal cations, flavonoids are propitious to human health. Computer-controlled high-performance liquid chromatography (HPLC) has become the analytical method of choice. Many systems have been developed for the detection and quantification of flavonoids across one, two, or three subclasses. A summary of the various HPLC and sample preparation methods that have been employed to quantify individual flavonoids within a subclass or across several subclasses are tabulated in this review.     

Isolation of some glycosides as aroma precursors in young leaves of Japanese pepper (Xanthoxylum piperitum DC.).

To clarify the formation mechanism for the major alcoholic aroma compounds in young leaves of Japanese pepper, the glycosides were isolated as aroma precursors. The presence of glycosides of the main alcoholic aroma constituents was indirectly determined by enzymatic hydrolysis and trifluoroacetylation (TFA) of the glycoside-containing fraction. After Amberlite XAD-2 column chromatography, ODS flash chromatography, and high-performance liquid chromatography (HPLC), two new compounds, namely, (3S,6S)-cis-linalool-3,7-oxide beta-D-glucopyranoside and 2-methylpropanyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside, were isolated. In addition, (3S,6R)-cis-linalool-3,6-oxide beta-D-glucopyranoside, which absolute configuration was the first determined, and six known glycosides, citronellyl beta-D-glucopyranoside, linalyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside, (Z)-3-hexenyl beta-D-glucopyranoside, benzyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside, dendranthemoside A, and 3,6-dihydroxy-5,6-dihydro-beta-ionol 9-beta-D-glucopyranoside, were isolated. All of these glycosides were isolated for the first time from the leaves of Japanese pepper. Their structures were established on the basis of spectral data and chemical evidence. The ratios of stereoisomers of the aglycon moieties of citronellyl beta-D-glucopyranoside and linalyl 6-O-beta-D-apiofuranosyl-beta-D-glucopyranoside were investigated by a chiral GC analysis and compared with those of free citronellol and linalool in the aroma concentrate.     

Bioevaluation of human serum albumin-hesperidin bioconjugate: insight into protein vector function and conformation

Hesperidin is a flavanone glycoside widely available for dietary intake in citrus fruits or citrus fruit derived products; however, exhaustive and reliable data are scarcely available for biological activity when it exerts protective health effects in humans. The principal intent of this work is to check binding domain and structural changes of human serum albumin (HSA), the primary carrier of flavonoids, in blood plasma association with hesperidin by employing molecular modeling, steady state and time-resolved fluorescence, and circular dichroism (CD) methods. From molecular modeling simulations, subdomains IIA and IIIA, which correspond to Sudlow's sites I and II, respectively, were earmarked to possess affinity for hesperidin, but the affinity of site I with flavanone is greater than that of site II. This corroborates the site-specific probe and hydrophobic 8-anilino-1-naphthalenesulfonic acid (ANS) displacement results placing the hesperidin at warfarin-azapropazone and indole-benzodiazepine sites. Steady state and time-resolved fluorescence manifested that static type, due to HSA-hesperidin complex formation (1.941 × 10(4) M(-1)), is the operative mechanism for the diminution in the tryptophan (Trp)-214 fluorescence. Moreover, via alterations in three-dimensional fluorescence and CD spectral properties, we can securely draw the conclusion that the polypeptide chain of HSA is partially destabilized after conjugation with hesperidin. We anticipate that this study can provide better knowledge of bioavailability such as absorption, biodistribution, and elimination, of hesperidin in vivo, to facilitate the comprehension of the biological responses to physiologically relevant flavanones.     

Varietal and interspecific influence on micronutrient contents in citrus from the Mediterranean area

To specify the genotypic variation of Mediterranean Citrus juices, the contents of carotenoids, flavonoids, and vitamin C were determined by high-performance liquid chromatography. A selection of orange varieties and Mandarin species from the Mediterranean area (Citrus sinensis, Citrus deliciosa Ten, and Citrus clementina Hort. ex Tan) was evaluated using carotenoid profiles and flavanones contents. Among the eight varieties of orange (Salustiana, Hamlin, Shamouti, Pera, Valencia, Maltaise, Sanguinelli, and Cara-cara) and two Mandarin species, only three cultivars (Pera, Sanguinelli, and Shamouti) and the two Mandarin species displayed a high content of vitamin A (374, 381, and 272 ER L(-1) for the three orange cultivars and 1156 and 960 retinol equivalent (RE) L(-1) for the Mandarins) due to a high content of beta-cryptoxanthin. These same Citrus were also rich in hesperidin (502, 537, 552, 767, and 754 mg L(-1), respectively). Principal component analysis allowed the Mediterranean orange varieties and Mandarin species to be differentiated on the basis of nutritional criteria. Strong correlations were observed between beta-cryptoxanthin and hesperidin (r = 0.92) and between beta-cryptoxanthin and beta-carotene (r = 0.98). In contrast, vitamin C content was not correlated with carotenoids and flavanone glycosides. The Mandarin and orange group was quite distinct. The orange varieties could be divided in two groups. In addition, a diversity tree allowed a genetic approach to differentiating Citrus cultivars on the basis of Euclidian distances. This representation showed that the hybrid Clementine was nearer to its parent Mandarin than to its parent orange, suggesting that beta-cryptoxanthin was a dominant genetic factor. With regard to vitamin A, Mandarin and its hybrid Clementine appeared to be the best Citrus species.     

Citrus flavanones: what is their role in cardiovascular protection?

Flavanones, including hesperidin and naringin, are polyphenolic compounds highly and almost exclusively present in citrus. Epidemiological studies reported an inverse relationship between their intake and the risk of cardiovascular diseases. Clinical and experimental data further showed their antihypertensive, lipid-lowering, insulin-sensitizing, antioxidative, and anti-inflammatory properties, which could explain their antiatherogenic action in animal models. Although flavanones may be promising compounds that are particularly active in cardiovascular disease prevention, clinical data are still scarce and most in vitro data have been obtained under nonphysiologically relevant conditions. Moreover, the mechanisms responsible for flavanone action are not fully elucidated. Therefore, further research is needed to better evaluate and understand the protective effects of flavanones in cardiovascular diseases.     

Phenols in citrus peel byproducts. Concentrations of hydroxycinnamates and polymethoxylated flavones in citrus peel molasses.

In addition to the main flavanone glycosides (i.e., hesperidin and naringin) in citrus peel, polymethoxylated flavones and numerous hydroxycinnamates also occur and are major phenolic constituents of the molasses byproduct generated from fruit processing. Although a small number of the hydroxycinnamates in citrus occur as amides, most occur as esters and are susceptible to alkaline hydrolysis. This susceptibility to alkaline hydrolysis was used in measuring the concentrations of hydroxycinnamates in citrus peel molasses. The highest concentrations of hydroxycinnamates occurred in molasses of orange [C. sinensis (L.) Osbeck] and tangerine (C. reticulata Blanco.) compared to grapefruit (C. paradisi Macf.) and lemon [C. limon (L.) Burm.]. Concentrations of two phenolic glucosides, phlorin (phloroglucinol-beta-O-glucoside) and coniferin (coniferyl alcohol-4-beta-O-glucoside), were also measured. Measurements of the polymethoxylated flavones in molasses from several tangerine and orange varieties showed that these compounds occurred in the highest amounts in Dancy tangerine, whereas samples from two other tangerine molasses contained significantly lower levels, similar to those in the molasses samples from late- and early/mid-season oranges.     

Changes in the levels of polymethoxyflavones and flavanones as part of the defense mechanism of Citrus sinensis (cv. Valencia Late) fruits against Phytophthora citrophthora

Phytophthora citrophthora causes serious losses in Citrus fruits through brown rot lesion. The effect of infection with P. citrophthora on Citrus sinensis (cv. Valencia Late) fruits was studied, with particular reference to the levels of the flavanones hesperidin and isonaringin and the polymethoxyflavones sinensetin, nobiletin, tangeretin, and heptamethoxyflavone, because flavonoids are most probably involved as natural defense or resistance mechanisms in this genus. Changes in the levels of these flavonoids were detected after infection. The hesperidin and isonaringin contents fell by 13 and 67%, respectively, whereas the contents of their corresponding aglycons, hesperetin and naringenin, increased, suggesting the hydrolyzing effect of this fungus on the glycosylated flavanones. The heptamethoxyflavone, nobiletin, sinensetin, and tangeretin levels increased by 48, 28, 26, and 24%, respectively. The in vitro study revealed that these compounds acted as antifungal agents, the most active being the aglycons (naringenin and hesperetin), followed by the polymethoxyflavones and flavanone glycosides. The participation of these flavonoids in the defense mechanism of this Citrus species is discussed.     

Liquid chromatographic methodology for the characterization of orange juice

Liquid chromatographic (LC) methodology potentially useful for the characterization of orange juice, with particular regard to detecting adulteration of orange juice by computer pattern recognition analysis, has been developed. After dilution with methanol the juice is extracted with hexane to remove the carotenoids, which are chromatographed on a C18 column with an acetonitrile-methanol-methylene chloride mobile phase and detection at 450 nm. Further extraction of the juice with methylene chloride isolates the methoxylated flavones, which are chromatographed by reverse phase LC with an acetonitrile-methanol-water mobile phase and detection at 280 nm. The flavanone glycosides remaining in solution are chromatographed on a C18 column with an acetonitrile-water mobile phase and detection at 280 nm. The precisions of the heights of the 32 LC peaks selected for pattern recognition analysis were determined from 5 replicate analyses of a single juice. Coefficients of variation of the replicates ranged from 0.3 to 4.5%, with an average of 2.1%. Adulteration of products with sodium benzoate-fortified pulpwash or grapefruit juice can be detected by this method. Pattern recognition analysis of the data obtained for 80 authentic and 19 adulterated orange juices showed that the method is potentially useful for distinguishing between authentic and adulterated products.     

HL-60 differentiating activity and flavonoid content of the readily extractable fraction prepared from citrus juices

Citrus plants are rich sources of various bioactive flavonoids. To eliminate masking effects caused by hesperidin, naringin, and neoeriocitrin, the abundant flavonoid glycosides which make up 90% of the conventionally prepared sample, the readily extractable fraction from Citrus juice was prepared by adsorbing on HP-20 resin and eluting with EtOH and acetone from the resin and was subjected to HL-60 differentiation assay and quantitative analysis of major flavonoids. Screening of 34 Citrus juices indicated that King (C. nobilis) had a potent activity for inducing differentiation of HL-60, and the active principles were isolated and identified as four polymethoxylated flavonoids, namely, nobiletin, 3,3',4',5,6,7, 8-heptamethoxyflavone, natsudaidain, and tangeretin. HPLC analysis of the readily extractable fraction also indicated that King contained high amounts of these polymethoxylated flavonoids among the Citrus juices examined. Principal component and cluster analyses of the readily extractable flavonoids indicated peculiarities of King and Bergamot.     

Antioxidant activity of pomegranate juice and its relationship with phenolic composition and processing

The antioxidant activity of pomegranate juices was evaluated by four different methods (ABTS, DPPH, DMPD, and FRAP) and compared to those of red wine and a green tea infusion. Commercial pomegranate juices showed an antioxidant activity (18-20 TEAC) three times higher than those of red wine and green tea (6-8 TEAC). The activity was higher in commercial juices extracted from whole pomegranates than in experimental juices obtained from the arils only (12-14 TEAC). HPLC-DAD and HPLC-MS analyses of the juices revealed that commercial juices contained the pomegranate tannin punicalagin (1500-1900 mg/L) while only traces of this compound were detected in the experimental juice obtained from arils in the laboratory. This shows that pomegranate industrial processing extracts some of the hydrolyzable tannins present in the fruit rind. This could account for the higher antioxidant activity of commercial juices compared to the experimental ones. In addition, anthocyanins, ellagic acid derivatives, and hydrolyzable tannins were detected and quantified in the pomegranate juices.     

Identification and occurrence of the novel alkaloid pentahydroxypentyl-tetrahydro-beta-carboline-3-carboxylic acid as a tryptophan glycoconjugate in fruit juices and jams

The novel carbohydrate-derived beta-carboline, 1-pentahydroxypentyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid, was identified in fruit- and vegetable-derived products such as juices, jams, and tomato sauces. This compound occurred as two diastereoisomers, a cis isomer (the major compound) and a trans isomer, ranging from undetectable amounts to 6.5 microg/g. Grape, tomato, pineapple, and tropical juices exhibited the highest amount of this alkaloid (up to 3.8 mg/L), whereas apple, banana, and peach juices showed very low or nondetectable levels. This tetrahydro-beta-carboline was also found in jams (up to 0.45 microg/g), and a relative high amount was present in tomato concentrate (6.5 microg/g) and sauce (up to 1.8 microg/g). This beta-carboline occurred in fruit-derived products as a glycoconjugate from a chemical condensation of D-glucose and L-tryptophan that is highly favored at low pH values and high temperature. Production, processing treatments, and storage of fruit juices and jams can then release this beta-carboline. Fruit-derived products and other foods containing this compound might be an exogenous dietary source of this glucose-derived tetrahydro-beta-carboline.     

Use of natural and modified cyclodextrins as inhibiting agents of peach juice enzymatic browning

Although cyclodextrins (CDs) have been successfully used as antibrowning agents in different fruit juices, no research has studied the effect of these compounds on enzymatic browning in peach juice. In this paper, the color of fresh peach juice was evaluated in the presence of two types of natural (alpha-CD and beta-CD) and a modified (maltosyl-beta-CD) CD, and the effectiveness of these compounds as browning inhibitors was determined using the color space CIELAB system. Moreover, to clarify the mechanism by which CDs inhibit peach juice enzymatic browning, the process was kinetically modeled in the absence and presence of CDs using a colorimetric method; the apparent complexation constants between the mixtures of diphenols present in peach juice and some types of CD were calculated. The results show that the highest affinity constant was presented by alpha-CD (Kc = 18.31 mM-1) followed by maltosyl-beta-CD (Kc = 11.17 mM-1), whereas beta-CD was incapable of inhibiting peach juice enzymatic browning. Keywords: Cyclodextrin; browning; peach; juice; color; polyphenol oxidase.     

Racemization at C-2 of naringin in sour oranges with increasing maturity determined by chiral high-performance liquid chromatography

Naringin is the major flavanone-7-O-glycoside of sour orange, and it is mainly responsible for the bitter taste of the fruit. The relative content of (2S)- and (2R)-naringin in the albedo of sour oranges during maturation in the entire season was determined by normal phase HPLC using Chiralcel OD-H as chiral stationary phase and n-hexane/ethanol doped with 0.5% of TFA as mobile phase. HPLC traces were complicated by the presence of (2S)-neohesperidin, and a software-guided analysis was developed to assess the relative amount of the C-2 diastereomers of naringin. A sigmoid curve was obtained showing variation from 94% of (2S)-naringin in very immature fruits to 69.7% in mature fruit samples. Spontaneous epimerization of (2S)-naringin in the ethanolic solutions of the albedo was noted after prolonged keeping for less acid samples. The separation of the C-2 diastereomers of other flavanoid-7-O-glycosides present in Citrus (neoeriocitrin, neohesperidin, and eriocitrin) was also obtained by chiral HPLC and afforded the identification, including stereochemistry, of the eluting peaks in the HPLC traces of the albedo extracts.     

Determination of saccharin, sodium benzoate, and caffeine in beverages by reverse phase high-pressure liquid chromatography.

A reverse phase high-pressure liquid chromatographic method is presented for the simultaneous separation and determination of sacchrain, sodium benzoate, and caffeine in soft drinks, fruit juices, fruit cocktails, fruit punches, coffee, and artificial sweetener concentrates. Decarbonated soft drinks, fruit punches, and artificial sweetener concentrates are injected directly into the chromatograph. Fruit juices and coffee solutions require filtration through a 0.45 mum pore membrane filter prior to injection. Samples are eluted from a mu-Bondapak/C18 column with 5% glacial acetic acid and are quantitated with an ultraviolet detector. The results of saccharin, sodium benzoate, and caffeine determinations in 34 soft drinks (representing 11 manufacturers and 20 flavors); 8 fruit juices, cocktails, and punches; 7 coffees; and 6 artificial sweetener concentrates are presented. Average recoveries of saccharin, sodium benzoate, and caffeine from soft drinks are 99.0, 99.3, and 100.2%, respectively.     

Detection of sugar syrups in apple juice by delta(2)H per thousand and delta(13)C per thousand analysis of hexamethylenetetramine prepared from fructose

An improved procedure for determining (13)C and (2)H isotope ratios, using gas chromatography-isotope ratio mass spectrometry (GC-IRMS), has been developed for identifying the addition of low cost commercial sugar syrups to apple juices and related products. Isotopic techniques are commonly used to identify the addition of low cost sugars to fruit juices and are difficult to circumvent as it is not economically viable to change the isotopic ratios of the sugars. The procedure utilizes the derivative hexamethylenetetramine, which is produced through chemical transformation of a sugar degradation product and provides position-specific (13)C and (2)H ratios that relate to the parent sugar molecule. The new procedure has advantages over methods using nitro-sugar derivatives in terms of analysis time and sensitivity. The differences between the delta(2)H per thousand and delta(13)C per thousand values of the 100 authentic apple juices and beet and cane commercial sugar syrups permit their addition to be reliably detected.