The phytochemical analysis and antioxidant activity assessment of orange peel (Citrus sinensis) cultivated in Greece-Crete indicates a new commercial source of hesperidin

The flavonoid content of several methanolic extract fractions of Navel orange peel (flavedo and albedo of Citrus sinensis) cultivated in Crete (Greece) was first analysed phytochemically and then assessed for its antioxidant activity in vitro. The chemical structures of the constituents fractionated were originally determined by comparing their retention times and the obtained UV spectral data with the available bibliographic data and further verified by detailed LC-DAD-MS (ESI+) analysis. The main flavonoid groups found within the fractions examined were polymethoxylated flavones, O-glycosylated flavones, C-glycosylated flavones, O-glycosylated flavonols, O-glycosylated flavanones and phenolic acids along with their ester derivatives. In addition, the quantitative HPLC analysis confirmed that hesperidin is the major flavonoid glycoside found in the orange peel. Interestingly enough, its quantity at 48 mg/g of dry peel permits the commercial use of orange peel as a source for the production of hesperidin. The antioxidant activity of the orange peel methanolic extract fractions was evaluated by applying two complementary methodologies, DPPH(*) assay and the Co(II)/EDTA-induced luminol chemiluminescence approach. Overall, the results have shown that orange peel methanolic extracts possess moderate antioxidant activity as compared with the activity seen in tests where the corresponding aglycones, diosmetin and hesperetin were assessed in different ratios.     

Flavonoid profiles of immature and mature fruit tissues of Citrus grandis Osbeck (Dangyuja) and overall contribution to the antioxidant effect

Citrus fruits are a valuable functional food and their peel is used in East Asian folk medicine. In this study, the polar components of the fruit tissues of Citrus grandis Osbeck were analyzed using high‐performance liquid chromatography–tandem mass spectrometry and compared with reported data. Among the 13 characterized compounds, eight flavonoids and one coumarin were identified for the first time in fruit tissues. The total amount of the identified components was the largest for the immature fruit peel, followed by mature fruit peel, mature fruit flesh, and immature fruit flesh. Naringin (2) and neohesperidin (3) were particularly rich in all samples. The antioxidant activity of the flavonoids extracted from fruit tissues increased in a dose‐dependent manner. The activity of the fruit peels was significantly higher than that of the fruit flesh. Copyright © 2014 John Wiley & Sons, Ltd.     

Bioflavonoid profile of citrus juices from Greece

High‐performance liquid chromatography with confirmation by UV–visible photodiode array detector–positive electrospray ionization–mass spectrometry [HPLC‐UV–vis‐DAD‐(+ESI)‐MS] with enhanced fragmentation by appropriate adjustment of the cone voltage was used to determine bioflavonoid content of five citrus species (tangerine, sanguine, sour orange, lemon and grapefruit) cultivated in Greece which come from citrus varieties analyzed for the first time. The main groups of bioflavonoids found in the juice of the citrus species according to HPLC retention times, spectral data and literature references were O‐glycosylated flavanones and flavones, C‐glucosylated flavones, O‐glucosylated flavones, O‐C‐glucosylated flavones like saponarin and a phenolic derivative. Copyright © 2012 John Wiley & Sons, Ltd.     

Analysis of antioxidant compounds in sweet orange peel by HPLC-diode array detection-electrospray ionization mass spectrometry

HPLC-diode array detection-electrospray ionization mass spectrometry was used to determine qualitatively and quantitatively the flavonoid content of several fractions and residues of extracts of Greek navel sweet orange peel (Citrus sinensis) from the region of southern Greece (Leonidi-Tripoli). The main groups of flavonoids found according to HPLC retention times, spectral data and literature references were polymethoxylated flavones, C-glycosylated flavones, O-glycosylated flavones, O-glycosylated flavanones, flavonols and phenolic acids and their derivatives. The ethyl acetate fraction which has been shown in previous work to possess the best radical scavenging activity among the others was found to contain C-glycosylated flavones, polymethoxylated flavones, O-glycosylated flavones, O-glycosylated flavanones, two phenolic acid derivatives and two unknown compounds, all in low concentrations. The group of C-glycosylated flavones was reported for the first time in the peel of Navel sweet orange. The C-glycosylated flavones found according to their spectral characteristics and literature were 6-C-beta-glucosyldiosmin, 6,8-di-C-glucopyranosylapigenin, 6,8-di-C-beta-glucosyldiosmin and two unknown. The results suggest that the ethyl acetate fraction of navel Citrus sinensis peel consists of significant antioxidant compounds and can be used as a food additive of natural origin or a pharmaceutical supplement using as a source of peel the byproducts of the orange juice industry.     

Ultra high performance liquid chromatography with ion‐trap TOF‐MS for the fast characterization of flavonoids in Citrus bergamia juice

We have developed a fast ultra HPLC with ion‐trap TOF‐MS method for the analysis of flavonoids in Citrus bergamia juice. With respect to the typical methods for the analysis of these matrices based on conventional HPLC techniques, a tenfold faster separation was attained. The use of a core–shell particle column ensured high resolution within the fast analysis time of only 5 min. Unambiguous determination of flavonoid identity was obtained by the employment of a hybrid ion‐trap TOF mass spectrometer with high mass accuracy (average error 1.69 ppm). The system showed good retention time and peak area repeatability, with maximum RSD% values of 0.36 and 3.86, respectively, as well as good linearity (R² ≥ 0.99). Our results show that ultra HPLC can be a useful tool for ultra fast qualitative/quantitative analysis of flavonoid compounds in citrus fruit juices.     

Efficient analysis of phytochemical constituents in the peel of Chinese wild citrus Mangshanju (Citrus reticulata Blanco) by ultra high performance liquid chromatography–quadrupole time‐of‐flight‐mass spectrometry

An efficient ultra high‐performance liquid chromatography coupled with quadrupole time‐of‐flight mass spectrometry method was developed for separation and profiling of phytochemical constituents of Chinese wild mandarin Mangshanju (Citrus reticulata Blanco). All constituents were well separated within 16 min. Based on retention times, accurate mass, MS^E fragments, and/or reference standards as well as databases, a total of 81 compounds were unambiguously identified or tentatively assigned including flavonoid glycosides, acylated flavonoid glycosides, flavones, polymethoxylated flavonoids, and limonoids as well as four other compounds. Among them, 22 polymethoxylated flavones and ten polymethoxylated flavanones/chalcones were identified in Mangshanju, more types than other citrus reported before. A basic procedure for identifying flavonoid‐O‐glycosides and the aglycones including polymethoxylated flavonoids was proposed. In addition, this method was successfully used to analyze another four mandarin germplasms, Cenxi suan ju, Xipi gousi gan, Nanfeng miju, and Or, showing that Mangshanju contained two characteristic compounds distinct from the other four citrus species. This study systematically profiled phytochemical constituents of Mangshanju, which was helpful for further utilization of Mangshanju owing to its abundant bioactive compounds.     

Mass spectrometric profiling of flavonoid glycoconjugates possessing isomeric aglycones

In fields such as food and nutrition science or plant physiology, interest in untargeted profiling of flavonoids continues to expand. The group of flavonoids encompasses several thousands of chemically distinguishable compounds, among which are a number of isobaric compounds with the same elemental composition. Thus, the mass spectrometric identification of these compounds is challenging, especially when reference standards are not available to support their identification. Many different types of isomers of flavonoid glycoconjugates are known, i.e. compounds that differ in their glycosylation position, glycan sequence or type of interglycosidic linkage. This work focuses on the mass spectrometric identification of flavonoid glycoconjugate isomers possessing the same glycan mass and differing only in their aglycone core. A non‐targeted HPLC‐ESI‐MS/MS profiling method using a triple quadrupole MS is presented herein, which utilizes in‐source fragmentation and a pseudo‐MS³ approach for the selective analysis of flavonoid glycoconjugates with isomeric/isobaric aglycones. A selective MRM‐based identification of the in‐source formed isobaric aglycone fragments was established. Additionally, utilizing the precursor scanning capability of the employed triple quadrupole instrument, the developed method enabled the determination of the molecular weight of the studied intact flavonoid glycoconjugate. The versatility of the method was proven with various types of flavonoid aglycones, i.e. anthocyanins, flavonols, flavones, flavanones and isoflavones, along with their representative glycoconjugates. The developed method was also successfully applied to a commercially available sour cherry sample, in which 16 different glycoconjugates of pelargonidin, genistein, cyanidin, kaempferol and quercetin could be tentatively identified, including a number of compounds containing isomeric/isobaric aglycones. Copyright © 2015 John Wiley & Sons, Ltd.     

The Chemical Constituents of the Peel of Citrus Tankan Hayata

Seven kinds of flavones, β-sitosterol, β-sitosterol-D-glucoside, hesperidin, limonin, ferulic acid and succinic acid were isolated from the fruit peel of Citrus tankan Hayata.     

Matrix solid-phase dispersion combined with micro-fractionation bioactivity evaluation screening polymethoxylated flavones from Citrus peels

Polymethoxyflavones were a unique class of natural and safe flavonoids containing two or more methoxy groups, which were also the most abundant edible part in Citrus peel. The optimum condition in the process of selective extraction of polymethoxylated flavones from Citrus peel by matrix solid-phase dispersion (MSPD) was as follows: SBA-15 as adsorbent, ethyl acetate as eluent, the mass ratio of adsorbent to sample 1:1, and the mixture of sample and adsorbent was ground for 3 min. Twelve antioxidants were successfully screened by micro-fractionation bioactivity evaluation assay, in which four of them were flavonoid glycosides, seven of them were polymethoxylated flavones, and one was phenylpropanoid. 1-sinapoly-β-D-glucopyranoside (1) was reported for the first time in Citrus peel. And antioxidant capacity of 1-sinapoly-β-D-glucopyranoside, 5, 7, 8, 3′, 4′, 5′-hexamethoxyflavone (6), hexamethoxyflavone (11), and 5, 6, 7, 4′-tetramethoxyflavone (7) were reported for the first time. Nobiletin (compound 8), 3, 5, 6, 7, 8, 3′, 4′-heptamethoxyflavone (9) and tangeretin (10) were isolated and purified by countercurrent chromatography combined with preparative liquid chromatography. Antioxidant activity evaluation indicated that the three isolated polymethoxylated flavones owned similar antioxidant activity. This study indicated that MSPD combined with micro-fractionation bioactive evaluation was efficient in screening bioactive compounds for rapid extraction and effective pinpointing bioactive substances in natural products.     

Practical guidelines for characterization of O‐diglycosyl flavonoid isomers by triple quadrupole MS and their applications for identification of some fruit juices flavonoids

Fifteen flavonoid O‐diglycosides with different interglycosidic linkage isomery and glycosylation position have been studied in order to analyze their fragmentation patterns. Initial separation was carried out using high performance liquid chromatography with diode array detection (HPLC/DAD) coupled to an electrospray ionization (ESI) interface and a triple quadrupole mass spectrometer. Some useful differences in their MS spectra have been found and discussed. As it has already been reported, [Y*]⁺/[Y₀]⁺ ratio for flavanones and [Y₁]⁺/[Y₀]⁺ ratio for other flavonoids is specific for each isomeric interglycosidic linkage. In this work it has also been observed that the abundance of these ions is dependent on the position of glycosylation. On the basis of these differences, systematic guidelines for our experimental conditions have been proposed for the differentiation of not only isomeric interglycosidic linkage but also glycosylation position using collision‐induced dissociation MS/MS (CID‐MS/MS) spectra in positive mode. These results have been successfully applied for the characterization of three diglycosyl flavonoids found in Citrus fruit juices and these conclusions have also been extrapolated for characterizing two triglycosides in the same fruits. Copyright © 2009 John Wiley & Sons, Ltd.     

Accumulation Pattern of Flavonoids during Fruit Development of Lonicera maackii Determined by Metabolomics

Lonicera maackii (Caprifoliaceae) is a large, upright shrub with fruits that contain many bioactive compounds. Flavonoids are common active substances in L. maackii. However, there is a dearth of information about the accumulation of these flavonoids and their possible medicinal value. We used targeted metabolomics analysis based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) to analyze five developmental stages of L. maackii fruit. A total of 438 metabolites were identified in the five developmental stages, including 81 flavonoids and derivatives. The 81 flavonoids included 25 flavones and derivatives, 35 flavonols and derivatives, two isoflavones, three cyanidins and derivatives, eight procyanidins, and eight flavanones. In addition, we outlined the putative flavonoid biosynthesis pathway and screened their upstream metabolites. More importantly, we analyzed the accumulation patterns of several typical flavones and flavonols. The results reported here improved our understanding of the dynamic changes in flavonoids during fruit development and contributed to making full use of the medicinal value of L. maackii fruit.     

Flavonoid compounds and antibacterial mechanisms of different parts of white guava (Psidium guajava L. cv. Pearl)

Abstract

The flavonoid compositions, extracted from leaves, peel and flesh of white guava (Psidium guajava L. cv. Pearl), were identified and quantified by UPLC-ESI-QTOF-MS, HPLC-ESI-MS/MS and HPLC. The main components of three extracts all were quercetin-glycosides, but the proportion and content of quercetin-hexoside and quercetin-pentoside in each extract were different. Based on the measurements of MIC, MBC value and time killing curve, it emerged that 3 flavonoid extracts of white guava had good antibacterial effects on four pathogenic bacteria. White guava leaves flavonoids (WGLF) concentrations of 5.00?mg/mL and 0.625?mg/mL could change the micro-morphology of Escherichia coli and Staphylococcus aureus. It suggested that the antibacterial mechanism of WGLF on gram-positive and gram-negative bacteria was to destroy the structure and function of the cell membrane. It is indicated that the flavonoid extracts from white guava is a potential natural antimicrobial agent.     

Screening and analysis of metabolites in rat urine after oral administration of Apocynum venetum L. extracts using HPLC–TOF‐MS

HPLC with diode array detection and ESI‐TOF‐MS was used for the study of the constituents in Apocynum venetum L. extracts and the metabolites in rat urine after oral administration of A. venetum L. extracts. A formula database of the known constituents in A. venetum L. was established, and 21 constituents were rapidly identified by accurately matching their molecular masses with the formulae of the compounds in the database. Furthermore, 34 metabolites were detected and elucidated in the rat urine. The scientific and plausible biotransformation pathways of the flavonoid components in A. venetum L. were also proposed together with the presentation of clues for potential mechanisms of bioactivity. This specific and sensitive HPLC–ESI‐TOF‐MS method can be used to identify the chemical components in the extracts of A. venetum L. and their metabolites in rat urine. This method can also be used to reveal the possible metabolic mechanisms of action of the extract components in vivo.     

Determination of polyphenols in three Capsicum annuum L. (bell pepper) varieties using high-performance liquid chromatography-tandem mass spectrometry: their contribution to overall antioxidant and anticancer activity

A mixture of polyphenol components was isolated from the fruits of C. annuum L. cv. Cupra, C. annuum L. cv. Orange glory, and C. annuum L. cv. ST4712 (CLST), via 70% methanol extraction followed by column chromatography over silica gel. The polyphenol components of the mixture were analyzed via HPLC-MS/MS and compared with the reported data. Three cinnamic acid derivatives and five flavonoid components in the fruits of the three varieties were identified for the first time in this study. The antioxidant activity and anticancer effect of the polyphenol mixtures of the three fruits were determined. The antioxidant and anticancer activities of CLST were substantially higher than those of C. annuum L. cv. Cupra and C. annuum L. cv. Orange glory. The high activities of CLST were attributed to the much higher concentration of quercetin derivatives in CLST.     

Antioxidative and Anti-Inflammatory Phytochemicals and Related Stable Paramagnetic Species in Different Parts of Dragon Fruit

In this study, we investigated the antioxidant and anti-inflammatory phytochemicals and paramagnetic species in dragon fruit using high-performance liquid chromatography (HPLC) and electron paramagnetic resonance (EPR). HPLC analysis demonstrated that dragon fruit is enriched with bioactive phytochemicals, with significant variations between each part of the fruit. Anthocyanins namely, cyanidin 3-glucoside, delphinidin 3-glucoside, and pelargonidin 3-glucoside were detected in the dragon fruit peel and fresh red pulp. Epicatechin gallate, epigallocatechin, caffeine, and gallic acid were found in the dragon fruit seed. Additionally, 25–100 mg × L⁻¹ of dragon fruit pulp and peel extracts containing enrichment of cyanidin 3-glucoside were found to inhibit the production of reactive oxygen species (ROS), reactive nitrogen species (RNS), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in cell-based studies without exerted cytotoxicity. EPR primarily detected two paramagnetic species in the red samples. These two different radical species were assigned as stable radicals and Mn²⁺ (paramagnetic species) based on the g-values and hyperfine components. In addition, the broad EPR line width of the white peel can be correlated to a unique moiety in dragon fruit. Our EPR and HPLC results provide new insight regarding the phytochemicals and related stable intermediates found in various parts of dragon fruit. Thus, we suggest here that there is the potential to use dragon fruit peel, which contains anthocyanins, as a natural active pharmaceutical ingredient.     

MALDI‐MS of flavonoids: a systematic investigation of ionization and in‐source dissociation mechanisms

Matrix assisted laser desorption ionization (MALDI) is a technique widely employed in the analysis of proteins and peptides, and nowadays it has also been applied to small molecules. There is little significant information regarding the in‐source dissociation processes on MALDI for natural products. Twenty‐six flavonoids (flavanones, flavones and flavonols) were analyzed by MALDI using different methods (with different matrices) and without matrix to comprehend the in‐source reactions and establish good analysis methods for these compounds. Depending on the class, structure and the laser intensity applied, methoxylated flavonoid aglycones can eliminate methyl radicals (˙CH₃) in the source, such as flavonols, but lithium 2,4‐dihydroxybenzoate matrix suppresses the ˙CH₃ eliminations and retro‐Diels–Alder cleavages in the source. All of the flavonoid O‐glycosides evaluated herein eliminated the sugar in source, even in the presence of the matrix, and its product radical ions ([M‐H‐sugar]^?˙) were observed in the negative mode. The flavone C‐glycosides suffered intense dissociation, which was reduced by the addition of a matrix and the application of low laser intensity, mainly in the negative mode. Depending on the hydroxyl substituents, the [M‐H‐H]^?˙ ion was observed with variable relative intensity in the spectra. Copyright © 2015 John Wiley & Sons, Ltd.     

Analysis and tentative structure elucidation of new anthocyanins in fruit peel of Vitis coignetiae Pulliat (meoru) using LC‐MS/MS: Contribution to the overall antioxidant activity

The skin of Vitis coignetiae Pulliat (meoru) grown wild in the Republic of Korea was analyzed for anthocyanins via HPLC coupled to ESI‐MS/MS in positive ion mode. Chromatographic separation was conducted via RP HPLC using a C₁₈ column, with a 50‐min gradient from 0 to 80% methanol in water containing 0.5% formic acid. A total of 18 anthocyanins were identified. Among them, nine compounds were newly determined by comparing the retention time (t_R) and mass fragmentation patterns with those of the previously reported anthocyanins for other grape varieties: malvidin hexose, peonidin 3‐galactoside, malvidin 3‐galactoside, cyanidin, petunidin, petunidin 3‐(6″‐coumaroyl)‐5‐diglucoside, peonidin, malvidin, and malvidin 3‐(6″‐coumaroyl)‐5‐diglucoside. The antioxidant activity of the V. coignetiae Pulliat anthocyanins was determined via 2,2‐diphenyl‐2‐picrylhydrazyl radical scavenging and 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation assays in a range of concentration from 25 to 500 mg/L. The capacity increased with concentration. The IC₅₀ values, defined as the concentration of sample required to scavenge 50% of free radicals, were calculated as follows: 189.63±11.31 mg/L and 141.29±6.70 mg/L for 2,2‐diphenyl‐2‐picrylhydrazyl and 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid) radical cation, respectively. The antioxidant activity of the V. coignetiae Pulliat anthocyanins is substantially higher than that of ascorbic acid and is similar to the effects of the extracts obtained from other grape varieties.     

Quantitative determination of dipyridamole in human plasma by high‐performance liquid chromatography–tandem mass spectrometry and its application to a pharmacokinetic study

Dipyridamole is a classic platelet inhibitor which has been a key medicine in clinical therapy of thrombosis and cerebrovascular disease. A rapid, selective and convenient method using high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) was developed for determination of dipyridamole in human plasma. After protein precipitation of 200 μL plasma with methanol, dipyridamole and diazepam (internal standard) were chromatographed on an Ultimate? XB‐C₁₈ (50 × 2.1 mm i.d, 3 μ) column with the mobile phase consisting of methanol–ammonium acetate (5 mM ; 80 : 20, v/v) at a flow rate of 0.25 mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring mode via positive eletrospray ionization source (ESI⁺). The retention times of dipyridamole and diazepam were 1.4 and 1.2 min, respectively. The method was validated over a concentration range of 0.0180–4.50 μg/mL (r² ≥ 0.99) with a lower limit of quantitation (LLOQ) of 0.0180 μg/mL for dipyridamole. The intra‐ and inter‐day precisions (RSD) of the assay at all three QC levels were 1.6–12.7% with an accuracy (RE) of ?4.3–1.9%, which meets the requirements of the FDA guidance. The HPLC‐MS/MS method herein described was proved to be suitable for pharmacokinetic study of sustained‐release dipyridamole tablet in volunteers after oral administration. Copyright © 2009 John Wiley & Sons, Ltd.     

Chemical constituents and antioxidant activity of essential oil and organic extract from the peel and kernel parts of Citrus japonica Thunb. (kumquat) from Iran

The constituents of essential oils and organic extracts from peel and kernels of Citrus japonica were analysed by GC and GC/MS. The content of essential oil in peel and kernel was 1.1 and 0.8% based on dry weight. The essential oil of C. japonica peel and kernel was characterised by a higher amount of limonene (51.0 and 47.1%) and germacrene D (12.1 and 6.3%), and the hexane extracts of its peel and kernel were characterised by a higher amount of dodecanol-1(12.9 and 20.8%) and linolenic acid (13.1 and 16.3%), respectively. The antioxidant activities of oils were evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) method. The results indicate that both oils from different parts of C. japonica possess considerable antioxidant activity. The fruit peel and kernel essential oil could thus be useful in the industries, chiefly in the food and pharmaceutical industries.