Simultaneous analysis of adrenergic amines and flavonoids in citrus peel jams and fruit juices by liquid chromatography: part 2

Citrus species are rich sources for various bioactive compounds such as flavonoids, adrenergic amines, limonoids, and coumarins. Six adrenergic amines and 20 flavonoids in the jams and juices of Citrus species were analyzed by high-performance liquid chromatography with a C18 reversed-phase column using gradient mobile phase of sodium acetate buffer (pH 5.5) and acetonitrile at a flow rate of 1.0 mL/min with detection at 254, 280, and 330 nm. Synephrine was present in levels from 3.65 to 60.66 mg/L in 48 Citrus juices and 0.018-1.02 mg per serving in 32 Citrus jams. The content of these amines and flavonoids varied to a large extent depending on the type of the Citrus species used. The method has been successfully applied to the determination of adrenergic amines and flavonoids in several samples of Citrus fruit juices and jams.     

分子印迹固相萃取/高效液相色谱法分离和富集枳实中4种黄烷酮

采用沉淀聚合法以橙皮素为模板分子,2-乙烯基吡啶为功能单体,二甲基丙烯酸乙二醇酯为交联剂,合成了橙皮素分子印迹聚合物。利用紫外光谱法确定了最佳功能单体与配比,优化了合成条件。采用傅立叶变换红外光谱、扫描电子显微镜、静态吸附对聚合物进行表征。实验结果表明,分子印迹聚合物的吸附性能明显优于空白印迹聚合物,且此聚合物对柚皮苷、橙皮苷、柚皮素和橙皮素的相对选择系数分别为1.40,1.39,1.59和2.89,表明该分子印迹聚合物对4种黄烷酮有较好的选择性。将印迹聚合物作为固相萃取填料,对枳实提取液进行分离和富集,结果表明上述4种黄烷酮的提取率分别为72.6%,61.1%,95.4%和93.5%,分离富集效果良好,大大提高了枳实中4种黄烷酮的提取效率。     

Enantiomeric Separation of Adrenergic Amines in Citrus Species, Related Genera and Dietary Supplements by Capillary Electrophoresis

Citrus aurantium L. (Rutaceae) fruit extracts have recently been used for weight loss. Among the adrenergic amines the most important active constituent is the sympathomimetic compound synephrine and commercially available extracts are standardized for their content of this active principle. A capillary electrophoresis method was developed for the quantitative and qualitative determination of d-synephrine, l-synephrine, d-octopamine, l-octopamine, tyramine, n-methyl tyramine and hordenine. The electrophoretic separation was performed using a 75 cm × 50 µm ID (66.5 cm effective length) fused silica capillary. The samples were injected by pressure for 5s at 50 mbar and the running voltage was 30 kV at the injector end of the capillary. The method developed was successively applied to the determination of the adrenergic amines in dietary supplements, in various Citrus species including Citrus aurantium, jams and juices. Synephrine was the main component and present in the levels from 0.02–0.17% in various Citrus species and 0.42–69.28 mg in dietary supplements claiming to contain Citrus aurantium. Parameters affecting the resolution between (+) and (−)-enantiomers, such as pH, cyclodextrin concentration, temperature, organic modifier, buffer concentration and capillary dimensions were reported.     

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.     

Chiral separation of diastereomeric flavanone-7-O-glycosides in citrus by capillary electrophoresis

The 2S- and 2R-diastereomers of major flavanone-7-O-glycosides found in sweet orange (Citrus sinensis), mandarine (Citrus deliciosa), grapefruit (Citrus paradisi), lemon (Citrus limon), and sour or bitter orange juice (Citrus aurantium) were separated for the first time by chiral capillary electrophoresis (CE) employing various buffers with combined chiral selectors. Native cyclodextrins (CDs), neutral and charged CD derivatives were examined as chiral additives to the background electrolyte (BGE). Separation efficiency has not proved satisfactory with one single CD as chiral selector in the buffer, a full and simultaneous separation could often be achieved only by using combined buffer with two different CDs. Chiral separation of major flavanones in sweet orange, mandarine and grapefruit juices raised more difficulties than in lemon and sour orange juices as narirutin will not readily build complexes with most CDs. Diastereomeric flavanones of mature and immature grapefruits were compared and some differences were found: naringin showed different diastereomeric ratio and 2S-prunin appeared only in immature grapefruit. Marmalade was also examined by chiral CE. Its major flavanones corresponded to flavanone pattern of mixed sour and sweet oranges.     

Chromatographic and electrophoretic methods for the analysis of phenethylamine [corrected] alkaloids in Citrus aurantium

Citrus aurantium (bitter orange) is a plant belonging to the family Rutaceae, whose fruit extracts have been used recently for the treatment of obesity. The most important biologically active constituents of the C. aurantium fruits are phenethylamine alkaloids (i.e. octopamine, synephrine, tyramine, N-methyltyramine and hordenine). Synephrine is a primary synthesis compound with pharmacological activities such as vasoconstriction, elevation of blood pressure and relaxation of bronchial muscle. Synephrine is present in the peel and the edible part of Citrus fruit. Of the adrenergic amines of natural origin, synephrine has been found to be the main constituent of C. aurantium fruits and extracts; the other alkaloids are either absent or present in only low concentrations. It is known that synephrine and the other amines found in C. aurantium have adverse effects on the cardiovascular system, owing to adrenergic stimulation. In light of the great commercial proliferation of C. aurantium herbal medicines in recent years, this review provides an overview of various extraction, separation and detection techniques employed for the qualitative and quantitative determination of the alkaloids in C. aurantium and related species. The application of chromatographic and electrophoretic methods for the separation and determination of these active components in C. aurantium plant material and derivatives are described. Since synephrine is a chiral compound, enantioselective chromatographic and electrophoretic techniques for the analysis of synephrine enantiomers in natural products are presented. Furthermore, examples of identification of these active compounds in complex matrices by hyphenated methods, such as gas chromatography-mass spectrometry and high-performance liquid chromatography-mass spectrometry, are described. The advantages and limitations of these separation and identification methods are assessed and discussed.     

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.     

Validation of a liquid chromatography ionspray mass spectrometry method for the analysis of flavanones, flavones and flavonols

The application of liquid chromatography/mass spectrometry (LC/MS) with a TurboIonspray (TIS) interface was investigated as a new method for the analysis of flavonoids. Eleven compounds belonging to three different classes of flavonoids were studied: eriocitrin, neoeriocitrin, naringin, narirutin, hesperidin, neohesperidin (flavanone glycosides), quercetin, kaempferol, galangin (flavonol aglycones), chrysin, apigenin (flavone aglycones). Chromatographic separations were performed under reversed-phase conditions using a C18 narrow-bore LC column; a mixture of an aqueous solution of formic acid (pH 2.4) and acetonitrile was used as the mobile phase. Isocratic elution was operated in the case of flavanones, whereas gradient elution was used for the simultaneous separation of flavones and flavonols. The adaptability of TIS to high flow applications allows the use of LC eluent flow rates at 200 μL/min without post-column splitting. Qualitative analysis was performed in negative-ion (NI) full-scan mode, whereas response linearity, detection limits and precision of the method were studied under NI selected ion monitoring (SIM) conditions. Characterization of isomers differing in the glycosylation was found to be possible on the basis of different mass spectra. Detection limits in the low-ng range (0.08-0.4 ng) were found, about twenty-fold lower than those reported previously. The method was applied to identify and determine the content of flavonoids in an orange juice sample. Copyright 1999 John Wiley & Sons, Ltd.     

Flavonoid profiling in three citrus varieties native to the Republic of Korea using liquid chromatography coupled with tandem mass spectrometry: contribution to overall antioxidant activity

A mixture of flavonoid components was isolated from the fruit peel of three varieties of citrus native to Republic of Korea, Citrus leiocarpa Hort. ex Tanaka (CLHT), Citrus aurantium L. (CAL) and Citrus erythrosa Hort. (CEH), via 70% methanol extraction followed by ethyl acetate elution over a silica gel cartridge. The flavonoid components of the mixture were analyzed via high‐performance liquid chromatography–tandem mass spectrometry (HPLC‐MS/MS) in positive‐ion mode and a comparison of the reported data. Among 17 characterized components, two flavanones, four flavones and two coumarin derivatives in the fruit peel of the three varieties were identified for the first time. The individual characterized components were quantified via HPLC‐UV. The flavanones dominated in CAL, whereas the flavones prevailed in CLHT and CEH. The antioxidant activity of the flavonoid mixture of the fruit peel was determined via DPPH^?, ABTS^?+ and reducing power assays. The antioxidant activity of CEH and CAL was greater than that of CLHT. Copyright © 2011 John Wiley & Sons, Ltd.     

Flavonoids inhibiting glycation of bovine serum albumin: affinity-activity relationship

Protein glycation leads to the formation of advanced glycation end-products (AGEs), which contribute to the pathogenesis of diabetic complications. The structure-activity relationship of dietary flavonoids for inhibiting the glycation of bovine serum albumin (BSA) in vitro was subjected to a detailed investigation. The structure-activity relationship revealed that: 1) the hydroxylation on ring B of the flavones enhanced the inhibition and the hydroxyl groups at the C-5 and C-7 positions of flavones favoured the inhibition; 2) the optimal number of hydroxyl groups on ring B of the flavonols was one (at the C-3 position) and the methylation of flavonols weakened the inhibition; 3) the methoxylation at the C-6 position and methylation at C-4′ position of genistein clearly enhanced the inhibition; 4) the hydroxyl groups at the C-5 and C-7 positions of flavanones were in favour of the inhibition; 5) the glycosylation of flavonoids significantly weakened the inhibition. Obvious linear affinity-activity relationships exist between the BSA-flavonoid interaction and flavonoids as BSA glycation inhibitors (R² = 0.76585). The flavonoids with a higher affinity to BSA exhibited a stronger inhibition of the glycation of BSA.     

Dietary Flavonoids: Cardioprotective Potential with Antioxidant Effects and Their Pharmacokinetic,Toxicological and Therapeutic Concerns

Flavonoids comprise a large group of structurally diverse polyphenolic compounds of plant origin and are abundantly found in human diet such as fruits, vegetables, grains, tea, dairy products, red wine, etc. Major classes of flavonoids include flavonols, flavones, flavanones, flavanols, anthocyanidins, isoflavones, and chalcones. Owing to their potential health benefits and medicinal significance, flavonoids are now considered as an indispensable component in a variety of medicinal, pharmaceutical, nutraceutical, and cosmetic preparations. Moreover, flavonoids play a significant role in preventing cardiovascular diseases (CVDs), which could be mainly due to their antioxidant, antiatherogenic, and antithrombotic effects. Epidemiological and in vitro/in vivo evidence of antioxidant effects supports the cardioprotective function of dietary flavonoids. Further, the inhibition of LDL oxidation and platelet aggregation following regular consumption of food containing flavonoids and moderate consumption of red wine might protect against atherosclerosis and thrombosis. One study suggests that daily intake of 100 mg of flavonoids through the diet may reduce the risk of developing morbidity and mortality due to coronary heart disease (CHD) by approximately 10%. This review summarizes dietary flavonoids with their sources and potential health implications in CVDs including various redox-active cardioprotective (molecular) mechanisms with antioxidant effects. Pharmacokinetic (oral bioavailability, drug metabolism), toxicological, and therapeutic aspects of dietary flavonoids are also addressed herein with future directions for the discovery and development of useful drug candidates/therapeutic molecules.     

Selective and efficient oxidative modifications of flavonoids with 2-iodoxybenzoic acid (IBX)

2-Iodoxybenzoic acid (IBX), a mild and efficient hypervalent iodine oxidant, has been utilised in different reaction conditions to perform several efficient oxidative modifications of flavonoids. Fine-tuning of the reaction conditions allowed remarkably selective modifications of these compounds. At room temperature, IBX proved to be an excellent reagent for a highly regioselective aromatic hydroxylation of monohydroxylated flavanones and flavones, generating the corresponding catecholic derivatives showing high antioxidant activity. At 90 °C, IBX efficiently dehydrogenated a large panel of methoxylated flavanones to their corresponding flavones exhibiting anticancer activity. IBX polystyrene has also been utilised to increase the recovery of highly polar compounds. Following the first oxidation, the reagent was recovered and reused in several runs without loss of efficiency and selectivity. The first example of an application of IBX polystyrene in a dehydrogenation reaction has been described.     

Evaluation of flavonoids and furanocoumarins from Citrus bergamia (Bergamot) juice and identification of new compounds

Bergamot juice (BJ) contains different classes of flavonoids (e.g. flavanones and flavones) that can exert beneficial effects on human health. The aim of this study was to evaluate the qualitative and quantitative composition of a BJ obtained from fruits harvested in Southern Italy (Calabria) at the end of their maturation period. The identity of several flavonoids and furanocoumarins was assessed by co-chromatography, UV spectra and molecular weight comparison. The unknown compounds were dissociated by induced collision (CID-MS) and their identity established through the characteristic ions product. By this approach a complete profile of about twenty compounds (furano-coumarins, flavonoids C- and O-glycosides) present in BJ was obtained. Furthermore, three acylated flavanones, present in amounts of 20.1+/-1.1, 89.3+/-2.2 and 190.1+/-3.1 mg/L, respectively, and which seem to correspond to di-oxalate derivatives of neoeriocitrin, naringin and neohesperidin, were identified for the first time in BJ. The other main flavanones were naringin, neohesperidin and neoeriocitrin, and their content was 167.5+/-1.8, 123.9+/-1.7 and 73.3+/-1.6 mg/L, respectively. Concerning flavones, the total amount in BJ was about 160 mg/L and the main ones were vicenin-2, stellarin-2, rhoifolin and neodiosmin. Bergapten and bergamottin were the primary furanocoumarins in BJ and their amounts were 9.0+/-0.4 and 18.2+/-0.5 mg/L, respectively.     

基于化学模式识别技术的枳实HPLC定量指纹图谱研究

建立了枳实的高效液相色谱(HPLC)指纹图谱分析方法.色谱柱为Tnature-ACCHROM C18色谱柱(4.6 mmx250 mm,5 μm);以乙腈-0.5％甲酸水溶液为流动相进行梯度洗脱,结合液相色谱-四极杆飞行时间质谱(HPLC-QTOF-MS)联用技术对枳实指纹图谱中的共有峰进行鉴定;采用相似度评价、聚类分...     

Flavonoids and their glycosides, including anthocyanins

This review describes more than 600 new examples of naturally occurring flavonoids found either as aglycones or glycosides, comprising flavones, flavonols, chalcones, dihydrochalcones, aurones, flavanones, dihydroflavonols, anthocyanidins and anthocyanins. The main topics addressed are source, identification, biological activity, biosynthesis, synthesis, and ecological or chemosystematic significance, and 514 references are cited.     

Determination of flavonoid aglycones in several food samples by mixed micellar electrokinetic chromatography

The application of mixed micellar electrokinetic chromatography to the separation of ten flavonoid aglycones (catechin, epicatechin, naringenin, morin, fisetin, quercetin, kaempferol, galangin, apigenin, and chrysin) belonging to four different classes (flavanols, flavanones, flavonols, and flavones), and expected to be prominent in commonly consumed foods, has been developed. A micellar system composed of 25 mM SDS and 25 mM sodium cholate buffered at pH 7.0 provided a simultaneous separation of all compounds in less than 20 min. The procedure could be easily adapted to the determination of some flavonoids from each of these classes in real complex samples (propolis, Ginkgo biloba, etc.). The LODs of these compounds were in the range of 1.2-4 microg/mL, and the peak area and migration time repeatabilities were below 6.0 and 3.1%, respectively.     

Rapid analysis of flavonoids based on spectral library development in positive ionization mode using LC-HR-ESI-MS/MS

Natural product screening in plants has always been a difficult task due to the complex nature of the plant material and diverse structures of the compounds present in them. Flavonoids are important and diverse class of plant secondary metabolites with numerous medicinal activities. The present study focuses on the development of a high-resolution tandem mass spectral library for the rapid and authentic identification of common flavonoids. A total of forty flavonoid standards belong to class flavones, isoflavones, flavanones, flavanols and anthocyanins were pooled into two solutions applying logP-based strategy. The flavonoids were analyzed using LC-QTOF-MS high-resolution mass spectrometer with optimization of different instrumental parameters to achieve good sensitivity. The library was built by incorporating names, molecular formulae, exact masses, and MS, and MS/MS spectra of analyzed flavonoids using Bruker Library Editor tool. The fragmentation pattern observed for the standard compounds were compared to the fragments reported in the literature. To assess the practical implications, an extract of tea sample was analyzed and screened using the developed library, which resulted in the identification of three common flavonoids based on their HR-ESI-MS/MS spectral features. The established LC-HR-MS/MS method can be used for the targeted identification of flavonoids in complex samples like food material from different botanical families.     

Synthesis of pyrroloquinolines as indole analogues of flavonols

7-Acetyl-4,6-dimethoxy-3-phenylindole 10 was converted into a range of 7-indolyl chalcones 13 by reaction with aryl aldehydes under basic conditions. Oxidation of the chalcones 13 with alkaline hydrogen peroxide gave the isolable epoxides 14, which were cyclized with further base treatment into the indole flavonols, or 5-hydroxy-6-oxopyrroloquinolines 15. The related compounds 25 and 26, examples of indole flavanones and flavones, respectively, were also synthesized. UV spectroscopic comparisons between flavonoids and indole flavonoids are discussed.     

Chemical ionization mass spectrometry with amines as reactant gases. I—amine chemical ionization mass spectrometry of flavonoid aglycones

Chemical ionization mass spectrometry of 34 flavones, isoflavones, flavanones, chalcones and aurones with aliphatic amines and ammonia as reactant gases have been investigated. Some unusual ions have been obtained and are discussed. This method can be used to determine the type of flavonoid and the location of some functional groups in the molecule.     

A Standardized Extract Prepared from Red Orange and Lemon Wastes Blocks High-Fat Diet-Induced Hyperglycemia and Hyperlipidemia in Mice

Citrus fruits are a rich source of high-value bioactive compounds and their consumption has been associated with beneficial effects on human health. Red (blood) oranges (Citrus sinensis L. Osbeck) are particularly rich in anthocyanins (95% of which are represented by cyanidin-3-glucoside and cyanidin-3-6″-malonyl-glucoside), flavanones (hesperidin, narirutin, and didymin), and hydroxycinnamic acids (caffeic acid, coumaric acid, sinapic, and ferulic acid). Lemon fruit (Citrus limon) is also rich in flavanones (eriocitrin, hesperidin, and diosmin) and other polyphenols. All of these compounds are believed to play a very important role as dietary antioxidants due to their ability to scavenge free radicals. A standardized powder extract, red orange and lemon extract (RLE), was obtained by properly mixing anthocyanins and other polyphenols recovered from red orange processing waste with eriocitrin and other flavanones recovered from lemon peel by a patented extraction process. RLE was used for in vivo assays aimed at testing a potential beneficial effect on glucose and lipid metabolism. In vivo experiments performed on male CD1 mice fed with a high-fat diet showed that an 8-week treatment with RLE was able to induce a significant reduction in glucose, cholesterol and triglycerides levels in the blood, with positive effects on regulation of hyperglycemia and lipid metabolism, thus suggesting a potential use of this new phytoextract for nutraceutical purposes.