isonaringin and naringin

The present study aimed to develop a strategy involving quantitative analysis of multicomponents by single marker in combination with high-performance liquid chromatography fingerprint qualitative analysis for performing the quality control of Aurantii Fructus. The content of 12 components (eriocitrin, neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, meranzin, poncirin, naringenin, nobiletin, tangeretin, and auraptene) in samples was determined using reliable relative correction factors that were obtained using naringin as an internal reference standard. The new method demonstrated good applicability, and no significant differences were observed between the external standard method and the new method as determined by calculating standard method difference. Qualitative evaluation of samples was conducted using similarity analysis, hierarchical cluster analysis, and quality fluctuation analysis. Chromatographic fingerprint data were divided into three groups by similarity and hierarchical cluster analyses, and seven components may have a more significant impact on the quality of Aurantii Fructus in quality fluctuation analysis. Overall, the study suggests that the qualitative and quantitative analyses of multicomponents using quantitative analysis of multicomponents by single marker combined with chromatographic fingerprinting can be considered good quality criteria for performing quality control and providing technical support for the further pharmacological and pharmaceutical research of Aurantii Fructus.

Topics: Chromatography, High Pressure Liquid; Citrus; Coumarins; Disaccharides; Flavanones; Flavones; Flavonoids; Fruit; Hesperidin

A large number of biologically active compounds are present in ripe citrus fruits. However, few studies have been focused on the changes in flavonoids and the evolution of antioxidant activity during citrus fruit growth. In this study, fruits of five citrus cultivars cultivated in China were sampled at 60-210 days post-anthesis (DPA) at intervals of 30 days. The amounts of main flavonoids in the peel and pulp were analyzed by HPLC and their activities were studied by DPPH, ABTS and FRAP. The results showed that the contents of hesperidin, diosmin, eriodictyol, rutin and nobiletin increased before 90 DPA and then decreased with the growth and development of fruits, but an opposite tendency was observed for naringin and narirutin. The antioxidant activities in citrus peel and pulp were found to be significantly correlated with some flavonoids. The results may be of guiding values in citrus production and utilization of citrus fruit by-products.

Topics: Antioxidants; Chromatography, High Pressure Liquid; Citrus; Disaccharides; Flavanones; Flavonoids; Fruit; Time Factors

Drug discovery from complex mixtures, like Chinese herbs, is challenging and extensive false positives make it difficult to obtain compounds with anti-Alzheimer's activity. In this study, a continuous method comprised of accelerated solvent extraction coupled with online two-dimensional countercurrent chromatography was developed for the efficient, scaled-up extraction and separation of six bioactive compounds from Citrus limon peels: neoeriocitrin, isonaringin, naringin, hesperidin, neohesperidin, and limonin. These active compounds were isolated and purified from the raw plant materials by two-dimensional countercurrent chromatography separation via two sets of an n-hexane/n-butanol/methanol/water solvent system: 0.23:1.00:0.25:1.13 and 0.47:1.00:0.38:1.46, v/v/v/v. The compounds were collected in yields of 0.22, 0.25, 0.10, 0.31, 0.29, and 0.28 mg/g, respectively, with purities of 95.79, 96.47, 97.69, 97.22, 98.11, and 98.82%, respectively. Subsequently, a simple and efficient in vitro method was developed for rapidly evaluating the acetylcholinesterase inhibitory activities of six bioactive components. Furthermore, the PC12 cell model and the in vitro metabolism of cytochromes P450 were employed to verify the monomers obtained from the continuous method. The results demonstrated that these six bioactive extracts from the C. limon peels were strong acetylcholinesterase inhibitors.

Topics: Animals; Cholinesterase Inhibitors; Citrus; Countercurrent Distribution; Cytochrome P-450 Enzyme System; Disaccharides; Flavanones; Flavonoids; Hesperidin; PC12 Cells; Plant Extracts; Rats; Solvents

In the present study, a green and efficient extraction method using deep eutectic solvents as extraction solvent was developed for extracting the four major active compounds narirutin, naringin, hesperidin and neohesperidin from Aurantii Fructus.. A series of tunable deep eutectic solvents were prepared and investigated by mixing choline chloride or betaine to different hydrogen-bond donors, and betaine/ethanediol was found to be the most suitable extraction solvent. To achieve the best extraction yield, the primary factors affecting the extraction efficiency, such as hydrogen-bond acceptor/hydrogen-bond donor ratio, water content in deep eutectic solvents, extraction temperature, solid/liquid ratio and extraction time, were investigated.. The optimal extraction conditions were 40% of water in betaine/ethanediol (1:4) at 60°C for heated extraction of 30 min and solid/liquid ratio 1:100 g/mL. Under the optimum extraction condition, the extraction yields of narirutin, naringin, hesperidin, and neohesperidin were 8.39 ± 0.61, 83.98 ± 1.92, 3.03 ± 0.35 and 35.94 ± 0.63 mg/g, respectively, which were much higher than those of methanol as extraction solvent (5.5 ± 0.48, 64.23 ± 1.51, 2.16 ± 0.15 and 30.14 ± 0.62 mg/g).. The present results showed that deep eutectic solvents could be promising green and efficient solvents for extraction of the bioactive ingredients from traditional Chinese medicine.

Topics: Chromatography, High Pressure Liquid; Disaccharides; Flavanones; Green Chemistry Technology; Hesperidin; Hydrogen Bonding; Reference Standards; Solvents; Spectrophotometry, Ultraviolet

Flavonoids, which comprise a large family of secondary plant metabolites, have received increased attention in recent years due to their wide range of features beneficial to human health. One of the most abundant flavonoid skeletons in citrus species is the flavanone naringenin, which is accumulated as glycosides containing terminal rhamnose (Rha) after serial glycosylation steps. The linkage type of Rha residues is a determining factor in the bitterness of the citrus fruit. Such Rha residues are attached by either an α1,2- or an α1,6-rhamnosyltransferase (1,2RhaT or 1,6RhaT). Although the genes encoding these RhaTs from pummelo (Citrus maxima) and orange (Citrus sinensis) have been functionally characterized, the details of the biochemical characterization, including the substrate preference, remain elusive due to the lack of availability of the UDP-Rha required as substrate. In this study, an efficient UDP-Rha in vivo production system using the engineered fission yeast expressing Arabidopsis thaliana rhamnose synthase 2 (AtRHM2) gene was constructed. The in vitro RhaT assay using the constructed UDP-Rha revealed that recombinant RhaT proteins (Cm1,2RhaT; Cs1,6RhaT; or Cm1,6RhaT), which were heterologously produced in fission yeast, catalyzed the rhamnosyl transfer to naringenin-7-O-glucoside as an acceptor. The substrate preference analysis showed that Cm1,2RhaT had glycosyl transfer activity toward UDP-xylose as well as UDP-Rha. On the other hand, Cs1,6RhaT and Cm1,6RhaT showed rhamnosyltransfer activity toward quercetin-3-O-glucoside in addition to naringenin-7-O-glucoside, indicating weak specificity toward acceptor substrates. Finally, naringin and narirutin from naringenin-7-O-glucoside were produced using the engineered fission yeast expressing the AtRHM2 and the Cm1,2RhaT or the Cs1,6RhaT genes as a whole-cell-biocatalyst.

Topics: Citrus; Citrus sinensis; Cloning, Molecular; Disaccharides; Enzymes; Flavanones; Flavonoids; Glucosides; Glycosides; Glycosylation; Glycosyltransferases; Humans; Plant Proteins; Quercetin; Recombinant Proteins; Rhamnose; Schizosaccharomyces; Substrate Specificity

The hydrolytic activity of the recombinant β-glucosidase from Pyrococcus furiosus for the flavanone glycoside hesperidin was optimal at pH 5.5 and 95 °C in the presence of 0.5% (v/v) dimethyl sulfoxide (DMSO) and 0.1% (w/v) Tween 40 with a half-life of 88 h, a Km of 1.6 mM, and a kcat of 68.4 1/s. The specific activity of the enzyme for flavonoid glycosides followed the order hesperidin > neohesperidin > naringin > narirutin > poncirin > diosmin > neoponcirin > rutin. The specific activity for flavanone was higher than that for flavone or flavonol. DMSO at 10% (v/v) was used to increase the solubility of flavanone glycosides as substrates. The enzyme completely converted flavanone glycosides (1 g/L) to flavanone aglycones and disaccharides via one-step reaction. The major flavanone in grapefruit peel, grapefruit pulp, or orange peel extract was naringin (47.5 mg/g), naringin (16.6 mg/g), or hesperidin (18.2 mg/g), respectively. β-Glucosidase from P. furiosus completely converted naringin and narirutin in 100% (w/v) grapefruit peel extract to 22.5 g/L naringenin after 12 h, with a productivity of 1.88 g L(-1) h(-1); naringin and narirutin in 100% (w/v) grapefruit pulp extract to 8.1 g/L naringenin after 9 h, with a productivity of 0.90 g L(-1) h(-1); and hesperidin in 100% (w/v) orange peel extract to 9.0 g/L hesperetin after 9 h, with a productivity of 1.00 g L(-1) h(-1). The conversion yields, concentrations, and productivities of flavanone aglycones in this study are the highest among those obtained from citrus extracts. Thus, this enzyme may be useful for the industrial hydrolysis of flavanone glycosides in citrus extracts.

Topics: beta-Glucosidase; Citrus; Detergents; Disaccharides; Flavanones; Flavonoids; Food Industry; Glycosides; Hesperidin; Hydrogen-Ion Concentration; Hydrolysis; Kinetics; Plant Extracts; Pyrococcus furiosus; Solvents; Substrate Specificity; Temperature

The phytochemical content and the antioxidant activity (AA) of physiological drop of the main citrus species grown in China were investigated. Among the flavonoids, hesperidin was found mostly in mandarin and sweet orange, naringin was found mostly in sour orange, pummelo, grapefruit and a hybrid (Gaocheng), narirutin was found in most varieties, neohesperidin was found in Gaocheng and Huyou, and nobiletin and tangeretin were found in most varieties. Hydroxycinnamic acids were the main phenolic acids present, ferulic acid and caffeic acid were the dominant in most cases. There was a greater amount of free (extractable) than bound (insoluble) phenolic acids. Levels of limonoids were higher in Foyou, Eureka lemon, and Gaocheng than those in the other cultivars. The highest level of synephrine was found in Ponkan and Weizhang Satsuma. AA was highest in Ponkan and Weizhang Satsuma and lowest in Huyou, pummel, and lemon. These results suggest that physiological drop of citrus fruits have good potential as sources of different bioactive compounds and antioxidants.. Physiological drop of citrus fruits may be a good resource of bioactive compounds including flavonoids, phenolic acids, limonoids, synephrine, and a good material of nutraceuticals.

Topics: Antioxidants; China; Citrus; Coumaric Acids; Dietary Supplements; Disaccharides; Flavanones; Flavones; Fruit; Hesperidin; Hydroxybenzoates; Limonins; Plant Extracts; Synephrine

A simple, rapid, and sensitive liquid chromatographic method has been established for the simultaneous analysis of three compounds (narirutin, hesperidin and naringin), in granule decoctions of Fructus Aurantii-type formulae. The compounds were separated in less than 10 min using a C18 column with gradient elution using (A) acetonitrile, (B) water, and (C) acetic acid at a flow rate of 0.3 mL/min, and with a PDA detector. The method was validated for specificity, accuracy, precision, and limits of detection. Good linear regression data (r2 > 0.9980) were obtained for all the calibration plots within the ranges tested. The method is an attractive alternative for quality control and clinical monitor of granule decoctions of Fructus Aurantii-type formulae.

Topics: Carbohydrate Sequence; Chemistry, Pharmaceutical; Chromatography, High Pressure Liquid; Citrus; Disaccharides; Drugs, Chinese Herbal; Flavanones; Flavonoids; Hesperidin; Indicators and Reagents; Limit of Detection; Molecular Sequence Data; Plant Extracts; Reference Standards; Regression Analysis; Reproducibility of Results; Solutions; Spectrophotometry, Ultraviolet

Grapefruits (Citrus paradisi Macfad) contain several phytochemicals known to have health maintaining properties. Due to the consumer's interest in obtaining high levels of these phytochemicals, it is important to understand the changes in their levels by common household processing techniques. Therefore, mature Texas "Rio Red" grapefruits were processed by some of the common household processing practices such as blending, juicing, and hand squeezing techniques and analyzed for their phytochemical content by high performance liquid chromatography (HPLC). Results suggest that grapefruit juice processed by blending had significantly (P < 0.05) higher levels of flavonoids (narirutin, naringin, hesperidin, neohesperidin, didymin, and poncirin) and limonin compared to juicing and hand squeezing. No significant variation in their content was noticed in the juice processed by juicing and hand squeezing. Ascorbic acid and citric acid were significantly (P < 0.05) higher in juice processed by juicing and blending, respectively. Furthermore, hand squeezed fruit juice had significantly higher contents of dihydroxybergamottin (DHB) than juice processed by juicing and blending. Bergamottin and 5-methoxy-7 gernoxycoumarin (5-M-7-GC) were significantly higher in blended juice compared to juicing and hand squeezing. Therefore, consuming grapefruit juice processed by blending may provide higher levels of health beneficial phytochemicals such as naringin, narirutin, and poncirin. In contrast, juice processed by hand squeezing and juicing provides lower levels of limonin, bergamottin, and 5-M-7-GC. These results suggest that, processing techniques significantly influence the levels of phytochemicals and blending is a better technique for obtaining higher levels of health beneficial phytochemicals from grapefruits. Practical Application:  Blending, squeezing, and juicing are common household processing techniques used for obtaining fresh grapefruit juice. Understanding the levels of health beneficial phytochemicals present in the juice processed by these techniques would enable the consumers to make a better choice to obtain high level of these compounds.

Topics: Ascorbic Acid; Beverages; Chromatography, High Pressure Liquid; Citric Acid; Citrus paradisi; Disaccharides; Flavanones; Flavonoids; Food Handling; Furocoumarins; Glycosides; Hesperidin; Limonins; Plant Extracts

An electrospray ionization-dual gate-ion mobility-quadrupole ion trap mass spectrometer was used to evaluate the separation characteristics of isomeric flavonoid diglycosides adducted with sodium, potassium, and silver. This instrumental configuration allows ions to be selectively accumulated within the ion trap on the basis of their gas phase conformation prior to mass analysis. For the metal cations examined, silver produced the most compact adducts with flavonoid diglycosides. Listed in order of increasing size, the trend of flavonoid diglycoside ion-neutral cross sections adducted with Na+, K+, and Ag+ was narirutin < naringin < hesperidin < neohesperidin < rutin. To examine the separation contribution of the carbohydrate group, hesperetin, the aglycone of hesperidin, and neohesperin were compared to quercetin, the aglycone of rutin. Separation of the flavonoid diglycosides indicated that quercetin-derived diglycosides drifted longer than their hesperetin-derived isomers. Combined with the observed collision assisted dissociation (CAD) data, these findings suggest that carbohydrate moiety plays a significant role in both the separation and metal chelating characteristics of flavonoid diglycosides.

Topics: Cations; Disaccharides; Flavanones; Flavonoids; Glycosides; Hesperidin; Isomerism; Potassium; Protons; Rutin; Sodium; Spectrometry, Mass, Electrospray Ionization

The rate constants and activation energy barriers DeltaG# of diastereomerization reaction of flavanones: naringin, narirutin, hesperidin and neohesperidin were determined. The stopped-flow HPLC (SFM-HPLC), dynamic HPLC (D-HPLC) and enantioselective HPLC combined with the classical kinetic method were applied for determination of these parameters. It was found that the rate constants of diastereomerization were about eight times higher for naringin and narirutin (1.9 x 10(-5) s(-1)) than for hesperidin and neohesperidin (2.4 x 10(-6) s(-1)). No significant differences in the rate of diastereomerization were found between neohesperidosides and corresponding rutinosides.

Topics: Chromatography, High Pressure Liquid; Disaccharides; Flavanones; Flavonoids; Hesperidin; Kinetics; Stereoisomerism

The separation of the major flavanone-7-O-glycoside constituents of Citrus was carried out by isocratic reversed phase capillary electrochromatography using a 75 microm i.d. silica fused column packed with 5 microm ODS silica gel. In comparison to HPLC mode, capillary electrochromatography resolution of flavanone glycosides was obtained with a high selectivity factor. Optimum separation conditions were found using a mixture of ammonium formate (pH 2.5)--acetonitrile (8:2, v/v) as the mobile phase by the short-end injection mode. Under these conditions all the investigated flavanones were baseline-resolved within short analysis time (i.e. between 5 and 10 min). A study, evaluating the intra- and inter-day repeatability as well as limit of detection and method linearity, was developed in accordance with the analytical procedures for method validation. The developed method was applied for the quantitative analysis of flavanone glycosides in commercial fruit juices (sweet orange, lemon and grapefruit).

Topics: Acetonitriles; Beverages; Chromatography, High Pressure Liquid; Citrus; Disaccharides; Electrophoresis, Capillary; Flavanones; Formates; Glycosides; Hesperidin; Reproducibility of Results

The main flavonoids in grapefruit juice, naringin and narirutin, were quantified by LC-MS with structural differentiation by LC-MS/MS. After human consumption of grapefruit juice, urine samples were collected for 24 hours and screened for flavonoid metabolites by LC-MS. The metabolite structures (glucuronides, sulfates, and glucuronide sulfates) were then confirmed via their unique fragmentation patterns by LC-MS/MS. To further verify the identity of the common aglycon (naringenin) shared by the metabolites, enzymatic hydrolysis was performed and the resulting products were analyzed. This work demonstrates that LC-MS and LC-MS/MS techniques can be used for fast metabolite screening without extensive sample preparation.

Topics: Adult; Beverages; Chemical Phenomena; Chemistry; Chromatography, High Pressure Liquid; Citrus paradisi; Disaccharides; Female; Flavanones; Flavonoids; Glycosides; Humans; Male; Spectrum Analysis

The original plant of Citrus hassaku HORT. Tanaka was found at Jyoudo-Temple in Inno-shima, Hiroshima Prefecture, Japan by Ekei Shounin in the Edo Era. Today, the fresh raw fruits of C. hassaku are cultivated as a seasonal food, and the most famous producing district of the C. hassaku fruit is Wakayama Prefecture, representing about 60% of the Japanese output. The immature fruits of C. hassaku and its three main flavanone glycosides (naringin, neohesperidin and narirutin) have shown inhibitory activities on a compound 48/80-induced histamine release from mast cells, and anti-allergic effects on dinitrofluorobenzen (DNFB)-induced triphasic skin reaction in mice. The contents of the three flavanone glycosides in the immature fruits were higher than those in mature fruits of C. hassaku to skin care for patients with atopitic dermatitis showed improving effects in 76% of the patients. Similar efficacy was obtained for patients with pollinosis.

Topics: Animals; Citrus; Dermatitis, Atopic; Disaccharides; Flavanones; History, 17th Century; Humans; Indoles; Japan; Phytotherapy; Plants, Medicinal; Rhinitis, Allergic, Seasonal; Sulfonamides