柚皮苷分子印迹传感器的制备与应用

以邻氨基酚为单体,无电化学活性的柚皮苷为模板分子,采用循环伏安法(扫速为100mV/s)在碳电极上往复扫描30次,电聚合出具有识别柚皮苷分子功能的敏感膜。以扫描电子显微镜(SEM)、X射线全反射(XRR)及电化学方法对该印迹传感器进行表征。结果表明,印迹传感器敏感膜与非印迹膜在形貌结构和电化学特性方面有明显的不同。此传感器对柚皮苷有较好的选择性,响应快(30s),在6.0×10-5～1.4×10-4mol/L范围内呈线性关系,且重现性好(RSD=1.8%,n=5);传感器对柚皮苷的检出限为1.6×10-5mol/L。     

Novel immunoassay and rapid immunoaffinity chromatography method for the detection and selective extraction of naringin in Citrus aurantium

In this work, a novel monoclonal antibody specific for naringin was prepared and characterized. Subsequently, an indirect competitive enzyme‐linked immunosorbent assay for naringin was developed, with an effective range from 4.8 to 156 ng/mL naringin. Next, an immunoaffinity column was obtained by coupling anti‐naringin monoclonal antibodies to CNBr‐activated Sepharose 4B and a rapid immunoaffinity chromatography assay for naringin was developed. The immunoaffinity column was used to separate naringin from Citrus aurantium. The results showed that 1 g of the dry Sepharose 4B can couple 10 mg of immunoglobulin G. And the immunoaffinity column can efficiently and specifically capture approximately 250 μg of naringin without cross reacting with its structurally similar compounds. Moreover, our results indicate that the application of immunoaffinity chromatography can simplify the pretreatment and the isolation process greatly compared to conventional methods, providing a potential method for extracting the target component from structurally similar compounds in natural products.     

Study on Adsorption and Separation of Naringin with Macroporous Resin

X-5 resin, with higher adsorption and easier desorption of naringin, was selected from five kinds of macroporous resins through static adsorption and desorption experiments. Effects of concentration, pH value, and flow rate of naringin extract on the adsorption of naringin by X-5 resin were studied. Meanwhile, the effect of these factors on the desorption of naringin from X-5 resin was also investigated. The experimental results show that the adsorption isotherm of naringin by X-5 resin can be described by the Langmuir isotherm equation. The static maximum adsorption capacity of naringin is 32.6 mg/g with naringin concentration at 2.7 g/L, while the dynamic adsorption capacity of naringin is 23.8 mg/g with naringin extract flow rate at two times that of resin volume per hour. The optimal eluant is 60% (v/v) ethanol-water with pH value of 10. The desorption ratio will rise to more than 85% when the flow rate of this optimal eluant is one to two times that of resin volume per hour. Translated from Journal of Central South University (Science and Technology)     

Determination of naringin and neohesperidin in orange juice by liquid chromatography with UV detection to detect the presence of grapefruit juice: Collaborative Study

Fifteen collaborating laboratories were sent 9 samples of citrus juice mixtures as blind duplicates for determination of naringin and neohesperidin by liquid chromatography. Two sample pairs were 100% orange juice and did not contain any naringin or neohesperidin. The remaining 7 sample pairs contained naringin at levels ranging from 3.9 to 46.5 ppm and neohesperidin at levels ranging from 0.14 to 35.6 ppm. Five sample pairs consisted of orange juice mixtures containing 1, 3, and 5% grapefruit juice; 5% sour orange; and 5% K-Early citrus variety. Two sample pairs were orange juice spiked with naringin, neohesperidin, sodium benzoate, and potassium sorbate. Data were received from 13 laboratories. Data from 1 collaborator were eliminated because the method protocol was not followed. Neohesperidin values from another laboratory were also not used because of problems with a coeluting component. Repeatability relative standard deviations ranged from 2.95 to 15.23% for naringin and from 3.00 to 11.74% for neohesperidin. Reproducibility relative standard deviations ranged from 11.34 to 31.94% for naringin and from 10.45 to 26.17% for neohesperidin. The method is reliable for detecting the presence of grapefruit juice in orange juice as indicated by a finding of > or =10 ppm naringin and < or =2 ppm neohesperidin. The method was adopted First Action by AOAC INTERNATIONAL.     

Determination of diastereomerization barrier of some flavanones by high-performance liquid chromatography methods

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.     

Extraction of naringin from pomelo peels as dihydrochalcone's precursor

A new method for the separation of naringin from pomelo peels was investigated by using ultrasonic-assisted extraction and macroporous resin purification technology. The ultrasonic extraction efficiency was dependent on agent's concentration, ratio of sample and solvent and ultrasonic time. Several parameters of macroporous resin-purified process, including resin selection, initial concentration, concentration of eluted agent and pH, were optimized. The experimental results showed that the naringin content in the mature pomelo peels was 2.20% and purification rate of naringin was 77.26% under optimum conditions of purification. The structure of synthetic naringin dihydrochalcone was determined by a series of spectroscopic methods, such as UV, NMR and MS.     

Preparation of Polymeric Membranes Entrapping β-Cyclodextrins and Their Molecular Recognition of Naringin

An amorphous polyetheretherketone known as PEEK-WC and a β-cyclodextrin derivative (O-octyloxycarbonyl-β-CD), insoluble in water, were used to prepare flat sheet membranes by the diffusion induced phase separation. It was found that the β-CD derivative entrapped in membranes is able to form a complex with naringin, a bitter component present in grapefruits. The recognition properties of the β-CD derivative immobilized in the PEEK-WC membranes towards naringin have been investigated as a function of the amount of β-CD derivative present in the casting solution. A significant improvement of the efficiency of theO-octyloxycarbonyl-β-CD was observed when an inclusion complex with naringin in the membrane was formed compared to when the β-CD derivative is dispersed in an aqueous solution of naringin. Washing with an alkaline solution allows an easy regeneration of the membranes and permits their reuse. No recognition properties were found for PEEK-WC membranes made without the β-CD derivative.     

免疫亲和色谱特异性剔除中药方剂四逆散中的柚皮苷

为了获得剔除柚皮苷(naringin)的中药方剂四逆散样品以供其药理活性探讨时使用,制备了抗柚皮苷抗体的免疫亲和色谱柱,用于特异性地剔除四逆散中的柚皮苷。首先合成了柚皮苷的完全抗原柚皮苷与牛血清白蛋白的结合物naringin-BSA,并用naringin-BSA对新西兰兔进行免疫获得抗血清,再将其纯化后与经CNBr活化的Sepharose 4B凝胶共价偶联制成免疫亲和色谱柱。将四逆散提取物样品溶液上样该色谱柱,洗脱,制得特异性剔除了柚皮苷的四逆散样品。由检测结果可知,naringin-BSA被成功合成。将其用于免疫新西兰兔,获得的抗血清的效价经酶联免疫吸附法（ELISA）测定达到1∶30000,抗体IgG的纯度达94%,交叉反应率低。在IgG与Sepharose 4B合成的IgG-Sepharose免疫亲和色谱柱中,IgG的偶联率为87%。用该免疫亲和色谱柱处理四逆散后,其中所含的柚皮苷几乎完全被剔除。结果证明,利用抗柚皮苷免疫亲和色谱,能特异性地剔除四逆散或其他样品中的柚皮苷成分。     

Modulation of the pharmacological activities of secretory phospholipase A2 from Crotalus durissus cascavella induced by naringin

In this work we have characterized the action of the naringin, a flavonoid found in grapefruit and known for its various pharmacological effects, which include antioxidant blood lipid lowering and anticancer activity, on the structure and biochemical activities of a secretory phospholipase A (sPLA2) from Crotalus durissus cascavella, an important protein involved in the releasinge of arachidonic acid in phospholipid membranes. sPLA2 was incubated with naringin (mol:mol) at 37 °C and a discrete reduction in the UV scanning signal and a modification of the circular dichroism spectra were observed after treatment with naringin, suggesting modifications of the secondary structure of the protein. This flavonoid was able to decrease enzymatic activity and some pharmacological effects, such as myonecrosis, platelet aggregation, and neurotoxic activity caused by sPLA2, however, the inflammatory effect was not affected by naringin. In addition, small angle X-ray scattering (SAXS) data were collected for sPLA2 and naringin-treated sPLA2 to evaluate possible modifications of the protein structure. These structural investigations have shown that sPLA2 is an elongated dimer in solution and after treatment with naringin a conformational change in the dimeric configuration was observed. Our results suggest that structural modification may be correlated with the loss of enzymatic activity and alterations in pharmacological properties.     

Mesoporous silica and chitosan based pH-sensitive smart nanoparticles for tumor targeted drug delivery

This study investigated inclusion formation and the physicochemical properties of naringin/cyclodextrin through a combined computational and experimental approach. Molecular dynamics simulations were applied to investigate the thermodynamics and geometry of naringin/cyclodextrin cavity docking. The complexes were investigated by UV, FT-IR, DSC, XRD, SEM, 2D-NOSEY and ¹H-NMR analyses. Clearly visible protons belonging to naringin and chemical shift displacements of the H3 and H5 protons in cyclodextrin were anticipated in the formation of an inclusion complex. Naringin solubility increased linearly with increasing cyclodextrin concentration (displaying an A_L profile). The simulations indicated that the phenyl group of naringin was located deep within the cyclodextrin cavity, while the glycoside group of naringin was on the plane of the wider rim of cyclodextrin. The simulation and molecular modeling results indicate that (2-hydroxypropyl)-β-cyclodextrin (HP-β-CD) provided the more stable inclusion complex. This result was also in good concordance with the stability constants that had been determined by the phase solubility method. The consistency of the computational and experimental results indicates their reliability.     

Controllable Regioselective Acylation of Flavonoids Catalyzed by Lipase in Microreactors

A method for regioselective acylation of flavonoids (naringin and naringin dihydrochalcone) in a flow microreactor using Lipozyme TL IM from Thermomyces lanuginosus as the catalyst has been developed. The important features of this method include mild reaction conditions, short reaction times (30 min), high yields, and high regioselectivities.     

Naringin Exhibited Therapeutic Effects against DSS-Induced Mice Ulcerative Colitis in Intestinal Barrier–Dependent Manner

Naringin is a kind of multi-source food additive which has been explored broadly for its various biological activities and therapeutic potential. In the present study, the protective effect and mechanism of naringin on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice were investigated. The results showed that naringin significantly alleviated DSS-induced colitis symptoms, including disease activity index (DAI), colon length shortening, and colon pathological damage. The tissue and serum secretion of inflammatory cytokines, as well as the oxidative stress, were decreased accordingly upon naringin intervention. Naringin also decreased the proteins involved in inflammation and increased the expression of tight junction (TJ) proteins. Moreover, naringin increased the relative abundance of Firmicutes/Bacteroides and reduced the content of Proteobacteria to improve the intestinal flora disorder caused by DSS, which promotes the intestinal health of mice. It was concluded that naringin can significantly ameliorate the pathogenic symptoms of UC through inhibiting inflammatory response and regulating intestinal microbiota, which might be a promising natural therapeutic agent for the dietary treatment of UC and the improvement of intestinal symbiosis.     

反相高效液相法测定枳实、枳壳中橙皮甙和柚皮甙的含量

 采用高效液相法测定了枳实和枳壳中的橙皮甙和柚皮甙含量。柱为HypersilODS1柱(250mm×4 6mmi d ),流动相为乙腈 0 5%乙酸溶液(体积比为22∶78),流速为1 0mL/min,检测波长为283nm。柚皮甙和橙皮甙的线性范围分别为0 060g/L～0 604g/L和0 0125g/L～0 125g/L,柚皮甙和橙皮甙的平均回收率分别为97 1%和95 3%。该方法具有操作简便、分析快速、准确等优点。     

高效液相色谱法测定柑橘汁中的柠檬苦素和柚皮苷

柑橘汁的苦味主要是由于柚皮苷和柠檬苦素的存在所致,其含量的测定可用于控制柑橘类果汁的质量。采用高效液相色谱法在KR100-5C18(4.6 mm i.d.×250 mm,5 μm)上,分别以乙腈-四氢呋喃-水(体积比为17.5∶17.5∶65)和甲醇-冰醋酸-水(体积比为40∶1∶59)为流动相（流速均为1 mL/min）,在207 nm和283 nm检测波长下分别测定了柠檬苦素和柚皮苷。实验结果表明,柠檬苦素在1.00～50.00 mg/L时线性关系良好(r=0.9992),检出限为0.07 μg,平均加标回收率为98.69%,相对标准偏差（RSD）为2.5%;柚皮苷在20.00～160.00 mg/L时线性关系良好(r=0.9988),检出限为0.14 μg,平均加标回收率为100.13%,RSD为1.5%。用该法检测柑橘汁样品中的柠檬苦素与柚皮苷,方法简便、快速、准确。     

Synthesis and characterization of a metal complex containing naringin and Cu, and its antioxidant, antimicrobial, antiinflammatory and tumor cell cytotoxicity

The antioxidant activity of flavonoids is believed to increase when they are coordinated with transition metal ions. However, the literature on this subject is contradictory and the outcome seems to largely depend on the experimental conditions. In order to understand the contribution of the metal coordination and the type of interaction between a flavonoid and the metal ion, in this study a new metal complex of Cu (II) with naringin was synthesized and characterized by FT-IR, UV-VIS, mass spectrometry (ESI-MS/MS), elemental analysis and 1H-NMR. The results of these analyses indicate that the complex has a Cu (II) ion coordinated via positions 4 and 5 of the flavonoid. The antioxidant, anti-inflammatory and antimicrobial activities of this complex were studied and compared with the activity of free naringin. The Naringin-Cu (II) complex 1 showed higher antioxidant, anti-inflammatory and tumor cell cytotoxicity activities than free naringin without reducing cell viability.     

First Order Derivative Voltammetry on the in situ Surfactant Modified Electrode for Naringin Quantification

Carboxylated multi‐walled carbon nanotubes based glassy carbon electrodes (MWNT‐COOH/GCE) modified in situ with surfactants (sodium dodecyl sulfate (SDS), cetylpyridinium bromide (CPB) and Triton X100) have been tested for the naringin determination. The effect of surfactant nature and concentration on the voltammetric characteristics of naringin has been evaluated. Anionic 100 μM SDS shows the highest 2.7‐fold increase of the oxidation currents in comparison to MWNT‐COOH/GCE. The irreversible electrooxidation of naringin is surface‐controlled process with the one electron and one proton transfer. Under conditions of first order derivative linear sweep voltammetry in Britton‐Robinson buffer (BRB) pH 8.0, the analytical ranges of 0.75–25 and 25–100 μM with the LOD and LOQ of 0.14 and 0.46 μM, respectively, have been obtained. The electrode response is selective in the presence of ascorbic, gallic and p‐coumaric acids as well as quercetin, catechin and rutin. The method has been applied for the naringin quantification in grapefruit juices.     

Simultaneous separation and sensitive detection of naringin and naringenin in nanoparticles by chromatographic method indicating stability and photodegradation kinetics

A simple, sensitive, precise and linear method by liquid chromatography was established for simultaneous determination and quantification of naringin and naringenin in polymeric nanoparticles. The method results in excellent separation in <11 min and with a peak purity of both flavonoids. The analyses were performed using a C₁₈ column (4.6 × 150 mm, 5 µm), at a 1 mL/min flow rate. The mobile phase consisted of a gradient of acetonitrile–water (pH 4.0; v/v) at a temperature of 25°C. The nanoparticles were prepared according to the method of interfacial deposition of a pre‐formed polymer. The method were validated in compliance with guidelines, and was found to be linear in the 1–40 µg/mL concentration range for both naringin and naringenin (r > 0.99). Repeatability was determined at three concentration levels, obtaining an RSD (%) <0.9%, and the accuracy of the method was >98%. The photodegradation kinetics was determined for naringin; the coefficient that best represents degradation was of first order and naringenin presented a zero‐order kinetics. To our knowledge, a rapid and sensitive method for naringin and naringenin in polymeric nanoparticles has not been published elsewhere and this method is applicable to simultaneous evaluation of flavonoids. Copyright © 2015 John Wiley & Sons, Ltd.     

Application of an efficient strategy based on MAE,HPLC‐DAD‐MS/MS and HSCCC for the rapid extraction,identification, separation and purification of flavonoids from Fructus Aurantii Immaturus

This study presents an efficient strategy based on microwave‐assisted extraction (MAE), HPLC‐DAD‐MS/MS and high‐speed counter‐current chromatography (HSCCC) for the rapid extraction, identification, separation and purification of active components from the traditional Chinese medicine Fructus Aurantii Immaturus. An LC‐DAD‐MS/MS method was applied for the screening and structural identification of main components in crude extract, and five components were preliminarily identified as neoeriocitrin, narirutin, naringin, hesperidin and neohesperidin according to their UV and mass spectra. An efficient MAE method for the extraction of the three most abundant components (narirutin, naringin and neohesperidin) was optimized by the combination of univariate and multivariate approaches. The crude extract was then separated and purified by HSCCC and a total of 61.6 mg of narirutin, 207.3 mg of naringin and 159.5 mg of neohesperidin at high purities of 98.1, 97.2 and 99.5%, respectively, were obtained from 1.42 g of crude extract. The recoveries of these compounds were 86, 93 and 89%, respectively. Copyright © 2009 John Wiley & Sons, Ltd.     

A straightforward access to neohesperidose from naringin by acetolysis. Comparative behaviour of some flavonoid neohesperidosides and rutinosides under acetolysis

A straightforward semisynthesis of neohesperidose was performed in three-steps with 43% overall yield starting from naringin, the main flavonoid of grapefruit. In addition, the behaviour of two pairs of flavonoid 7-O-rhamnoglucosides (neohesperidosides naringin and rhoifolin; rutinosides hesperidin and diosmin) under same acetolysis conditions was compared. Results demonstrated the crucial influence of the oxidation state of the aglycone (flavanone or flavone), and of the nature of the disaccharidic moiety on the course of the reaction.     

Optical isomer separation of flavanones and flavanone glycosides by nano-liquid chromatography using a phenyl-carbamate-propyl-β-cyclodextrin chiral stationary phase

In this paper a phenyl-carbamate-propyl-β-cyclodextrin stationary phase was employed for the enantioseparation of several flavonoids, including flavanones and methoxyflavanones by using nano-liquid chromatography (nano-LC). The same stationary phase was also used for the diastereoisomeric separation of two flavanone glycosides. The compounds: flavanone, 2′-hydroxyflavanone, 4′-hydroxyflavanone, 6-hydroxyflavanone, 7-hydroxyflavanone, 4′-methoxyflavanone, 6-methoxyflavanone, 7-methoxyflavanone, hesperetin, hesperidin, naringenin and naringin were studied using reversed, polar organic and normal elution modes. The effect of the nature and composition of the mobile phase (organic modifier type, buffer and water content in the reversed phase mode) on the enantioresolution (R_s), retention factor (k) and enantioselectivity (α) were investigated. Baseline resolution of all studied flavonoids, with the exception of 2′-hydroxyflavanone and naringin, was achieved in reversed phase mode using a mixture of MeOH/H₂O at different ratios as mobile phase. Good results, in terms of peak efficiency and short analysis time, were obtained adding 1% triethylammonium acetate pH 4.5 buffer to MeOH/H₂O mixture. The separation of the studied compounds was also performed in polar organic mode. By using 100% of MeOH as mobile phase, the resolution was achieved for the studied analytes, except for 7-hydroxyflavanone, 2′-hydroxyflavanone, naringenin, hesperidin and naringin. Normal mode was tested employing a mixture of EtOH/hexane/TFA as mobile phase achieving the enantiomeric and diastereomeric separation of only hesperetin and hesperidin, respectively. The use of nano-LC technique for the resolution of flavanones optical isomers allowed to achieve good resolutions in shorter analysis time compared to the results reported in literature with conventional HPLC.