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.     

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。     

Liquid Chromatography for Separation and Quantitative Determination of Adrenergic Amines and Flavonoids from Poncirus trifoliatus Raf. Fruits at Different Stages of Growth

The fruits of Poncirus trifoliatus Raf. (Rutaceae) have been used against allergic diseases for generations and still occupy an important place in traditional oriental medicine. They have also been used to treat gastric and duodenal ulcers. Chemical analysis of extracts of Poncirus trifoliatus Raf. fruit at different stages of maturation revealed variations in the concentrations of flavonoids present. Fourteen flavonoids (neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, neoponcirin, poncirin, naringenin, hesperetin, sinensetin, nobiletin, heptamethoxyflavone, 5-O-demethylnobiletin, and tangeretin) and five amines (synephrine, octopamine, N-methyltyramine, hordenine, and tyramine) in extracts from the fruit of Poncirus trifoliatus Raf. were analyzed by HPLC with a C₁₈ reversed-phase column. The concentrations of the four flavonoids naringin, poncirin, narirutin, and neoponcirin was maximum during the first stage of growth and gradually decreased until the fruits were fully developed. The paper also discusses the anatomical variations observed at different stages of development of the fruit.     

Dissolution profiles of principal ingredients in Kampo medicinal powders by high-performance liquid chromatography

We describe here a method using HPLC for the simultaneous determination of albiflorin, paeoniflorin, glycyrrhizin and six flavanone glycosides (liquiritin, liquiritin apioside, naringin, neohesperidin, hesperidin and narirutin) in the Kampo medicines, Shigyaku-san and Haino-san. All nine components were separated in less than 40 min by linear gradient elution using a mobile phase containing aqueous phosphoric acid and acetonitrile. The dissolution of these components from powders of Shigyaku-san in aqueous solution at pH 1.80, 4.08 and 6.89 was examined. All of the components except glycyrrhizin were dissolved entirely within 5 min regardless of pH. Dissolution of glycyrrhizin was dependent on the pH of the aqueous solution, and increased with increasing pH.     

Simultaneous separation of flavanone glycosides and polymethoxylated flavones in citrus juices using liquid chromatography

We present a simultaneous liquid chromatographic method for the separation of two flavonoid compound families, flavanone glycosides (FGs) and polymethoxylated flavones (PMFs), which are usually found in citrus fruit species and varieties. This technique permits the quantitation of six FGs (narirutin, naringin, hesperidin, neohesperidin, didymin, poncirin) and six PMFs (sinensetin, hexamethoxyflavone, nobiletin, scutellarein, heptamethoxyflavone and tangeretin). This technique, to be used to characterize a citrus juice by its polyphenolic profile, has been applied to the determination of flavonoid compounds in grapefruit- and orange juice. Differentiation of orange juice varieties and mixtures containing tangor juice using polyphenolic profiles and flavonoid content has been achieved.     

Overall rate constant measurements of the reaction of hydroxy- and chloroalkylperoxy radicals derived from methacrolein and methyl vinyl ketone with nitric oxide

The overall rate constants of the reactions of NO with hydroxy- and chloroalkylperoxy radicals, derived from the OH- and Cl-initiated oxidation of methacrolein and methyl vinyl ketone, respectively, were directly determined for the first time using the turbulent-flow technique and pseudo-first-order kinetics conditions with high-pressure chemical ionization mass spectrometry for the direct detection of peroxy radical reactants. The individual 100 Torr, 298 K hydroxyalkylperoxy + NO rate constants for the methacrolein [(0.93 +/- 0.12) (2sigma) x 10(-11) cm3 molecule(-1) s(-1)] and methyl vinyl ketone [(0.84 +/- 0.10) x 10(-11) cm3 molecule(-1) s(-1)] systems were found to be identical within the 95% confidence interval associated with each separate measurement, as were the chloroalkylperoxy + NO rate constants for both methacrolein [(1.17 +/- 0.11) x 10(-11) cm3 molecule(-1) s(-1)] and methyl vinyl ketone [(1.14 +/- 0.14) x 10(-11) cm3 molecule(-1) s(-1)]. However, the difference in the rate constants between the hydroxyperoxy + NO and chloroalkylperoxy + NO systems was found to be statistically significant, with the chloroalkylperoxy + NO rate constants about 30% higher than the corresponding hydroxyalkylperoxy + NO rate constants. This substituent effect was rationalized via a frontier molecular orbital model approach.     

Differentiation of Four Pairs of Isomers by Comparing Their Relative Abundances of Fragment Ions

Based on the LC-ESI/MSn technique, the four pairs of isomers of psoralen and isopsoralen, imperatorin and isoimperatorin, neohesperidin and hesperidin, naringin and narirutin were distinguished mainly by comparison of the relative abundances of their major fragment ions. Because of the effect of the hydrogen on the C8 of psoralen, the intensity of the fragment ion formed by the loss of CO2 was relatively increased. By comparing the abundance of the product ions formed by the loss of CO and 2CO, the isomers ...     

Origin identification on the commercial samples of Aurantii Fructus

A total of 50 commercial samples of Aurantii Fructus, originating from Poncitrus trifoliata for Aurantii Fructus Immaturus, Citrus aurantium, and C. wilsonii for Aurantii Fructus Maturus, respectively, were collected from Taiwan and China herbal markets. Contents of the constituents in the samples were determined within 60 min using a developed HPLC method, which had been validated in terms of precision, repeatability, and accuracy. The results of the analyses showed that the constituents were closely related to the species and also the degree of maturity of the fruits, especially in the case of hesperidin (HE), naringin (NG), neohesperidin (NE), naringenin-7-glucoside (NGC), narirutin (NR), and quercetin (QU). The mature fruits (C. aurantium and C. wilsonii) contained chiefly NG, HE, and NE and the immature ones (P. trifoliata) had NG, NR, and QU majorly. Within the mature samples, the ratios of HE/NE and NGC/NE values were higher than 2.94 and 0.21 in C. aurantium and lower than 1.31 and 0.02 in C. wilsonii, respectively. A flowchart that is useful for identifying Aurantii Fractus was devised, based on the various chemical identification methods.     

HPLC Fingerprint with Multi-components Analysis for Quality Consistency Evaluation of Traditional Chinese Medicine Si-Mo-Tang Oral Liquid Preparation

Si-Mo-Tang(SMT) oral liquid preparation, a traditional Chinese medicine, was prepared from four crude herbal drugs, Fructus Aurantii Submaturus, Radix Aucklandiae, Semen Arecae and Radix Linderae Aggregatae. A combinative method using HPLC fingerprint and quantitative analysis was developed and validated for quality consistency evaluation of SMT. Individual HPLC chromatograms were evaluated against the mean chromatogram generated via a similarity evaluation computer program. Data from chromatographic fingerprints were also processed with principal component analysis(PCA) and hierarchical cluster analysis(HCA). Additionally, six components (naringin, isonaringin, hesperidin, neohesperidin, norisoboldine and potassium sorbate) in SMT were simultaneously determined to interpret the quality consistency. For fingerprint analysis, 20 peaks were selected as the characteristic peaks to evaluate the similarities of 26 SMT collected from different manufacturers. Among the 20 characteristic peaks, 10 peaks were assigned to be naringin, hesperidin, neohesperidin, isonaringin, neoeriocitrin, tangeretin, nobiletin, norisoboldine, 5-(ethoxymethyl)furan-2-carbaldehyde and potassium sorbate, respectively. The results of similarity analysis, PCA and HCA, indicate that the samples from different manufacturers were consistent with each other in composition. The results from the quantitative data show that the contents of six compounds were significantly different in SMT oral liquid preparations from different manufacturers. The combinative method of chromatographic fingerprint with quantitative analysis developed here offered an efficient way for the quality consistency evaluation of the traditional Chinese medicine SMT.     

Kinetic and mechanistic studies of allicin as an antioxidant

We have undertaken a detailed study of the antioxidant activity of allicin, one of the main thiosulfinates in garlic, in order to obtain quantitative information on it as a chain-breaking antioxidant. The antioxidant actions of allicin against the oxidation of cumene and methyl linoleate (ML) in chlorobenzene were studied in detail using HPLC. The hydroperoxides formed during the course of the inhibited oxidation of ML were analyzed as their corresponding alcohols by HPLC, and it is apparent that an allylic hydrogen atom of the allicin is responsible for the antioxidant activity. Furthermore, it is clear that the radical-scavenging reactions of allicin proceed via a one-step hydrogen atom transfer based on the results of the reaction with 2,2-diphenyl-1-picrylhydrazyl (DPPH) in the presence of Mg2+ and calculation of the ionization potential value. In addition, we determined the stoichiometric factor (n), the number of peroxyl radicals trapped by one antioxidant molecule, of allicin by measuring the reactivity toward DPPH in chlorobenzene, and the value of n for allicin was about 1.0. Therefore, we measured the rate constants, k(inh), for the reaction of allicin with peroxyl radicals during the induction period of the cumene and the ML oxidation. As a result, we found that allicin reacts with peroxyl radicals derived from cumene and ML with the rate constants k(inh) = 2.6 x 10(3) M(-1)s(-1) and 1.6 x 10(5) M(-1)s(-1) in chlorobenzene, respectively. Our results demonstrate for the first time reliable quantitative kinetic data and the antioxidative mechanism of allicin as an antioxidant.     

Free-radical-induced oxidative and reductive degradation of sulfa drugs in water: absolute kinetics and efficiencies of hydroxyl radical and hydrated electron reactions

Absolute rate constants and degradation efficiencies for hydroxyl radical and hydrated electron reactions with four different sulfa drugs in water have been evaluated using a combination of electron pulse radiolysis/absorption spectroscopy and steady-state radiolysis/high-performance liquid chromatography measurements. For sulfamethazine, sulfamethizole, sulfamethoxazole, and sulfamerazine, absolute rate constants for hydroxyl radical oxidation were determined as (8.3 +/- 0.8) x 10(9), (7.9 +/- 0.4) x 10(9), (8.5 +/- 0.3) x 10(9), and (7.8 +/- 0.3) x 10(9) M(-1) s(-1), respectively, with corresponding degradation efficiencies of 36% +/- 6%, 46% +/- 8%, 53% +/- 8%, and 35% +/- 5%. The reduction of these four compounds by their reaction with the hydrated electron occurred with rate constants of (2.4 +/- 0.1) x 10(10), (2.0 +/- 0.1) x 10(10), (1.0 +/- 0.03) x 10(10), and (2.0 +/- 0.1) x 10(10) M(-1) s(-1), respectively, with efficiencies of 0.5% +/- 4%, 61% +/- 9%, 71% +/- 10%, and 19% +/- 5%. We propose that hydroxyl radical adds predominantly to the sulfanilic acid ring of the different sulfa drugs based on similar hydroxyl radical rate constants and transient absorption spectra. In contrast, the variation in the rate constants for hydrated electrons with the sulfa drugs suggests the reaction occurs at different reaction sites, likely the different heterocyclic rings. The results of this study provide fundamental mechanistic parameters, hydroxyl radical and hydrated electron rate constants, and degradation efficiencies that are critical for the evaluation and implementation of advanced oxidation processes (AOPs).     

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。     

Analytical monitoring of citrus juices by using capillary electrophoresis

A capillary electrophoretic method was developed to analyze simultaneously most citrus juice components in a single procedure. After filtration, sample components are separated with an uncoated capillary tubing and a 35 mM sodium borate buffer (pH 9.3) containing 5% (v/v) acetonitrile. Analyses were run at 21 kV and 23 degrees C. Compounds monitored regularly were the biogenic amine synephrine, some flavonoids (didymin, hesperidin, narirutin, neohesperidin, and naringin), the polyphenol phlorin, 3 UV-absorbing amino acids (tryptophan, phenylalanine, and tyrosine), ascorbic acid, an unidentified peak generated by heat and storage, and the preservatives sorbate and benzoate that can be added to citrus products. Separation can be achieved in 20 min, and each compound can be subsequently quantitated. Didymin, narirutin, and phlorin peaks were used with an artificial neural network to assess the volume of added pulp wash, a by-product of juice preparation. This method allows rapid monitoring of citrus juices, giving information on quality, freshness, and possible adulteration of the product. Similar procedures could be used to monitor other fruit juices and quantitate diverse juice blends.     

Desorption of polycyclic aromatic hydrocarbons from soot surface: pyrene and fluoranthene

The kinetics of thermal desorption of two four-ring polycyclic aromatic hydrocarbons, fluoranthene, and pyrene from well-characterized laboratory-generated kerosene soot surface was studied over the temperature range 260-320 K in a low-pressure flow reactor combined with an electron-impact mass spectrometer. Two methods were used to measure the desorption rate constants: monitoring of the surface-bound fluoranthene and pyrene decays due to desorption using off-line HPLC measurements of their concentrations in soot samples, and monitoring of the desorbed molecules in the gas phase using in situ mass spectrometric detection. Results obtained with the two methods were in good agreement and yielded the following Arrhenius expressions for the desorption rate constants: k(des) (fluoranthene) = 4 x 10(14) exp[-(93900 +/- 1700)/RT] and k(des) (pyrene) = 6 x 10(14) exp[-(95200 +/- 1800)/RT] (k(des) are in units of s(-1), and activation energies are in J mol(-1)). In addition, the combined uptake coefficient of fluoranthene and pyrene on soot (calculated using specific surface area) was estimated to be near 5 x 10(-3) at T = 310 K.     

Repair reactions of pyrimidine-derived radicals by aliphatic thiols

Pyrimidinyl radicals of various structures (Pyr*) were generated in aqueous and alcohol-containing solutions by means of pulse radiolysis to determine the rate constants of their repair reactions by different thiols (RSH = cysteamine, 2-mercaptoethanol, cysteine, and penicillamine): Pyr* + RSH --> PyrH + RS*. C5-OH and C6-OH adduct radicals of the pyrimidines react with thiols with k9 = (1.2-10.0) x 10(6) dm3 mol(-1) s(-1). Similar repair rate constants were found for uracil- and thymine-derived N1-centered radicals, k31 = (1.5-6.1) x 10(6) dm3 mol(-1) s(-1). However, pyrimidine radical anions protonated at their C6 position and C6-uracilyl radicals, with carbonyl groups at their C5 position, react with thiols faster, with k24 = (0.5-7.6) x 10(7) dm3 mol(-1) s(-1) and k14 = (1.4-4.8) x 10(7) dm3 mol(-1) s(-1), respectively. Quantum chemical calculations, at the B3LYP/6-31G(d,p) and self-consistent reaction field polarizable continuum model level point to the combined effects of the energy gap between interacting molecular orbitals, charge distribution within different pyrimidine-derived radicals, and the coefficients of the atomic orbitals as the possible reasons for the differences in the rate constants of repair.     

The kinetics of Z/E isomerization of methyl oleate catalyzed by photogenerated thiyl radicals.

The reaction of beta-mercaptoethanol with methyl oleate in tertbutyl alcohol was investigated under photochemical conditions. The time-dependent Z/E isomerization of the unsaturated moiety as well as the thiol-adduct formation were analyzed on the basis of radical generation rates and established rate constants. This provides precise room-temperature rate constants for the reversible thiyl radical addition to both Z and E isomers. The rate constants for HOCH2CH2S* addition to the Z and E forms were found to be 1.6 x 10(5) and 2.9 x 10(5) M(-1) S(-1), respectively. For the beta-elimination of the thiyl radical from the alkyl radical adduct, the rate constants were 1.7 x 10(7) and 1.6 x 10(8) S(-1), the faster associated with the formation of the E isomer. DFT-BB1K calculations on 2-butene assign this large preference of fragmentation for the E isomer to a lower activation barrier for the formation of the E transition state from the equilibrium radical adduct structure.     

Quenching of I(2P1/2) by NO2, N2O4, and N2O

Quenching of excited iodine atoms (I(5p5, 2P1/2)) by nitrogen oxides are processes of relevance to discharge-driven oxygen iodine lasers. Rate constants at ambient and elevated temperatures (293-380 K) for quenching of I(2P1/2) atoms by NO2, N2O4, and N2O have been measured using time-resolved I(2P1/2) --> I(2P3/2) 1315 nm emission. The excited atoms were generated by pulsed laser photodissociation of CF3I at 248 nm. The rate constants for I(2P1/2) quenching by NO2 and N2O were found to be independent of temperature over the range examined with average values of (2.9 +/- 0.3) x 10(-15) and (1.4 +/- 0.1) x 10(-15) cm3 s(-1), respectively. The rate constant for quenching of I(2P1/2) by N2O4 was found to be (3.5 +/- 0.5) x 10(-13) cm3 s(-1) at ambient temperature.     

From alcohols to sugars-how does the reactivity of alpha-hydroxyalkyl radicals change with structure? A quantitative examination by pulse radiolysis

Rate constants are reported for the 1-electron reduction of the azo dye Orange II in water (pH 7.0) by 10 different alpha-hydroxy radicals. The radicals were created by pulse radiolysis of aqueous solutions of the corresponding alcohol/sugar. The rate constants varied from 1 x 10(8) to 2.7 x 10(9) mol(-1) dm(3) s(-1) and radicals with beta-hydroxy groups had the lowest rate constant. The reaction was found to be controlled by the reduction potentials of the radicals, with steric influences having little effects. Good fits of the data were obtained using the Marcus equation with lambda =140 kJ/mol.     

Room-temperature decomposition of 2,2'-azobis(isobutyronitrile) in emulsion gels with and without silica

The decomposition of 2,2'-azobis(isobutyronitrile) (AIBN) at room temperature was studied in emulsion gels, with and without silica, using UV/visible spectroscopy. The emulsion gels consisted of toluene, AIBN, an aqueous solution of a surfactant (either hexadecyltrimethylammonium bromide, CTAB, or sodium dodecyl sulfate, SDS), and, in some cases, fumed silica. The AIBN compositions were determined in the opaque emulsion gels by converting them to transparent microemulsions. The decomposition rate constants for AIBN were determined to be 3.7 x 10(-8) s(-1) (+/-0.6 x 10(-8) s(-1)) for the emulsion gels and 10.2 x 10(-8) s(-1) (+/-1.3 x 10(-8) s(-1)) for the silica-containing emulsion gels. These rate constants were significantly greater than that in toluene solution, which, by our technique, is below our measurement threshold (effectively 0).