Effects of Concentration of 5,5 - Dimethylhydantoin as Com- plexing Agent and pH Value of

酶法合成淀粉接枝聚己内酯共聚物的核磁分析1徐玲，杨成，段彬，蒋超江南大学化学与材料工程学院，江苏无锡（214122）E-mail：ckxl0571@摘要：在有机溶剂DMSO中，以猪胰脂肪酶为催化剂，合成了淀粉接枝聚己内酯共聚物。

利用红外光谱初步验证接枝情况，再采用1H-NMR、13C-NMR核磁共振光谱表征产物微结构，完成了1H-NMR、13C-NMR谱带的归属，并计算了己内酯的接枝率及主要接枝位置，实验结果己内酯接枝率达30％以上，主要接枝在C2、C3上。

关键词：淀粉，脂肪酶，开环聚合，己内酯，核磁共振1. 引言随着环境污染问题的越来越严重，完全生物可降解材料的研究引起了人们广泛的关注。

淀粉及其衍生物凭借其优良的性能成为研究的热点。

其中，淀粉接枝聚己内酯是近几年研发的新产物[1,2]。

目前报道的合成方法基本是利用钛酸四丁酯、辛酸亚锡等引发剂引发己内酯开环聚合并接枝到淀粉基上的化学合成，但所得产物易引入杂质、色泽偏黄、淀粉葡萄糖单元上三个羟基都有可能接上聚己内酯，位置选择性差。

酶催化作为一种生物催化方法，与传统的化学催化相比具有反应条件温和、位置选择性好、产物结果单一、纯度高、色泽浅等优点[3]，故本文选择以脂肪酶为催化剂，DMSO为溶剂，淀粉、己内酯为底物，尝试性合成淀粉-聚己内酯接枝共聚物。

应用1H-NMR、13C-NMR核磁共振光谱对合成产物进行微结构表征，分析谱带归属，计算己内酯的接枝率及主要接枝位置。

2. 实验2.1 原料ε-己内酯单体：Fluka Chemie GmbH CH-9471 Buchs(瑞士)，纯度≥99％；玉米淀粉：百灵威公司；猪胰脂肪酶：Sigma–Aldrich公司；二甲亚砜、甲醇、丙酮等，AR，国药集团化学试剂有限公司。

2.2 方法2.2.1 淀粉脱脂处理：乙醚为抽提液，用脂肪抽出器抽提12h，真空干燥后备用。

2.2.2 pH记忆酶的制备[ 4]：取适量酶浸泡在浓度为0.2mol/L不同pH值的磷酸缓冲溶液中，搅匀，冻干后备用。

Calculate the quantity, in mg, of thiamine hydrochloride (C12H17ClN4OS·HCl) in the assay material on the basis of the ali-quots taken. Where indicated, the quantity, in mg, of thiamine mononitrate (C12H17N5O4S) may be calculated by multiplyingthe quantity of C12H17ClN4OS·HCl found by 0.9706.á541ñ TITRIMETRYDirect Titrations—Direct titration is the treatment of a soluble substance, contained in solution in a suitable vessel (the titrate), with an appropriate standardized solution (the titrant), the endpoint being determined instrumentally or visually with the aid of a suitable indicator.The titrant is added from a suitable buret and is so chosen, with respect to its strength (normality), that the volume added is between 30% and 100% of the rated capacity of the buret. [N OTE—Where less than 10 mL of titrant is required, a suitable microburet is to be used.] The endpoint is approached directly but cautiously, and finally the titrant is added dropwise from the buret in order that the final drop added will not overrun the endpoint. The quantity of the substance being titrated may be calculated from the volume and the normality or molarity factor of the titrant and the equivalence factor for the substance given in the individual monograph.Residual Titrations—Some Pharmacopeial assays require the addition of a measured volume of a volumetric solution, in excess of the amount actually needed to react with the substance being assayed, the excess of this solution then being titrated with a second volumetric solution. This constitutes a residual titration and is known also as a “back titration.” The quantity of the substance being titrated may be calculated from the difference between the volume of the volumetric solution originally added, corrected by means of a blank titration, and that consumed by the titrant in the back titration, due allowance being made for the respective normality or molarity factors of the two solutions, and the equivalence factor for the substance given in the individual monograph.Complexometric Titrations—Successful complexometric titrations depend on several factors. The equilibrium constant for formation of the titrant-analyte complex must be sufficiently large that, at the endpoint, very close to 100% of the analyte has been complexed. The final complex must be formed rapidly enough that the analysis time is practical. When the analytical reaction is not rapid, a residual titration may sometimes be successful.In general, complexometric indicators are themselves complexing agents. The reaction between metal ion and indicator must be rapid and reversible. The equilibrium constant for formation of the metal-indicator complex should be large enough to produce a sharp color change but must be less than that for the metal-titrant complex. Indicator choice is also restricted by the pH range within which the complexation reaction must be carried out and by interference of other ions arising from the sample or the buffer. Interfering ions may often be masked or “screened” via addition of another complexing agent. (The masking technique is also applicable to redox titrations.)Oxidation-Reduction (Redox) Titrations—Determinations may often be carried out conveniently by the use of a reagent that brings about oxidation or reduction of the analyte. Many redox titration curves are not symmetric about the equivalence point, and thus graphical determination of the endpoint is not possible; but indicators are available for many determinations, and a redox reagent can often serve as its own indicator. As in any type of titration, the ideal indicator changes color at an endpoint that is as close as possible to the equivalence point. Accordingly, when the titrant serves as its own indicator, the difference between the endpoint and the equivalence point is determined only by the analyst's ability to detect the color change. A common example is the use of permanganate ion as an oxidizing titrant since a slight excess can easily be detected by its pink color. Other titrants that may serve as their own indicators are iodine, cerium (IV) salts, and potassium dichromate. In most cases, however, the use of an appropriate redox indicator will yield a much sharper endpoint.It may be necessary to adjust the oxidation state of the analyte prior to titration through use of an appropriate oxidizing or reducing agent; the excess reagent must then be removed, e.g., through precipitation. This is nearly always the practice in the determination of oxidizing agents since most volumetric solutions of reducing agents are slowly oxidized by atmospheric oxy-gen.Titrations in Nonaqueous Solvents—Acids and bases have long been defined as substances that furnish, when dissolved in water, hydrogen and hydroxyl ions, respectively. This definition, introduced by Arrhenius, fails to recognize the fact that prop-erties characteristic of acids or bases may be developed also in other solvents. A more generalized definition is that of Bröns-ted, who defined an acid as a substance that furnishes protons, and a base as a substance that combines with protons. Even broader is the definition of Lewis, who defined an acid as any material that will accept an electron pair, a base as any material that will donate an electron pair, and neutralization as the formation of a coordination bond between an acid and a base.The apparent strength of an acid or a base is determined by the extent of its reaction with a solvent. In water solution all strong acids appear equally strong because they react with the solvent to undergo almost complete conversion to oxonium ion and the acid anion (leveling effect). In a weakly protophilic solvent such as acetic acid the extent of formation of the ace-tate acidium ion shows that the order of decreasing strength for acids is perchloric, hydrobromic, sulfuric, hydrochloric, andnitric (differentiating effect).Acetic acid reacts incompletely with water to form oxonium ion and is, therefore, a weak acid. In contrast, it dissolves in a base such as ethylenediamine, and reacts so completely with the solvent that it behaves as a strong acid. The same holds for perchloric acid.This leveling effect is observed also for bases. In sulfuric acid almost all bases appear to be of the same strength. As the acid properties of the solvent decrease in the series sulfuric acid, acetic acid, phenol, water, pyridine, and butylamine, the bases become progressively weaker until all but the strongest have lost their basic properties. In order of decreasing strength, the strong bases are sodium 2-aminoethoxide, potassium methoxide, sodium methoxide, and lithium methoxide.Many water-insoluble compounds acquire enhanced acidic or basic properties when dissolved in organic solvents. Thus the choice of the appropriate solvent permits the determination of a variety of such materials by nonaqueous titration. Further-more, depending upon which part of a compound is the physiologically active moiety, it is often possible to titrate that part by proper selection of solvent and titrant. Pure compounds can be titrated directly, but it is often necessary to isolate the active ingredient in pharmaceutical preparations from interfering excipients and carriers.The types of compounds that may be titrated as acids include acid halides, acid anhydrides, carboxylic acids, amino acids,enols such as barbiturates and xanthines, imides, phenols, pyrroles, and sulfonamides. The types of compounds that may be titrated as bases include amines, nitrogen-containing heterocyclic compounds, oxazolines, quaternary ammonium com-pounds, alkali salts of organic acids, alkali salts of weak inorganic acids, and some salts of amines. Many salts of halogen acids may be titrated in acetic acid or acetic anhydride after the addition of mercuric acetate, which removes halide ion as the unionized mercuric halide complex and introduces the acetate ion.For the titration of a basic compound, a volumetric solution of perchloric acid in glacial acetic acid is preferred, although perchloric acid in dioxane is used in special cases. The calomel-glass electrode system is useful in this case. In acetic acid sol-vent, this electrode system functions as predicted by theory.For the titration of an acidic compound, two classes of titrant are available: the alkali metal alkoxides and the tetraalkylam-monium hydroxides. A volumetric solution of sodium methoxide in a mixture of methanol and toluene is used frequently, al-though lithium methoxide in methanol-benzene solvent is used for those compounds yielding a gelatinous precipitate on titra-tion with sodium methoxide.The alkali error limits the use of the glass electrode as an indicating electrode in conjunction with alkali metal alkoxide ti-trants, particularly in basic solvents. Thus, the antimony-indicating electrode, though somewhat erratic, is used in such titra-tions. The use of quaternary ammonium hydroxide compounds, e.g., tetra-n -butylammonium hydroxide and trimethylhexade-cylammonium hydroxide (in benzene-methanol or isopropyl alcohol), has two advantages over the other titrants in that (a)the tetraalkylammonium salt of the titrated acid is soluble in the titration medium, and (b) the convenient and well-behaved calomel-glass electrode pair may be used to conduct potentiometric titrations.Because of interference by carbon dioxide, solvents for acidic compounds need to be protected from excessive exposure to the atmosphere by a suitable cover or by an inert atmosphere during the titration. Absorption of carbon dioxide may be deter-mined by performing a blank titration. The blank should not exceed 0.01 mL of 0.1 N sodium methoxide VS per mL of sol-vent.The endpoint may be determined visually by color change, or potentiometrically, as indicated in the individual monograph.If the calomel reference electrode is used, it is advantageous to replace the aqueous potassium chloride salt bridge with 0.1 N lithium perchlorate in glacial acetic acid for titrations in acidic solvents or potassium chloride in methanol for titrations in basic solvents.Where these or other mixtures are specified in individual monographs, the calomel reference electrode is modified by first removing the aqueous potassium chloride solution and residual potassium chloride, if any, by rinsing with water, then elimi-nating residual water by rinsing with the required nonaqueous solvent, and finally filling the electrode with the designated nonaqueous mixture.In nearly all cases, except those where silver ion might interfere, a silver-silver chloride reference electrode may be substitu-ted for the calomel electrode. The silver-silver chloride electrode is more rugged, and its use helps to eliminate toxic mercury salts from the laboratory. Generally, a salt bridge may be used to circumvent interference by silver ion.The more useful systems for titration in nonaqueous solvents are listed in Table 1.Table 1. Systems for Nonaqueous TitrationsType ofAcidic (for titrationof bases and theirsalts)Relatively Neutral(for differentialtitration of bases)Basic (for titrationof acids)Relatively Neutral(for differentialtitration of acids) Solvent1Glacial Acetic Acid Acetonitrile Dimethylformamide AcetoneAcetic Anhydride Alcohols n-Butylamine AcetonitrileFormic Acid Chloroform Pyridine Methyl Ethyl KetonePropionic Acid Benzene Ethylenediamine Methyl Isobutyl KetoneSulfuryl Chloride Toluene Morpholine tert-Butyl AlcoholChlorobenzeneEthyl AcetateDioxaneIndicator Crystal Violet Methyl Red Thymol Blue Azo VioletQuinaldine Red Methyl Orange Thymolphthalein Bromothylmol Bluep-Naphtholbenzein p-Naphtholbenzein Azo Violet p-HydroxyazobenzeneAlphezurine 2-G o-Nitroaniline Thymol BlueMalachite Green p-HydroxyazobenzeneElectrodes Glass–calomel Glass–calomel Antimony–calomel Antimony–calomelGlass–silver–silver chloride Calomel–silver–silverchlorideAntimony–glass Glass–calomelMercury–mercuric acetate Antimony–antimony2Glass–platinum2Platinum–calomelGlass–calomel1 Relatively neutral solvents of low dielectric constant such as benzene, toluene, chloroform, or dioxane may be used in conjunction with any acidic or basic solvent in order to increase the sensitivity of the titration end-points.2 In titrant.Indicator and Potentiometric Endpoint Detection—The simplest and most convenient method by which the equivalence point, i.e., the point at which the stoichiometric analytical reaction is complete, may be determined is with the use of indica-tors. These chemical substances, usually colored, respond to changes in solution conditions before and after the equivalence point by exhibiting color changes that may be taken visually as the endpoint, a reliable estimate of the equivalence point.A useful method of endpoint determination results from the use of electrochemical measurements. If an indicator electrode, sensitive to the concentration of the species undergoing titrimetric reaction, and a reference electrode, whose potential is in-sensitive to any dissolved species, are immersed in the titrate to form a galvanic cell, the potential difference between the elec-trodes may be sensed by a pH meter and used to follow the course of the reaction. Where such a series of measurements is plotted correctly (i.e., for an acid-base titration, pH versus mL of titrant added; for a precipitimetric, complexometric, or oxida-tion-reduction titration, mV versus mL of titrant added), a sigmoid curve results with a rapidly changing portion (the “break”) in the vicinity of the equivalence point. The midpoint of this linear vertical portion or the inflection point may be taken as the endpoint. The equivalence point may also be determined mathematically without plotting a curve. However, it should be no-ted that in asymmetrical reactions, which are reactions in which the number of anions reacting is not the same as the number of cations reacting, the endpoint as defined by the inflection of the titration curve does not occur exactly at the stoichiometric equivalence point. Thus, potentiometric endpoint detection by this method is not suitable in the case of asymmetric reactions, examples of which are the precipitation reaction,2Ag+ + CrO4–2and the oxidation-reduction reaction,5Fe+2 + MnO4–.All acid-base reactions, however, are symmetrical. Thus, potentiometric endpoint detection may be employed in acid-base ti-trations and in other titrations involving symmetrical reversible reactions where an indicator is specified, unless otherwise direc-ted in the individual monograph.Two types of automatic electrometric titrators are available. The first is one that carries out titrant addition automatically and records the electrode potential differences during the course of titration as the expected sigmoid curve. In the second type, titrant addition is performed automatically until a preset potential or pH, representing the endpoint, is reached, at which point the titrant addition ceases.Several acceptable electrode systems for potentiometric titrations are summarized in Table 2.Table 2. Potentiometric Titration Electrode SystemsTitration Indicating Electrode Equation 1Reference Electrode Applicability 2Acid-base Glass E = k + 0.0591 pH Calomel or silver–silver chlorideTitration of acids and basesPrecipitimetric (silver)Silver E = E° + 0.0591 log [Ag +]Calomel (with potassium ni-trate salt bridge)Titration with or of silver in-volving halides or thiocya-nateComplexometric Mercury–mercury(II) E = E° + 0.0296(log k ¢ − pM)Calomel Titration of various metals(M), e.g., Mg +2, Ca +2 Al +3,Bi +3, with EDTAOxidation–reduction Platinum E = E° + (0.0591/n) × log [ox]/[red]Calomel or silver–silver chloride Titrations with arsenite, bro-mine, cerate, dichromate,exacyonoferrate(III), iodate,nitrite, permanganate, thio-sulfate1 Appropriate form of Nernst equation describing the indicating electrode system: k = glass electrode constant; k ¢ = constant derived from Hg–Hg(II)–EDTA equi-librium; M = any metal undergoing EDTA titration; [ox] and [red] from the equation, ox + n e ®¬red.2Listing is representative but not exhaustive.Blank Corrections—As previously noted, the endpoint determined in a titrimetric assay is an estimate of the reaction equiv-alence point. The validity of this estimate depends upon, among other factors, the nature of the titrate constituents and theconcentration of the titrant. An appropriate blank correction is employed in titrimetric assays to enhance the reliability of theendpoint determination. Such a blank correction is usually obtained by means of a residual blank titration , wherein the required procedure is repeated in every detail except that the substance being assayed is omitted. In such instances, the actual volumeof titrant equivalent to the substance being assayed is the difference between the volume consumed in the residual blank titra-tion and that consumed in the titration with the substance present. The corrected volume so obtained is used in calculatingthe quantity of the substance being titrated, in the same manner as prescribed under Residual Titrations . Where potentiometricendpoint detection is employed, the blank correction is usually negligible.á551ñ VITAMIN E ASSAYINTRODUCTIONThe following liquid chromatographic procedures are provided for the determination of vitamin E as an active pharmaceutical ingredient, as a dietary supplement ingredient, or as a component in compendial dosage forms in the forms of alpha toco-pherol (C 29H 50O 2), alpha tocopheryl acetate (C 31H 52O 3), or alpha tocopheryl acid succinate (C 33H 54O5).Throughout this assay, protect solutions containing, and derived from, the test specimen and the Reference Standard from the atmosphere and light, preferably by the use of a blanket of inert gas and low-actinic glassware.Where vitamin E (alpha tocopherol, alpha tocopheryl acetate, or alpha tocopheryl acid succinate) is specified in the following procedure, use the chemical form present in the formulation and the relevant USP Reference Standard.ASSAY• P ROCEDURE 1•This procedure can be used to determine vitamin E in:•Oil-Soluble Vitamins Tablets•Oil-Soluble Vitamins Capsules•Oil-Soluble Vitamins with Minerals Tablets•Oil-Soluble Vitamins with Minerals Capsules•Oil- and Water-Soluble Vitamins Tablets•Oil- and Water-Soluble Vitamins Capsules•Oil- and Water-Soluble Vitamins with Minerals Tablets•Oil- and Water-Soluble Vitamins with Minerals Capsules•This is a neutral procedure that involves the use of dimethyl sulfoxide to dissolve the excipients in the sample, fol-lowed by a liquid–liquid extraction of vitamin E with hexane. The hexane extract is then evaporated in vacuum todryness, and the residue is reconstituted in methanol prior injection into the chromatograph.•Unless specified in the individual monographs, the System suitability solution , Standard solution , Sample solutions , and reagent solutions are prepared as follows.Solution A:Phosphoric acid solution (1 in 100) in waterMobile phase: Methanol and Solution A (19:1)。

Ratio: A240/A445, 1.30–1.50for Labeling 〈7〉, Labels and Labeling for Injectable Prod-•B. The retention time of the major peak of the Sample ucts•(CN 1-May-2016).solution corresponds to that of the Standard solution, asADDITIONAL REQUIREMENTS obtained in the Assay.ASSAYChange to read:•P ROCEDURE[N OTE—Use freshly prepared Standard solution and Sam-•P ACKAGING AND S TORAGE:•Preserve as described in Pack-ple solution, protected from light.]aging and Storage Requirements 〈659〉, Injection Packaging, Mobile phase: Acetonitrile and water (3:2)Sterile solids packaging; protect from light.•(CN 1-May-2016).Standard solution: 250µg/mL of USP Dactinomycin RS•L ABELING: Label it to include the statement “Protect from in Mobile phaselight.”Sample solution: 250µg/mL of dactinomycin from•USP R EFERENCE S TANDARDS〈11〉Dactinomycin for Injection diluted with Mobile phase.USP Dactinomycin RSFilter, if necessary, to obtain a clear solution. [N OTE—USP Endotoxin RSPrepare the solution by adding a suitable aliquot of Mo-bile phase to one container of Dactinomycin forInjection.]Chromatographic system(See Chromatography 〈621〉, System Suitability.)Mode: LC Add the following:Detector: UV 254 nmColumn: 3.9-mm × 30-cm; packing L1Flow rate: 2.5mL/minv Dalteparin SodiumInjection size: 10µLSystem suitability [9041-08-1].Sample:Standard solution[N OTE—The retention time for dactinomycin is 6 min.]Suitability requirementsColumn efficiency: NLT 1200 theoretical platesTailing factor: NMT 2Relative standard deviation: NMT 3.0%AnalysisSamples:Standard solution and Sample solutionCalculate the percentage of C62H86N12O16 in the por-tion of Dactinomycin for Injection taken:Result = (r U/r S) × (C S/C U) × 100r U= peak response from the Sample solutionr S= peak response from the Standard solutionC S= concentration of USP Dactinomycin RS in theStandard solution (µg/mL)DEFINITIONC U= nominal concentration of dactinomycin in the Dalteparin Sodium is the sodium salt of a low molecularSample solution (µg/mL)weight heparin obtained by nitrous acid depolymerization Acceptance criteria: 90.0%–120.0%of heparin from porcine intestine or intestinal mucosa.Heparin source material used in the manufacture of SPECIFIC TESTSDalteparin Sodium complies with the compendial require-•P H 〈791〉: 5.5–7.5, in the solution constituted as directedments stated in the Heparin Sodium monograph.in the labelingDalteparin Sodium is produced by a validated manufac-•L OSS ON D RYING〈731〉: Dry a portion in vacuum at aturing and purification procedure under conditions shown pressure not exceeding 5mm of mercury at 60° for 3 h:to minimize the presence of species containing the N–NO it loses NMT 4.0% of its weight.group. The majority of the components have a 2-O-sulfo-α-L-idopyranosuronic acid structure at the non-reducing Change to read:end and a 6-O-sulfo-2,5-anhydro-D-mannitol structure atthe reducing end of their chains. The weight-average mo-•O THER R EQUIREMENTS: It meets the requirements under lecular weight (M w) ranges between 5600 Da and 6400•Injections and Implanted Drug Products 〈1〉•(CN 1-May-2016).Da, with a characteristic value of about 6000 Da. The •B ACTERIAL E NDOTOXINS T EST〈85〉: NMT 100.0 USP Endo-percentage of chains lower than molecular weight 3000 toxin Units/mg of dactinomycin.Da is NMT 13.0%, and the percentage of chains higher •S TERILITY T ESTS〈71〉: Meets the requirements when than molecular weight 8000 Da ranges between 15.0% tested as directed for Test for Sterility of the Product to be and 25.0%. The degree of sulfation is NLT 1.8/disaccha-Examined, Membrane Filtration, each container being con-ride unit. The potency is NLT 110 and NMT 210 Anti-stituted aseptically by injecting Sterile Water for Injection Factor X a International Units (IU)/mg of activity, calculated through the stopper, and the entire contents of all the on the dried basis. The anti-factor IIa activity is NLT 35 containers being collected aseptically with the aid of IU/mg and NMT 100 IU/mg, calculated on the dried ba-200mL of Fluid A before filtering.sis. The ratio of anti-factor Xa activity to anti-factor IIaactivity is between 1.9 and 3.2.Change to read:IDENTIFICATION•A.1H NMR S PECTRUM•C ONSTITUTED S OLUTION: At the time of use, it meets the Standard solution: Dissolve 15mg of USP Dalteparin requirements for •Injections and Implanted Drug Products Sodium RS in 0.7mL of deuterium oxide with deuter-〈1〉, Specific Tests, Completeness and clarity of solutions and ated trimethylsilylpropionic (TSP) acid sodium salt. Thesample is freeze-dried to remove exchangeable P MonographsRedissolve the sample and repeat the freeze-drying step centage of chains lower than the molecular weight twice more before transferring the sample into an NMR3000 Da (M3000) is NMT 13.0%, and the percentage of tube.chains higher than the molecular weight 8000 Da Sample solution: Dissolve 15mg of Dalteparin Sodium(M8000) ranges between 15.0% and 25.0%.in 0.7mL of deuterium oxide (99.9%) with deuterated•C. A NTI-F ACTOR X A TO A NTI-F ACTOR II A R ATIOTSP. The sample is freeze-dried to remove exchangea-(See Anti-Factor Xa and Anti-Factor IIa Assays for Unfrac-ble protons. Redissolve the sample and repeat the tionated and Low Molecular Weight Heparins 〈208〉, Anti-freeze-drying step twice more before transferring the Factor Xa and Anti-Factor IIa Assays for Low Molecular sample into an NMR tube.Weight Heparins.)Instrumental conditions Acceptance criteria: The ratio of the numerical value of (See Nuclear Magnetic Resonance Spectroscopy 〈761〉.)the anti-factor Xa activity, in Anti-Factor X a IU/mg, to Mode: NMR, pulsed (Fourier transform)the numerical value of the anti-factor IIa activity, in Frequency: NLT 500MHz for 1H Anti-Factor II a IU/mg, as determined by the Anti-Factor Temperature: 30°Xa Activity and Anti-Factor IIa Activity assays, is NLT 1.9 System suitability and NMT 3.2, respectively.Samples:Standard solution and Sample solution•D. I DENTIFICATION T ESTS—G ENERAL: Meets the require-Transfer the Standard solution and the Sample solution to ments for Sodium ContentNMR tubes of 5mm in diameter. Using a pulsedASSAY(Fourier transform) NMR spectrometer operating at•A NTI-F ACTOR X A A CTIVITYNLT 500MHz for 1H, acquire a free induction decay(See Anti-Factor Xa and Anti-Factor IIa Assays for Unfrac-(FID) with NLT 32 scans using a 90° pulse, an acquisi-tionated and Low Molecular Weight Heparins 〈208〉, Anti-tion time of NLT 2 s, and at least a 10-s delay. ForFactor Xa and Anti-Factor IIa Assays for Low Molecular each sample, an initial short spectrum is collected (1Weight Heparins, Anti-Factor Xa Activity for Low Molecular scan), and the water resonance is then suppressed byWeight Heparin.)selective irradiation during the relaxation delay. FinalAnalysis: Proceed as directed in the chapter.spectra are recorded over 32 scans. For all samples,Acceptance criteria: The potency is NLT 110 and NMT the TSP methyl signal should be set to 0.00ppm. Re-210 Anti-Factor X a IU/mg on the dried basis.cord the 1H NMR spectrum of the Standard solution.Collect the 1H NMR spectrum with a spectral window OTHER COMPONENTSof at least 10 to −2ppm and without spinning. The•N ITROGEN D ETERMINATION, Method II 〈461〉: 1.5%–2.5% Standard solution shall be run at least daily when the on the dried basisSample solution is being run. All spectra are phased,•S ODIUM C ONTENTand linear baseline correction is applied to all spectra Cesium chloride solution: 1.27mg/mL of cesium chlo-before peak identification.ride in 0.1 M hydrochloric acidSuitability requirements Standard solution A: 0.0025% of sodium chloride in Chemical shift: The TSP methyl signal should be set Cesium chloride solutionto 0.00ppm for all samples.Standard solution B: 0.0050% of sodium chloride in Chemical shifts for system suitability: The ppm val-Cesium chloride solutionues for the methyl group of N-acetyl, the H-2 of N-Standard solution C: 0.0075% of sodium chloride in sulfo glucosamine, the H-2 of glucuronic acid plus Cesium chloride solution3-O-sulfo glucosamine, the H-1 of iduronic acid, and Sample solution: Transfer 50.0mg of Dalteparin So-the H-1 of 3-O sulfo glucosamine of dalteparin in the dium to a 100-mL volumetric flask, and dissolve in and Standard solution are present at 2.05, 3.28, 3.39,dilute with Cesium chloride solution to volume.5.01, and 5.51, respectively. Two additional signals,Analysiscorresponding to the H-1 of the 2-O-sulfo iduronic Samples:Cesium chloride solution, Standard solution A, acid linked to the terminal 2, 5-anhydromannitol and Standard solution B, Standard solution C, and Sample the H-1 of 2-O-sulfo iduronic acid are located at solution5.18–5.22ppm. The ppm values of these signals do Concomitantly determine the absorbances of the Ce-not differ by more than ±0.03ppm, Standard solution.sium chloride solution (blank), the Sample solution, and [N OTE—Depending on specific sample makeup and the Standard solutions at 330.3 nm, using a sodium instrument parameters, including the field strength hollow-cathode lamp and an air–acetylene flame. Us-of the NMR instrument, the two signals associated ing the absorbances of Standard solutions A, B, and C, with the H-1 of 2-O-sulfo iduronic acid at determine the sodium content in the Sample solution5.18–5.22ppm may appear well separated or as a after an appropriate blank correction.main signal with a shoulder.]Acceptance criteria: 10.5%–13.5% on the dried basis AnalysisSample:Sample solution IMPURITIESRecord the 1H NMR spectra of the Sample solution.•L IMIT OF N ITRITESAcceptance criteria: The ppm values for the methyl Mobile phase: Dissolve 13.6g of sodium acetate trihy-group of N-acetyl, the H-2 of N-sulfo glucosamine, the drate in 900mL of water in a 1000-mL volumetric flask.H-2 of glucuronic acid plus 3-O-sulfo glucosamine, the Adjust with orthophosphoric acid to a pH of 4.3, and H-1 of iduronic acid and the H-1 of the 2-O-sulfo dilute with water to 1000mL. Filter through a 0.45-µm iduronic acid linked to the terminal anhydromannitol,membrane.the H-1 of 2-O-sulfo iduronic acid and the H-1 of 3-O Nitrite stock standard solution: Dissolve 0.075g of so-sulfo glucosamine of dalteparin in the Sample solution dium nitrite in a 1000-mL volumetric flask with carbon are present at 2.05, 3.28, 3.39, 5.01, 5.18–5.22, and dioxide-free water (0.05g/L of nitrite).5.51, respectively. The ppm values of these signals do Nitrite standard solution: Dilute 1mL of Nitrite stocknot differ by more than ±0.03ppm.standard solution in a 100-mL volumetric flask with car-•B. M OLECULAR W EIGHT D ISTRIBUTION AND W EIGHT-A VERAGE bon dioxide-free water (500 ng/mL of nitrite).M OLECULAR W EIGHT Calibration standard solutions: Dilute Nitrite standard (See Low Molecular Weight Heparin Molecular Weight De-solution in carbon dioxide-free water to prepare four so-terminations 〈209〉.)lutions with the final nitrite concentrations of 2.5, 5, Acceptance criteria: The weight-average molecular15, and 25 ng/mL.weight (M w) ranges between 5600 Da and 6400 Da,with a characteristic value of about 6000 Da. The per-Sample solution: Weigh 80.0mg of Dalteparin Sodium r SA= response of boron from Standard solution A into a 20-mL volumetric flask, and dissolve in carbon r C= response of boron from the Calibration solution dioxide-free water.r B= response of boron from the BlankChromatographic system Acceptance criteria: NMT 1ppm(See Chromatography 〈621〉, System Suitability.)SPECIFIC TESTSMode: LC•A NTI-F ACTOR II A A CTIVITYDetector: Electrochemical detector containing a work-(See Anti-Factor Xa and Anti-Factor IIa Assays for Unfrac-ing electrode (glassy carbon type) with the potentialtionated and Low Molecular Weight Heparins, 〈208〉, Anti-of +1.00 V against a silver–silver chloride referenceFactor Xa and Anti-Factor IIa Assays for Low Molecular electrodeWeight Heparins, Anti-Factor IIa Activity for Low Molecular Column: 3-mm × 15-cm; 5-µm packing L92Weight Heparin.)Column temperature: 30±5°Acceptance criteria: NLT 35 and NMT 100 Anti-Factor Column regeneration: 1M sodium chloride (NaCl) atII a IU/mg on the dried basis0.5mL/min for about 1 h. After regeneration, wash•M OLAR R ATIO OF S ULFATE TO C ARBOXYLATE the column with water and re-equilibrate with MobileMobile phase: Carbon dioxide-free water phase.Sample solution: 50mg of Dalteparin Sodium in 10mL Flow rate: 0.5mL/minof carbon dioxide-free waterInjection volume: 25µLChromatographic systemRun time: 10 min(See Chromatography 〈621〉, System Suitability.) System suitabilityMode: LCSamples:Calibration standard solutions and SampleDetector: IonsolutionColumn: Two columns: one 1.5-cm × 2.5-cm column, Suitability requirementspacked with an anion-exchange resin L64packing, and Column efficiency: NLT 4000 theoretical plates forone 1.5-cm × 7.5-cm column, packed with a cation-the nitrite peak for all Calibration solutions and Sampleexchange resin L65packing.1 The outlet of the anion-solution runsexchange column is connected to the inlet of the cat-Tailing factor: Between 0.8 and 1.2 for all Calibrationion-exchange column.solutions and Sample solution runsFlow rate: 1mL/minRelative standard deviation: Inject Calibration stan-Analysisdard solutions with 25 ng/mL concentration at leastSample:Sample solutionsix times. Calculate the relative standard deviation %[N OTE—Regenerate the anion-exchange column and the (%RSD) of the nitrite peak areas of the last six injec-cation-exchange column with 1N sodium hydroxide tions. The %RSD is NMT 2%.and 1N hydrochloric acid, respectively, between two Analysisinjections.]Samples:Calibration standard solutions and SampleWith the valve in the inject position, inject the Sample solutionsolution into the anion-exchange column, and collect Plot the areas of the nitrite peaks from the chromato-the eluate from the cation-exchange column in a grams of the Calibration standard solutions against re-beaker at the outlet until the ion detector reading re-spective concentrations of nitrite. Draw a best-fit re-turns to the baseline value. Quantitatively transfer the gression line through the points. The correlationeluate to a titration vessel containing a magnetic stir-coefficient is NLT 0.995. Calculate the concentrationring bar, and dilute with carbon dioxide-free water to of nitrite from the areas of the nitrite peak in theabout 60mL. Position the titration vessel on a mag-chromatogram of the Sample solution.netic stirrer, and immerse the electrodes. Note the ini-Acceptance criteria: NMT 5ppmtial conductivity reading, and titrate with approxi-•B ORONmately 0.1 N sodium hydroxide added in 100-µL [N OTE—Use only plastic labware, avoid glass.]portions. [N OTE—Prepare the sodium hydroxide solu-Blank: 1% (v/v) solution of nitric acid in watertion in carbon dioxide-free water.] Record the buret Calibration solution: Prepare a 11.4-µg/mL solution ofreading and the conductivity meter reading after each USP Boric Acid RS in the Blank.addition of the sodium hydroxide solution.Standard solution A: Dissolve 0.2500g of USP LowPlot the conductivity measurements on the y-axis Molecular Weight Heparin for Boron Analysis RS inagainst the volumes of sodium hydroxide added on about 2mL of water, add 100µL of nitric acid, andthe x-axis. The graph will have three linear sections—dilute with the Blank to 10.00mL.an initial downward slope, a middle slight rise, and a Standard solution B: Dissolve 0.2500g of USP Lowfinal rise. For each of these sections, draw the best-fit Molecular Weight Heparin for Boron Analysis RS instraight lines using linear regression analysis. At the about 2mL of Blank, add 10µL of a 5.7-mg/mL solu-points where the first and second straight lines inter-tion of USP Boric Acid RS, and dilute with the Blank tosect and where the second and third lines intersect,10.00mL. This solution contains 1µg/mL of boron.draw perpendiculars to the x-axis to determine the Sample solution: Dissolve 0.2500g of Dalteparin So-volumes of sodium hydroxide taken up by the sample dium in about 2mL of water, add 100µL of nitric acid,at those points. The point where the first and second and dilute with the Blank to 10.00mL.lines intersect corresponds to the volume of sodium Analysishydroxide taken up by the sulfate groups (V S). The Samples:Blank, Calibration solution, Standard solutionpoint where the second and third lines intersect corre-A, Standard solution B, and Sample solutionsponds to the volume of sodium hydroxide consumed Boron is determined by measurement of the emissionby the sulfate and the carboxylate groups together from inductively coupled plasma (ICP) at 249.733 nm(V T).or a suitable wavelength. Use an appropriate appara-Calculate the molar ratio of sulfate to carboxylate: tus with settings that have been optimized as directedby the manufacturer.Result = V S/(V T−V S) Calculate the content of boron in Dalteparin Sodiumusing the following correction factor:1The procedure is based on analyses performed with two columns: one 1.5-cm × 2.5-cm packed with anion-exchange resin Dowex 1X8 (200–400 mesh)and the other 1.5-cm × 7.5-cm packed with cation-exchange resin DowexF = (r SB – r SA) × 2/(r C – r B)50WX2 (100–200 mesh).r SB= response of boron from Standard solution BAcceptance criteria: The molar ratio of sulfate to car-Percentageboxylate is NLT 1.8.Concentra-(%,•P H 〈791〉: 5.5–8.0 for a 1.0% solution in water tion for comparison•L OSS ON D RYING 〈731〉Standard (µg RS perwith test Sample: 1gsolutionDilution mL)specimen)Analysis: Dry the Sample under vacuum at 70° for 6 h.A (1 in 2)500 1.0Acceptance criteria: NMT 10%B (1 in 4)2500.5•B ACTERIAL E NDOTOXINS T EST 〈85〉: It contains NMT 0.01C (1 in 10)1000.2USP Endotoxin Unit/IU of anti-factor Xa activity.D(1 in 20)500.1ADDITIONAL REQUIREMENTS•P ACKAGING AND S TORAGE : Preserve in tight, light-resistant Test solution—Dissolve an accurately weighed quantity of containers, and store below 40°, preferably at room Danazol in Solvent to obtain a solution containing 50mg temperature.per mL.•L ABELING : Label to state the number of Anti-factor X a In-Procedure—Apply separately 5µL of the Test solution and ternational Units of activity per mg.5µL of each Standard solution to a suitable thin-layer chro-•USP R EFERENCE S TANDARDS 〈11〉matographic plate (see Chromatography 〈621〉) coated with USP Boric Acid RSa 0.25-mm layer of chromatographic silica gel mixture. Posi-USP Dalteparin Sodium RS tion the plate in a chromatographic chamber and develop USP Endotoxin RSthe chromatograms in a solvent system consisting of a mix-USP Low Molecular Weight Heparin for Bioassays RSture of cyclohexane and ethyl acetate (7:3) until the solvent USP Low Molecular Weight Heparin for Boron Analysis RS front has moved about three-fourths of the length of the USP Low Molecular Weight Heparin Molecular Weight plate. Remove the plate from the developing chamber, mark Calibrant RSthe solvent front, and allow the solvent to evaporate in v USP39warm, circulating air. Examine the plate under short-wave-length UV light. Expose the plate to iodine vapors for 5min-utes. Compare the intensities of any secondary spots ob-served in the chromatogram of the Test solution with those of the principal spots in the chromatograms of the Standard solutions: the sum of the intensities of secondary spots ob-Danazoltained from the Test solution corresponds to not more than 1.0% of related compounds, with no single impurity corre-sponding to more than 0.5%.Assay—Dissolve about 100mg of Danazol, accuratelyweighed and previously dried, in about 50mL of alcohol ina 100-mL volumetric flask, swirl until dissolved, dilute with alcohol to volume, and mix. Transfer 2.0mL of this solution C 22H 27NO 2337.46to a 100-mL volumetric flask, dilute with alcohol to volume,Pregna-2,4-dien-20-yno[2,3-d ]isoxazol-17-ol, (17α)-.and mix. Similarly, dissolve an accurately weighed quantity 17α-Pregna-2,4-dien-20-yno[2,3-d ]isoxazol-17-ol of USP Danazol RS in alcohol to obtain a Standard solution [17230-88-5].having a known concentration of about 20µg per mL. Con-comitantly determine the absorbances of both solutions in » Danazol contains not less than 97.0percent 1-cm cells at the wavelength of maximum absorbance at and not more than 102.0percent of C 22H 27NO 2,about 285 nm, using alcohol as the blank. Calculate the quantity, in mg, of C 22H 27NO 2 in the portion of Danazol calculated on the dried basis.taken by the formula:Packaging and storage—Preserve in tight, light-resistant containers.5C (A U /A S )USP Reference standards 〈11〉—in which C is the concentration, in µg per mL, of USPUSP Danazol RS Danazol RS in the Standard solution; and A U and A S are the Identification—absorbances of the solution of Danazol and the Standard A: Infrared Absorption 〈197K 〉.solution, respectively.B: Ultraviolet Absorption 〈197U 〉—Solution: prepared as directed in the Assay .Specific rotation 〈781S 〉: between +21° and +27°.Test solution: 10mg per mL, in chloroform.Danazol CapsulesLoss on drying 〈731〉—Dry it at a pressure not exceeding 5mm of mercury at 60° to constant weight: it loses not » Danazol Capsules contain not less thanmore than 2.0% of its weight.90.0percent and not more than 110.0percent of Chromatographic purity—the labeled amount of C 22H 27NO 2.Solvent—Prepare a mixture of chloroform and methanol (9:1).Packaging and storage—Preserve in well-closed contain-Standard solutions—Dissolve an accurately weighed quan-ers.tity of USP Danazol RS in Solvent to obtain a solution having USP Reference standards 〈11〉—a known concentration of 1mg per mL. Dilute quantita-USP Danazol RStively with Solvent to obtain Standard solutions having the Identification—Shake the contents of a sufficient number following compositions:of Capsules, equivalent to about 50mg of Danazol, with 50mL of chloroform, and filter. Evaporate the filtrate on a steam bath with the aid of a stream of nitrogen to dryness:the IR absorption spectrum of a potassium bromide disper-sion of the residue, previously dried, exhibits maxima at the。

新疆医科大学 硕士学位论文 大蒜辣素生物合成、稳定性研究及相关物质分析 姓名：林守峰 申请学位级别：硕士 专业：药物化学 指导教师：李新霞;陈坚 2010-04摘 要　大蒜辣素生物合成、稳定性研究及相关物质分析研究生：林守峰 导师：李新霞 陈坚 教授摘要目的 ： 本研究以大蒜中提取的蒜氨酸和蒜酶为原料，生物合成制备大蒜辣素。

在前期建立的蒜酶活力测定方法和蒜氨酸、大蒜辣素测定方法基础上，研究蒜酶催 化裂解蒜氨酸的配比，从而为蒜氨酸/蒜酶二元制剂的研究建立基础；考察大蒜辣素 溶液的稳定性，研究和探讨影响稳定性因素。

由于生物合成大蒜辣素所使用的蒜氨 酸和蒜酶原料为中试生产得到，原料中的杂质可能会对制备得到的大蒜辣素溶液的 纯度造成影响，通过液质联用方法，对于大蒜辣素溶液中的杂质和相关物质及降解 产物进行结构解析，分析杂质来源，从而提出改进和优化生物合成大蒜辣素的工艺 条件。

方法：1.综合考虑所使用蒜氨酸原料的含量和经济性，通过 HPLC 对反应产物 进行测定，确定蒜酶催化蒜氨酸生物合成大蒜辣素工艺中的原料配比、反应温度、 投料方式等。

2.参考欧洲药典中潜在大蒜辣素含量测定方法，HPLC 测定考察三个温 度条件下的大蒜辣素溶液的稳定性，计算半衰期作为评价稳定性的标准。

3.通过液质 联用测定大蒜辣素溶液和蒜氨酸原料，建立 HPLC-MS 分析方法，进行相应物质的结 构解析和确证。

结果： 1.确定大蒜辣素生物合成工艺中的蒜氨酸和蒜酶配比为 1mg 蒜氨酸需要 1.3U 蒜酶，综合蒜酶活性和大蒜辣素稳定性确定 2５℃为反应温度，由于 蒜酶为自杀式酶，生成的大蒜辣素抑制蒜酶活力，故经考察分次将蒜酶溶液滴加到 蒜氨酸中用最少的蒜酶量制备大蒜辣素，结合膜分离技术滤除蒜酶等大分子物质。

2.影响大蒜辣素稳定性的因素除了温度原因之外， 还有空气中的氧气对于亚砜基氧化 作用等，采用隔绝空气等方法提高稳定性。

3.HPLC-MS 确定了大蒜辣素溶液中的杂 质为甲基丙烯基硫代亚硫酸脂；并且不同含量的蒜氨酸原料合成的大蒜辣素溶液含 有不等量的蒜氨酸和大蒜辣素的同分异构体；确定了大蒜辣素的部分降解产物。

Toxicology237(2007)126–133Toxic effects of di(2-ethylhexyl)phthalate on mortality,growth, reproduction and stress-related gene expression inthe soil nematode Caenorhabditis elegansJi-Yeon Roh,In-Ho Jung,Jai-Young Lee,Jinhee Choi∗Faculty of Environmental Engineering,College of Urban Science,University of Seoul,90Jeonnong-dong,Dongdaemun-gu,Seoul130-743,Republic of KoreaReceived16March2007;received in revised form19April2007;accepted2May2007Available online18May2007AbstractIn this study,di(2-ethylhexyl)phthalate(DEHP)toxicities to Caenorhabditis elegans were investigated using multiple toxic endpoints,such as mortality,growth,reproduction and stress-related gene expression,focusing on the identification of chemical-induced gene expression as a sensitive biomarker for DEHP monitoring.The possible use of C.elegans as a sentinel organism in the monitoring of soil ecosystem health was also tested by conducting the experiment on the exposure of nematode tofield soil. Twenty-four-hour median lethal concentration(LC50)data suggest that DEHP has a relatively high potential of acute toxicity to C. elegans.Decreases in body length and egg number per worm observed after24h of DEHP exposure may induce long-term alteration in the growth and reproduction of the nematode population.Based on the result from the C.elegans genome array and indicated in the literatures,stress proteins,metallothionein,vitellogenin,xenobiotic metabolism enzymes,apoptosis-related proteins,and antioxidant enzyme genes were selected as stress-related genes and their expression in C.elegans by DEHP exposure was analyzed semi-quantitatively.Expression of heat shock protein(hsp)-16.1and hsp-16.2genes was decreased by DEHP exposure.Expression of cytochrome P450(cyp)35a2and glutathione-S-transferease(gst)-4,phase I and phase II of xenobiotic metabolism enzymes, was increased by DEHP exposure in a concentration-dependent manner.An increase in stress-related gene expressions occurred concomitantly with the deterioration on the physiological level,which suggests an increase in expression of those genes may not be considered as a homeostatic response but as a toxicity that might have physiological consequences.The experiment with the soil from the landfill site suggests that the potential of the C.elegans biomarker identified in laboratory conditions should be calibrated and validated for its use in situ.©2007Elsevier Ireland Ltd.All rights reserved.Keywords:Caenorhabditis elegans;Di(2-ethylhexyl)phthalate;Stress-related gene expression;Ecotoxicity monitoring1.IntroductionDi(2-ethylhexyl)phthalate(DEHP)is widely used in flexible polyvinyl chloride(PVC)as a plasticizer to∗Corresponding author.Tel.:+82222105622;fax:+82222442245.E-mail address:jinhchoi@uos.ac.kr(J.Choi).soften plastic materials.Owing to its utility and cost effectiveness,DEHP has been used in a broad range of applications,such as wires and cables,floor tiles,garden hoses,containers,footwear and clothing(De Jonge et al., 2002;Chao and Cheng,2007).Due to the widespread use of DEHP,it is ubiquitous in the environment and many environmental species are thus exposed to vari-ous levels of DEHP in their natural habitat.At present,0300-483X/$–see front matter©2007Elsevier Ireland Ltd.All rights reserved. doi:10.1016/j.tox.2007.05.008J.-Y.Roh et al./Toxicology237(2007)126–133127hazard or risk assessments of DEHP conducted by inter-national authorities are available(WHO,1992;EPA, 1999;EU,2001;ATDSR,2002),some of which include an assessment of the ecological risk of DEHP to aquatic life.However,few studies have been performed on the ecotoxicity of soil organism.Caenorhabditis elegans,a free-living nematode that lives mainly in the liquid phase of soils,is thefirst multicellular organism to have its genome completely sequenced.The genome showed an unexpectedly high level of conservation with the vertebrate genome,which makes C.elegans an ideal system for biological studies, such as those in genetics,molecular biology and devel-opment biology(Brenner,1974;Bettinger et al.,2004; Leacock and Reinke,2006;Schafer,2006;Schroeder, 2006).C.elegans is also a good animal model for the study of ecotoxicology.Due to its abundance in soil ecosystems,its convenient handling in the labora-tory,and its sensitivity to different kinds of stresses, C.elegans is frequently used in ecotoxicological stud-ies utilizing various exposure media,including soil and water(Peredney and Williams,2000;Willams et al., 2000;Boyd and Williams,2003a).The range of C.ele-gans studies in ecotoxicology focuses on organism-level endpoints,such as mortality,behavior,growth or repro-duction.However,using these classical test endpoints, it is difficult tofind significant effects.Therefore,more specific and sensitive systems than classical ecotoxico-logical tests are needed.In this study,to identify a suitable tool to develop a screening system for ecotoxicity monitoring,DEHP toxicities to C.elegans were investigated using multiple toxic endpoints,such as mortality,growth,reproduc-tion and stress-related gene expression,focusing on the identification of chemical-induced gene expression as a sensitive biomarker for DEHP toxicity.C.elegans whole genome microarray was conducted for screen-ing the differentially expressed gene list by DEHP exposure.Tested stress-related genes were selected according to the results of microarray data and liter-ature.Alteration on stress-related gene expression by DEHP exposure was examined in a semi-quantitative manner.The response of greenfluorescent protein (gfp)transgenic nematode,incorporated full-length heat shock protein(hsp)-16.2and hsp-16.48genes to DEHP exposure was also examined to test a possibility of transgenic worm as a biosensor of environmen-tal monitoring.Additionally,in situ application of C. elegans toxicity indicator was investigated on the nema-todes exposed to soil from landfill sites,using the same endpoints applied for the exposure of laboratory condition.2.Materials and methodsanismsThe wild-type C.elegans Bristol strain N2was used in this study.C.elegans were maintained on nematode growth medium(NGM)plates seeded with Escherichia coli strain OP50,at20◦C,using the standard method previously described by Brenner(1974).Young adults(3days old)from an age-synchronized culture were used in all the experiments.Worms were incubated at20◦C for24h without a food source,and were then subjected to the analysis.2.2.Sample preparationFour types of endpoints(mortality,growth,reproduction, and stress-related gene expression)were assessed for exposure to DEHP.Pure analytical-grade DEHP(Sigma–Aldrich Chem-ical,St.Louis,MO,USA)was used in the experiment and it was dissolved in dimethyl sulfoxide(DMSO,Sigma–Aldrich Chemical).Nematodes were exposed to DEHP prepared in a K-medium(0.032M KCl,0.051M NaCl)(Williams and Dusenbery,1990).Three replicates for each concentration and a control were conducted for all the test types.The DEHP concentrations in a K-medium were nominal values.Soil toxicity testing with C.elegans was performed as described in the American Society for Testing and Materials (ASTM,2001).Briefly,for each sample,2.33g of soil as loaded in to a35mm×10mm petridish.The moisture was adjusted to35%(dry weight)using K-medium,worms were added and were incubated at20◦C for24h without a food source.The worms were then recovered with centrifugation/flotation using Ludox(Sigma–Aldrich).Three replicates were conducted for all the test types.2.3.Lethal toxicity testsEach test consisted of four concentrations and a control,in which10±1of young C.elegans adults were transferred onto 24-well tissue culture plates containing1ml of the test solution for each of thefive wells.The worms were exposed for24h at20◦C.After the24h,the numbers of live and dead worms were determined through visual inspection and by probing the worms with a platinum wire under a dissecting microscope.2.4.Measurement of growth and reproductionFollowing the24h incubation with exposure to sub-lethal concentrations of DEHP,growth and reproduction were assessed.Growth was assessed by measuring the length of the worms that had been killed by the heat through microscopy, with a scaled lens in each sample.The average length of the unexposed control worm was in the range of1.0–1.2mm. Reproduction was assessed by counting the eggs of each worm through the microscopic inspection of the transparent C.ele-gans body in each sample.Although this procedure differs from128J.-Y.Roh et al./Toxicology237(2007)126–133more commonly used reproduction tests of offspring counting from an age-synchronized single worm,this simple detection method seems appropriate for the rapid screening of the repro-duction effect(Roh et al.,2006).The average number of eggs per worm in the unexposed controls was in the range of10–25. One hundred worms were examined per treatment for growth and reproduction experiments.2.5.RNA extractionFollowing the24h incubation with exposure to sub-lethal concentrations of DEHP,nematodes were harvested for the preparation of RNA.Total RNA was prepared by phenol–chloroform extraction,according to the manufacturer’s standard protocol.RNA concentrations were determined by the absorbance at260nm.The quality of total RNA was estimated based on the ratio of the optical densities from RNA samples measured at260and280nm.2.6.Microarray analysisFive micrograms of the total RNA extracted from nema-todes exposed to DEHP and the control was used for reverse and in vitro transcription followed by application to a GeneChip®C.elegans Genome Array(Affymetrix,Santa Clara,CA, USA),which contains22,500probe sets against22,150unique C.elegans transcripts.The arrays were scanned with the GeneChip scanner3000(Affymetrix),controlled by GeneChip Operating Software(GCOS,Affymetrix).2.7.Semi-quantitative reverse transcription-polymerase chain reactionThe two-step reverse transcription-polymerase chain reac-tion(RT-PCR)method was used with RT Premix(Bioneer Co., Seoul,Korea)and PCR Premix kits(Bioneer Co.),using a PTC-100thermal cycler(MJ Research,Lincoln,MA,USA). The primers were designed on the basis of the sequences retrieved from the C.elegans database(). Actin mRNA was used for expression-level normalization of the studied genes.The PCR products were separated through electrophoresis on1.5%agarose gel(Promega,Madison,WI, USA)and were visualized with ethidium bromide(Bioneer Co.).All the tests were replicated at least three times,and the relative densities of each band were determined with the use of a Kodak EDAS290image analyzer(Kodak,Rochester,NY, USA),with a TFX-20.M UV transilluminator(Vilber Lourmat, Marne la Vallee,France).2.8.Detection of greenfluorescence protein transgenic C. elegansThe transgenic strains(hsp-16.2::gfp and hsp-16.48::gfp)of C.elegans were developed as previously described by Hong et al.(2004).Transgenic C.elegans were incubated for24h with 2mg/l of DEHP,as well as,with soil from landfill sites,and the fluorescence signal was examined from20independent trans-genic worms per treatment.Fluorescence was observed using a Leica DM IRB microscope(Leica,Wetzlar,Germany),and the image was taken using a Leica DC300FX camera(Leica). Levamisole(Sigma–Aldrich Chemical)treatment(2mM)was used to take pictures of the live worms.2.9.Analysis of DEHP from soil of Korean landfill sitesSoil was collected from Korean landfill sites,namely Jeonju (J),Gwangju(G)and Mokpo(M).Reference soil(R)was sampled from the clean area.Soil samples were dried in the air at room temperature.DEHP was extracted using the Pres-surized Solvent Extraction(SPE)method and was analyzed using6890Gas Chromatography–5973N Mass Spectroscopy systems(Agilent,Santa Clara,CA,USA).2.10.Data analysisMedian lethal concentration(LC50)were derived through Probits analysis.The statistical differences between the con-trol and treated worms were determined with the aid of the parametric t-test.3.ResultsAcute toxicity of DEHP on C.elegans was investigated using mortality as endpoint(Table1). Twenty-four-hour LC50of DEHP in C.elegans was estimated as22.55mg/l.Based on the results of the acute toxicity test,three concentrations corresponding to 1/1000,1/100,and1/10of the24h LC50were selected for laboratory sublethal exposure conditions,that is, 0.02,0.2and2mg/l.The changes in the worms’body lengths and in the number of eggs per worm were investigated as a growth and a reproduction indicator,respectively(Fig.1).The worms,which had been exposed to0.02and0.2mg/l of DEHP,showed a decrease in their body length,as well as,in the number of eggs per worm.Suitability of the DNA microarray technique for the ecotoxicological approach was investigated in C. elegans exposed to0.2mg/l of DEHP.DEHP-induced genes expression was screened using C.elegans Genome Arrays.Major up-and down-regulated known genes by Table1Estimation of24h LC10,LC50and LC90of DEHP in C.elegans24-h LC(mg/l)Interval of confidence(95%) LC10 3.6500.016<LC10<11.33LC5022.55 4.200<LC50<56.63LC90139.455.75<LC90<5611LC means lethal concentration.J.-Y.Roh et al./Toxicology237(2007)126–133129Fig.1.Growth and reproduction indicators examined in the young adult of Caenorhabditis elegans exposed to DEHP for24h.Growth was assessed by measuring the length of the worms that had been killed by the heat through microscopy,with a scaled lens in each sample.Reproduction was assessed by counting the eggs of each worm through the microscopic inspection of the transparent C.elegans body in each sample.DEHP exposure in C.elegans were shown in Table2.For stress-related gene expression profiling analysis,based on the result from the microarray and what is found in the literature,we investigated alteration on the gene expression of heat shock proteins(hsp-16.1,hsp-16.2, hsp-16.48,hsp-70),metallothioneins(mt-1,mt-2),vitel-logenins(vit-2,vit-6),xenobiotic metabolism enzymes (cyp35a2,gst-4),tumor suppressor and apoptosis pro-teins(cep-1,ape-1),and antioxidant enzymes(sod-1, ctl-2)in C.elegans by DEHP exposure.The stress-related gene expression profile was inves-tigated in the young C.elegans adults exposed to DEHP for24h(Fig.2).Expression of hsp-16.1and hsp-16.2decreased at0.02and0.2mg/l of DEHP expo-sure.Expression of mt-2increased at DEHP-treated C.elegans.However,due to high data variation,sta-tistical significance was not observed.Expression of cyp35a2and gst-4,phase I and phase II of xenobiotic metabolism enzymes,was increased by DEHP expo-sure in a concentration-dependent manner.Expression of genes related to vitellogenes,apoptosis,or antioxidant enzymes was not changed by DEHP exposure.As shown in Fig.3,the hsp-16.2and hsp-16.48 gene expression levels were assayed using gfp-basedTable2Screening of DEHP induced gene expression using C.elegans whole genome microarrayUp-regulated genes Down-regulated genesWormbase no.Sequence description Flod change Wormbase no.Sequence description Flod change K02D7.3Cuticular collagen17Y71D11A.5Ion channel protein0.03C39E9.3Collagen 6.4W03G1.7Sphingomyelin phosphodiesterase0.03R08F11.7Peroxidase 6.2F36D3.9Cysteine protease0.07B0365.6C-type lectin domain 5.8C06A8.9Glutamate receptor0.07CEC2033Major sperm protein 5.0F21F8.2Protease0.08K08E7.9Multidrug resistance protein 4.2C58111GTP-binding protein0.08C33A12.6UDP-glucuronysltransferase 3.6C52B9.9AMP-binding protein0.1K09F5.2vit-1 3.0C37H5.1Annexin0.13F09G2.3Permease 2.9C08H9.1Serine carboxypeptidase0.15T11F9.3Zinc metalloprotease 2.9T07G12.1Calmodulin0.16F34H10.1Ubiquitin/ribosomal protein 2.7K08C7.5Flavin-containing monoxygenase0.16C03G6.15Cytochrome P450 2.7F53B7.2G-protein coupled receptor0.18F28A12.4Peptidase 2.6F48E3.7Acetylcholine receptor0.18F22D6.10Collagen 2.6Y46H3A.3Heat shock protein0.19ZK1248.17Major sperm protein 2.6C42C1.2Protein phosphatase0.19F08A8.3Acyl-coenzyme A oxidase 2.5E02H9.5Glycosyl hydrolase0.23R07B1.3Membrane glycoprotein 2.5T27E4.8Heat shock protein HSP16-10.28W06D12.3Fatty acid desaturase 2.4T27E4.9Heat shock protein0.3K08B4.3Glucuronosyltransferase 2.4K03A1.4Calmodulin calcium-binding sites0.31F44G3.2Arginine kinase 2.4AU113943Carbonic anhydrase0.31130J.-Y.Roh et al./Toxicology 237(2007)126–133Fig.2.Stress-related gene expression profiling in the young adult of C.elegans exposed to DEHP for 24h.Stress related gene mRNA was amplified by RT-PCR method using the primers designed on the basis of the sequences retrieved from the C.elegans database ( ).Actin mRNA was used for expression-level normalization of the studied genes.The PCR products were separated through electrophoresis on 1.5%agarose gel and were visualized with ethidium bromide.All the tests were replicated at least threetimes.Fig.3.Response of hsp-16.2::gfp and hsp-16.48::gfp transgenic C.elegans exposed to DEHP for 24h.Transgenic C.elegans were incubated for 24h with 2mg/l of DEHP and the fluorescence signal was examined from 20independent transgenic worms per treatment.Fluorescence was observed using a Leica DM IRB microscope,and the image was taken using a Leica DC 300FX camera.reporter transgenic nematodes.Transgenic nematodes were exposed to 2mg/l of DEHP and the fluorescence signals from both gfp transgenic lines increased after DEHP exposure.The response of hsp-16.48::gfp,how-ever,was greater than that of hsp-16.2::gfp.Concomitantly with bioassay using C.elegans ,the analysis of DEHP was conducted on three Korean landfill sites (Table 3).The contamination levels of DEHP in soil from landfill sites were between 6and 20mg/kg soil.DEHP was not detected in the soil from the referencesite.Fig.4.Mortality,growth and reproduction indicators (A),stress-related gene expression profiling measured in the young adult of C.elegans exposed for 24h to DEHP contaminated soil from Korean landfill sites (B).Response of hsp-16.2::gfp and hsp-16.48::gfp transgenic C.elegans exposed for 24h to DEHP contaminated soil from Korean landfill sites (C).The soils were named as R,G,M,J (R:reference site soil,G:Gwangju landfill soil,M:Mokpo landfill soil,J:Jeonju landfill soil).J.-Y.Roh et al./Toxicology237(2007)126–133131Table3Analysis of DEHP on soil from Korean landfill sites(number=3; mean±standard error of the mean)R a G b M c J dDEHP(mg/kg)ND e 6.27±0.6520.05±1.3410.73±1.37a R:Reference soil site.b G:Gwangju landfill site.c M:Mokpo landfill site.d J:Jeonju landfill sites.e ND:non detected.Fig.4shows the response of C.elegans on mortal-ity,growth,reproduction,stress-related gene expression and the response of transgenic C.elegans exposed to soil from Korean landfill sites for24h.Increase in mortality occurred in the most severe extent(about30%)in soil from the M landfill site,which showed the highest DEHP contaminated level among the three sites(20mg/kg). Growth parameters did not change in the worm exposed to soil from the landfill site.The number of eggs per worm slightly increased in the nematode that had been exposed to soil from the J landfill site.Differently from that in Fig.2,worms that had been exposed to soil from landfill sites did not show any statistically significant change in studied stress-related gene expression.Theflu-orescence signals from both gfp transgenic lines slightly increased in transgenic nematode exposed to soil from the landfill site.4.DiscussionsC.elegans is an attractive animal model for the study of the ecotoxicological relevance of chemical-induced molecular-level responses(Menzel et al.,2005;Reichert and Menzel,2005).In this study,the utility of molecu-lar parameters,such as stress-related gene expression, as biomarkers in C.elegans were investigated to iden-tify specific and sensitive tools to develop a screening system for ecotoxicity monitoring.And the possible use of C.elegans as a sentinel organism in the monitoring of soil ecosystem health was also tested by conduct-ing the experiment on the exposure of nematode tofield soil.Although DEHP is a widely used environmental compound,little study has been performed on its eco-toxicological properties.Twenty-four-hour LC50data (Table1)suggest that DEHP has a relatively higher potential of acute toxicity to C.elegans than previ-ously studied metals have(Roh et al.,2006).Mortality is a reliable ecotoxicological endpoint.However,such a high level of exposure hardly occurred in the real environment.More sensitive indicators,physiological-level alterations,such as growth,reproduction,feeding, movement,or behavior,have been used as endpoints for chemical-induced toxicity testing in C.elegans(Dhawan et al.,2000;Anderson et al.,2001,2004;Kohra et al., 2002;Tominaga et al.,2003).The effects of xenobiotics on the growth and reproduction of the test organisms are broadly accepted test parameters,and were found to be more sensitive indicators of toxicity than lethality,as also shown in this study(Fig.1).The decreases in body length and egg number per worm observed after24h of DEHP exposure may induce alteration in the growth and reproduction of the nematode population in the long term.However,due to the low concentration of xenobi-otics in the environment,it is hard tofind a correlation between the occurrence of contaminants and a physio-logical effect of a test organism in the environment,even when using reliable ecotoxicological endpoints,such as,growth or reproduction,which emphasizes the need for understanding the sublethal effects at the biochem-ical and molecular levels where the toxicant-induced responses are initiated.The effect of DEHP to the C.elegans whole genome gene expression was ing concentra-tion corresponding to the1/100of LC50value,which was0.2mg/l of DEHP,it was possible to show that a strong and differential gene induction or repression was detectable in response to DEHP exposure;they were cyp superfamily,hsp,vit,multidrug-resistance protein, etc.(Table2).In case of some genes,such as C-type lectin,collagene,and major sperm protein genes,it is unclear what physiological meaning the over-expression has.Stress-related genes were selected from the array results and previously reported literature.Expression of selected stress-related genes to DEHP exposure was investigated at three different sublethal concentrations, using the semi-quantitative RT-PCR method.It is obvi-ous that members of the cyp family,gst,hsp,and vit genes should be included in a selection of stress-related genes.Among the group of genes,which encode proteins belonging to different metabolic pathways,the met-allothionein,apoptosis,and antioxidant enzyme genes were also included in the stress-related gene selection. As screened in the microarray result,the expression of hsp-16.1and hsp-16.2genes was decreased by DEHP exposure.DEHP exposure led to increases in the expres-sion of some stress-related genes tested,including mt-2, cyp35a2and gst-4.In particular,it has been reported that almost all cyp35forms in C.elegans are moder-ately or strongly inducible by different xenobiotics in a cyp450gene-expression screening experiment(Menzel et al.,2001,2005).Increase in stress-related gene expres-sions occurred concomitantly with this deterioration on132J.-Y.Roh et al./Toxicology237(2007)126–133the physiological level,which suggests that the increase in the expression of those genes may not be considered as a homeostatic response but as toxicity that might lead to physiological consequences(Figs.1and2).C.elegans also offers the advantage of transgenetic approaches,which may allow the development of a sensi-tive biosensor for environmental monitoring(Stringham and Candido,1994;Jones et al.,1996;Chu et al.,2005). In our previous study(Roh et al.,2006),gfp trans-genic nematode,incorporated full-length hsp-16.2and hsp-16.48genes were developed,since hsps are thought to play roles in various stress conditions.As shown in their response to the exposure to four metals(Roh et al.,2006),semi-quantitatively assayed using gfp-based reporter hsp-16.2and hsp-16.48transgenic nematodes were not very sensitive towards DEHP exposure.Even though,transgenic nematode seems to have a consider-able potential as a biosensor for toxicity monitoring,to develop a sensitive biosensor using transgenic C.ele-gans,the responses of a broad range of stress-related gene promoters to various classes of chemicals should be screened and validated with environmentally relevant low-concentration samples.Bioassay methods for soil toxicity monitoring have also been developed and frequently used in C.ele-gans(Peredney and Williams,2000;Boyd and Williams, 2003b).As a soil-dwelling organism,C.elegans has a potential as a bioindicator for the detection of soil con-tamination because of its abundance in soil ecosystems and its sensitivity to different kinds of stresses,which was tested in this study using the same endpoints used for laboratory K-media exposure condition(Fig.4).Increase in egg number per worm in the nematode exposed to soil from landfill sites suggests a mixture of contaminants, including DEHP,might have a stimulatory effect on the reproduction of the nematode.Expression of stress-related genes did not affect C.elegans exposed to soil from the landfill site,which were different from the response of nematodes exposed to DEHP prepared in K-media in laboratory conditions.The common feature of responses to DEHP of C.elegans between labora-tory andfield conditions was the increase in gst-4gene expression.But,as gst is known to use a broad range of substrates,it is difficult to consider it as to DEHP-specific biomarker.Increase influorescence signal of hsp-16.2::gfp and hsp-16.48::gfp transgenic nematodes might be interpreted as a response of heat-shock protein upon exposure to mixture contaminants.The experiment with the soil from the landfill site suggests that the poten-tial of the C.elegans biomarker identified in laboratory conditions should be calibrated and validated for their use in situ.The data obtained from this study can comprise an important contribution to the knowledge of the toxi-cology of DEHP in C.elegans,about which little data are available.A link or correlation between a vali-dated toxicity endpoint(e.g.,growth and reproduction) and upstream-induced gene expression is interest-ing,particularly for ecotoxicological purposes.Direct experimental demonstrations of the wider relationships between molecular-/biochemical level effects and their subsequent consequences at higher levels of biologi-cal organization are needed in order to establish causal relationships.The characterization of the causal rela-tionships between the molecular level responses and the effects at higher biological levels will help to define the sublethal hazards of chemicals in C.elegans. AcknowledgementThis work was accomplished through the generous support of the Korea Research Foundation Grant funded by the Korean Government(MOEHRD)(Grant No. KRF-2005-204-D00009).Appendix A.Supplementary dataSupplementary data associated with this arti-cle can be found,in the online version,at doi:10.1016/j.tox.2007.05.008.ReferencesAnderson,G.L.,Boyd,W.A.,Williams,P.L.,2001.Assessment of sublethal endpoints for toxicity testing with the nematode Caenorhabditis elegans.Environ.Toxicol.Chem.20,833–838. Anderson,G.L.,Cole,R.D.,Williams,P.L.,2004.Assessing behav-ioral toxicity with Caenorhabditis elegnas.Environ.Toxicol.Chem.23,1235–1240.ASTM,2001.Standard Guide for Conducting Laboratory Soil Toxi-city Test with the Nematocde Caenorhabditis elegans.E2172-01.American Society for Testing and Materials,West Conshohocken, PA.ATDSR,2002.Toxicological Profile for Di-(2-ethylhexyl)phthalate (DEHP).Department of Health and Human Services,Public Health Service,Agency for Toxic Substances and Disease Registry, Atlanta.Bettinger,J.C.,Carnell,L.,Davies,A.G.,McIntire,S.L.,2004.The use of Caenorhabditis elegans in molecular neuropharmacology.Int.Rev.Neurobiol.62,195–212.Boyd,W.A.,Williams,P.L.,2003a.Availability of metals to the nematode Caenorhabditis elegans:toxicity based on total concen-trations in soil and extracted fractions.Environ.Toxicol.Chem.22,1100–1106.Boyd,W.A.,Williams,P.L.,parison of the sensitivity of three nematode species to cooper and their utility in aquatic and soil toxicity tests.Environ.Toxicol.Chem.22,2768–2774.。

复方五仁醇胶囊入血成分鉴定及含量测定生垦堕垫堂垄查堡箜堂箜塑旦!塑里:垒坚:!:!:?937?究其原因,可能因为每种药物含有十分复杂的多种活性成分,从而以不同的作用机制发挥效应有关.本研究观察到藿香正气液对大鼠胃及肠道的运动着有明显的促进作用,提示该药物有较好的促胃肠动力作用和较好的应用前景.实验结果表明藿香正气液能显着降低大鼠血浆及胃肠组织匀浆中VIP的含量,同时减少胃窦黏膜,肌间神经丛及空肠黏膜,黏膜下和肌间神经丛中的VIP-IR阳性产物,两者变化趋势一致,且VIP的这些变化以给予藿香正气液1h大鼠胃肠动力显着增强时尤为明显,提示VIP释放减少可能是受藿香正气液的影响所致.而VIP的释放减少在藿香正气液促动力效应中可能起一定作用.藿香正气液可能通过减少胃肠道VIP的释放,降低VIP对胃肠道的抑制作用,而促进胃肠运动.参考文献:Y amamotoH,KuwaharaA,FuiimuraM,eta1.Motoractivi—tyofvascularlyperfusedratduodenum[J].Neurogastroen—terolMotil,1999,11:23—241.SchemannM,SchaafC,MaderM.Neurochemicalcodingof entericneuronsintheguineapigstomach[J~.JCompNeurol,1995,353:161-178.SimsSM,JiaoY,HaroldGP.Regulationofintracellularcal—eiuminhumaneSClphagealsmoothmuscle~J].AMJPhysiol,1997,273:C1679—1689.杨国汉.藿香正气散的方证研究与实践[J].贵阳中医学院学报,2002,24(2):48.杨国汉.藿香正气液治疗d,JL功能性消化不良的疗效初探[J].实用中医药杂志,2002,18(10):3.杨国汉.藿香正气液治疗d,JL功能性消化不良的临床观察[J].中国药房,2003,14(4):2300.徐叔云.药理实验方法学[M].第2版.北京:北京人民卫生出版社.1985.857.郗树清,张墨玲,马丽云,等.大鼠脑内胃动素的免疫组织化学观察[J].天津医科大学,1996.2:12—13.杜卓民主编.实用组织学技术[M].第2版.北京:人民卫生出版社,1998.329—330.[收稿日期]2006一叭一23复方五仁醇胶囊入血成分鉴定及含量测定窦志华,丁安伟,王陆军,罗琳,张兵,施忠(1.南京中医药大学,江苏南京210029;2.南通市第三人民医院,江苏南通226006;3.南通大学,江苏南通226006)[摘要]目的:鉴定复方五仁醇胶囊入血成分并进行含量测定,为该制剂血清药理学研究结果验证积累数据.方法:灌胃给药制备含药血清,高效液相法测定含药血清的色谱图,与体外制剂及对照品色谱图进行比较,分析,鉴定入血成分,并对鉴定出的主要成分进行含量测定.结果:灌胃给药后血清中产生了13个与药物因素有关的成分,其中8个为制剂原形成分,包括五味予醇甲,五味予甲素和五味予乙素,其余5个为制剂成分的代谢产物.血清中五味予醇甲,五味予乙素的平均含量分别为11.0632,7.4909rag?L～.结论:五味予醇甲,五味予乙素在血清中含量较高,可能为复方五仁醇胶囊重要的药效成分.[关键词]复方五仁醇胶囊;含药血清;入血成分;含量测定[中图分类号]927.2[文献标识码]A[文章编号]1001—5213(2006)08-0937.04 TheidentificationanddeterminationofconstituentsofcompoundWurenchuncapsulesmigratingtobloodDOUZhi—hua,DINGAn-wei,WANGLun2,LUOLingo,ZHANGBingo,SHIZhongo(1.NanjingUni versi—tyofTraditionalChineseMedicine,JiangsuNanjing210029,China;2.ThirdPeople'sHospit alofNantongCity.JiangsuNan—tong226006,China;3.NantongUniversity,JiangsuNantong226006,China) ABSIRACI"..ORIF.L-'rIVEToidentifytheconstituentsmigratingtothebloodofratsafteror aladministrationofcompound Wurenchuncapsulesanddeterminethecontentoftheseconstituents,thusaccumulatedatafor thevalidationofserumDham1a—cologyofthispreparation.METIIODSSerumcontainingdrugwerepreparedaftertheprepar ationoraladministeredtoratsand theehromatogramofthisserurnwasdeterminedbyHPLC.Bycomparingtheehromatogram ofserumcontainingdrugwiththe onesofpreparationandcontrolcompounds,constituentsmigratingtobloodwereidentifieda ndthemainonesweredetemned.RESULTSThirteendrug-inducedconstituentsinbloodweredetected,eightofthemwerethe onescontainedintheprepara-tion,includingsehisandrin,deoxysehizandrinandschisandrinB,fiveofthemweremetabolit es.Theaveragecontentofschisandrin,andschisandrinBinbloodwas11.0632,7.4909rng?L一respectively.CONCLUSIONSchisandrinandschisan一[作者简介]窦志华,男,博士,副主任中药师,电话:135****6303,E-mail:******************[通讯作者]丁安伟,男,博士,教授,电话:025—51997178,E-mail:**************.cn]]]]]]]]r=1938中国医院药学杂志2(HJ6年第26卷第8期ChinHospPharmJ,2006Aug-V ol26,No.O一8 drinBaccountforhigherthanotherinserumcontainingdrugandtheylikelybetheeffectivesu bstancesincompoundWuren—chuncapsules.KEYWORDS:compoundWurenchuncapsules;serumcontainingdrug;constituentsmigra tingtoblood;determination复方五仁醇胶囊由五味子,柴胡,叶下珠,三七4味药组成,是一种治疗急慢性肝炎的制剂l_1],不但具有组方简单的特点,而且临床疗效确切.我们选定该制剂为研究对象进行含药血清谱(指纹图谱)效一药效关系分析,对中药复方药效物质基础的方法学进行探讨.本试验在对含药血清药源性成分进行鉴定的基础上,测定其中两个含量较高成分的含量,为用对照品对血清药理学研究结果进行验证积累数据.1材料Agilent1100Series高效液相色谱仪(美国安捷伦公司);五味子醇甲,五味子乙素对照品(江西南昌中药固体制剂制造技术国家工程研究中心.批号1171—040208,1173—040110);五味子酯甲,五味子甲素(中国药品生物制品检定所,批号1529-200001,0764:-2000107);复方五仁醇胶囊(南通市第三人民医院);甲醇,乙腈为色谱纯;硫酸铵为分析纯;水为娃哈哈纯净水;SD大鼠[SPF级,体重(248.4±16.3)g,旱舍各半,南通大学实验动物中心,许可证号SYKX(苏)2002—0022].2方法与结果2.1溶液的制备2.1.1含药血清,空白血清供试品溶液的制备取SD大鼠,禁食12h(自由饮水),每天按照0.45mL? (100g)体重,分两次分别灌胃给予0.5CMC-Na和复方五仁醇胶囊甲醇提取物的0.5CMC-Na混悬液(给药剂量相当于临床等效剂量的10 倍),连续3d,末次给药后1h3戊巴比妥钠0.15 mL?(100g)一'腹腔注射麻醉,颈总动脉取血,置冰箱4℃冷藏过夜,分离血清.分别取各含药血清,空白血清1mL,置离心管中,加入硫酸铵0.36g,置微型旋涡混合仪快速混匀,分次加甲醇(每次1mL,总计5mL)同法充分混匀,16000r?min离心10 min,上清液置80℃水浴锅上蒸干,用微量移液器精密加入甲醇0.5mL,并使残渣充分溶解,16000r min离心10min,上清液即为含药血清,空白血清供试品溶液,高效液相色谱进样前0.45m微孔滤膜过滤.2.1.2供试品溶液的制备精密称取复方五仁醇胶囊内容物0.5g,置25mL量瓶中,加甲醇适量,浸泡5h,超声处理30min,甲醇定容至刻度,摇匀,过滤,取续滤液进样前0.45m微孑L滤膜过滤.2.1.3成分比对用对照品溶液的制备精密称取五味子醇甲,五味子酯甲,五味子甲素及五味子乙素适量,甲醇溶解并稀释,配制成分别含五味子醇甲0.134g?L～,五味子酯甲0.107g?L～,五味子甲素0.123g?I～,五味子乙素0.099g?L的混合溶液,进样前0.45m微孔滤膜过滤.2.2色谱条件2.2.1成分比对测定色谱条件LichrosphereC, (4.6nM'nx250nM'n,5ttm)色谱柱,Phenomenex DescriptionClH保护柱(4.0Lmm×3.0mm),水(A)一乙腈(B)梯度洗脱(()～20min,15～55B;20~40min,55～60B;40~50min,60～7【)9,6B;50～60min,70～90B;6()～70min,90～100B),流速1.0mL?min～,柱温:30C,检测波长:210nm.2.2.2含量测定色谱条件色谱柱,流速,柱温同上,水(A)一甲醇(B)梯度洗脱(0～15min,40～70B;15～2Omin,70B;20～25min,70～85B;25～35min,85～90B;35～45min,909,6B),检测波长:254nm.2.3血中移行成分认定"2.2.1"项条件下分别吸取制剂供试品溶液,成分比对用对照品溶液10L及各含药血清,空白血清供试品溶液20L,注入高效液相色谱仪,记录70min色谱图.结果显示,复方五仁醇胶囊给药后血中出现了13个与药物相关的成分,将其色谱峰编号为1～13,其中6～13号峰代表的8个成分为制剂原形成分,通过对照品谱图比对,认定6,10,12号峰分别为五味子醇甲,五味子甲素和五味子乙素.见图1.2.4含药血清中五味子醇甲,五味子乙素的含量测定2.4.1线性关系考察分别精密称取五味子醇甲,五味子乙素对照品2.56,1.99mg,置10mL量瓶中,加甲醇溶解并稀释至刻度,摇匀,作为对照品储备液.取空白血清7份,每份1mL,分别加入稀释10倍后的对照品储备液0.1,0.2,0.4,0.6,0.8,1.0,1.2mL,按"2.1.1"项下方法处理(相应扣除加入甲醇的体积),配制成五味子醇甲,五味子乙素含量分别为0.00512g?L和0.00398g?L～,0.01024g?L和0.00796g?L～,0.02048g?L中国医院药学杂志2006年第26卷第8期ChinHospPharmJ,2006Aug?V ol26—,No—.08?939?01020]0405060t/rain图1空白血清(A),含药血清(B),对照品(C)及复方五仁醇胶囊(D)高效液相图谱比较a.五味子醇甲;b.五味子醋甲}c五味子甲素;d五味子乙素Fig1ComparisonofthechromatogranmofthecontrolserLim(A), theserLimcontainingdrug(B),controlcompounds(C)andcom—poundWurenchuncapsules(D)a.schi~ndfin;b.schisanthefin)cdeoxyschizandfin)dsehisan—dfinB和0.01592g?L～,0.03072g?L和0.02388g?L～,0.04096g?L和0.03184g?L～,0.0512g?L和0.0398g?L～,0.06144g?L和0.04776g?L的系列混合对照品溶液.分别取此系列混合对照品溶液10L进样,测定其峰面积值,以峰面积对进样量(g)求得线性回归方程.五味子醇甲:y=1210.18X+7.39,r=0.9993,线性范围为0.0512～0.6144g;五味子乙素:Y=752.60X+14.83,r=0.9997,线性范围为0.0398～0.4776/lg.2.4.2方法的专属性考察"2.2.2"项条件下分别吸取空白血清,含药血清供试液20L,五味子醇甲,五味子乙素含量为0.03072g?L和0.02388gL的混合对照品溶液10L,注入高效液相色谱仪,结果显示,可知内源性物质不干扰测定,在选定的色谱条件下,五味子醇甲和五味子乙素分离度良好,保留时间分别为21.1min和34.8min.见图2.i2.....一..I.^....——.....～A图2空白血清(A),含药血清(B)及对照品(C)含量测定高效液相图谱比较1一五味子醇甲;2一五味子乙素Fig2Comparisonofthedeterminationchromatogramsofthecon—trolserum(A),theserumcontainingdrug(B)andcontroltom—pounds(C)1一Schisandfinl2一SchisandfinB含量分别为0.01024g?L和0.00796g?L～,0.03072g?I和0.02388g?L～,0.0512g?L和0.0398g?L),分别进样10L测定峰面积(第1天每个浓度连续进5针,第2天起每天各进1针,共测定5d),得日内,日间精密度.见表1.表1精密度和回收率试验结果(±s)Tab1Theresultsofprecisionandrecoverytest(x-±s)2.4.4回收率试验相对回收率试验:将"2.4.3"项下测得的低,中,高3个浓度的对照品溶液峰面积代人当日回归方程,计算五味子醇甲,五味子乙素含量,与相应实际含量比较,得相对回收率.见表1.绝对回收率试验:精密量取对照品储备液0.5mL置5mL量瓶中,加甲醇溶解并稀释至刻度,摇匀,分别量取0.2,0.6,1.0mL蒸干,分别精密量取甲醇0.5 mL充分溶解,配制成低,中,高3种浓度的对照品溶液(五味子醇甲,五味子乙素含量分别为0.01024 g?L和0.00796g?L～,0.03072g?L和0.02388g?L～,0.0512g?L和0.0398g?L),分别进样10L测定峰面积并计算平均值,作为对照组,同时用"2.4.3"项下制备的低,中,高3个浓度的对照品溶液分别进样10L,测定峰面积,每个浓度重复5次,作为样品组,将样品组峰面积与对照组峰面积比较,计算绝对回收率.见表1.2.4.5样品含量测定取含药血清5份,按"2.1.1"项下方法制备含药血清供试品溶液,20止注入液相色谱仪,测定峰面积,计算五味子醇甲,五味子乙素含量,结果见表2.表2含药血清含量测定结果(=2)Tab2Theresultsofdeterminationoftheserumcontaining drug(=2)编号含量/mg?L一1111.9986211.6821310.0977410.8740510.6635平均含量ID/%含量/rag?L一1平均含量ID/6.97458.979511.06326.988.12827.490914.387.16356.20883讨论2.4.3精密度试验取空白血清3份,按"线性关本含量测定前已经分别进行了制剂和含药血清系考察"系列混合对照品溶液制备方法制备低,中,指纹图谱研究(论文另发),以上含药血清色谱图上高3个浓度的对照品溶液(五味子醇甲,五味子乙素标出的13个峰即为其指纹图谱的全部共有峰,表明940中国医院药学杂志2006年第26卷第8期ChinHospPharmJ'2006A.复方五仁醇胶囊给药后血中出现13个移行成分具有普遍性.复方五仁醇胶囊给药后血中出现了8个制剂原形成分,其中五味子醇甲和五味子乙素的峰面积较大,含量较高,推测这两种成分为复方五仁醇胶囊的君药五味子保肝降酶的主要药效成分,与文献报道的研究结果一致L2].在含药血清指纹图谱研究过程中比较了高氯酸法,沸水浴法,硫酸铵法3种含药血清的预处理方法,结果显示硫酸铵处理较为理想.从本次实验方法学考察的绝对回收率试验结果看,血清预处理过程中五味子醇甲,五味子乙素的损失率分别在309,6和409,6以上.虽然从相对回收率试验结果看不影响含量测定结果,但也说明此法的局限性,有可能使其他含量较低的入血成分没有鉴定出来,有待于改进方法深入研究.参考文献:[1]窦志华,施忠,李伟红,等.复方五仁醇胶囊的研制EJ].时珍国医国药,2005,16(10):953,957.[23王胜春,胡咏武,王俊琴,等.五灵胶囊药效作用的有效成分[J].第四军医大学,2004,25(17):1587—1591.[收稿日期]2005—11—11甲磺酸多沙唑嗪片的人体药动学研究谢平',张毕奎,朱运贵,李焕德,陈本美,刘晓磊',冯胜(1.中南大学湘雅二医院,湖南长沙410009;2中南大学药学院,湖南长沙410078;3中南大学现代分析测试中心生物分析化学研究室,湖南长沙410009;4.中南大学药学院2006届实习生)[摘要]目的:研究甲磺酸多沙唑嗪片在中国健康人体内的药动学.方法:液相色谱质谱联用法(HPLC-MS)测定12名健康男性受试者单剂量口服4mg甲磺酸多沙唑嗪片后的血药浓度,由DAS2.0药学与统计程序处理计算药动学参数.结果:建立的分析方法在0.5~100g?L范围内线性关系良好(r=0.9977),平均回收率为98.33,日内RSD为8.43,日间RSD为9.5.12名受试者单次服用4rag甲磺酸多沙唑嗪片后,体内呈二室模型药动学特征,主要的药动学参数￡…c…K,tl/2fl及V/F分别为30h,47.66g?L～,0.763,2619h和5.40L.结论:分析方法准确,灵敏度高,主要药动学参数与文献报道相似.[关键词]甲磺酸多沙唑嗪;药动学;高效液相色谱一质谱联用[中图分类号]R965[文献标识码]A[文章缩号]1001—5213(2006)08-0940-03 PharmaeokineticsofdoxazosinmesilatetabletinhealthyChinesevolunteersXIEPing',ZHANGBi—kuP一,ZHUYun-gui'一,LIHuan-de'一,CHENBen-mei,LIUXiao—lei',FENGSheng(1.PharmacyDepartmentofSecondXiangyaHospitalofCentralSouthUniversity,Hu nanChangsha410011,"China;2.SchoolofPharmaceuticalSciencesofCentralSouthUmiversity,HunanChangsha410078 ,China;3.BioanalyticalChemistryResearchLaboratory,ModemAnalyticalTestCenterofCentralSouthUniversity,HunanChangsha410009,China)ABSTRACT:0珏Ⅱ℃nⅦTostudypharmacokineticsofdoxazosinmesilatetabletsinhealthyChinesevolunteers.ME1 1)DsAftergivendoxazosinmesilatetablets4nagto12healthymalevolunteers.doxazosinconcent rationsinplasmaweredeter—minedbyHPLC-M_sThepharmacokineticsparametersweregottenwiththehelpofDAS2.0 .RESULTSThecalibrationcurvewaslinearwithintherangeof05—100g?L(r=0.9977),therecoverywas98.33.theRSDsofintra-andinter-daywere9.5and835.Afterasingleoraldoseof4nagdoxazosintesttablets,themainpharmaco kineticsparameterswereasfollows:￡,G,K.,ti/2B,andV/Fwere30h,47.66g?L～,0.763?h～,26.19h,and5.40L.CONCLUSIO NThemethodisrapidandsensitive.Pharmacokineticsparametersareconsistentwiththeliteraturer eport.KEYW0砌】s:doxazosinmesilate;pharmacokinetics;HPUMS甲磺酸多沙唑嗪(doxazosinmesyllate,Dox)是新一代a肾上腺素受体阻滞剂Ⅲ,目前还未见国内有关多沙唑嗪血药浓度分析方法及其药动学方面的研究报道.本实验用高效液相色谱一质谱联用法对[作者简介]谢平,女,学士,主管药师,电话:0731—5292128,E-mail:***************[通讯作者]张毕奎,男,副主任药师,电话:0731—5292097,E-mail,zhhk68~yahoo.oon1.CI'I。

在生物学或生物化学实验室使用的去污剂都是作用比较温和的表面活性剂（=表面活性成分），是用来破坏细胞膜（裂解细胞）以释放细胞内的可溶性物质。

它们可以破坏蛋白质-蛋白质、蛋白质-脂质、脂质-脂质之间的连接，使蛋白质发生结构上的变性，防止蛋白质结晶，另外在免疫学实验中还可避免非特异性吸附。

去污剂根据其特性可以分为好几类，因此科学研究中去污剂的选择很关键，取决于后续研究的具体内容。

实际应用中有众多不同的去污剂可以选择。

为了某些特殊的应用，新的去污剂被不断开发出来[]。

在这篇综述中，对一些最常用的去污剂的特点和应用进行了论述。

去污剂是由一个疏水尾端基团和一个极性亲水头端基团组成的有机化合物（图一A）。

在一定的温度条件下，以特定浓度溶解于水时，去污剂分子会形成胶束，疏水基团部分位于胶束内部，而极性亲水基团则在其外部（图一B）。

因此，胶束的疏水中心会结合到蛋白的疏水区域。

一个胶束中，去污剂分子的聚集数目，是用来评价膜蛋白溶解度的一个重要参数[]。

去污剂分子疏水区域的长度和其疏水性成正比，且去污剂的疏水区域非常恒定，而极性头端亲水基团是可变的，可据其特点，把去污剂分为三类：离子型（阴离子或阳离子型），两性离子型和非离子型（见表一）。

在特定的温度下，表面活性剂分子缔合形成胶束的最低浓度，称之为临界胶束浓度（CMC）。

当去污剂低于临界胶束浓度时，只有单体存在；当高于临界胶束浓度时，胶束、单体以及其余不溶于水的非胶束相共存。

同样，胶束形成的最低温度称为临界胶束温度（CMT）。

因此，温度和浓度是去污剂两相分离和溶解性的重要参数。

一般来说，低亲脂或憎油的去污剂的临界胶束浓度会较高。

种类化合物离子去污剂十二烷基硫酸钠（SDS），脱氧胆酸钠，胆酸钠，肌氨酸非离子去污剂tritonX-100，十二烷基麦芽糖苷，洋地黄皂苷，tween20，tween80两性离子去污剂CHAPS离液剂尿素表一：去污剂的分类。

离子去污剂离子去污剂是由一个亲水链和一个阳离子或阴离子的极性头端基团组成。

Res Chem Intermed(2012)38:2191–2204DOI10.1007/s11164-012-0536-7Kinetic model for formation of DMPO-OH in waterunder ultrasonic irradiation using EPR spin trapping methodMasaki Kubo•Kazuhiro Sekiguchi•Naomi Shibasaki-Kitakawa•Toshikuni YonemotoReceived:22November2011/Accepted:14March2012/Published online:31March2012ÓSpringer Science+Business Media B.V.2012Abstract The rate of the formation of the2-hydroxy-5,5-dimethyl-1-pyrrolidiny-loxy(DMPO-OH)radical in water during ultrasonic irradiation was evaluated both experimentally and theoretically.The hydroxyl radical(OH radical)was indirectly detected using5,5-dimethyl-1-pyrroline N-oxide(DMPO)as the spin trapping com-pound,and the generated DMPO-OH by the reaction between the OH radical and DMPO was measured by an electron paramagnetic resonance.The rate of change in the concentration of the DMPO-OH decreased with time,suggesting that not only the formation reaction of DMPO-OH but also the degradation reaction would take place by ultrasonic irradiation.The formation rate of the DMPO-OH was higher with ultrasonic power intensity and lower with reaction temperature.Based on the exper-imental results,a kinetic model for the formation of the DMPO-OH was proposed by considering the formation reaction,the ultrasonic degradation,and spontaneous degradation of DMPO-OH.The model well described the effect of the ultrasonic power intensity and the reaction temperature on the formation rate of DMPO-OH.The rate of the formation of the DMPO-OH was evaluated with the aid of the kinetic model. Keywords Ultrasonic irradiationÁHydroxyl radicalÁElectron paramagnetic resonanceÁSpin trapping methodÁKinetic modelIntroductionAdvanced oxidation process(AOP)is one of the water treatment technologies to eliminate hazardous organic compounds with powerful oxidizing species[1–3].The hydroxyl radical(OH radical)is the most powerful oxidant and enhances the M.Kubo(&)ÁK.SekiguchiÁN.Shibasaki-KitakawaÁT.YonemotoDepartment of Chemical Engineering,Tohoku University,Aoba-yama6-6-07,Sendai,Miyagi980-8579,Japane-mail:kubo@pcel.che.tohoku.ac.jp2192M.Kubo et al. degradation of hazardous organic compounds,such as chlorinated organic compounds[3],phenol[4,5]and phenolic compounds[6],and dyes[7,8].Ultrasonic irradiation is proposed as one of the techniques to generate the OH radical.The ultrasonic irradiation to water results in the formation and collapse of microscale bubbles[9,10].The local high temperature caused by the collapsed bubbles forces the decomposition of water to generate the OH radical[11].The generation of the OH radical is an important factor for the rate of degradation of organic compounds.The generation rate of the OH radical may depend not only on operational conditions,such as ultrasonic power intensity,reaction temperature,and dissolved gas concentration,but also on the configuration of the ultrasonic irradiation system, such as type of ultrasonic irradiation system,the shape of the probe and reaction vessel,and so on[12].It is desired to evaluate the rate of the generation of the OH radical quantitatively for the design of the reactor and the determination of reaction conditions.However,the lifetime of the OH radical is too short to directly measure its concentration[13].A spin trapping technique in conjunction with electron paramagnetic resonance (EPR)has been widely used for the indirect detection of short-lived radicals[14].A spin trapping compound is used to convert a short-lived radical into a relatively longer-lived radical product,i.e.,a spin adduct,easily detected by EPR.Makino et al.[15,16]measured the concentration of the spin adduct of5,5-dimethyl-1-pyrroline N-oxide(DMPO),2-hydroxy-5,5-dimethyl-1-pyrrolidinyloxy(DMPO-OH),under ultrasonic irradiation with a frequency of50kHz to evaluate the amount of the OH radical.Nam et al.[17]reported that the concentration of DMPO-OH under ultrasonic irradiation with a frequency of20kHz increased early in the stage and then approached the plateau value.They concluded that the degradation of the DMPO-OH also took place during the ultrasonic irradiation.Their result suggested that the existing amount of DMPO-OH estimated from the measured concentration was not equal to the integrated amount of DMPO-OH formed.In order to accurately evaluate the generation rate of the OH radical,it is necessary to elucidate the mechanism of formation and degradation of the DMPO-OH. Yanagida et al.[18]used the high frequency ultrasound of1,000kHz and observed that the decay of the concentration of the DMPO-OH.They proposed that‘‘the unknown scavenger’’was generated to decrease the concentration of the DMPO-OH. Nakamura et al.[19]also used the high frequency ultrasound of1,650kHz and investigated kinetics of formation and decay of DMPO-OH.They concluded that DMPO-OH formation was saturated even though the OH radical was generated continuously and the concentration of DMPO-OH decreased after stopping ultrasonic irradiation.The kinetic model is a useful tool for the evaluation of each reaction rate.The model including both the formation and degradation reactions is expected to provide a rigorous estimation of the rate of DMPO-OH formation.We focused on the formation of DMPO-OH by low frequency ultrasound,which was effective for the degradation of the organic compounds in water[20].The time course of the concentration of DMPO-OH during ultrasonic irradiation was measured by an EPR spin trapping method.The effect of the ultrasonic power intensity and the reaction temperature on the behavior of the formation ofDMPO-OH was investigated.The kinetic model for the formation of DMPO-OH was proposed,including both the formation and degradation reactions of DMPO-OH,and then the formation rate of DMPO-OH was estimated.Materials and methods Ultrasonic irradiation experimentDMPO and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL)were obtained from Sigma-Aldrich Chemical,St.Louis,MO,USA,and were used without further purification.The general expression of the reaction between DMPO and free radicals (R )is shown in Fig.1.DMPO reacts with short-lived free radicals (R )to convert into longer-lived spin adducts.The shape of EPR signal of spin adduct is associated with the structure of the free radical,so that the free radical can be identified by the EPR spectrum.The schematic illustration of the apparatus used for the ultrasonic irradiation is shown in Fig.2.A horn-type ultrasonic homogenizer (Sonifier 250;Branson Ultrasonics,Danbury,CT,USA)operating at a constant frequency of 20kHz was used.The diameter and surface area of the probe was 1.3cm and 1.19cm 2,Fig.2Experimental apparatusEPR spin trapping method 21932194M.Kubo et al. respectively.Three sizes of reaction vessels made of stainless steel(SUS304)were used;vessel1was32mm inner diameter(i.d.)and75mm height,vessel2was40mm i.d.and82.5mm height,vessel3was50mm i.d.and93mm height.The solution volumes were25,50,and100cm3for vessels1,2,and3,respectively.The vessels were placed in a temperature-controlled water bath whose temperature was regulated using a water circulator(CTE-82A;,Yamato Science,Tokyo,Japan).To avoid the degradation of DMPO by light,the reaction system was covered by a blackout curtain.A10mol/m3DMPO aqueous solution was poured into the reaction vessel.The DMPO concentration was high enough to achieve the excess amount of DMPO compared to the amount of generated the OH radical,according to the results of Makino et al.[16]and Num et al.[17].The ultrasonic horn was then immersed in the solution.The reaction was started by irradiating the ultrasound to the solution. The ultrasonic power intensity was defined as the input power of the ultrasound per volume of the solution and regulated in the range of0.25–3W/cm3.The temperature of the reaction solution was regulated in the range of293–313K by balancing the heat generation due to the ultrasonic irradiation and the heat emission to the water bath.All the experiments were conducted under air atmosphere. EPR measurementsDuring the ultrasonic irradiation,small amounts of the solution were taken with capillary tube at specified time intervals.The EPR spectrum was measured at room temperature using an EPR spectrometer(e-scan;Bruker BioSpin,Rheinstetten, Germany)with microwave of3.6mW(9.8GHz),modulation frequency of86kHz, modulation amplitude of0.306mT,afield set of3,498G with a scan range of60G, and number of scans of64.The concentration of the spin-adduct was determined from the EPR spectrum.TEMPOL was used as the standard.In order to elucidate the spontaneous degradation of the formed spin adducts,the time course of the concentration of the spin-adduct after stopping the ultrasonic irradiation was measured at293K.Measurement of the concentration of DMPOThe concentration of DMPO was measured during the ultrasonic irradiation to verify whether the ultrasonic degradation of DMPO took place or not.The solution was taken for specific time intervals and was diluted appropriately with water.The absorbance of the DMPO in the solution was measured at227nm using a spectrophotometer(U-2010;Hitachi,Tokyo,Japan).Results and discussionUltrasonic irradiation of the DMPO aqueous solutionFigure3shows the EPR spectra.Abscissa is the magneticfield.The relative value of the magneticfield is important,since the absolute value of the magneticfield is dependson the setup of the each EPR equipment.Figure 3a shows the spectrum of the aqueous solution of 5910-3mol/m 3TEMPOL,the standard material for the quantification of radical concentration.Figure 3b,c show the spectra of the DMPO aqueous solution before and after the ultrasonic irradiation at 3W/cm 3for 60min,respectively.Before the ultrasonic irradiation,only a signal of the noise of the measurement system was observed in Fig.3b.After the ultrasonic irradiation,a quartet signal with the intensity ratio of 1:2:2:1,the characteristic spectrum of DMPO-OH,was appeared as shown in Fig.3c.Therefore,only the OH radical was considered to evolve.During the ultrasonic irradiation,water is directly decomposed to generate hydrogen and the OH radical asH 2O !H ÁþÁOHð1ÞThe experiment was conducted in the air atmosphere,so that there existed dissolved oxygen in the aqueous solution.The hydrogen radical rapidly reacts with the oxygen molecule to form a hydroperoxy radical as [21]H ÁþO 2!HO 2Áð2ÞThe hydroperoxy radical disappears by the bimolecular reaction as2HO 2Á!H 2O 2þO 2ð3ÞThe reactions (2)and (3)were very fast so that the hydrogen radical and hydro-peroxy radical disappeared before reacting with DMPO.Therefore,the OH radical was considered to be alone detected.-5 10405 104(a) TEMPOL (5x10-3 mol/m 3)-5 10405 104(b) DMPO solution without irradiation-5 10405 104347034803490350035103520Magnetic field [G](c) DMPO solution after irradiation (3W/dm 3, 60min)Fig.3ESR spectra of DMPO solution and DMPO-OH.a 5mol/dm 3TEMPOL solution,the standard for the quantification of radical concentration,b DMPO solution without ultrasonic irradiation,c DMPO solution after ultrasonic irradiation at 3W/cm 3for 60minEPR spin trapping method 2195Figure 4shows the time course of the concentration of DMPO during the ultrasonic irradiation at the ultrasonic power intensity of 3W/cm 3and the reaction temperature of 293K,at which the rate of formation reaction of DMPO-OH is most rapid in this study.The data denoted by triangles and inverse triangles were obtained under the same condition.Those data almost overlapped each other,so that the reproducibility of the data was good.The concentration of DMPO was almost constant during the ultrasonic irradiation.There existed an excess amount of DMPO so that the decrease of the concentration of DMPO by the reaction itself was negligibly small.Time course of DMPO-OH and effect of solution volumeFigure 5shows the time courses of DMPO-OH concentration for the two reactors whose solution volumes were 25and 50cm 3,respectively,at 1.0W/cm 3of ultrasonic power intensity and 293K of reaction temperature.The data almost overlapped each other,so that the same ultrasonic power intensity for different solution volume was found to give the same results.The concentration of DMPO-OH increased with the ultrasonic irradiation time.The rate of change in the concentration decreased with time.The order of magnitude of the concentration of DMPO-OH,10-3mol/m 3,was much lower than the concentration of DMPO,10mol/m 3.In the formation reaction of DMPO-OH,DMPO +ÁOH !DMPO ÀOHð4ÞThe concentration of DMPO was practically constant.Furthermore,the OH radical is highly reactive so that the reaction (4)occurs instantaneously.Thus,the formation rate of DMPO-OH was considered to be proportional to the generation rate of the OH radical.Since the ultrasonic power intensity and the reaction temperature were fixed,and also the concentration of dissolved oxygen was constant,the generation246810120501001502001st run 2nd runD M P O c o n c e n t r a t i o n [m o l /m 3]Ultrasonic irradiation time [min]Fig.4Time course of theconcentration of DMPO at 3W/cm 3of ultrasonic powerintensity and 293K of reaction temperature.Filled triangle and open inverted triangle :two sets of experimental data obtained under the same conditions2196M.Kubo et al.rate of the OH radical as well as the formation rate of DMPO-OH were constant during the ultrasonic irradiation.The constant formation rate of DMPO-OH made us expect the concentration of DMPO-OH to increase linearly.In practice,however,the rate of change in the concentration of DMPO-OH decreased with time.This result showed a possibility of the degradation reaction of DMPO-OHDMPO -OH !Decomposed productsð5Þby ultrasonic irradiation as Nam et al.[17]suggested.It is also known that DMPO-OH decomposes spontaneously [22].Thus,the formation reaction of DMPO-OH as well as the ultrasonic-induced degradation and spontaneous degradation were considered to occur simultaneously.The concentration of DMPO-OH approached a plateau value.This result suggested that the formation and degradation reactions of DMPO-OH reached a dynamic equilibrium at the plateau domains.Further discussion on the mechanism of DMPO-OH formation and degradation is in the ‘‘Application of the model ’’section.Effect of ultrasonic power intensityFigure 6shows the time courses of the concentration of DMPO-OH for various ultrasonic power intensities at a reaction temperature of 293K.The concentration of DMPO-OH was higher with the ultrasound power intensity.This was because the higher ultrasound power intensity induced a larger number of local high temperature and pressure regions,giving a higher generation rate of the OH radical.The lines in25 cm 350 cm 30123456306090irradiation time [min]D M P O -O H c o n c e n t r a t i o n [m m o l /m 3]Solution volumeFig.5Effect of solution volume on time courses of DMPO-OH concentration at 1.0W/cm 3of ultrasonic power intensity and 293K of reaction temperature.Solution volume:filled square 25cm 3,open square 50cm 3EPR spin trapping method 21972198M.Kubo et al. thefigure are the calculated results by the kinetic model and are discussed in the ‘‘Application of the model’’section.Effect of reaction temperatureFigure7shows the effect of the reaction temperature on the time courses of the concentration of DMPO-OH at the ultrasonic power intensity of1W/cm3.The formation rate of DMPO-OH decreased with the reaction temperature,contrary to most chemical reactions.The negative temperature dependence of the reaction rate was explained by the temperature dependence of the vapor pressure of the water. The water vapor pressure increases with the reaction temperature and the influx content of water vapor into the cavitation bubble also increases[23,24].When the cavitation bubble collapses,the temperature rise is suppressed due to the heat capacity of the existing vapor in the bubbles,so that the higher reaction temperature gives a smaller reaction rate,i.e.,a smaller formation rate of DMPO-OH.The lines in thefigure are the calculated results by the kinetic model and are discussed in the ‘‘Application of the model’’section.Stability of DMPO-OHFigure8shows the time course of the concentration of DMPO-OH at293K after stopping the ultrasonic irradiation.The abscissa denotes the dimensionlessconcentration normalized by the concentration when the ultrasonic irradiation was stopped.The concentration of DMPO-OH decreased with time after stopping the ultrasonic irradiation.This result suggested that DMPO-OH decomposed spontaneously.Figure9shows the plot of the logarithm of the dimensionless concentration of DMPO-OH against time.A linear relationship was obtained between the log(C/C0) and time,so that the spontaneous degradation of DMPO-OH wasfirst order with respect to its concentration.Construction of the kinetic modelThe kinetic model for the formation and degradation of DMPO-OH is constructed according to the experimental results.The following reaction scheme is taken into account.Generation of the OH radical:H2OÀ!k1UltrasoundHÁþÁOHð1ÞFormation of DMPO-OH:DMPOþÁOH!DMPOÀOHð4ÞUltrasonic degradation of DMPO-OH:DMPO-OHÀ!k2Ultrasound Productsð5ÞEPR spin trapping method2199Spontaneous degradation of DMPO-OH:DMPO-OH À!k 3Productsð6ÞThe spontaneous degradation of DMPO-OH,reaction (6),was first order withrespect to its concentration from the result in Fig.9.For reactions (1),(4),and (5),the following assumptions are set.(1)The formation rate of the OH radical is proportional to the ultrasonic power intensity,P .(2)The formation of DMPO-OH is an instantaneousreaction.2200M.Kubo et al.(3)The rate of the ultrasonic decomposition of DMPO-OH is proportional to theproduct of the ultrasonic power intensity and the concentration of DMPO-OH.The rate of change in the concentration of DMPO-OH is then expressed as dC DMPOÀOHdt¼k1PÀk2PC DMPOÀOHÀk3C DMPOÀOHð7Þi.c.;C DMPO-OH=0at t=0.Thefirst term on the right hand side denotes the formation rate by the reaction between DMPO and the OH radicals,while the second and third terms denote the degradation rates by the ultrasound induced and spontaneous reactions,respectively. k1and k2are the reaction rate constants related to the formation and degradation reactions by the ultrasound,respectively.k3is the reaction rate constant for the spontaneous degradation reaction.Integrating Eq.(7)givesC DMPOÀOH¼k1Pk2Pþk31ÀexpÀðk2Pþk3Þtf g½ ð8ÞAn exponential temperature dependence is applied to the rate constants,k1,k2, and k3.k i¼k i;0expÀE i RTi¼1;2;3ð9ÞHere,k i,0is the frequency factor,E i is the activation energy,R is the universal gas constant,and T is the temperature.Substituting Eq.(9)into Eq.(8)givesC DMPOÀOH¼Pk1;0expÀE1RTÀÁPk2;0expÀE2ÀÁþk3;0expÀE3ÀÁÂ1ÀexpÀPk2;0expÀE2RTþk3;0expÀE3RT&'t!!ð10ÞIn the case of the spontaneous degradation of DMPO-OH after stopping the ultra-sonic irradiation,the rate of change in the concentration of DMPO-OH is written asdC DMPOÀOHdt¼Àk3C DMPOÀOHi.c.:t¼0;C DMPOÀOH¼C DMPOÀOH;0ð11ÞIntegrating Eq.(11)and substituting Eq.(9)for k3givesC DMPOÀOH C DMPOÀOH;0¼expÀk3;0expÀE3RT&'t!ð12ÞEqs.(10)and(12)are used in the calculation.There are six unknown constants;k1,0, k2,0,k3,0,E1,E2,and E3.These constants were estimated byfitting the calculated values to eight sets of the experimental data of the time courses of the concentration of DMPO-OH simultaneously;i.e.,five different values of ultrasonic powerintensity,two another reaction temperatures during ultrasonic irradiation,and one other experiment after stopping the ultrasonic irradiation.The sum of the squares of the relative errors between the experimental data and the calculated values was determined by the following equation asSSE¼X C DMPOÀOH;calc:ÀC DMPOÀOH;exp:C DMPOÀOH;calc:þC DMPOÀOH;exp:ðÞ=2&'2m ið13ÞThe numbers of data points were different from one another,so that these data numbers,m i,were used to calculate the value of SSE.The value of SSE was minimized using the Simplex method[25]to estimate the optimum values of the unknown constants.Application of the modelThe calculated results are shown by the lines in Figs.6,7,8and9.The lines well describe the effect of the ultrasonic power intensity,the reaction temperature,and the behavior of the spontaneous degradation.Thus,the validity of the assumptions for the kinetic model was verified.The rate of change in the concentration of DMPO-OH is expressed as Eq.(7).In the case of the dynamic equilibrium of the reactions,that is,the formation rate of DMPO-OH is the same as the degradation rate,the rate of change in the concentration of DMPO-OH is zero,so that the plateau concentration of DMPO-OH is written as.C DMPOÀOH¼k1Pk2Pþk3ð14ÞEq.(14)well describes the dependence of plateau concentration of DMPO-OH on the ultrasonic power intensity.The estimated values of the constants are listed in Table1.The values of the constants related to the reactions induced by the ultrasonic irradiation,E1and E2, were both negative.Those negative values are caused by the temperature dependence of the water vapor pressure,as previously discussed in‘‘Effect of reaction temperature’’section.Table1Estimated constantsConstant Estimated valuek1,0 1.77910-14mol/(W s)k2,0 3.89910-12m3/(W s)k3,0 1.77910-2s-1E1-13.3kJ/molE2-13.3kJ/molE312.5kJ/molThe half-life of DMPO-OH was 108min.This value was consistent with that obtained by the chemical reaction in homogeneous solution,2h (i.e.,120min)[22].In order to elucidate the validity of the estimated constants,a sensitivity analysis of these constants was conducted.The values of SSE were calculated under the condition where the value of one of the constants was changed to double or half of the estimated constant without changing the value of the other constants.The estimated constants,the values for sensitivity analysis,SSE ,and the deviation of SSE for sensitivity analysis from that for fitting procedure are shown in Table 2.The deviations of SSE were not zero for all the constants,so that the sensitivities of these constants were considered to be high.That is,the estimated values of these constants were reasonable,not only the constants related to ultrasonic irradiation,k 1,0,k 2,0,E 1,and E 2but also the constants related to spontaneous decomposition,k 3,0and E 3.The rate of the formation of DMPO-OH,r DMPO-OH ,can be expressed as the product of k 1and P ,so that r DMPO ÀOH ¼1:77Â10À14exp 1:33Â104RTÂP mol =m 3s ÀÁÂÃ:ð15ÞConclusionsThe EPR spin trapping method was used to measure the amount of DMPO-OH formed by ultrasonic irradiation of water.Only the OH radical was detected underTable 2Sensitivity of estimated constants;estimated constants,values for sensitivity analysis,SSE ,and deviation of SSEConstant Estimated value Value for sensitivity analysisSSE Deviation 3.53910-14mol/(W s)3.151[-]5,085%k 1,0 1.77910-14mol/(W s)0.061[-]Minimum 0.88910-14mol/(W s) 3.153[-]5,089%7.78910-12m 3/(W s) 1.564[-]2,474%k 2,0 3.82910-12m 3/(W s)0.061[-]Minimum 1.94910-12m 3/(W s)0.938[-]1,444%3.54910-2s -10.602[-]890.2%k 3,0 1.77910-2s -10.061[-]Minimum 0.88910-2s -10.214[-]252.2%-26.6kJ/mol 27.491[-]45,142%E 1-13.3kJ/mol 0.061[-]Minimum -6.6kJ/mol 21.300[-]34,953%-26.6kJ/mol 27.061[-]44,434%E 2-13.3kJ/mol 0.061[-]Minimum -6.6kJ/mol 3.850[-]6,236%24.9kJ/mol 0.623[-]925%E 312.5kJ/mol 0.061[-]Minimum 6.2kJ/mol7.381[-]12,046%the air atmosphere.The time courses of the concentration of DMPO-OH showed that the DMPO-OH increased with time and the rate of change in the concentration decreased.The higher ultrasonic power intensity and lower reaction temperature resulted in a higher formation rate of DMPO-OH.DMPO-OH was found to decompose both ultrasonically and spontaneously.The kinetic model for the formation of DMPO-OH was constructed by considering the ultrasonic formation of DMPO-OH and the ultrasonic degradation and spontaneous degradation of DMPO-OH.The kinetic model well described the effect of the ultrasonic power intensity and the reaction temperature on the rate of the formation of DMPO-OH and the behavior of the spontaneous degradation of DMPO-OH.The model provides the rate of the formation of DMPO-OH during the ultrasonic irradiation.Acknowledgment This research was supported in part by Grant-in-Aid for Young Scientists(B)No. 20760619from the Ministry of Education,Culture,Sports,Science and Technology(MEXT). 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