Free radical studies of ellagic acid, a natural phenolic antioxidant

Ellagic acid, a plant-derived polyphenol, inhibits gamma-radiation (hydroxyl radical) induced lipid peroxidation in rat liver microsomes in a dose- and concentration-dependent manner. Its antioxidant capacity has been estimated using the 1,1-diphenyl-2-picrylhydrazyl radical assay. To understand the actual mechanisms involved in antioxidant activity and the free radical scavenging ability,a nanosecond pulse radiolysis technique has been employed. The rate constants for the reactions of several reactive oxygen species and reactive nitrogen species such as hydroxyl, peroxyl, and nitrogen dioxide radicals have been found to be in the range of 10(6)-10(9) M(-1) s(-1). The ellagic acid radicals have been characterized by the absorption spectra and decay kinetics. Studies on the reactions of ellagic acid with the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) radical and the radicals of ellagic acid with ascorbate have been used to estimate its one-electron reduction potential. Ellagic acid has also been found to be a good scavenger of peroxynitrite. Using stopped-flow reaction analyzer with absorption detection, the rate constant for this reaction has been determined to be 3.7 x 10(3) M(-1) s (-1). The electron spin resonance spectra of the oxidized ellagic acid radicals have been recorded by horseradish peroxidase and hydrogen peroxide method.     

A simplified spectrophotometric method for routine analysis of saccharin in commercial noncaloric sweeteners

A simple, rapid, and sensitive spectrophotometric method for routine analysis of saccharin in commercial noncaloric sweeteners is proposed. This method is based on the reaction of saccharin with tetrachloro-p-benzoquinone (p-chloranil) accelerated by hydrogen peroxide and conducted in an ethanol:acetone (4:1) medium, producing a violet-red compound (lambda(max) = 550 nm). Beer's law is obeyed in a concentration range of 2.05 x 10(-4) to 3.00 x 10(-3) M with an excellent correlation coefficient (r = 0.9998). The detection limit was 1.55 x 10(-5) M, and the effect of interferences on the spectrophotometric measurements was evaluated. The proposed procedure was applied successfully to the determination of saccharin in noncaloric sweeteners. Recoveries were within 99.2-104.3% with standard deviations ranging from to 0.5-1.6%. Results of the proposed method compare very favorably with those given by the high-performance liquid chromatography method recommended by the Food and Drug Administration.     

A new green analytical procedure for monitoring sub-nanogram amounts of chlorpyrifos on fruits using flow injection chemiluminescence with immobilized reagents

A novel green method using flow injection chemiluminescence with controlled-reagent-release technology has been investigated for the rapid and sensitive monitoring of sub-nanogram amounts of chlorpyrifos. The analytical reagents involved in chemiluminescence (CL) reaction, luminol and periodate, were both immobilized on an anion-exchange column. The CL signals produced by the reaction between luminol and periodate, which were eluted from the column through water injection, were decreased in the presence of chlorpyrifos. The decrease of CL intensity was linear over the logarithm of concentration of chlorpyrifos ranging from 0.48 to 484.0 ng x mL(-1) (r(2) = 0.9969), and the limit of detection was 0.18 ng x mL(-1) (3sigma). At a flow rate of 2.0 mL x min(-1), the determination of chlorpyrifos, including sampling and washing, could be performed in 0.5 min with a relative standard deviation of less than <3.0%. The proposed method was applied successfully in an assay of remnant chlorpyrifos on fruits such as orange and shaddock with the recovery of 94.4-107.4%. The change of the concentration of chlorpyrifos in a water sample was also investigated, and the variation rate was 99.96% during 35 h in the open air.     

Study of 2-(2-pyridyl)benzothiazoline as a novel fluorescent probe for the identification of superoxide anion radicals and the determination of superoxide dismutase activity in scallion genus foods

This paper presents a novel spectrofluorometric method using the novel fluorescent probe 2-(2-pyridyl)benzothiazoline for the determination of superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by IR and (1)H NMR spectra. It could specially identify and trap superoxide anion radicals (O2(.-)), and then was oxidized by O2(.-) to form a strong fluorescence product. On the basis of this reaction, the spectrofluorometric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species, because the probe can be oxidized to afford a highly fluorescent product only by O2(.-) excluding hydrogen peroxide and hydroxyl radical. As a kind of simple, rapid, precise, and sensitive technique, it could avoid the errors caused by detection time and was applied to the measurement of SOD activity in scallion genus foods with satisfactory results.     

Antioxidant mechanism of flavonoids. Solvent effect on rate constant for chain-breaking reaction of quercetin and epicatechin in autoxidation of methyl linoleate.

The rate of oxygen depletion, as measured by electron spin resonance spectroscopy (oximetry using a spin probe), in a homogeneous solution of peroxidating methyl linoleate (initiated by an azo initiator) in the presence or absence of antioxidants was converted to second-order rate constants for the inhibiting reaction of quercetin and epicatechin. In the non-hydrogen-bonding solvent chlorobenzene at 50 degrees C, k(inh) had values of 4.3 x 10(5) M(-)(1) s(-)(1) for quercetin and 4.2 x 10(5) M(-)(1) s(-)(1) for epicatechin, respectively. In the hydrogen-accepting "water-like" solvent tert-butyl alcohol, the values were 2.1 x 10(4) and 1.7 x 10(4) M(-)(1) s(-)(1), respectively. The solvent effect (factor of 20) is more significant than for alpha-tocopherol (factor of 4), and the two flavonoids have efficiencies comparable to that of alpha-tocopherol in scavenging peroxyl radicals in the nonpolar solvent but not in the hydrogen-bonding solvent.     

Flow injection potentiometric system for the simultaneous determination of inositol phosphates and phosphate: phosphorus nutritional evaluation on seeds and grains

A simple flow injection potentiometric (FIP) system, which uses a tubular cobalt electrode, has been developed for phosphorus nutritional evaluation of seeds and grains. Inorganic phosphorus, P(i), is determined using a 1 x 10(-2) mol.L(-1) potassium phthalate buffer solution adjusted at pH 4. A sensitivity of 47 mV/decade and an operating range from 10 to 1000 mg.L(-1) (1 x 10(-4)-1 x 10(-2) M) of dihydrogen phosphate are obtained. The inositol phosphates amount, which is referred to the organic phosphorus, P(org), is directly determined from extracts using a 1 x 10(-2) mol.L(-1) Tris-HCl buffer solution adjusted at pH 8. A sensitivity of 127 mV/decade and an operating range of 10-1000 mg.L(-1) (2.5 x 10(-4)-5 x 10(-3) M) of P(org) (expressed as inositol hexakisphosphoric acid monocalcium) are achieved. Some samples of seed and grain are analyzed by an ICP-OES and a spectrophotometric method to compare results to the developed flow system; no significant differences at the 95% confidence level are observed using a paired t test. Other samples such as animal nursing feed, soybean meal, and corn are also analyzed with the proposed FIP system, showing a good correlation to the ICP-OES values.     

Orthophosphate, phytate, and total phosphorus determination in cereals by flow injection analysis

A flow injection spectrophotometric procedure with enzymatic hydrolysis was developed for determination of orthophosphate, phytate and total phosphorus in cereal samples. Phosphorus species were extracted from cereals with 0.05 mol L(-1) potassium hydrogen phthalate buffer solution at pH 5.7. Orthophosphate was directly determined in the extracts by molybdenum blue spectrophotometric method. The phytate was hydrolyzed by the enzyme phytase coupled to a solid phase packed into an enzymatic reactor, and the resulting hydrolyzed orthophosphate was also determined by spectrophotometry at 650 nm. After optimization for phosphorus species extraction and enzymatic hydrolysis, a linear calibration graph was obtained up to 196 x 10(-6) mol L(-1) orthophosphate (P conc = -2.67 + 0.52x, r = 0.9998). Measurements are characterized by relative standard deviation of 1.6% for a standard of 72 x 10(-6) mol L(-1) orthophosphate and no baseline drift was observed during 4 h operation periods. It provides 72 measurements per hour, with 2.4 x 10(-)6) mol L(-1) and 7.9 x 10(-6) mol L(-1) as detection and quantification limits, respectively.     

Polyvinyl chloride matrix membrane electrodes for manual and flow injection analysis of chloroquine in pharmaceutical preparations.

Two types of polyvinyl chloride (PVC) matrix membrane electrodes responsive to the antimalarial drug chloroquine have been constructed, electrochemically evaluated, compared and used in pharmaceutical analysis. Type 1 is the classic PVC model with chloroquine-tetraphenylborate (TPB) sensor; Type 2 is a coated silver disk without internal filling solution. Both electrode types exhibited rapid linear potentiometric response to the logarithmic concentration of diprotonated chloroquine cation in the 10(-1) - 10(-6)M range with calibration slopes 28-30 mV/concentration decade over the pH range 1.8-6.2. These electrodes were sensitive enough to permit determination of chloroquine phosphate at concentrations as low as 5 microgram/mL with good accuracy and precision. Determination of chloroquine in various pharmaceutical preparations using direct potentiometry and potentiometric titration with NaTPB gave an average recovery of 98.8% of the nominal values (SD 0.5%). The Type 2 electrode was also assessed in a flow-through sandwich cell for flow injection analysis. Results were compared with data obtained by the U.S. Pharmacopeia method.     

Chemiluminescence investigation of detection of rutin in medicine and human urine using controlled-reagent-release technology.

A novel continuous-flow sensor based on chemiluminescence (CL) detection was developed for the determination of rutin in pharmaceutical preparations and human urine by controlled-reagent-release technology. The analytical reagents involved in the CL reaction, including luminol and hexacyanoferrate(III), were both immobilized on an anion-exchange column in a flow-injection system. The CL signal produced by the reaction between luminol and hexacyanoferrate(III), which were eluted from the column through sodium phosphate injection, was decreased in the presence of rutin. CL intensity was inhibited by rutin; the decrement of CL intensity was linear over the logarithm of the rutin concentration range of 1.0-400 ng x mL(-1), and the detection limit was 0.35 ng x mL(-1) (3 sigma). The whole process, including sampling and washing, could be completed in 1.5 min with a relative standard deviation of <3.5%. The flow sensor showed remarkable stability and could be easily reused >450 time; the sensor proposed was applied successfully to the determination of rutin in pharmaceutical preparations and human urine.     

Evaluation of the antioxidant and pro-oxidant effects of tea catechin oxypolymers

Tea catechin oxypolymers (TCOP) were prepared by oxidizing tea catechin (TC, the content of EGCG was >85%) with H2O2. Their antioxidant and pro-oxidant effects were tested using a deoxyribose assay, a photoreduction of NBT assay, a lipoxygenase assay, a POV assay, and animal tests. The scavenging effects of TCOP to both the hydroxyl radical and superoxide radical were stronger than that of TC, and also they had no pro-oxidant effect; the rate constant for reactions of TC and TCOP for hydroxyl radical were 1.0 x 10(10) and (1.4-2.8) x 10(10) M(-1) x S(-1), respectively. TCOP can inhibit lipid peroxidation and lipoxygenase effectively, and it also can activate red cell SOD and reduce the MDA content in serum of mice very significantly. These results suggested that the antioxidant activity of TCOP was not less than or even more notable than that of TC.     

Accurate determination of oosporein in fungal culture broth by differential pulse polarography

A simple and accurate differential pulse polarographic method has been developed for the determination of oosporein in the culture broth of the fungus Beauveria brongniartii. This hydroxybenzoquinone derivative is the only major secondary metabolite secreted by this entomopathogenic fungus, which is used as biological pest control agent (BCA) against Melolontha melolontha larvae. It can be found in the host organism as well as in the formulated product. The polarographic behavior of oosporein was examined in various buffer systems over the pH range 3-10. In Britton-Robinson buffer/methanol solution (3:7 v/v, pH 5.5) the differential pulse polarograms exhibited reproducible peaks at E(p) = -0.18 V vs silver/silver chloride/potassium chloride (3 M). Under these conditions, a plot of peak height vs concentration of oosporein was found to be linear over the range 5.9 x 10(-)(7) to 2.5 x 10(-)(5) M (0.18-7.74 microg mL(-)(1); r = 0.9998). The detection limit was calculated to be 54 ng mL(-)(1). To evaluate the concentration of oosporein, the standard addition method was applied. The analysis of oosporein in the culture broth led to a mean value of 524.9 microg mL(-)(1) broth with a relative standard deviation (S(rel)) of +/-2.6%. The proposed polarographic method is accurate, not time-consuming, and it is of low cost because no separation steps are necessary.     

Oxidative degradation of triazine derivatives in aqueous medium: a radiation and photochemical study

Pulse and steady state radiolysis techniques have been used to determine the bimolecular rate constants and to investigate the spectral nature of the intermediates and the degradation induced by hydroxyl radicals ((*)OH) with 1,3,5-triazine (T), 2,4, 6-trimethoxy-1,3,5-triazine (TMT), and 2,4-dioxohexahydro-1,3, 5-triazine (DHT) in aqueous medium. A competitive kinetic method with KSCN as the (*)OH scavenger was used to determine the rate constants for the reaction of (*)OH with T, TMT, and DHT. The bimolecular rate constants are 3.4 x 10(9), 2.06 x 10(8), and 1.61 x 10(9) dm(3) mol(-)(1) s(-)(1) respectively, for T, TMT, and DHT at pH approximately 6. The transient absorption spectra obtained from the reaction of (*)OH with T, TMT, and DHT have single absorption maxima at 320, 300, and 300 nm, respectively, and were found to undergo a second-order decay. The formation of TOH(*) [C(6)OH-N(5)-yl radical], TMTOH(*) [N(5)OH-C(6)-yl radical], and DHT(*) [C(6)-yl radical] is proposed from the initial attack of (*)OH with T, TMT, and DHT, respectively. A complete degradation of TMT (10(-3) mol dm(-3)) was obtained after absorbed doses of 5 kGy in N(2)O-saturated solutions and 16 kGy in aerated solutions. A similar degradation pattern was obtained with DHT in N(2)O-saturated solutions. Complete degradation was observed with an absorbed dose of 7 kGy. On the basis of the results from both pulse and steady state radiolysis, a possible reaction mechanism involving (*)OH-mediated oxidative degradation is proposed. A complete photodecomposition of DHT was also observed in the presence of ferric perchlorate using ultraviolet light at low pH. Photoinduced electron transfer between Fe(III) and DHT in the Fe(III)-DHT complex and subsequent formation of DHT(*) are proposed to be the major processes that lead to the complete degradation of DHT at pH 3.     

Determination and differentiation of Flos Chrysanthemum based on characteristic electrochemical profiles by capillary electrophoresis with electrochemical detection

A high-performance capillary electrophoresis with electrochemical detection (CE-ED) method has been developed for the analysis of bioactive ingredients in Flos Chrysanthemum in this work. The effects of several factors such as the acidity and concentration of running buffer, the separation voltage, the applied potential, and the injection time were investigated. Under the optimum conditions, the eight analytes could be well separated within 20 min at the separation voltage of 14 kV in a 50 mmol/L Borax running buffer (pH 9.2). A 300 microm diameter carbon disk electrode has a good response at a potential of +950 mV (vs SCE) for all analytes. Good linear relationship was established over 3 orders of magnitude with detection limits (S/N = 3) that ranged from 1.9 x 10(-7) to 3.0 x 10(-8) g/mL. This proposed method has been successfully applied for the determination and differentiation of six kinds of popular Flos Chrysanthemum samples based on their characteristic electrochemical profiles, and the results are satisfactory.     

改良茚三酮比色法测定土壤蛋白酶活性的研究

通过引入Ca2 为蛋白酶激活剂、以Pb(CH3 COO) 2 和Na2 C2 O4—CH3 COOH混合试剂作为除杂剂、将抗坏血酸与KIO3 结合起来以提高茚三酮比色法测定氨基酸的灵敏度和稳定性,并使培养和测定两个过程的pH缓冲体系有机结合起来,从而全面改进了土壤蛋白酶活性的测定方法。用该方法测定了潮土、黄棕壤和红壤的蛋白酶活性,并与常用的Folin比色法进行了试验比较,证明该方法适合于测定中性、碱性土壤的蛋白酶活性并优于Folin比色法     

Fenton-dependent damage to carbohydrates: free radical scavenging activity of some simple sugars

The antioxidant properties of simple carbohydrates were studied in a chemical system. Hydroxyl radicals generated by a Fenton reaction induce damage on simple carbohydrates with a consequent free radical scavenging activity. Carbohydrate activities were measured by different methods as spin-trapping of hydroxyl radical and electron paramagnetic resonance detection and 1,1-diphenyl-2-picrylhydrazyl quenching. Carbohydrate damage was evaluated in a Fenton system by measuring the reactive substances to thiobarbituric acid, by their decreased detection with an HPLC test, and by a gas chromatographic determination of formic acid from sugar oxidation. Different intensities of damage and scavenging were found according to molecular structure, and some hyphotheses on the carbohydrate action against free radicals were attempted. The assayed disaccharides were shown to be more active toward and less damaged by hydroxyl radical than monosaccharides.     

Isolation and characterization of aminopeptidase (Jc-peptidase) from Japanese cedar pollen (Cryptomeria japonica)

An aminopeptidase, Jc-peptidase, was purified from Japanese cedar pollen by seven steps, including precipitation with ammonium sulfate, ion-exchange chromatography, gel filtration, hydrophobic interaction chromatography on phenyl-agarose, and high-performance liquid chromatography. Purified Jc-peptidease has a molecular weight of 42 kDa and hydrolyzes the synthetic substrates of L-phenylalanyl-4-methylcoumaryl-7-amide (Phe-MCA) with Km = 5 x 10(-5) M, Tyr-MCA with Km = 7 x 10(-4) M, Leu-MCA with Km = 1 x 10(-3) M, and Met-MCA with Km = 1 x 10(-3) M. Other MCA analogues such as Arg-MCA or Glu-MCA failed to serve as its substrates. The activity was inhibited in the presence of phebestin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl-L-valyl]-L-phenylalanine, with Ki = 4.7 x 10(-5) M, or bestatin, [(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, with Ki = 1.1 x 10(-4) M. According to amino acid sequence analysis, the N-terminal amino group seems to be blocked. The physiological function of the aminopeptidase (Jc-peptidase) has not been clarified in vivo.     

Imazethapyr aqueous photolysis, reaction quantum yield, and hydroxyl radical rate constant

The recent introduction of imidazolinone-tolerant rice varieties allow imazethapyr to be used in commercial rice. Little is known about imazethapyr photodegradation in the rice field. Laboratory studies were conducted to determine the direct and indirect photolysis rates for imazethapyr and to evaluate the photolysis of imazethapyr in three rice paddy waters. The reaction quantum yield (phi I) for imazethapyr was determined to be 0.023 +/- 0.002, while the hydroxyl radical rate constant (K(I)*OH) was 2.8 x 10(13) M(-1) h(-1). These results show that imazethapyr is susceptible to both direct and indirect photolysis reactions in water. The results also show that imazethapyr photolysis in paddy water will be affected by turbidity because of its impact on the availability of sunlight to drive direct and indirect photolysis reactions.     

Kinetic solvent effects on phenolic antioxidants determined by spectrophotometric measurements

The effects of polar (acetonitrile and tert-butyl alcohol) and apolar (cyclohexane) solvents on the peroxyl-radical-trapping antioxidant activity of some flavonoids, catechol derivatives, hydroquinone, and monophenols have been studied. The inhibition rate constants k(inh) of the antioxidants have been determined by following the increase in absorbance at 234 nm of a dilute solution of linoleic acid at 50 degrees C containing small amounts of antioxidant and radical initiator. Despite the low concentration of linoleic acid, the peroxidation process has been confirmed to be a free radical chain reaction described by the classical kinetic laws for this process. However, in the evaluation of k(inh), a careful analysis of the peroxidation curve, absorbance versus time, must be done because the final oxidation products of phenols may absorb at 234 nm. Phenols with two ortho-hydroxyls are the most active antioxidants, with inhibition rate constants in the range of (3-15) x 10(5) M(-1) x s(-1) (in cyclohexane). Nevertheless, it has been observed that in tert-butyl alcohol (a strong hydrogen bond acceptor) the rate constants dramatically decline to values not detectable by the present kinetic method. In acetonitrile (a weaker hydrogen bond acceptor) instead, the phenols with two ortho-hydroxyls scavenge the peroxyl radicals with rate constants close to those in cyclohexane. From the kinetic solvent effect, the equilibrium constant of the first solvation step of hydroquinone with tert-butyl alcohol has been determined at 50 degrees C, K(1) = 2.5 +/- 0.5 M(-1).     

Determination of phenolic constituents of biological interest in red wine by capillary electrophoresis with electrochemical detection

A simultaneous determination of trans-resveratrol, (-)-epicatechin, and (+)-catechin in red wine by capillary electrophoresis with electrochemical detection (CE-ED) is reported. The effects of the potential of the working electrode, pH and concentration of running buffer, separation voltage, and injection time on CE-ED were investigated. Under the optimum conditions, the analytes could be separated in a 100 mmol/L borate buffer (pH 9.2) within 20 min. A 300 microm diameter carbon disk electrode has a good response at +0.85 V (vs SCE) for all analytes. The response was linear over 3 orders of magnitude with detection limit (S/N = 3) ranging from 2 x 10(-7) to 5 x 10(-7) g/mL for all analytes. This method has been used for the determination of these analytes in red wine without enrichment, and the assay result was satisfactory.     

抗氧化剂对辐照猪肉理化和感官品质的影响

为减少猪肉辐照灭菌后产生的异味和脂肪氧化,研究了叔丁基对苯二酚、虾青素、维生素E、茶多酚4种抗氧化剂对辐照猪肉理化和感官品质的影响。采用2 g/L抗氧化剂浸泡处理,透氧包装,辐照剂量2.6 kGy,冷藏10 d。分析测定辐照猪肉感官品质、过氧化值、硫代巴比妥酸反应物、挥发性物质、抗氧化剂对羟自由基清除能力,筛选出适合猪肉辐照的高效抗氧化剂。结果表明：叔丁基对苯二酚在储藏期可以很好地减轻辐照异味并抑制脂肪氧化,效果优于虾青素、维生素E、茶多酚。叔丁基对苯二酚和维生素E可以有效地降低脂肪辐照后产生的挥发性物质含量。叔丁基对苯二酚（0.5 g/L）对羟自由基的清除率为52.5%,高于其他抗氧化剂,可抑制猪肉辐照过程中羟自由基参与的反应。