Investigation of plant extracts for the protection of processed foods against lipid oxidation. Comparison of antioxidant assays based on radical scavenging, lipid oxidation and analysis of the principal antioxidant compounds

 Antioxidant activities of plant extracts from spices, coffee, tea, grape skin, and tomato peel slurry were evaluated using a number of analytical methods including the quantification of principal compounds. Similar rankings in the activities of these extracts were obtained by evaluating their efficiencies as scavengers of stable free radicals: Fremy's salt, galvinoxyl or α,α-diphenyl-β-picrylhydrazyl (DPPH). Similar results were obtained with the lipid oxidation assays based on thermal acceleration (formation of conjugated dienes in methyl linoleate at 40  °C or the Rancimat test at 100  °C with lard). Rankings of the extract activity obtained by scavenging of hydroxyl radicals generated in the Fenton reaction were similar to those obtained by an oxygen consumption assay with linoleic acid as substrate and metmyoglobin as catalyst. However, the results of the latter two assays differed from those of the other assays. In the overall ranking, coffee and rosemary extracts were amongst the most potent extracts whereas the tomato peel slurry showed no activity. Received: 16 February 2000 / Revised version: 7 July 2000     

A strategy for high-speed countercurrent chromatography purification of specific antioxidants from natural products based on on-line HPLC method with radical scavenging assay

We have proposed a novel and first strategy of high-speed countercurrent chromatography (HSCCC) purification for the efficient and effective discovery of antioxidant from natural product based on on-line HPLC method with radical scavenging assay. To achieve a strategy for HSCCC purification, the antioxidants in materials are identified by on-line HPLC with DPPH radical scavenging assay. Then, the optimal condition of target peaks would be investigated for the two-phase solvent system, and purified by HSCCC. In this study, the specific antioxidants in red cabbage, perilla and elderberry pigments were evaluated by on-line HPLC with DPPH radical scavenging assay, and purified by HSCCC technique. Specific antioxidants could be rapidly pinpointed in complex mixtures by on-line HPLC with DPPH radical scavenging assay. Then, the optimal two-phase solvent systems were investigated using these HPLC peaks. Finally, the purification of these nine antioxidants form three mixtures were performed by HSCCC. Using mass spectrometric analysis, these antioxidants were confirmed to cyanidin-based anthocyanin from red cabbage and elderberry pigments, and luteolin-based flavones from perrilla pigment. Due to the advantages derived from on-line HPLC with DPPH radical scavenging assay and HSCCC technique, a rapid, efficient and effective strategy has been developed for the discovery of antioxidants from natural products.     

Biological screening of 100 plant extracts for cosmetic use (II): anti-oxidative activity and free radical scavenging activity

Methanol aqueous extracts of 100 plants were screened for anti-oxidative activity using Fenton's reagent/ethyl linoleate system and for free radical scavenging activity using the 1,1-diphenyl-2-picryl hydrazyl free radical generating system. The results suggest that 14 plants - Alpinia officinarum, Areca catechu, Brassica alba, Cannabis sativa, Curcuma longa, Curcuma aromatica, Eugenia caryophyllata, Evodia officinalis, Paeonia suffruticosa, Rhaphanus sativus, Rheum palmatum, Rhus verniciflua, Trapa bispinosa, Zanthoxylum piperitum - may be potential sources of anti-oxidants. Eight plants - Citrus aurantium, Cornus officinalis, Gleditsia japonica, Lindera strychnifolia, Phragmites communis, Prunus mume, Schizandra chinensis, Terminalia chebula - may be the potential source of free radical scavengers from natural plant.     

HPLC法同时检测复方产品中西洋参、红景天和刺五加的皂苷含量

采用高效液相色谱法建立了同时检测复方产品中人参皂苷Rb1、人参皂苷Re、红景天苷、刺五加苷B和刺五加苷E含量的方法。以60%乙醇作为提取溶剂,用色谱柱Kromasil 100-5C18（250×4.6mm,5μm）进行分离,以0.1%磷酸-乙腈混合液为流动相进行梯度洗脱,结果表明,在此条件下5种皂苷得到了良好的分离。人参皂苷Rb1、人参皂苷Re、红景天苷、刺五加苷B和刺五加苷E分别在80⁸00、80⁸00、40⁴00、20¹60μg/m L和40³20μg/m L范围内呈现良好的线性关系,平均加标回收率在99.5%¹06.8%之间,相对标准偏差为0.5%².3%。本方法具有重现性好、回收率高等优点,适用于复方产品中人参皂苷Rb1、人参皂苷Re、红景天苷、刺五加苷B和刺五加苷E的同时检测。      

New approach for evaluation of the antioxidant capacity based on scavenging DPPH free radical in micelle systems

A simple and sensitive method was developed to evaluate the antioxidant capacity using a 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) incorporated in surfactants. Parameters possibly affected the DPPH scavenging activity were investigated including the type of surfactant, concentration of surfactant, solution pH and concentration of buffer. The optimum micelle system for determining the antioxidant capacity was 2 mM CTAB in 0.1 M acetate buffer (pH 4.6). The IC₅₀ values of various antioxidants were calculated and compared to those prepared in methanol. Furthermore, the rate of the reaction between DPPH and antioxidants was investigated. The rate constants observed in the micelle system were significantly faster than those in methanol, and this allowed shorter analysis time. This method was validated through linearity, limit of detection and precision by comparing with conventional methanolic assay. The purposed method was applied to evaluate the antioxidant capacity from aqueous extracted plant samples with satisfactory results.     

毛豆腐提取物的自由基清除活性研究

利用20%、40%、60%乙醇溶液（v/v）、pH4.5水、pH6.5水作为提取溶剂,对发酵时间为3、5、7、9d的毛豆腐分别进行提取,测定提取物中蛋白质的提取率、水解度,以及采用DPPH、ABTS法评价提取物的体外自由基清除活性。研究结果表明,随着毛豆腐发酵时间的延长,提取物中蛋白质的提取率、水解度均逐渐增加,并且提取物的自由基清除活性也明显地提高;通过比较还发现,毛豆腐乙醇提取物的自由基清除活性高于水提取物,且随着乙醇浓度的提高,提取物的自由基清除活性提高。排阻色谱分析结果证明,发酵9d后毛豆腐中蛋白质充分水解,5种提取物的主要成分为肽与氨基酸。     

RP-HPLC测定油用牡丹籽饼粕和籽壳中单萜苷的含量

建立RP-HPIC同时测定油用牡丹籽饼粕和籽壳中6种单萜苷类化合物含量的方法。采用乙腈(A)和磷酸二氢钾缓冲盐(B)(pH=3)为流动相梯度洗脱,梯度为0 min(A,15%)→10 min(A,15%)→22 min(A,25%);检测波长:4"-羟基白芍苷和氧化芍药苷为260 nm,白芍苷、芍药苷、6-O-β-D-吡喃葡萄糖-8-O-苯甲酰基-9α-甲氧基-牡丹酮和白芍苷R1为232 nm,柱温30℃。结果表明,单萜苷类物质在油用牡丹籽饼粕中的含量远远大于在其籽壳中的含量,牡丹籽饼粕可以作为单萜苷类物质的一个重要来源。该方法准确,实用性好,可用于牡丹籽饼粕中单萜苷类成分的定性与定量分析。     

Antimicrobial activity of some plant extracts and essential oils against foodborne pathogens in vitro and on the fate of inoculated pathogens in chocolate

The efficacy of commercially available plant extracts and essential oils used extensively as flavour ingredients in confectionery products were used as antimicrobials in laboratory media against the following microorganisms: Escherichia coli O157:H7, Salmonella Enteritidis, Salmonella Typhimurium, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus. Using the disc diffusion method, inhibition zones in diameter >20 mm were observed by adding 10 μl of each antimicrobial substance on the following microorganisms: lemon flavour applied on E. coli O157:H7, lemongrass essences against S. aureus, plum using a B. cereus strain and strawberry flavour using a L. monocytogenes strain. E. coli O157:H7 strains were the most susceptible microorganisms inhibited by 18 extracts, followed by S. Typhimurium and S. aureus which were inhibited by 17 extracts. Lemon flavour, lemongrass essences, pineapple and strawberry flavour inhibited the foodborne pathogens at the lowest concentration (5 ml/100 ml). Plant extracts and essential oils with potent antimicrobial activities were tested in chocolate held at different temperatures (7 and 20 °C) in dry or humidified environment, which resulted in different a_w values of the product (i.e. 0.340, 0.450, and 0.822), in order to determine their efficacy on the fate of the inoculated pathogens. The most inhibitory action was observed by lemon flavour applied on chocolate inoculated with E. coli cocktail culture after storage at 20 °C for 9 days. Plant extracts tested on chocolate show an enhanced inhibitory effect during storage at 20 °C indicating that their application may provide protection in case of storage at the above temperature or even higher.     

Development of a rapid colorimetric method for the determination of chlorogenic acid in freeze-dried potato tubers

After-cooking blackening (ACB) in potato cultivars results from the formation of complex compounds of chlorogenic acid and iron ions. At present the estimation of chlorogenic acid content represents the most reliable method for predicting the extent of ACB in material in a potato breeding programme to allow subsequent selection. A number of methods for determining chlorogenic acid levels, including high performance liquid chromatography, sodium nitrite and other colorimetric and chromatographic assays, are evaluated. A rapid screening method for the direct determination of chlorogenic acid levels is described, and its application within a breeding programme and the results obtained from a number of cultivars are discussed.     

A rapid method for the measurement of Leucaena spp proanthocyanidins by the proanthocyanidin (butanol/HCl) assay

Proanthocyanidin (PA) extraction, sample preparation and proanthocyanidin assay (butanol/HCl) reaction conditions were evaluated for measuring PA in Leucaena spp leaf material. The optimal conditions for extracting PA from leaf tissue are described, with short sequential sonications in 70% aqueous acetone being as efficient as prolonged sequential mechanical agitation. In methanol–based extracts, after back extraction to remove pigments, increasing the water content of the reagent/sample matrix suppressed colour development. The addition of low concentrations of Fe³⁺ to the butanol/HCl reagent enhanced colour yield, but higher Fe³⁺ concentrations suppressed colour development. The presence of ascorbic acid in the sample extract was shown to increase colour development. Varying the reagent: sample extract ratio from 4:1 to 6:1 significantly decreased colour yield, but neither ratio was different from 5:1. Optimum conditions for the PA assay were as follows: a water content of 8%, the omission of Fe³⁺, a reagent: sample extract ratio of 5:1 and the addition of ascorbic acid to the stock PA standard solution to match that contributed by the extract in the final mixture. Sample preparation procedures, using back extraction to remove pigments and non-PA phenolics with diethyl ether and ethyl acetate, respectively, were time-consuming and subject to PA losses. The measurement of PA directly in the 70% aqueous acetone extract eliminated these PA losses, but the PA assay required additional optimisation for direct analysis of crude acetone extracts. In the final optimised procedure, PA was extracted by sequential sonication with 70% aqueous acetone containing 5·26 mM sodium metabisulphite as the antioxidant. These extracts were directly analysed by the butanol/HCl reaction using a reagent: sample extract ratio of 5:1, the omission of Fe³⁺ from the butanol/HCl reagent and the addition of sodium metabisulphite to match that contributed by the extract. This produced consistent linear calibration curves over the range 25–1000 μg PA with an average recovery of 101%. © 1998 Society of Chemical Industry.     

Validation of HPLC method for quantitative determination of Tinosorb S and three other sunscreens in a high protection cosmetic product

A chromatographic method (high performance liquid chromatography) with a diode array detector was developed for simultaneous assay of Tinosorb S (bis-ethylhexyloxyphenol methoxyphenyl triazine) with three other sunscreen agents [benzophenone-3, butyl methoxydibenzoylmethane (avobenzone) and ethylhexyl methoxycinnamate] in high protection sunscreen. Separations were performed on a RP-18 Nucleodur Gravity column (150 x 4.6 mm, 5 mum) eluted with a ternary gradient mixture constituted of tetrahydrofuran, acetonitrile and an aqueous solution of acetic acid. The quantitative analysis was achieved with internal calibration performed with octyl dimethyl para-aminobenzoate (PABA) at 330 nm. In accordance with the analytical references (SFSTP, ICH, ISO...), the accuracy of the method was evaluated using a statistical approach of the validation parameters (specificity, response function, linearity, precision and trueness). For each studied ultraviolet filter, an accuracy profile was determined on a predicted range. These profiles show a graphical representation of the recovery percentage and confidence limits centred on 100%. The method is validated and can be used for analysis in cosmetic sunscreen products.     

基于柱后衍生发芽糙米中γ-氨基丁酸HPLC检测方法的建立及应用

建立了一种能与17种常见氨基酸分离的发芽糙米中γ-氨基丁酸的检测方法,即采用Hypercarb column色谱柱,以邻苯二甲醛作为衍生试剂进行柱后衍生,检测波长为338 nm,γ-氨基丁酸的定量线性范围为0.2～50 mg/L,线性方程为A=0.304 3C+0.065 3,相关系数R2为0.999 9。确定了发芽糙米中γ-氨基丁酸的提取条件,发芽糙米经20%甲醇-水提取后,用1.0%甲酸稀释,进样检测,回收率可达97.7%。按照上述提取与检测方法,对不同种糙米制品中γ-氨基丁酸含量进行了测定,发芽糙米中γ-氨基丁酸含量明显高于普通糙米,发芽糙米经高温挤压与模拟蒸煮处理后,其γ-氨基丁酸含量均无明显变化。     

Automated HPLC method for the measurement of free amino acids including cysteine in musts and wines; first applications

An analytical method for the determination of free amino acids and especially of cysteine in musts and wines is reported. This technique involves the use of a pre‐column derivatisation with iodoacetic acid (IDA) and o‐phthaldialdehyde (OPA). Isoindole‐type fluorescent derivatives were formed in alkaline solution from OPA, 2‐sulphanylethanol and the primary amine group of the amino acids. The derivatives were separated by HPLC using a linear multistep solvent gradient and were detected by spectrofluorimetry. This method is suitable for the separation of most free amino acids including cysteine. It allows high recovery and satisfies the necessary requirements with respect to accuracy, repeatability and sensitivity. The first application of this analytical method to the measurement of cysteine in musts and wines is reported. © 2001 Society of Chemical Industry     

Development of an improved extraction and HPLC method for the measurement of ascorbic acid in cows' milk from processing plants and retail outlets

An improved extraction (2.5% HPO₃, 5 mm dithiothreitol) and HPLC quantification methodology using a C–18 column at 35 °C and 0.1 m acetic acid (98%) and acetonitrile (2%) mobile phase was developed to quantify total ascorbic acid (AA) in commercial whole/semi‐skim/skim raw/pasteurised/UHT milk packaged in opaque bags, transparent plastic, cardboard and Tetra Brik^?. AA content ranged from 0.21 to 10 and from 3.4 to 16 mg L^?1 in milk from retail outlets and processing plants, respectively, and was higher in organic milk. For same processor/lot samples, pasteurised milk showed higher AA content than UHT milk. This was not true for retail outlets samples. AA content was similar for whole/semi‐skim and semi‐skim/skim milk, but not for whole/skim comparisons. Among UHT samples, the AA content trend was whole<semi‐skim<skim and lower for UHT milk in opaque plastic and Tetra Brik^? container. After 14 days at 4 °C in the dark, AA losses ranged 35–83% depending on milk type and preservation method with a higher AA retention in unopened containers.     

一株槐树内生真菌发酵产物对羟自由基所致小鼠肝组织过氧化的体外作用

为了研究初筛获得的具有较高抗氧化活性和清除O2-.能力的槐树内生真菌菌株No.7发酵液提取物对.OH导致的氧化损伤的抑制作用及其清除.OH的效果,采用Fe2+-H2O2和Fe2+-VC两种羟自由基发生系统,以硫代巴比妥酸法测定了小鼠肝匀浆及肝线粒体中脂质过氧化物丙二醛(MDA)含量,以分光光度法测定了小鼠肝线粒体的膨胀程度。结果表明,槐树内生真菌No.7发酵液提取物具有清除.OH的作用,能抑制小鼠肝脏脂质过氧化,从而降低线粒体氧化损伤及肿胀。     

A rapid HPLC post-column reaction analysis for the quantification of ergothioneine in edible mushrooms and in animals fed a diet supplemented with extracts from the processing waste of cultivated mushrooms

For establishing an efficient and sensitive method for the quantitative determination of 2-thiol-l-histidine-betaine (ergothioneine, ERG) in edible mushrooms and the blood and muscles of animals, a technique using reversed-phase separation and post-column reaction between 2′-dipyridyl disulphide and ERG was developed. A corresponding derivative 2-thiopyridone, detected at 343 nm, was used for estimating ERG concentration. The flow rate, temperature, pH, and composition of the solution were optimised. A low limit of quantification (1.41 ppm) and a simpler sample preparation made this technique more rapid compared to other methods using liquid chromatography–mass spectrometry. The coefficient of variation (CV) values for the reproducibility and recovery of ERG were within the acceptable values of 6% and 97.5–100.0%, respectively. The efficiency of this methodology was compared with that of spectrophotometric and mass-spectrometric quantitative methods, and was assessed in the light of previous studies. The ERG contents in different mushrooms were 12.69–234.85 mg/kg wet weight basis. Dietary supplementation with extracts from mushroom processing waste significantly improved ERG bioavailability in the blood of yellowtail fish and muscle tissue of cattle.     

New method for the rapid identification of tetracycline residues in foods of animal origin — using the Premi®Test in combination with a metal ion chelation assay

A post-screening classification assay for tetracycline compounds has been developed and integrated into the previously reported optimized Premi®Test methodology. The new post-antimicrobial screening assay is based on a metal ion chelation using calcium and sodium chloride and has been shown to be specific towards the tetracycline class. The assay is both cost-effective and complementary to the post-screening procedures that have previously been developed for the β-lactam and sulfonamide compounds. A validation study was conducted in accordance with 2002/657/EC (Commission Decision). The method is rugged and applicable to a range of tetracyclines of differing antimicrobial potencies over a wide concentration range. A blind trial was undertaken in which all antimicrobial residues in the unknown samples were successfully identified by the analyst following the integrated Premi®Test procedure for the classification of antimicrobial compounds.     

日本核电站事故对蘑菇的放射性污染(137Cs)特征及去除污染的研究进展

日本福岛核电站事故导致大量人工放射性物质释放到环境中.蘑菇可以将周围环境的放射性物质富集在子实体内,使其在生态系统中循环,而消费者可能误买误食受污染蘑菇,造成放射性物质在人体内蓄积.通过查阅大量文献发现:蘑菇中放射性铯来源有多种,主要源自其生长的底物;蘑菇中放射性铯含量高低,与距事故发生地的距离和蘑菇自身的营养机制有着...     

Validation of HPLC method for the simultaneous and quantitative determination of 12 UV-filters in cosmetics

The aim of the study was the validation of a high-performance liquid chromatography (HPLC) method for the simultaneous and quantitative determination of twelve commonly used organic UV-filters (phenylbenzimidazole sulfonic acid, benzophenone-3, isoamyl p -methoxycinnamate, diethylamino hydroxybenzoyl hexyl benzoate, octocrylene, ethylhexyl methoxycinnamate, ethylhexyl salicylate, butyl methoxydibenzoylmethane, diethylhexyl butamido triazone, ethylhexyl triazone, methylene bis -benzotriazolyl tetramethylbutylphenol and bis -ethylhexyloxyphenol methoxyphenyl triazine) contained in suncare products. The separation and quantitative determination was performed in <30 min, using a Symmetry Shield® C18 (5 μm) column from Waters and a mobile phase (gradient mode) consisting of ethanol and acidified water. UV measurements were carried out at multi-wavelengths, according to the absorption of the analytes.