DPPH Assay Adapted to the FIA System for the Determination of the Antioxidant Capacity of Wines: Optimization of the Conditions Using the Response Surface Methodology

The objective of the present work was to adapt the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay to a system of flow injection analysis (FIA) of merging zones to determine the antioxidant capacity of wine samples. The conditions of the FIA system proposed here were optimized with the response surface methodology using central composite rotatable design. The optimization parameters studied here were the carrier flow rate, the length of the sampling and reagent loops, and the length of the reaction coil. The experimental data were fitted to a polynomial model, and the optimal conditions were: flow rate of 1.12 mL?min^?1, loop length of 18.5 cm, and reaction coil length of 234.60 cm. The limits of detection and quantification were 2.65 and 8.82 μM of Trolox, respectively. The method proposed had an analytical frequency of 41 analyses per hour and a reagent consumption economy of 57.3 % for DPPH in relation to the conventional method. The results of the antioxidant activities of the wine samples obtained with the proposed method were correlated and compared to those of the conventional method.     

Determination of free fatty acids in palm oil samples using non-aqueous flow injection titrimetric method

Flow injection (FI) non-aqueous titrimetric methods for the determination of free fatty acids (FFA) in palm oil samples are described. Single-line and two-line FI manifolds using phenolphthalein (PHP) and bromothymol blue (BTB) as indicators were developed. The method is based on the monitoring of the changes of absorbance of the indicators used from basic–acidic–basic form (pink–colourless–pink for PHP, blue–yellow–blue for BTB) as a result of the neutralization of KOH that was used as carrier stream by the injected FFA sample. FI parameters such as carrier and reagent concentration, flow-rate, length of reaction coil, size of mixing chamber and injected volume were optimized. The single-line manifold with PHT as indicator is recommended for the determination of samples with acidity degree (a.d.) higher than 0.4, but the oil samples need to be diluted with 2-propanol before their injection. For lower acidities (a.d. < 0.4), a two-line manifold with BTB as indicator is recommended. The two-line manifold allows direct injection of oil samples (no off-line dilution required). The optimized FIA method is linear over the range 0.4–10.0 a.d. (based on palmitic acid) for single-line manifold and 0.11–0.50 a.d. for the two-line manifold. Sample throughput of 35–74 and 21–46 samples h⁻¹ for single-line and two-line manifolds, respectively, were achieved. Fifty different samples of palm oils were tested using the appropriate FIA manifolds, and results were compared with the standard PORIM procedure which involves manual titration. Good correlations between the two methods were obtained (r², at least 0.92) UV–VIS absorption spectra indicate that the absorption of these oil samples were minimum at the detection wavelengths (562 nm for PHP and 627 for BTB), indicating that the method is negligibly interfered from the background colour of the samples.     

Fast and reliable analyses of sulphite in fruit juices using a supramolecular amperometric detector encompassing in flow gas diffusion unit

A new compact system encompassing in flow gas diffusion unit and a wall-jet amperometric FIA detector, coated with a supramolecular porphyrin film, was specially designed as an alternative to the time-consuming Monier-Williams method, allowing fast, reproducible and accurate analyses of free sulphite species in fruit juices. In fact, a linear response between 0.64 and 6.4 ppm of sodium sulphite, LOD = 0.043 ppm, relative standard deviation of ±1.5% (n = 10) and analytical frequency of 85 analyses/h were obtained utilising optimised conditions. That superior analytical performance allows the precise evaluation of the amount of free sulphite present in foods, providing an important comparison between the standard addition and the standard injection methods. Although the first one is most frequently used, it was strongly influenced by matrix effects because of the unexpected reactivity of sulphite ions with the juice matrixes, leading to its partial consumption soon after addition. In contrast, the last method was not susceptible to matrix effects yielding accurate results, being more reliable for analytical purposes.     

Determination of polyphenolic content and antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng

The total polyphenol content and the antioxidant activity of kudingcha made from Ilex kudingcha C.J. Tseng were determined by Folin–Ciocalteu reagent, and DPPH, FRAP, and TEAC methods, respectively. The crude extract (CE) of kudingcha and its four fractions of chloroform (CfF), ethyl acetate (EaF), n-butanol (nBF), and water (WtF) were prepared and subjected to antioxidant evaluation and analysis by high performance liquid chromatography. The extracts of kudingcha contained large amounts of caffeoylquinic acid (CQA) derivatives, including 3-CQA, 5-CQA, 3,4-diCQA, 3,5-diCQA, and 4,5-diCQA, and showed potent antioxidant activity. The antioxidant activities of CE and its fractions decreased in the order of EaF > nBF > CE > WtF > CfF, according to the DPPH assay and FRAP assay, which were the same, with the exception of the rank order of CfF and WtF, as the TEAC assay. Furthermore, a satisfactory correlation between total polyphenol content and antioxidant activity was observed.     

Screening of 70 medicinal plant extracts for antioxidant capacity and total phenols

The total phenolic content and related total antioxidant capacity of 70 medicinal plant infusions was analyzed. Infusions were prepared in common way in which teas are prepared for human consumption. The total phenolics were measured by Folin–Ciocalteau assay. The total antioxidant capacity was estimated by Ferric Reducing/Antioxidant Power (FRAP) assay. To make practical comparison of relative antioxidant potential of phenolics extracted from selected medicinal plants, the phenol antioxidant coefficient (PAC) was calculated for each infusion. The total phenolic content of medicinal plant infusions ranges from 9 to 2218 mg/L. The FRAP range from 0.06 to 25 mM/L. There was significant linear correlation between total phenolic content and FRAP. According to their antioxidant capacity, 70 medicinal plant extracts can be divided in five groups: (a) very low FRAP (<1 mM/L) n = 9; (b) low FRAP (1–5 mM/L), n = 37; (c) good FRAP (5–10 mM/L), n = 15; (d) high FRAP (10–20 mM/L), n = 8; and (e) very high FRAP (>20 mM/L), n = 1 medicinal plant extract. The PAC was ranging from 1.1 to 3.9 (average 2.4). The best results were obtained for Melissae folium infusions: high phenolic concentration, very high FRAP (>20 mM/L) and PAC > 3. The effect of infusion time and infusion temperature on the phenolic content, FRAP, and free radical scavenging ability was tested. DPPH radical scavenging ability of Melissae folium phenolics was similar to (+)-catechin but not as good as for quercetin. Compared to Trolox and vitamin C, Melissae folium phenolics were more efficient free ABTS radical scavengers. The results indicate that Melissae folium infusions could be an important dietary source of phenolic compounds with high antioxidant capacity comparable with red wine or beverages like tea.     

Validation of a quantitative assay for the total content of lipophilic and hydrophilic antioxidants in foods

One of the main methods used to assess total antioxidant content in foods is the Ferric Reducing Ability of Plasma (FRAP) assay. The FRAP assay has previously not been extensively validated. In the present study, 39 pure compounds, such as different polyphenols, ascorbic acid, tocopherols, tocotrienols and carotenoids dissolved in water/methanol, water/2-propanol and 2-propanol, were tested. Our results demonstrate that the FRAP assay can quantitate satisfactorily most hydrophilic and lipophilic compounds with antioxidant properties. The reaction was followed for 60 min. The most extensive reaction occurred within the first 4 min for most compounds and foods. The lipophilic tocopherols and tocotrienols were easily quantitated and reached endpoint within 4 min while carotenoids were somewhat more demanding due to low solubility and slower reaction kinetics. We conclude that the FRAP assay, with 4 min reaction time, is suitable for high-throughput screening of total antioxidant content of edible items.     

Flow injection spectrophotometry using natural reagent from Morinda citrifolia root for determination of aluminium in tea

A flow injection (FI) spectrophotometric method with using natural reagent extracted from Morinda citrifolia root has been developed for determination of aluminium. The extract contained anthraquinone compounds which could react with Al³⁺ to form reddish complexes which had maximum absorption wavelength at 499.0 nm. The extract could be used as a reagent in FI system without further purification to obtain pure compound. A sensitive method for determination of aluminium in concentration range of 0.1-1.0 mg L⁻¹, with detection limit of 0.05 mg L⁻¹ was achieved. Relative standard deviations of 1.2% and 1.7% were obtained for the determination of 0.1 and 0.6 mg L⁻¹ Al³⁺ (n = 11). Sample throughput of 35 h⁻¹ was achieved with the consumption of 3 mL each of carrier and reagent solutions per injection. The developed method was successfully applied to tea samples, validated by the FAAS standard method. The method is simple, fast, economical and could be classified as a greener analytical method.     

Effect of drying method on the antioxidant capacity of six Lamiaceae herbs

The present study investigated the changes in total phenols (TP), rosmarinic acid content and antioxidant capacity of six Lamiaceae herbs (rosemary, oregano, marjoram, sage, basil and thyme) after three drying treatments (air-, freeze- and vacuum oven-drying) stored for 60 days at −20 °C and compared to fresh samples. Ferric reducing antioxidant property (FRAP) and oxygen radical absorbance capacity (ORAC) were used as markers for antioxidant capacity. Air-dried samples had significantly (p < 0.05) higher TP, rosmarinic acid content and antioxidant capacity than had freeze-dried and vacuum oven-dried samples throughout the storage period. Fresh samples had the lowest values for the parameters tested. Vacuum oven-drying resulted in higher TP and FRAP values in rosemary and thyme during 60 days of storage than did freeze-drying. In ORAC assay, the difference was significantly higher only in thyme. Storage did not show any effect on the dried samples for the parameters tested.     

Changes in bioactive compounds and antioxidant capacity of fresh-cut cashew apple

In this study, ascorbic acid, total polyphenols and proanthocyanidins of fresh-cut cashew apple were quantified. Antioxidant capacity was determined in whole juice and in polyphenols extracts by three methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power (FRAP) and ??-carotene bleaching. Effect of cutting and storage for 24 h at 2 °C, 27 °C and 40 °C on these compounds were also evaluated. Cashew apple presented 163 mg of ascorbic acid per 100 g of fresh weight (FW). Soluble and hydrolysable polyphenols contents were 12.79 mg GAE/100 g FW and 18.53 mg GAE/100 g FW and proanthocyanidins were 9.27 mg/100 g FW. Antioxidant capacity of juice and polyphenols extract was high for DPPH method. Storage temperatures affected bioactive compounds on cut cashew apple. The content of ascorbic acid decreased in all temperatures. Proanthocyanidins were more sensitive to 40 °C than to other temperatures. The content of polyphenols and antioxidant capacity of juice by DPPH assay did not change. However, the reducing power was lower in samples kept at high temperatures. A strong positive correlation between ascorbic acid and FRAP (r = 0.99) and a negative correlation between DPPH and FRAP (r = − 0.79) were observed. No correlations were found between polyphenols and antioxidant capacity indicating the importance of phenolic composition in the extracts. The results confirm the importance of temperature and injury on the quality of cashew apple.     

Detection of sulphathiazole in honey samples using a lateral flow immunoassay

A lateral flow immunoassay (LFIA) was developed in the competitive reaction format and applied to test sulphathiazole (STZ) residues in honey samples. To prepare the assay test, a hapten conjugate and goat antirabbit antiserum as capture and control reagent, respectively, were dispensed on nitrocellulose membrane. Polyclonal antiserum against sulphathiazole was conjugated to colloidal gold nanoparticles and used as the detection reagent. The visual limit of detection (cut-off value) of the sulphathiazole LFIA was 15 ng/g, reaching qualitative results within 10 min. The assay was evaluated with STZ spiked honey samples from different geographical origins (n = 25). The results were in good agreement with those obtained from liquid chromatography separation and mass spectroscopy detection (LC–MS), indicating that the LFIA test might be used as a qualitative method for the determination of sulphathiazole residues without expensive equipment. The test was also highly specific, showing no cross-reactivity to other chemically similar antibiotics. To our knowledge, this is the only work where a development of LFIA tests for the detection of sulphathiazole residues is performed.     

Evaluation of antioxidant activity and total phenolics of selected Czech honeys

Honey serves as a good source of natural antioxidants, which are effective in reducing the risk occurrence of heart disease, cancer, cataracts, different inflammatory processes and immune-system decline. In the fresh selected Czech honey samples originated mainly from the region North Moravia antioxidant activity and total polyphenol content were determined. A total of 40 honey samples (multifloral, lime, rape, raspberry, mixture and honeydew honeys) native to different stations gained in the period from May by August year 2006 were analysed. Total phenolics (TP) content was determined by the modified Folin-Ciocalteau method [TP was expressed as mg of gallic acid equivalent (GA eq.) per kg of honey]. For evaluation of the antioxidant activity (AOA) three different methods were used, the ferric reducing antioxidant power (FRAP) assay, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay and the 2,2′-azinobis(3-ethylbenzothiazolin)-6-sulfphonate (ABTS) assay. AOA was expressed in mg ascorbic acid equivalent (AA eq.) per kg of honey. The results indicated that TP and AOA in Czech honey varied greatly between the honey kinds, location and time of the harvest. Average TP ranged from 89.9 mg GA eq. kg^-1 in lime honey to 215.2 mg GA eq. kg⁻¹ in honeydew honey. Antioxidant activity determined by the DPPH, ABTS and FRAP methods was lowest in floral honeys. The highest values were obtained for honeydew and mixture honeys. ABTS and FRAP assays have been shown to be the optimal methods for AOA determination in honey. A positive linear correlation between AOA and TP was observed (in FRAP assay R² = 0.852). It indicates that phenolics are one of the main components responsible for antioxidant behaviour of honey. The obtained results support and extend complete knowledge about the content of bioactive phenolics and antioxidant activity in the Czech honeys.     

Automated flow pH-method for the determination of total free fatty acids content in edible oils

An automated flow pH-method for the determination of titratable acidity (TA) in edible oil without titration is proposed, based on pH measurements on an oil sample emulsion in a suitable reagent. The developed flow pH system involves two constant rate flows: the flow of oils samples and the flow of reagent: triethanolamine + KNO₃ + H₂O + i-PrOH, in which oils is insoluble. The flows are mixed in the mixer forming the emulsion in which the free fatty acids (FFA) extraction is started. Then, the emulsion is passed to the coil for completion of FFA extraction and thermostation and then forwarded to pH-cell for pH measurement. Using pH values and linear dependence of pH vs. log (TA) allows the TA determination.The optimum parameters for the automated flow pH-system have been found, i.e., volumes of the mixer and the coils, the rate of sample and reagent, pH and TA ranges. The results of the TA determination in the edible oils were statistically compared with the standard titration method.     

Determination of nitrofurans residues in animal feeds by flow injection chemiluminescence procedure

A simple flow injection chemiluminescence (FI-CL) method was developed for the rapid and sensitive determination of nitrofurans, including furazolidone, nitrofurantoin and nitrofurazone, in animal feeds based on its chemiluminescence induced by potassium permanganate in sulphuric acid medium. The method involves the injection of nitrofuran samples or standards into H₂SO₄ carrier stream, which then merges at a T-piece with a reagent stream consisting of KMnO₄ in the H₂SO₄ carrier solution. The elicited chemiluminescence intensity of the resulting reaction mixture was measured by photomultiplier tube operated at a voltage of 950 V. Optimum CL signals were given using 2.5 × 10⁻⁵ mol L⁻¹ potassium permanganate in 0.1 mol L⁻¹ sulphuric acid as an oxidant stream and a carrier stream of 0.1 mol L⁻¹ sulphuric acid with a total flow rate of 7.0 mL min⁻¹. Results detailing the optimisation of the analytical signal, calibration, and common interferences of animal feeds were also discussed. The proposed FI-CL method was successfully applied to the determination of nitrofurans in animal feeds, with excellent recoveries, as the determination is free from interference. The method validation has been compared versus HPLC method for animal feed samples.     

Evaluation of a simple and promising method for extraction of antioxidants from sea buckthorn (Hippophaë rhamnoides L.) berries: Pressurised solvent-free microwave assisted extraction

The pressurised solvent-free microwave assisted extraction (PSFME) technique has been developed and optimised for extraction of antioxidants from Hippophaë rhamnoides L. berries using a two-level full factorial design. The effects of factors (extraction time, irradiation power, number of cycles) and their first order interactions were evaluated from antioxidant activity of extracts using the 2,2′-diphenyl-1-picrylhydrazil (DPPH) free radical scavenging method, the ferric reducing ability of plasma (FRAP) assay, and the estimation of total phenolic content using the Folin–Ciocalteu method. The best extraction conditions were obtained, in a laboratory scale extractor of 50 mL filled with 4 g fresh berries, using a 1000 W microwave power applied during 50 s and repeated five cycles. PSFME was then compared to other common extraction techniques such as pressing, maceration and pressurised liquid extraction. It is appeared that PSFME leads to the most active and richest extract in phenolic content including molecules such as quercetin and isorhamnetin not extracted with other techniques. Furthermore PSFME respect green chemistry, it is rapid, cheap and does not need sample preparation and/or evaporation step.     

Development of an enzyme-linked immunosorbent assay for diniconazole in agricultural samples

Diniconazole hapten was synthesized and conjugated to bovine serum albumin (BSA) by the carbodiimide method to produce an immunogen and to ovalbumin (OVA) by mixed anhydride method to produce a coating antigen. Polyclonal antibody against diniconazole was generated by immunizing New Zealand rabbits with the immunogen. Under optimised conditions (20% methanol, 0.4 mol/L Na⁺, pH 7.5), an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed for detecting diniconazole. The 50% inhibitory concentration (IC₅₀) was 0.071 ± 0.013 mg/L and the limit of detection (LOD) was 1.28 ± 0.80 μg/L. Triazole fungicide analogues of diniconazole were tested and did not obviously cross-react, except for uniconazole (2.25%). The recoveries obtained after addition of standard diniconazole to agricultural samples, including water, soil, pear, grape, tomato and wheat flour, ranged from 70% to 120%. The ic-ELISA developed could successfully be applied to analysis of diniconazole residues in agriculture samples.     

Amperometric detection of ascorbic acid in honey using ascorbate oxidase immobilised on amberlite IRA-743

A differential amperometric method for the specific determination of ascorbic acid in honey was developed by association of a flow injection analysis (FIA) system and a tubular reactor containing the ascorbate oxidase enzyme immobilised. A gold electrode modified by electrochemical deposition of palladium was employed as working electrode. Ascorbic acid was quantified in seven samples of commercial honeys using a potential of +0.60 V vs. Ag/AgCl_(sat). The linear dynamic range in ascorbic acid extends from 1 to 50 μmol L⁻¹, at pH 7.0. At flow rate of 1.5 mL min⁻¹ and injecting 250 μL sample volumes, a sampling frequency of 180 determinations per hour is afforded. The detection and quantification limit of this method are 0.14 and 0.49 μmol L⁻¹, respectively. The samples analyses were compared with the volumetric method, and showed an excellent correlation between the methods.     

Antioxidant power of Iranian propolis extract

Propolis is a resinous natural hive product derived from plant exudates collected by honey bees. Due to biological and pharmacological activities, it has been extensively used in folk medicine. The present study was designed to measure the antioxidant power of ethanolic extracts of propolis samples from different parts of Iran with “ferric reducing ability of plasma” (FRAP) assay and compare the results with Trolox at concentrations of 100, 1000 and 2000 μg/ml. FRAP values of propolis ethanolic extracts were in the range of 31.5 ± 14.6 to 1650 ± 72 μM, whereas the values of Trolox ranged from 125.25 ± 9.95 to 3381.64 ± 113.83 μM. The FRAP values of Tehran propolis ethanolic extract and Trolox at concentration of 100 μg/ml did not show any significant difference (P > 0.05). Total flavonoid and polyphenol contents of ethanolic extracts of propolis samples, determined by using aluminum nitrate and Folin–Ciocalteu colorimetric methods, were in the range of 1.22 ± 0.33–7.79 ± 0.39 g/100 g and 3.08 ± 0.02–8.46 ± 0.03 g/100 g crude extract of propolis, respectively. The result of this experiment may show that propolis as a natural source of antioxidant compounds may be of use in prevention of free radical-related diseases.     

Evaluation of the phenolic content,antioxidant activity and colour of Slovenian honey

Honey samples from the seven most common honey types in Slovenia were screened for total phenolic content by the modified Folin–Ciocalteu method, for potential antioxidant activity using the ferric reducing antioxidant power (FRAP) assay and by the 1,1-diphenyl-2-picrylhydrazyl (DPPH) method for antiradical activity. In addition the colour characteristics of honey samples were analysed. The results of the study showed that total phenolic content, antioxidant activity and colour parameters differ widely among different honey types. Phenolic content expressed as gallic acid equivalent ranged from 44.8 mg/kg in acacia honey to 241.4 mg/kg in fir honey. Antioxidant activity was the lowest in the brightest acacia and lime honeys and the highest in darker honeys, namely fir, spruce and forest. The colour of the Slovenian honeys, analysed in this study was very variable and ranged from pale yellow to dark brown. Correlations between the parameters analysed were found to be statistically significant (p < 0.05).     

Flow injection analysis of total curcuminoids in turmeric and total antioxidant capacity using 2,2′-diphenyl-1-picrylhydrazyl assay

A simple flow injection analysis procedure is proposed for the determination of curcuminoids content in turmeric extracts. The method is based on the formation of a coloured complex between 4-aminoantipyrine and curcuminoids, in the presence of an oxidising reagent such as potassium hexacyanoferrate (III) in alkaline media. Conditions selected as a result of these trials were implemented in a flow injection analytical system in which the influence of injection volume, flow rate, reagent concentration and mixing coil length, was evaluated. Under the optimum conditions the total amount of curcuminoids could be determined within a concentration range of 5–50 μg mL⁻¹ which can be expressed by the regression equation y = 0.003x − 0.0053 (r² = 0.9997). The limits of detection and quantitation were found to be 0.6 μg mL⁻¹ and 1.8 μg mL⁻¹, respectively. The reproducibility of analytical readings was indicative of standard deviations <2%. The sample was extracted and analysed by using the proposed method. The percentage recoveries were found to be 94.3–108.0. The proposed system was applied to the determination of curcuminoids content in turmeric. The total curcuminoid contents in turmeric extract were found to be 0.9–4.3% (w/w). The development method is simple, economic, rapid and especially suitable for quality control in pharmaceutical plants.     

Extraction and application of antioxidants from black glutinous rice

This research aimed to determine optimum extraction condition of black glutinous rice crude extract and to determine its application as an antioxidant in fish oil enriched mayonnaise. Black glutinous rice flour was extracted twice with 70:30 acetone-water mixture (v/v) at pH 2 and 6.8 for 2, 4 and 8 h of total extraction times. Total phenolic content (TPC), total monomeric anthocyanin content (TMA) and antioxidant activities as determined by ferric reducing antioxidant power (FRAP) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity assays of the crude extracts were measured. The extraction with pH 6.8 solvent for 4 h yielded the crude extract with significantly highest antioxidant activities analyzed by both FRAP and DPPH tests (p ≤ 0.05) although its TPC and TMA were not greatest. The freeze-dried extract from this condition was then added into fish oil enriched mayonnaise at 500 mg/kg and 1000 mg/kg (oil weight basis). Conjugated diene hydroperoxides (CDH), thiobarbituric acid reactive substance (TBARs) and color in CIELAB system of the mayonnaise samples stored at 30 °C were determined up to 30 days. The samples contained 1000 mg/kg crude extract had lowest rate of CDH and TBARs increase but had greatest extent of color deterioration, possibly due to anthocyanin degradation and Maillard reaction.