ABILITY OF VARIOUS CHEMICALS TO REDUCE COPPER AND TO INACTIVATE MUSHROOM TYROSINASE1

Treatment of mushroom tyrosinase with reducing agents such as hydrogen peroxide, ascorbic acid, phenylhydrazine, gallic acid, ferrocyanide and NH₂OH resulted in inactivation of the enzyme. Under the conditions tested, 50% inactivation of the enzyme was obtained with 4 μM H₂O₂, 20 μM ascorbic acid, 40μM phenylhydrazine, 6 mM gallic acid, 12 mM ferrocyanide and 22 mM NH₂OH. The ability of the reducing agents to reduce Cu²⁺ in a chemical model system was determined and it was found that gallic acid, phenylhydrazine, ascorbic acid and NH₂OH are relatively good reductants of Cu²⁺ while H₂O₂ and ferrocyanide are relatively poor ones. The copper content of mushroom tyrosinase before and after inactivation by each of the reducing agents was determined. The copper content of the enzyme inactivated by H₂O₂, NH₂OH, phenylhydrazine, ferrocyanide, gallic acid and ascorbic acid was 100%, 90%, 90%, 85%, 85% and 76% compared to that of the control (enzyme not treated). It was concluded that the degree of inactivation of mushroom tyrosinase by the reducing agents was not correlated with the decrease in the copper content of the enzyme nor with their ability to reduce Cu²⁺ in a chemical model system.     

Analysis of tryptophan oxidation by fluorescence spectroscopy: Effect of metal-catalyzed oxidation and selected phenolic compounds

The suitability of using fluorescence spectroscopy to assess tryptophan (TRYP) depletion by quantifying the loss of natural TRYP fluorescence during metal-catalyzed oxidation (MCO) was assessed. To analyze the effect of the oxidation conditions, N-acetyl-l-tryptophanamide (NATA) solutions were oxidized in the presence of Cu²⁺ and Fe²⁺ (40 and 80 μM), with and without 0.8 mM H₂O₂. Also, the effect of selected phenolic compounds (PhC), namely, catechin, genistein, quercetin, rutin, gallic acid and chlorogenic acid (0.08, 0.8 and 8.0 μM), on TRYP MCO was studied. Oxidation systems catalyzed by Fe²⁺ had a major oxidative potential as a result of a higher amount of free radicals formed through the Fenton reaction. PhC could exert both antioxidant and pro-oxidant effects depending on their concentration, chemical structure of the PhC and the oxidation system. In general, all PhC acted as pro-oxidant agents at the highest dose. The major pro-oxidant effect was displayed by gallic and chlorogenic acid in the presence of Fe²⁺, Cu²⁺ or Cu²⁺/H₂O₂. Quercetin, rutin and gallic acid showed antioxidant effect against TRYP oxidation at low concentrations in Fe²⁺/H₂O₂ systems. Plausible mechanisms for the antioxidant and pro-oxidant effects of PhC on TRYP oxidation are discussed in the present article. The antioxidant efficiency of PhC or PhC-rich extracts must be studied before being used as functional food ingredients.     

Pomological features, nutritional quality, polyphenol content analysis, and antioxidant properties of domesticated and 3 wild ecotype forms of raspberries (Rubus idaeus L.)

Abstract: The raspberry (Rubus idaeus L.) is an economically important berry crop that contains many phenolic compounds with potential health benefits. In this study, important pomological features, including nutrient content and antioxidant properties, of a domesticated and 3 wild (Yayla, Yavuzlar, and Yedigöl) raspberry fruits were evaluated. Also, the amount of total phenolics and flavonoids in lyophilized aqueous extracts of domesticated and wild ecotypes of raspberry fruits were calculated as gallic acid equivalents (GAEs) and quercetin equivalents (QE). The highest phenolic compounds were found in wild Yayla ecotype (26.66 ± 3.26 GAE/mg extract). Whilst, the highest flavonoids were determined in wild Yedigöl ecotype (6.09 ± 1.21 QA/mg extract). The antioxidant activity of lyophilized aqueous extracts of domesticated and wild ecotypes of raspberry fruits were investigated as trolox equivalents using different in vitro assays including DPPH^?, ABTS^?+, DMPD^?+, and O^??₂ radical scavenging activities, H₂O₂ scavenging activity, ferric (Fe³⁺) and cupric ions (Cu²⁺) reducing abilities, ferrous ions (Fe²⁺) chelating activity. In addition, quantitative amounts of caffeic acid, ferulic acid, syringic acid, ellagic acid, quercetin, α‐tocopherol, pyrogallol, p‐hydroxybenzoic acid, vanillin, p‐coumaric acid, gallic acid, and ascorbic acid in lyophilized aqueous extracts of domesticated and wild ecotypes of raspberry fruits were detected by high‐performance liquid chromatography and tandem mass spectrometry (LC‐MS‐MS). The results clearly show that p‐coumaric acid is the main phenolic acid responsible for the antioxidant and radical scavenging activity of lyophilized aqueous extracts of domesticated and wild ecotypes of raspberry fruits.     

Lucerne tannins. I. Content and composition during growth

Composition and content of polyphenols of lucerne leaf and stalk were investigated during growth depending on the time of season at one growth stage. (+)-Gallocatechin, (–)-epigallocatechin gallate, (–)-epigallocatechin, (–)-epicatechin, (–)-gallocatechin gallate, gallic acid, gallotannin and shikimic acid were estimated by qualitative chromatographic analyses on Whatman Chromedia paper SG-81. The content of some polyphenol groups was determined by quantitative analyses and amounts were found of 0.06 to 0.50%, gallic acid; 1.10 to 2.36%, gallotannins; 0.31 to 1.10%, catechins in the dry matter of lucerne depending upon growth stage and cuts. Polyphenoloxidase activity from 7.80 to 18.70 units was established in the investigated samples.     

Ranking the efficacies of selected red wine phenolic anti-oxidants using reversed-phase HPLC

Several studies have assessed total anti-oxidant activity of wine or individual components in isolation using chemical-based assays. In this study, a quantitative approach was developed to assess the relative anti-oxidant efficacies of selected red wine phenolics via peak reduction, using reversed-phase HPLC. Both intact red wine and phenolic standard solutions were challenged with five oxidant model systems as follows: (1) hydrogen peroxide (H₂O₂); redox-active metal ions (2) Fe³⁺ and (3) Cu²⁺; and the Fenton reagents (4) H₂O₂ + Fe²⁺; and (5) H₂O₂ + Cu⁺. Treatment with oxidants (1–3) resulted in loss of 47–60% of phenolic standards, which increased to 66–89% for treatment with the Fenton systems, with quercetin exhibiting the optimal anti-oxidant activity. For intact red wine, treatment with oxidants (1–3) led to all phenolic compounds being oxidised (27–77% loss), with caffeic acid and quercetin as the most effective anti-oxidants. For both Fenton systems (4–5), activities in red wine were considerably enhanced for caffeic acid and quercetin, which exhibited the highest anti-oxidant efficacies with 100% peak reduction, while p-coumaric acid and gallic acid were less effective anti-oxidants with peak reductions of 60–68%. The ranking, facilitated by this new quantitative approach, allows comparison of the individual efficacies of the anti-oxidants in a complex matrix.     

Biochemical properties and potential endogenous substrates of polyphenoloxidase from chufa (Eleocharis tuberosa) corms

Polyphenoloxidase (PPO) was partially purified from chufa corms through ammonium sulphate precipitation and dialysis. Biochemical properties of chufa PPO were analysed using exogenous substrate catechol. Optimal pH and temperature for PPO activity were 5 and 45 °C. Ethylenediaminetetraacetic acid disodium salt and l-cysteine could not inhibit the PPO activity. However, sodium thiosulphate pentahydrate exhibited the strongest inhibiting effect, followed by ascorbic acid and anhydrous sodium sulphite. Except for K⁺, other metal ions such as Zn²⁺, Cu²⁺, Fe³⁺, Ca²⁺, Fe²⁺ and Na⁺ accelerated the enzymatic reaction between catechol and PPO. Kinetic analysis showed that the apparent K_m and V_max values were around 10.77 mM and 82 units/ml min. In addition, (−)-gallocatechin gallate, (−)-epicatechin gallate and (+)-catechin gallate isolated and identified from chufa corms were supposed to be the potential endogenous PPO substrates due to their ortho-diphenolic or pyrogallolic structures. These polyphenols might be catalysed by PPO, resulting in the browning of chufa corms after fresh-cut processing.     

Identification of Galactaric Acid as an Intermediate Product of Breast Milk Digestion

The product forming after oxidative acid digestion of the breast milk and the usage of ethyl ether or petroleum ether to remove the fat by solvent extraction has been identified as galactaric (mucic) acid, C₆H₁₀O₈. This compound cannot be discarded, as its capability to form stable complexes with most important bioactive ions (Cu²⁺, Zn²⁺ Mg²⁺, and others) can lead to their vigorous sequestration from nitric acid solution before being measured by spectroscopic techniques.     

Fractionation of green tea extracts: correlation of antimutagenic effect with flavanol content

The present study was undertaken to evaluate the role of individual flavanols in the antimutagenic potential of green tea. Aqueous extracts of green tea were fractionated into four fractions, each of which was fully defined with respect to its content of (-)-epigallocatechin gallate, (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epicatechin and gallic acid. The ability of each fraction to antagonize the mutagenicity of four model mutagens, namely N-nitrosopyrrolidine, benzo(a)pyrene, 2-aminoanthracene and Glu-P-1 (2-amino-6-methyldipyrido[1,2-a: 3,2-d]imidazole), was investigated in the Ames test. No correlation could be established between any of the flavanols and antimutagenic potential. Similarly, no correlation was evident between the flavanol content of each fraction and its ability to inhibit CYP1A, as exemplified by the O-dealkylations of methoxy- and ethoxy-resorufin. Furthermore, no relationship could be established between CYP2B activity, as exemplified by the O-depentylation of pentoxyresorufin and the antimutagenic potential of green tea. Using a modified Ames test procedure, the ability of each tea fraction to scavenge the metabolically generated reactive intermediates of the model mutagens was investigated, this being an additional mechanism of the antimutagenic potential of green tea. Generally, fractions with high flavanol content were more effective scavengers. It is concluded that the contribution of flavanols to the antimutagenic activity of green tea is, at best, limited. ©1997 SCI     

Preparation of Amylose-g-Poly (Styrene)

A study on the grafting of styrene on amylose has been made using H₂O₂ as an initiator and Cr³⁺, Fe²⁺, Co²⁺, Cu²⁺, Zn²⁺, and Ag⁺ ions as catalyst. It has been shown that with Fe²⁺ ions maximum grafting yield was obtained. The optimum conditions for the preparation of amylose-g-poly (styrene) have been set up.     

Effects of metal ions (Cu,Fe and Fe) on HPLC analysis of catechins

Catechins [(−)-epicatechin (EC), (−)-epigallocatechin (EGC), (−)-epicatechin gallate (ECG) and (−)-epigallocatechin gallate (EGCG)] were analysed by HPLC using an ODS column, an electrochemical detector (0.75 V vs. Ag/AgCl) and an eluting solvent composed of water containing buffer (84% v/v), acetonitrile (12% v/v) and ethylacetate (4% v/v) in the presence of metal ions (Cu²⁺, Fe²⁺ and Fe³⁺). HPLC peaks were affected by metal ions: the peak intensities of ECG and EGCG decreased, but the peak intensities of EC and EGC were not affected seriously. Fe²⁺ most markedly decreased the peak intensities of EGCG. EDTA was added to mask metal ions and an optimum condition was proposed. The effects of the metal ions on HPLC analysis are discussed from the viewpoints of metal complex formation with catechins and oxidation of catechins on the basis of ultraviolet–visible (UV–vis) spectrophotometry, electrospray ionisation mass spectrometry (ESI-MS) and cyclic voltammetry (CV).     

Effect of reaction pH on the photodegradation of model melanoidins

The effect was studied of the pH of the amino-carbonyl reaction on the photodegradation of model melanoidins. Nondialyzable model melanoidins were prepared from glucose and lysine with or without initial pH control to 7.0.: 2 mol/l phosphate buffer (buffer-melanoidin) and pH adjustment at the reaction start with NaHCO₃ (NaHCO₃-melanoidin). Melanoidin was also prepared from glucose and the lysine-Cu²⁺complex to investigate the difference of complexes between melanoidin and Cu²⁺ (Cu²⁺-melanoidin). Each melanoidin solution was irradiated with a Xe or tungsten-halogen lamp under dissolved oxygen or by the continuous supply of oxygen in a Cu²⁺/O₂ or ascorbic acid/Cu²⁺/O₂ system. The effects of the concentrations of Cu²⁺ and melanoidin, reaction pH value, and metal ion species on the decolorization rate in the Cu²⁺/O₂ system were investigated. The most effective factor for decolorization was found to be the melanoidin concentration. The decolorization rate was negligible when 14 g/l of Cu²⁺-melanoidin was photodegraded in the ascorbic acid/Cu²⁺/O₂ system under dissolved oxygen, although depolymerization was observed. Photodegradation of NaHCO₃-melanoidins in the Cu²⁺/O₂ system by the continuous supply of oxygen resulted in an increased decolorization rate, decreased molecular mass, production of low-molecular-weight compounds, release of free lysine, and pI change. The buffer- and Cu²⁺-melanoidins did not show changes in chemical characteristics similar to those of the NaHCO₃-melanoidin.     

Kinetic Properties of (+)-Catechin Oxidation by a Basic Peroxidase Isoenzyme from Strawberries

The ability of a partially-heat stable strawberry (Fragaria × ananassa) peroxidase to oxidize (+)-catechin was studied and its oxidation followed the accepted model for peroxidase oxidations. Compound I (CoI) and compound II (CoII) were the main intermediates in the catalytic cycle. The reactivity of strawberry peroxidase with H₂O₂ [K₁= 2.63 μM^-1 s^-1] and with (+)-catechin [k₃= 0.57 μM^-1 s^-1] suggested that (+)-catechin is an excellent o-diphenol for CoII reduction. The strong cooperativity of this peroxidase isoenzyme indicates its great efficacy in oxidation of (+)-catechin at low H₂O₂ concentrations.     

Antioxidant and pro-oxidant properties of ascorbic acid and gallic acid

The antioxidant and pro-oxidant properties of ascorbic acid (AA) and gallic acid (GA) were investigated. AA and GA, at a concentration of 1.65 mM, accelerate the oxidation of deoxyribose induced by Fe³⁺–EDTAJH₂O₂. The reducing power of these two compounds increased upon increasing the concentration. AA and GA showed no chelating ability toward iron (II). At a concentration of 4.17 mM, AA and GA exhibited 42.1 and 43.9% scavenging effects on DPPH radicals, respectively. They exhibited 60% scavenging effects on hydrogen peroxide at a concentration of 4.17 mM. No toxicity was found in AA and GA toward human lymphocytes. AA, at 0.82 mM, and GA, at 0.6 mM, exhibited the maximal DNA damage, the means of tail DNA% were 14.8 and 28.8%, respectively. When AA and GA were mixed with H₂O₂, they exhibited a slight inhibitory effect on DNA damage induced by H₂O₂ on pre-incubating both the compounds with human lymphocytes for 30 min before exposure to H₂O₂. The antioxidant activities of AA and GA at a higher concentration were mainly due to the scavenging of hydrogen peroxide in this system. The pro-oxidant mechanism for AA and GA acid is most likely due to the strong reducing power and weak metalchelating ability.     

Effectiveness of Sanitizing Agents in Inactivating Escherichia coli in Golden Delicious Apples

Research was undertaken to develop improved methods of sanitizing apples contaminated with Escherichia coli. Unwaxed Golden Delicious apples, inoculated with non pathogenic E. coli, were washed with 200 ppm Cl₂, commercial washing formulations, 5% H₂O₂, or combinations of H₂O₂ with commercial formulations at ca. 20°C or 50°C. Heated commercial formulations achieved a 2.5 log reduction in E. coli load, compared to a 2 log reduction for 200 ppm Cl₂. However, heated combinations of H₂O₂ with acidic surfactants achieved a 3–4 log reduction. Residual H₂O₂ in treated apples dissipated within several hours. These results demonstrate the efficacy of H₂O₂ in decontaminating apples containing E. coli.     

Purification,structural data and biological properties of polysaccharide from Prunus amygdalus gum

This work demonstrates the efficiency of almond gum polysaccharides (AGPs) as bioactive compounds. AGPs were first extracted using H₂O₂, in the presence of NaOH, at different times and temperatures. The optimal extraction conditions were 4% H₂O₂ and 2 N NaOH, for 7 h at 50 °C, leading to an extraction yield of 58.2% (w/w). After a purification step, the retained AGPs were characterised using high‐performance liquid chromatography showing a molecular weight of 99.3 kDa. The monosaccharide composition of AGPs were assessed using gas chromatography–mass spectrometry. AGPs were found to be a complex heteropolysaccharide with a repeating unit mainly composed of galactose, arabinose, xylose, mannose, rhamnose, and glucuronic acid with the respective ratios: 45:26:7:10:1:11. The acidic nature of the polysaccharide is due to the presence of glucuronic acid. Total antioxidant activity, free radical‐scavenging activity and reducing power assay of AGPs were investigated. The obtained results showed high antioxidant activities of AGPs. Furthermore, beyond 60 mg mL^?1, AGPs exhibited bacterial growth inhibition for five pathogenic strains: Escherichia coli, Staphylococcus aureus, Enterococcus feacalis, Pseudomonas aeruginosa and Salmonella typhimurium.     

Accumulation of Iron in Lactic Acid Bacteria and Bifidobacteria

Lactobacillus acidophilus, L. delbrueckii var. bulgaricus, L. plantarum and Streptococcus thermophilum, all used extensively in the food industry, were tested for their ability to internalize and/or oxidize ferrous iron (Fe²+). For comparison some experiments were performed with bifidobacteria, B. thermophilum and B. breve. All organisms except L. bulgaricus could transport Fe²⁺ into the cell, where it was partially oxidized to the ferric form (Fe(III)). In addition, L. acidophilus and L. bulgaricus could oxidize Fe²⁺ to Fe(III) extracellularly through the elaboration of H₂O₂ into the medium when the experiments were carried out in air. L. bulgaricus elaborated H₂O₂ only in the presence of glucose, whereas L. acidophilus released H₂O₂ in absence of glucose. We concluded that lactic acid bacteria, like bifidobacteria, can exert some beneficial effects in animal organisms, or in food processing and storage, by making Fe²⁺ unavailable to harmful microorganisms.     

Impact of trolox,quercetin, genistein and gallic acid on the oxidative damage to myofibrillar proteins: The carbonylation pathway

The carbonylation pathway involves the oxidative deamination of lysine residues to yield a carbonyl compound (α-aminoadipic semialdehyde) that can be further oxidised to α-aminoadipic acid and form Schiff bases structures. The effect of trolox and other phenolic compounds (PhC) (namely genistein, quercetin and gallic acid) on the protein carbonylation pathway occurred during the oxidation of myofibrillar proteins (MP) catalysed by a Fe³⁺/H₂O₂ system was studied. Trolox and PhC can exert either antioxidant or pro-oxidant capacities depending on their concentration, the oxidation conditions and the target in proteins. In general, quercetin and genistein showed an antioxidant activity towards lipid oxidation and the carbonylation pathway at different concentrations under the analysed conditions. Plausible mechanisms for the antioxidant and pro-oxidant effects of trolox and PhC on MP are discussed. Further research is needed to shed light on the effect of PhC mixtures on both lipid and protein oxidation.     

Gastric H+, K+-ATPase Inhibition,and Antioxidant Properties of Selected Commonly Consumed Vegetable Sources

Oxidative stress and upregulation of gastric H⁺, K⁺-ATPase enzyme activity have been known to cause ulcer pathogenicity for which safer drugs are yet to be identified. Aqueous extracts of seven commonly consumed vegetable sources were screened for inhibition of H⁺, K⁺-ATPase and antioxidant activities. Results indicated that Z. officinale (Ginger) followed by M. arvensis (Pudina) are potent gastroprotective sources with inhibition of H⁺, K⁺-ATPase of IC₅₀ of 18.3 ± 0.7 and 25.2 ± 0.9 μg gallic acid equivalents/ml respectively, which is almost equivalent or better than the known inhibition of H⁺, K⁺-ATPase—Omeprazole (IC₅₀ ?27 μg/ml). Further, all these vegetable extracts showed multi-potent antioxidant activity, such as free radical scavenging, reducing power ability, and inhibition of lipid peroxidation, which are required to inhibit complex steps of ulcerations. On the basis of the absolute amounts and potency of inhibition of H⁺, K⁺-ATPase as well as antioxidant activity of individual phenolic acids, the relative percentage contribution of phenolic acids from different vegetable extracts to both inhibition of H⁺, K⁺-ATPase and antioxidant activity was calculated and data revealed that gentisic and protocatechuic acid contributes significantly to both inhibition of H⁺, K⁺-ATPase and antioxidant activity.     

Cream formation and main chemical components of green tea infusions processed from different parts of new shoots

The formation of green tea cream and its chemical components were investigated. The green tea infusions were processed from different parts of new shoots: the bud and the first leaf, the second leaf and its implicative stem, the third leaf and its implicative stem, and the fourth leaf and its implicative stem, which were named as first part, second part, third part and fourth part, respectively. The results showed that the formation of tea cream and its settlement slowed down gradually from the first part to the fourth part, and that the amount of tea cream also decreased accordingly. The amount of tea cream was influenced remarkably by the chemical components in the green tea infusion. The main components of green tea cream were polyphenols (29.86 –78.66%), total sugar (14.47–27.62%) and caffeine (2.35–10.43%). Catechins (12.8–42.5%) were the main components of polyphenols which participated in tea cream formation. The main components in the catechins were found to be (−)-epigallocatechin (EGC), (−)-epigallocatechin gallate (EGCG), (−)-epicatechin (EC) and (−)-epicatechin gallate (ECG). Seven minerals were found in the tea cream, including Ca²⁺, K⁺, Mg²⁺, Mn²⁺, Na⁺, Zn²⁺ and Ni²⁺.     

Isolation of methyl syringate as a specific aflatoxin production inhibitor from the essential oil of Betula alba and aflatoxin production inhibitory activities of its related compounds

Methyl syringate was isolated from the essential oil of Betula alba as an aflatoxin production inhibitor. It inhibited aflatoxin production of Aspergillus parasiticus and Aspergillus flavus with IC₅₀ values of 0.9 and 0.8 mM, respectively, without significantly inhibiting fungal growth. Methyl syringate reduced mRNA levels of genes (aflR, pksA, and omtA) encoding proteins required for aflatoxin biosynthesis. Methyl gallate, methyl 3,4,5-trimethoxybenzoate, and methyl 3-O-methylgallate inhibited both aflatoxin production and fungal growth of A. parasiticus and A. flavus. However, their acids and syringic acid did not inhibit aflatoxin production and growth of A. parasiticus significantly, although gallic acid inhibited aflatoxin production of A. flavus with selectivity. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of methyl syringate was much weaker than that of gallic acid. These results showed that methyl syringate has a unique inhibitory activity toward aflatoxin production with a different mode of action from that of gallic acid.