Ö. İrfan Küfrevioğlu

Determination of antioxidant activity of lichen Cetraria islandica (L) Ach.

The study was aimed at evaluating the antioxidant activity of aqueous extract of C. islandica. The antioxidant activity, reducing power, superoxide anion radical scavenging and free radical scavenging activities were studied. The antioxidant activity increased with the increasing amount of extracts (from 50 to 500 microg) added to linoleic acid emulsion. About 50, 100, 250, and 500 microg of aqueous extract of C. islandica showed higher antioxidant activity than 500 microg of alpha-tocopherol. The samples showed 96, 99, 100, and 100% inhibition on peroxidation of linoleic acid, respectively. On the other hand, the 500 microg of alpha-tocopherol showed 77% inhibition on peroxidation on linoleic acid emulsion. Like antioxidant activity, the reducing power, superoxide anion radical scavenging and free radical scavenging activities of C. islandica depends on concentration and increasing with increased amount of sample. The results obtained in the present study indicate that C. islandica is a potential source of natural antioxidant.

Carbonic anhydrase inhibitors. Inhibition of human erythrocyte isozymes I and II with a series of antioxidant phenols

The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II, with a series of phenol derivatives was investigated by using the esterase assay, with 4-nitrophenyl acetate as substrate. 2,6-Dimethylphenol, 2,6-diisopropylphenol (propofol), 2,6-di-t-butylphenol, butylated hydroxytoluene, butylated hydroxyanisole, vanillin, guaiacol, di(2,6-dimethylphenol), di(2,6-diisopropylphenol), di(2,6-di-t-butylphenol), and acetazolamide showed K(I) values in the range of 37.5-274.5 microM for hCA I and of 0.29-113.5 microM against hCA II, respectively. All these phenols were non-competitive inhibitors with 4-nitrophenylacetate as substrate. Some antioxidant phenol derivatives investigated here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms which have not been yet assayed for their interactions with such agents.

On the in vitro antioxidative properties of melatonin

Abstract:The aim of this study is to examine possible in vitro antioxidant effects of melatonin. Thus, the total in vitro antioxidant activity of melatonin was studied using a thiocyanate method. Additionally, the reducing power, the superoxide anion scavenging activity and free radical scavenging activity of melatonin were determined. Melatonin exhibited potent antioxidant activity in a linoleic acid emulsion system. The antioxidant activity increased with increasing concentrations of melatonin (50–500 μg). The 50, 100, 250 and 500 μg melatonin doses showed 41, 60, 86 and 99% inhibition of peroxidation of linoleic acid, respectively. On the other hand, a 500‐μg dose of α‐tocopherol showed 34% inhibition of peroxidation of linoleic acid. Like the total antioxidant activity, the reducing power of melatonin increased in a dose‐dependent manner. The reducing power of melatonin was statistically significant versus control, but lower than butylated hydroxytoluene (BHT) or quercetin. Additionally, melatonin had potent superoxide radical scavenging activity and exhibited a higher superoxide radical scavenging activity than quercetin or BHT but lower than butylated hydroxyanisole (BHA). Melatonins direct free radical scavenging actions may account, at least in part, for its ability to reduce lipid peroxidation. Melatonin may have utility in protecting stored foods from free radical‐induced deterioration.

In vitro inhibition of salicylic acid derivatives on human cytosolic carbonic anhydrase isozymes I and II

The inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes, hCA I and II, with a series of salicylic acid derivatives was investigated by using the esterase method with 4-nitrophenyl acetate as substrate. IC(50) values for sulfasalazine, diflunisal, 5-chlorosalicylic acid, dinitrosalicylic acid, 4-aminosalicylic acid, 4-sulfosalicylic acid, 5-sulfosalicylic acid, salicylic acid, acetylsalicylic acid (aspirin) and 3-metylsalicylic acid were of 3.04 microM, 3.38 microM, 4.07 microM, 7.64 microM, 0.13 mM, 0.29 mM, 0.42 mM, 0.56 mM, 2.71 mM and 3.07 mM for hCA I and of 4.49 microM, 2.70 microM, 0.72 microM, 2.80 microM, 0.75 mM, 0.72 mM, 0.29 mM, 0.68 mM, 1.16 mM and 4.70 mM for hCA II, respectively. Lineweaver-Burk plots were also used for the determination of the inhibition mechanism of these substituted phenols, most of which were noncompetitive inhibitors with this substrate. Some salicylic acid derivatives investigated here showed effective hCA I and II inhibitory activity, and might be used as leads for generating enzyme inhibitors eventually targeting other isoforms which have not been assayed yet for their interactions with such agents.

Metal chelating and hydrogen peroxide scavenging effects of melatonin.

Abstract: Antioxidant activity of a molecule is attributed to various mechanisms such as prevention of chain initiation, binding of transition metal ion catalysts and decomposition of peroxides. This study was aimed at evaluating the metal chelating and hydrogen peroxide (H2O2) scavenging activity of melatonin. The metal chelating and H2O2 scavenging activity increased with increasing concentrations of melatonin (20–60 μg/mL). α‐Tocopherol, butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) were used as standards. Sixty micrograms per milliliter concentration of melatonin exhibited 95% chelating effect on ferrous ions and scavenged 83% of H2O2. On the other hand, the same concentration of α‐tocopherol, BHA, and BHT exhibited 58, 61, and 72% inhibition, respectively, of the formation of the Fe2+–ferrozine complex and scavenged 48, 20, and 23%, respectively, of H2O2. Based on these results, it is concluded that melatonin is an effective metal chelating agent and scavenger of H2O2. These properties may be major reasons for the melatonins ability to inhibit lipid peroxidation.

In Vitro Inhibition of Human Carbonic Anhydrase I and II Isozymes with Natural Phenolic Compounds

Inhibition of two human cytosolic carbonic anhydrase (hCA, EC 4.2.1.1) isozymes I and II with some natural phenolic derivatives was investigated using the esterase assay with 4‐nitrophenyl acetate as substrate. Resveratrol, catechin, silymarin, dobutamin, and curcumin showed KI values in the range of 4.47–9.47 mm for hCA I and of 2.86–7.44 μm against hCA II, respectively. These natural product phenols were generally competitive inhibitors with 4‐nitrophenylacetate as substrate. Some natural phenols investigated here showed effective hCA II inhibitory effects, in the same range as the clinically used sulfonamide acetazolamide, and might be used as leads for generating enzyme inhibitors possibly targeting other CA isoforms that have not been yet assayed for their interactions with such agents.

Purification and characterization of polyphenol oxidase from nettle (Urtica dioica L.) and inhibitory effects of some chemicals on enzyme activity.

Polyphenol oxidase (PPO) of nettle (Urtica dioica L.) was extracted and purified through (NH4)2SO4 precipitation, dialysis, and CM-Sephadex ion-exchange chromatography and was used for its characterization. The PPO showed activity to catechol, 4-methylcatechol, L-3,4-dihydroxyphenylalanine (L-DOPA), L-tyrosine, p-cresol, pyrogallol, catechin and trans-cinnamic acid. For each of these eight substrates, optimum conditions such as pH and temperature were determined and L-tyrosine was found to be one of the most suitable substrates. Optimum pH and temperature were found at pH 4.5 and 30°C respectively and Km and Vmax values were 7.90 × 10−4 M, and 11290 EU/mL for with L-tyrosine as substrate. The inhibitory effect of several inhibitors, L-cysteine chloride, sodium azide, sodium cyanide, benzoic acid, salicylic acid, L-ascorbic acid, glutathione, thiourea, sodium diethyl dithiocarbamate, β-mercaptoethanol and sodium metabisulfite were tested. The most effective was found to be sodium diethyl dithiocarbamate which acted as a competitive inhibitor with a Ki value of 1.79 × 10−9 M. In addition one isoenzyme of PPO was detected by native polacrylamide slab gel electrophoresis.

Kinetic and docking studies of phenol-based inhibitors of carbonic anhydrase isoforms I, II, IX and XII evidence a new binding mode within the enzyme active site

Carbonic anhydrases (CAs, EC 4.2.1.1) are inhibited by sulfonamides, inorganic anions, phenols, coumarins (acting as prodrugs) and polyamines. A novel class of CA inhibitors (CAIs), interacting with the CA isozymes I, II (cytosolic) and IX, XII (transmembrane, tumor-associated) in a different manner, is reported here. Kinetic measurements allowed us to identify hydroxy-/methoxy-substituted benzoic acids as well as di-/tri-methoxy benzenes as submicromolar-low micromolar inhibitors of the four CA isozymes. Molecular docking studies of a set of such inhibitors within CA I and II allowed us to understand the inhibition mechanism. This new class of inhibitors binds differently compared to all other classes of inhibitors known to date: they were found between the phenol-binding site and the coumarin-binding site, filling thus the middle of the enzyme cavity. They exploit different interactions with amino acid residues and water molecules from the CA active site compared to other classes of inhibitors, offering the possibility to design CAIs with an interesting inhibition profile compared to the clinically used sulfonamides/sulfamates.

One-step purification of lactoperoxidase from bovine milk by affinity chromatography

Sulphanilamide was determined to be a new inhibitor of lactoperoxidase (LPO) with an IC(50) of 0.848.10(-5)M. The K(i) for sulphanilamide was determined to be 3.57.10(-5)M and sulphanilamide showed competitive inhibition, which makes it a suitable ligand for constructing a Sepharose 4B-L-tyrosine affinity matrix. The affinity matrix was synthesised by coupling sulphanilamide as the ligand and L-tyrosine as the spacer arm to a cyanogen bromide (CNBr)-activated-Sepharose 4B matrix. Lactoperoxidase was purified 409-fold from the synthesized affinity matrix in a single step, with a yield of 62.3% and a specific activity of 40.9 EU/mg protein. The enzyme activity was measured using ABTS as a chromogenic substrate (pH 6.0). The degree of LPO purification was monitored by SDS-PAGE and its R(z) (A(412)/A(280)) value. The R(z) value for the purified LPO was found to be 0.7. Maximum binding was achieved and K(m) and V(max) values were determined.

A Study on the In Vitro Antioxidant Activity of Juniper (Juniperus communis L.) Fruit Extracts

Abstract This study aimed at evaluating the in vitro antioxidant activity of water and ethanol extracts of juniper (Juniperus communis L., Family Cupressaceae) fruit. The antioxidant properties of both Juniper extracts were studied using different antioxidant assays, including reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities. Both the water and the ethanol extracts exhibited strong total antioxidant activity. The concentrations of 20, 40, and 60 µg/mL of water and ethanol extracts of juniper fruit showed 75%, 88%, 93%, 73%, 84%, and 92% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, 60 µg/mL of standard antioxidant such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and α‐tocopherol exhibited 96, 96, and 61 inhibitions on peroxidation of linoleic acid emulsion, respectively. However, both extracts of juniper had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities at these same concentrations (20, 40, and 60 µg/mL). Those various antioxidant activities were compared to BHA, BHT, and α‐tocopherol as standard antioxidants. In addition, total phenolic compounds in both aqueous and ethanolic juniper extracts were determined as gallic acid equivalents. Accordingly, these results indicate that juniper has in vitro antioxidant properties and these may be major reasons for the inhibition of lipid peroxidation properties.