Reevaluation of the 2-deoxyribose assay for determination of free radical formation.

BACKGROUND: The 2-deoxyribose (2-DR) degradation assay is a widely used test for determining anti/pro-oxidant properties of molecules and plant extracts. Most reports use reaction blanks omitting 2-DR or thiobarbituric acid (TBA). However, when studying Fe(II)-mediated reactions, we verified that these blanks are not appropriate. Fe(III)--a product of these reactions--causes a relevant artifact in the assay, where 2-DR is oxidized by Fe(III). METHOD: 2-DR degradation was determined at 532 nm as TBA-reactive substances. RESULTS AND CONCLUSION: HPLC determinations indicated that Fe(III) added after or before TBA generates considerable amounts of malondialdehyde (2-DR degradation product) in comparison with assays employing Fenton reagents or Fe(II) autoxidation. Addition of catalase and thiourea has no effect on Fe(III)-induced 2-DR degradation indicating lack of ROS involvement. This Fe(III)-mediated 2-DR damage is dependent on iron and 2-DR concentrations, but not on H2O2, buffer composition or iron-chelators. Depending on the assay conditions Fe(III)-interference accounts for 20% to 90% of 2-DR degradation mediated by Fe(II). SIGNIFICANCE: A new reaction blank is proposed herein-based on the use of Fe(III)-for the assay. The lack of such correction has caused the underestimation of antioxidant capacity of various compounds in many studies in the last 2 decades.

Antioxidant properties of methanol extract and its solvent fractions obtained from selected Indian red seaweeds.

In vitro antioxidant activities of three selected Indian red seaweeds - viz., Euchema kappaphycus, Gracilaria edulis and Acanthophora spicifera were evaluated. Total phenolic content and reducing power of crude methanol extract were determined. The antioxidant activities of total methanol extract and five different solvent fractions (viz., petroleum ether (PE), ethyl acetate (EA), dichloromethane (DCM), butanol (BuOH) and aqueous) were also evaluated. EA fraction of A. spicifera exhibited higher total antioxidant activity (32.01 mg ascorbic acid equivalent/g extract) among all the fractions. Higher phenolic content (16.26 mg gallic acid equivalent/g extract) was noticed in PE fraction of G. edulis. Reducing power of crude methanol extract increased with increasing concentration of the extract. Reducing power and hydroxyl radical scavenging activity of E. kappaphycus were higher compared to standard antioxidant (alpha-tocopherol). The total phenol content of all the seaweeds was significantly different (P<0.05). In vitro antioxidant activities of methanol extracts of all the three seaweeds exhibited dose dependency; and increased with increasing concentration of the extract.

Antiradical activity of water soluble components in common diet vegetables.

The antiradical activity of water-soluble components contained in mushrooms (Psalliota campestris), onions (Allium cepa), white cabbage (Brassica oleracea var. alba), and yellow bell peppers (Capsicum annuum) against hydroxyl radicals was tested in a chemical and biological system. The vegetable juices were obtained by centrifugation of a vegetable homogenate processed at 2 degrees C or heated at 102 degrees C. The chemical system consisted of a buffered reaction mixture composed of Fe(III)-EDTA, 2-deoxy-D-ribose, ascorbic acid, and H(2)O(2) generating the hydroxyl radical. The antiradical activity was expressed as an inhibition of deoxyribose degradation. The biological system consisted of IMR32 neuroblastoma cells exposed to H(2)O(2) in the presence or absence of the vegetable juices. Cells were pretreated for either 24 h or 1 h with the vegetable juices, and reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used as a cell viability assay. All vegetable juices inhibited the degradation of deoxyribose and increased the viability of H(2)O(2) treated cells. Raw mushroom juice proved to be the most active in both cases. Boiling significantly affected the activity of mushroom juice, but did not change significantly the effect on onions and yellow bell peppers, and partially increased the activity of white cabbage juice. Mushroom antiradical activity was also confirmed by a cytofluorimetric analysis.

Inhibition of macrophage migration by nucleotide-containing streptococcal preparations.

Certain extracts of streptococcal cell walls are known to inhibit macrophage migration in vitro. In this study, we attempted to identify the streptococcal components responsible for this phenomenon. Trypsinized cell walls and cytoplasm from groups A and B streptococci were extracted with hot formamide followed by acetone precipitation. Subsequent gel filtration in aqueous solutions yielded a fraction devoid of C-carbohydrate and containing mostly oligonucleotides, apparently derived from streptococcal cytoplasm. This fraction significantly inhibited the migration of peritoneal exudate cells from rats sensitized to groups A and B streptococci. It was noteworthy that no inhibition of migration was observed with cells from nonsensitized animals or control rats injected with BCG or complete Freund adjuvant. Similarly, no inhibition was obtained with formamide extracts of calf thymus RNA. Although the inhibition does not show specificity for streptococcal groups, it seems to have immunological specificity since prior sensitization with streptococci is required for migration inhibition.