Antioxidant activity evaluation by physiologically relevant assays based on haemoglobin peroxidase activity and cytochrome c-induced oxidation of liposomes (original) (raw)

2015, Natural Product Research

Two new protocols for exploring antioxidant-related chemical composition and reactivity are described: one based on a chronometric variation of a hemoglobin ascorbate peroxidase assay and one based on cytochrome c-induced oxidation of lecithin liposomes. Detailed accounts are given on their design, application, critical correlations with established methods, and mechanisms. These assays are proposed to be physiologically relevant and bring new information regarding a real sample, both qualitative and quantitative. The well-known assays used for evaluation of antioxidant (re)activity are revisited and compared with these new methods. Principal component analysis (PCA) allow straightforward comparisons of these antioxidant assays based on mechanism and reinforce the need to use more than a single parameter in examining such reactivity. Extracts of the Hedera helix L. are examined as test case, with focus on seasonal variation and on leaf, fruit and flower with respect to chromatographic, spectroscopic and reactivity properties. Keywords: antioxidant (re)activity assays; hemoglobin ascorbate peroxidase assay; liposome peroxidation; principal component analysis; Hedera helix. Experimental section 1.1. Chemicals. AAPH (2,2'-azobis-2-methyl-propanimidamide dihydrochloride), DPPH (di(phenyl)-(2,4,6-trinitrophenyl)iminoazanium), beta-carotene, rutin, kaempferol, tween, linoleic acid, methanol, ethanol, trolox, soy lecithin, chloroform, horse heart cytocrom c, ascorbic acid, sodium hydroxide are of high analytical purity and obtained from several companies (Sigma, Fluka, Merck). Standards: chlorogenic acid, p-coumaric acid, caffeic acid, rutin, apigenin, quercitrin, isoquercitrin, hyperoside, kaempferol, quercetol, myricetol and fisetin from Sigma (Germany); ferulic acid, sinapic acid, gentisic acid, patuletin and luteolin from Roth (Germany); and cichoric acid and caftaric acid from Dalton (USA). Methanol of HPLC analytical-grade and hydrochloric acid of analytical-grade were purchased from Merck (Germany). Methanolic stock solutions (100 mg mL-1) of the above standards were prepared and stored at 4ºC, and protected from daylight. They were appropriately diluted with double distilled water before being used as working solutions. 1.2. Extract preparation. Ivy (Hedera helix L.) was collected from the A. Borza Botanical Garden of Cluj-Napoca (46°45′36″N and 23°35′13″E) and was identified by Dr. M. Parvu, Babes-Bolyai