Antioxidant effects of Origanum majorana L. on superoxide anion radicals

A purified compound with antioxidant properties (28 mg), T3b, was isolated from a methanol extract (10 g) of Origanum majorana L. by sequential procedures, with silica gel column, thin-layer, and LH-20 Sephadex gel column chromatographies. The in vitro scavenging activity of T3b on superoxide anion radical (O₂⁻) was investigated and compared to those of seven commercially available synthetic or natural antioxidants. Of those, the strongest scavenging action was observed in T3b with an IC₅₀ of 1.44 μg/ml. The T3b also exhibited significant inhibitory effects on 12-O-tetradecanolyphorbol-13-acetate (TPA)-induced O₂⁻ generation and hydrogen peroxide formation in differentiated HL-60 cells, indicating that the isolated compound is a potent chemopreventer. The purified compound from O. majorana L. was shown to possess both O₂⁻ scavenging activity and an inhibitory effect against TPA-induced O₂⁻ generation.     

Multifunctional peptides from egg white lysozyme

Hen’s egg white lysozyme (HEWL) is one of the major egg white proteins with well demonstrated antimicrobial activity. Bioactive peptides other than antimicrobial peptides from HEWL have not been reported; therefore, the purpose of the study was to explore new bioactivities of lysozyme-derived bioactive peptides. HEWL was hydrolysed with Alcalase and fractionated by cation-exchange chromatography. The Alcalase HEWL hydrolysate and its fractions were analyzed for inhibitory activities against calmodulin-dependent phosphodiesterase (CaMPDE) and antioxidant activities using oxygen radical absorbance capacity-fluorescein (ORAC-FL), 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt radical cation (ABTS⁺) and 2,2-di(4-tert-octylphenyl)-1-picrylhydrazyl (DPPH) radical scavenging methods. The fractionated peptides had higher CaMPDE inhibition activity, ORAC-FL value and ABTS⁺ scavenging activity than those of the hydrolysate. Peptide sequences in the most overall active fractions were characterized by LC–MS/MS. Our results showed that HEWL hydrolysate and its peptide fractions may serve as useful ingredients in the formulation of functional foods and nutraceuticals.     

An investigation of antioxidant capacity of fruits in Singapore markets

The antioxidant capacity of a group of fruits obtained in the Singapore markets was investigated. A total of 27 fruit pulps were tested for their general antioxidant capacity based on their ability to scavenge 2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) free radical. The contribution of l-ascorbic acid (AA) to the total antioxidant activity of fruits was investigated by using RP-HPLC. The antioxidant capacity of the fruit pulp was measured by monitoring the change of absorbance of the free radical solution at 414 nm in the test reaction mixture following addition of the fruit extract, as compared with AA. The results were expressed as mg of AA equivalents per 100 g, i.e. the quantity of AA required to produce the same scavenging activity as the extract in 100 g of sample (l-ascorbic acid equivalent antioxidant capacity, AEAC). Total antioxidant capacities of AA acid, trolox, hydroquinone, pyrogallol and several fruits were also evaluated based on its ability to scavenge the 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radical. Results obtained were compared with those of ABTS assay. Every mol of AA, trolox or hydroquinone, was found to reduce about 2 mol of ABTS⁺ or DPPH. However, 4 mol of DPPH or 7 mol of ABTS⁺ were scavenged by every mol of pyrogallol. A good correlation of AEAC was observed between the two methods. Both methods have been recommended to be useful tools to evaluate antioxidant capacities of fruits. According to the AEAC value of binary extract solution of fruits in the ABTS model, ciku shows the highest antioxidant capacity, followed by strawberry, plum, star fruit, guava, seedless grape, salak, mangosteen, avocado, orange, solo papaya, mango, kiwi fruit, cempedak, pomelo, lemon, pineapple, apple, foot long papaya, rambutan, rambutan king, banana, coconut pulp, tomato, rockmelon, honeydew, watermelon and coconut water. The AA contribution to AEAC of fruits varied greatly among species, from 0.06% in ciku to 70.2% in rambutan.     

Isolation,identification and activity of natural antioxidants from horehound (Marrubium vulgare L.) cultivated in Lithuania

In an earlier screening of Lithuanian plants, horehound (Marrubium vulgare) showed good antioxidant activity and as this species is used in herbal teas and cough pastilles it was selected for further investigation. Some fractions of the aerial parts were strong scavengers of the model free radicals DPPH and ABTS⁺. Activity in the β-carotene bleaching assay and the rapeseed oil oxidation assay was lower. Several active compounds were observed in the crude methanol–water extract, and in butanol and methyl tert-butyl ether fractions using HPLC with on-line radical scavenging detection. After multi-step fractionation of these fractions five compounds possessing radical scavenging activity were purified and their structures were elucidated by NMR and MS as 5,6-dihydroxy-7,4′-dimethoxyflavone (syn. ladanein), 7-O-β-glucopyranosyl luteolin, 7-O-β-glucuronyl luteolin, verbascoside and forsythoside B. Their activities were tested in off-line DPPH and ABTS⁺ free radical scavenging assays, and compared with the antioxidants rosmarinic acid and Trolox.     

Factors influencing ultraviolet and electron beam irradiation-induced free radical damage of ascorbic acid

The oxidation of an unsaturated fatty acid and antioxidant vitamin by ultraviolet (UV) and electron beam (e⁻ beam) irradiation was studied. Hydroxyl radical (OH), generated by Fe-EDTA and hemoglobin, under heat incubation or UV illumination, was compared. L-ascorbic acid (LAA; 10, 100 and 500 mM) oxidation was measured after irradiation at 0–10 kGy, in the presence and absence of myoglobin. Moreover, both UV and e⁻ beam irradiation at 0–20 kGy were used to oxidize a linoleic acid emulsion, both with and without LAA. The presence of hemoglobin generated less OH, than Fe-EDTA under similar heating conditions. Generation of OH increased with illumination by UV. LAA was oxidized by e⁻ beam irradiation, with an increasing rate of oxidation occurring at lower concentrations of LAA and higher dosages of irradiation. LAA (10 mM) exhibited pro-oxidant activity in the linoleic acid emulsion when exposed to both forms of irradiation, while 500 mM LAA exhibited antioxidant activity.     

Evaluation of glycosidic bond cleavage and formation of oxo groups in oxidized barley mixed-linkage β-glucans using tritium labelling

This study investigated the formation of oxo groups in pure solutions of barley mixed-linkage (1 → 3),(1 → 4)-β-d-glucans (MLGs) incubated in the presence of hydrogen peroxide or Fenton reaction-generated hydroxyl radicals (OH), and it gives a fingerprint of products obtained after enzymic and acidic hydrolysis of OH-attacked MLG. Hydroxyl radical, but not hydrogen peroxide, introduced a range of NaB³H₄-reducible functions into MLG chains. Driselase or lichenase digestion of NaB³H₄-reduced MLGs released a complex mixture of ³H-labelled products due to the presence of unusual and incompletely digestible residues in OH-attacked polysaccharide chains. Complete acid hydrolysis of OH-treated MLGs yielded a mixture of ³H-aldoses (mainly glucose, mannose, galactose and allose) deriving from random OH attack at positions 2, 3 or 4 to form glycosulose residues which were NaB³H₄-reducible to epimeric mixtures of ³H-aldose residues. Furthermore, the production of [³H]glucitol demonstrated the radical-mediated cleavage of mid-chain glucose residues to create new reducing termini. Oxidative scission of MLGs by hydroxyl radical caused a decrease in molecular weight of about 96%, which was partially inhibited by the addition of DMSO, an OH scavenger. The results can be a starting point for developing an assay to detect changes due to OH attack of MLGs in vivo, or during food processing and storage, based on the chemical or enzymic release of unusual sugar residues such as allose as diagnostic products after reduction/labelling treatment and partial purification (or selective digestion by lichenase) of polymeric material.     

Antioxidant properties of cold-pressed black caraway,carrot, cranberry,and hemp seed oils

Cold-pressed black caraway, carrot, cranberry, and hemp seed oils were extracted with methanol and evaluated for radical-scavenging activities against ABTS⁺ and DPPH, chelating activity, oxygen radical absorbing capacity (ORAC), and total phenolic contents (TPC). All the oil extracts had significant antioxidant activities. The ORAC value ranged from 28 to 220 μmol TE/g oil for the cold-pressed hemp, carrot, and black caraway seed oils, whereas the ABTS⁺ – scavenging capacity ranged 8.9–30.8 μmol TE/g oil for the four cold-pressed edible seed oils. The greatest TPC, 3.53 mg gallic acid equivalent (GE) per gramme of oil, was detected in the cold-pressed black caraway seed oil extract, while the lowest TPC, 0.44 mg GE/g, was observed in the cold-pressed hemp seed oil extract. In addition, methanol extracts of the cold-pressed black caraway and cranberry seed oils were evaluated for their inhibitory capacities on human LDL oxidation by measuring the reduction of the thiobarbituric acid-reactive substance production (TBARS). Both oil extracts significantly suppressed the lipid peroxidation in human LDL, with TBARS reductions of 2.84 and 3.77 mg/g for cranberry and black caraway seed oil extracts, respectively. These results suggest that cold-pressed black caraway, cranberry, carrot and hemp seed oils may serve as dietary sources of natural antioxidants for health promotion and disease prevention, and the cold-pressed black caraway seed oil may be used as a natural antioxidative food additive for improving food quality and stability.     

Antioxidant activity of dulse (Palmaria palmata) extract evaluated in vitro

Palmaria palmata (dulse) is traditionally consumed as a snack food and garnish; but, little is known about its potential as a source of antioxidants. A 1-butanol soluble fraction extracted from dulse exhibited OH scavenging activity ± EDTA (non-site and site specific activity) in a deoxyribose assay. EC₅₀ concentrations of dulse extract to quench DPPH and ABTS⁺ free radicals were 12.5 and 29.5 mg/ml. Dulse extract inhibited (p < 0.05) conjugated diene production in a linoleic acid emulsion at 24, 48 and 52 h, 38 °C; and inhibited (p = 0.044) thiobarbituric acid reactive substances (TBARS) production at 52 h. One milligram dulse extract exhibited reducing activity = 9.68 μg l-ascorbic acid and total polyphenol content = 10.3 μg gallic acid; the dulse extract did not chelate transition metal ions. The antioxidant activity of the dulse extract was associated with aqueous/alcohol-soluble compounds characterized by phenolic functional groups with reducing activity.     

Evaluation of grape (Vitis vinifera L.) stems from Portuguese varieties as a resource of (poly)phenolic compounds: A comparative study

Winery industry is a major agro-economic activity in Southern European countries, which entails the production of high amounts of by-products with environmental/economic consequences in the local area. New potential resources of phytochemicals with biological activity are being claimed to substitute used drugs and synthetic protective compounds. The present study describes the phenolic content of grape (Vitis vinifera L.) stems from red (n 4) and white (n 3) cultivars grown in Northern Portugal under continental climate. The HPLC–DAD–ESI/MSⁿ analysis revealed the presence of six proanthocyanidins, five flavonols, three anthocyanins, two hydroxycinnamic acids, and one stilbene. Nine compounds are described for the first time in grape stems. Caftaric acid, quercetin-3-O-glucuronide, malvidin derivatives, and epicatechin were the main metabolites, representing from 54% to 75% of the total phenolic content, in all cultivars analyzed. The quantitative analysis showed that red varieties were richer in proanthocyanidins, flavonols, hydroxycinnamic acid derivatives, and anthocyanins than the white cultivars, whereas white varieties presented the higher content in quercetin-3-O-rutinoside and Σ-viniferin (resveratrol dimer). Both red and white stem extracts proved to be strong radical scavengers by DPPH, ABTS⁺, FRAP, ORAC_FL, and O₂⁻ antioxidant assays, with stems of red varieties displaying better results for all tests developed. These data point out that grape stems are a rich source of health-promoting phytochemicals for feed, food or nutraceutical developments after further processing and optimization.     

Antioxidant activity of lycopene extracted from tomato paste towards trichloromethyl peroxyl radical CCl3O2·

The objectives of this paper were to evaluate the antioxidant activity of lycopene towards the trichloromethyl peroxyl radical (CCl₃O₂) and to obtain a precise rate constant using a pulse radiolysis technique. Lycopene was extracted and purified from tomato paste and its purity reached 95.0% (HPLC analysis). CCl₃O₂ was generated from a pulsed aqueous air-saturated solution of 2-propanol (2 M), acetone (1 M) and CCl₄ (10⁻² M). The observed spectrum showed an intense bleaching of the lycopene ground-state absorption in the region 350–450 nm and the bleaching rate constant was determined to be 1.17×10⁹ M⁻¹s^−1, which indicated a rapid interaction between lycopene and CCl₃O₂.     

Purification and identification of three novel antioxidant peptides from protein hydrolysate of bluefin leatherjacket (Navodon septentrionalis) skin

Skin protein from a bluefin leatherjacket (Navodon septentrionalis) processing by-product was hydrolyzed by trypsin, flavourzyme, neutrase, papain, alcalase, and pepsin, and protein hydrolysate (BSH) prepared using alcalase showed the highest DPPH, HO, and O₂⁻ · scavenging activities among all hydrolysates. Using ultrafiltration and consecutive chromatography, three novel peptides with strong antioxidant properties were purified from BSH, and their sequences were determined as Gly-Ser-Gly-Gly-Leu (GSGGL, BSP-A), Gly-Pro-Gly-Gly-Phe-Ile (GPGGFI, BSP-B), and Phe-Ile-Gly-Pro (FIGP, BSP-C) with molecular weights of 389.41, 546.63, and 432.52 Da, respectively. BSP-C exhibited the highest scavenging activities on DPPH (EC₅₀ 0.118 mg/ml), HO (EC₅₀ 0.073 mg/ml), and O₂⁻ · (EC₅₀ 0.311 mg/ml) among the three peptides. In addition, BSP-C could effectively inhibit autooxidation in a linoleic acid model system. The antioxidant activities of BSP-A, BSP-B, and BSP-C might be due to the small molecular sizes and the hydrophobic and/or aromatic amino acid residues in their amino acid sequences. The present results suggested that peptides purified from the skin protein hydrolysate of bluefin leatherjacket were excellent antioxidants and could be effectively used as food ingredients and additives, and pharmaceuticals.     

Free-radical scavenging capacity and antioxidant activity of selected plant species from the Canadian prairies

Ethanolic extracts from the roots of wild licorice (Glycyrrhiza lepidota), narrow-leaved echinacea (Echinacea angustifolia), senega (Polygala senega), leaves of bearberry (Arctostaphylos uva-ursi) and aerial parts of two varieties of horsetail (Equisetum spp.) were prepared and evaluated for their free-radical scavenging capacity and their antioxidant activity, by a number of chemical assays. Assays employed included a β-carotene-linoleic acid (linoleate) model system, reducing power, scavenging effect on the DPPH free radical and capacity to scavenge hydroxyl free radicals (HO), by use of electron paramagnetic resonance (EPR) spectroscopy. The bearberry-leaf extract consistently exhibited the highest antioxidant activity based on the tests performed, and seems to be a promising source of natural antioxidants. The polyphenolic constituents appear to be responsible, at least in part, for the extract's radical-scavenging capacity. Research is progressing to characterize the antioxidant compounds in the bearberry-leaf extract and their mode of action in imparting antioxidant activity to various food systems.     

Liposomal stabilization of ascorbic acid in model systems and in food matrices

Ascorbic acid (AA) was encapsulated into dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylcholine/cholesterol (DPPC/chol) liposomes. We have shown that co-encapsulation of citric and ascorbic acids into liposomes results in considerable stabilization of the latter in model systems containing catalytic concentrations of copper ions. The rate of AA oxidation was decreased by up to 300-fold that of free AA. Incorporation of cholesterol into membranes resulted in lower stabilization efficacy at room temperature and better thermal stability at higher temperatures than pure DPPC liposomes. Total AA, extra-liposomal AA and leakage of AA out of liposomes during thermal treatment were evaluated using the free radical 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS). A modification of the ABTS based assay for liposome permeability makes it applicable to reducing agents and antioxidants as well as AA. Encapsulated AA was also stabilized in real food matrices. The rate of AA oxidation in apple juice, relative to that of free AA, was decreased by two orders of magnitude when encapsulated in DPPC liposomes and by more than one order of magnitude in DPPC/chol liposomes. Liposomal stabilization of AA in a fermented milk product was less pronounced.     

NADH oxidation and superoxide production by caprine milk xanthine oxidoreductase

Xanthine oxidoreductase (XOR) was purified from caprine milk where more than 90% exists in demolybdo ‘inactive’ form. The dehydrogenase form of the enzyme, oxidises nicotinamide adenine dinucleotide (reduced) (NADH), in the presence of oxygen, and generates superoxide anion radical (O₂⁻) significantly faster than does the oxidase form. The corresponding forms of human and bovine milk enzymes behaved similarly. The steady-state kinetics of NADH oxidation and O₂⁻ production, in the absence and presence of NAD⁺, by the dehydrogenase form of XOR from the three species, are analysed. Allopurinol, oxypurinol and amflutizole blocked the reducing substrates that act at the molybdenum site, and all of which were ineffective in the case of NADH oxidase activity. However, and as expected, diphenyleneiodonium was a powerful inhibitor of NADH oxidation. The possible physiological and pathological significance of reactive oxygen species, especially O₂⁻, arising from NADH oxidation by caprine XOR is discussed.     

Evaluation of antioxidant activity of parsley (Petroselinum crispum) essential oil and identification of its antioxidant constituents

Antioxidant capacities of the essential oil extracted from parsley (Petroselinum crispum) were evaluated by three different in vitro assays: β-carotene bleaching assay, DPPH free radical scavenging assay and Fe²⁺-metal chelating assay. Results showed that the parsley oil (PO) possessed a certain degree of antioxidant activities in terms of β-carotene bleaching capacity and free radical scavenging activity, but its metal chelating capacity was negligible. The antioxidant EC₅₀ values of the β-carotene bleaching assay and DPPH free radical scavenging assay of the crude PO dissolved in methanol were measured in about 5.12 and 80.21 mg/mL, respectively. However, these values were much weaker than those of BHT in 0.01 and 0.58 mg/mL, and of α-tocopherol in 0.01 and 0.10 mg/mL. Isolation and identification of the inherent antioxidants in PO involved using various chromatographic techniques including silica gel open column chromatography, normal phase-HPLC and GC–MS. Myristicin in PO was found as a dominant compound (32.75%) that exhibited a moderate antioxidant activity. Apiol was the second dominant compound (17.54%), but it might be the major contributor to the antioxidant activity of PO. These results suggest that the PO and its two major components can be potential alternative natural antioxidants.     

Concentration-dependence of prooxidant and antioxidant effects of aloin and aloe-emodin on DNA

To understand the toxic effects and conflicting reports of aloin and aloe-emodin, we investigated their antioxidant and prooxidant effects on free radical-induced DNA breaks over a wide range of concentrations. Aloin, at concentrations ranging from 1.25 to 2.5 mM, prevented OH-induced DNA breaks by 5–30% over control values. In contrast, addition of aloin at lower concentrations (8–300 μM) increased DNA damage by about 24–74%, indicating its prooxidant effect. Aloe-emodin showed little antioxidant effect at any tested concentration, but addition of higher concentrations (1.25–2.5 mM) of aloe-emodin had a prooxidant effect on DNA (about 29–35%). The greater reducing activity of aloin at low concentrations, reducing Fe³⁺ to Fe²⁺, which enhanced the generation of OH from the Fenton reaction, may play a key role in its prooxidant effect on DNA. At higher concentrations, however, free radical-scavenging activity of aloin gradually predominated over its reducing power, resulting in the protection of DNA. Predomination of reducing power over scavenging activity resulted in the prooxidant effect of aloe-emodin at high concentrations. The relationship between structures and anti- or pro-oxidant effects of aloin and aloe-emodin is also discussed.     

Scavenging ability on ROS of alpha-lipoic acid (ALA)

The scavenging ability of alpha-lipoic acid (ALA) towards superoxide anions, hydroxyl radicals and hydrogen peroxide was evaluated by means of chemiluminescence (CL). In neutral or acid conditions, ALA could scavenge superoxide anions and its scavenging efficiency depended on its concentration. In neutral or alkaline conditions, the CL intensity of the reaction of ALA with hydroxyl radical (OH radicals) decreased after the first 10 min and then increased with time, suggesting that ALA could scavenge OH radicals at the beginning, but yielded unknown intermediate products that could lead to the increase of CL intensity at later stages. The increase of CL intensity in the reaction of hydrogen peroxide with ALA may also be due to these products.     

Structure–antioxidant activity relationships of flavonoids isolated from different plant species

In the course of our phytochemical studies of different plants from developing countries we isolated and structurally characterized several flavonoid derivatives (compounds 1–26), both aglycones and glycosides, typical of the species investigated. The aim of this study was to evaluate varieties of medicinal plants that were growing in developing countries, known in traditional medicine as anti-inflammatory remedies, with respect to their flavonoidic isolated constituents, assuming that their anti-inflammatory activity could be explained, at least in part, by the presence of antioxidant principles. The antioxidant activities of compounds 1–26 were evaluated by measuring their ability to scavenge the radical cation 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS⁺) and the superoxide anion, to inhibit β-carotene oxidation in a lipid micelle system, and to inhibit xanthine oxidase activity, showing some structure–activity relationships.     

Antioxidant activity of brown pigment and extracts from black sesame seed (Sesamum indicum L.)

The antioxidant activities of brown pigment, extract of n-hexane and extract of supercritical carbon dioxide extraction of black sesame seeds were investigated in this study. Kinetics of anti-radical activity showed that the reaction between DPPH and brown pigment of sesame seed was rapid and reached the steady state in 10 min. Extracts from supercritical carbon dioxide extraction and n-hexane extraction reacted with DPPH slowly and the absorbance became stable after 35 min. However, α-tocopherol and trolox were rapid while the kinetic behaviour of BHA was intermediate. The brown pigment of sesame seed also showed a lower EC₅₀ (13.5 μg ml⁻¹) than the other two extracts and α-tocopherol, which was about 7–10 fold of the antioxidant activity of the supercritical carbon dioxide extract and the n-hexane extract. The ferric thiocyanate (FTC) method also showed that the brown pigment of sesame seed provided higher inhibition activity against lipid peroxidation at 200 μg ml⁻¹ than did the supercritical carbon dioxide extract an n-hexane extract at 1 mg ml⁻¹. However, the activities of the supercritical carbon dioxide extract and n-hexane extracts were significantly higher than that of α-tocopherol. In the linoleic acid system, the brown pigment of black sesame seed showed an equal antioxidant activity to BHA and higher than trolox and α-tocopherol. The results indicated that the brown pigment of sesame seed possessed excellent antioxidant activity.     

Chemical characterization and antioxidant capacity of the araçá-pera (Psidium acutangulum): An exotic Amazon fruit

The “araçá-pera” (Psidium acutangulum DC) is an exotic guava consumed by the Brazilian Amazon population. This paper describes 22 compounds from this fruit by UHPLC–HRMS and NMR methods, being one disaccharide, five monosaccharides, two organic acids, one trihydroxycinnamic acid glucopyranosyl, one tannine digalloyl glucopyranosyl, five triterpenoid acids, and six fatty acids. It also quantifies the level of ascorbic acid (AA) by HPLC–MS (74.32 ± 1.23 mg/100 g of fresh fruit), and defines the chemical antioxidant activities by DPPH and ABTS⁺ assays (24.96 ± 0.75, 90.57 ± 0.63 mg of vitamin C/100 g fresh fruit, respectively), and a cell-based assays (76–100%). These results have shown that this exotic guava can be consumed as a nutraceutical ingredient, as well as be used in the production of functional foods in the Amazonian diet to prevent chronic and oxidative diseases.