Antioxidant activities of extract and fractions from Tuber indicum Cooke & Massee

The antioxidant activities of ethanolic crude extract (ECE) and its four different solvent sub-fractions (namely, petroleum ether fraction (PEF), ethyl acetate fraction (EAF), n-butyl alcohol fraction (BAF) and the rest fraction (RF)) from Tuber indicum were investigated using several in vitro antioxidant assays. ECE and four sub-fractions possessed different antioxidant and radical-scavenging activities in different assays. BAF showed the most potent radical-scavenging activity on DPPH radicals, superoxide anion radicals and reducing power, with EC₅₀ values of 1.38, 0.96, 16.0 mg/ml, respectively. EAF exhibited the highest hydroxyl radical-scavenging and ferrous ion chelating activities with EC₅₀ values of 3.31 and 0.70 mg/ml, respectively. The total phenolics (TP) and total flavonoids (TF) were also determined. BAF had the highest TP and TF contents, and the next was EAF. These results showed that the amounts of phenolics and flavonoids were in accordance with higher effectiveness in scavenging radicals and chelating ferrous ions.     

Antioxidative activities and chemical characterization of polysaccharides extracted from the basidiomycete Schizophyllum commune

Antioxidant properties of hot water extract (HWE), hot water extracted polysaccharides (HWP) and hot alkali extracted polysaccharides (HWAE) were obtained from fruiting bodies of the wild basidiomycete Schizophyllum commune. All extracts contained both α- and β-glucans as determined by Megazyme β-glucan assay kit. Glucose was found by TLC and NMR to be the prevalent monosaccharide in all extracts. Total phenol contents were in descending order of HWP ≈ HWE > HWAE. Median effective concentrations (EC₅₀ values) of antioxidant activities were 8.3 ± 0.1, 6.9 ± 0.0 and 8.9 ± 0.1 mg/mL, and of the DPPH scavenging activity 0.8 ± 0.0, 0.6 ± 0.0 and 1.8 ± 0.0 mg/mL, for HWE, HWP and HWAE, respectively. EC₅₀ values of reducing power were 7.6 ± 0.1, 7.9 ± 0.0 and 12.5 ± 0.1 mg/mL, whereas those of the chelating abilities on ferrous ions were 3.1 ± 0.0, 4.6 ± 0.1 and 4.9 ± 0.1 mg/mL. The EC₅₀ values of the antioxidant activity, of the DPPH scavenging, and of the reducing power were correlated with total polysaccharide as well as with total phenol content. The antioxidant activities of all the extracts may be caused by both polysaccharides and polyphenols or by a complex of both.     

A comparative study of tocopherols composition and antioxidant properties of in vivo and in vitro ectomycorrhizal fungi

In aerobic organisms, the free radicals are constantly being produced during the normal cellular metabolism. The antioxidant properties of many organisms and particularly of wild mushrooms with their content in antioxidant compounds such as tocopherols, can detoxify potentially damaging forms of activated oxygen. Herein, a comparative study of tocopherols composition and antioxidant properties of in vivo (fruiting bodies) and in vitro (mycelia) ectomycorrhizal fungi: Paxillus involutus and Pisolithus arhizus. Tocopherols were determined by high performance liquid chromatography (HPLC) coupled to a fluorescence detector. The antioxidant properties were studied in terms of DPPH radical-scavenging activity, reducing power and inhibition of β-carotene bleaching. Fruiting bodies revealed the highest antioxidant properties, including scavenging effects on free radicals (EC₅₀ = 0.61 and 0.56 mg/ml) and inhibition of lipid peroxidation capacity (EC₅₀ = 0.40 and 0.24 mg/ml for P. involutus and P. arhizus, respectively), than mycelia produced in vitro cultures. Nevertheless, mycelia revealed higher levels of total tocopherols than fruiting bodies, and particularly P. arhizus mycelium proved to be a powerful source of γ-tocopherol (154.39 μg/g dry weight).     

Effect of gamma irradiation on the extraction yield, total phenolic content and free radical-scavenging activity of Nigella staiva seed

In the present study, the radiation processing of Nigella sativa seed samples was carried out at dose levels of 2, 4, 8, 10, 12 and 16 kGy. The extraction yield, total phenolic content and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity of both control and irradiated samples extracted in acetone, methanol and water were assessed. The results showed that the extraction yields increased with an increase in radiation dose for all the test solvents. At 16 kGy the increases were 3.7%, 4.2%, 5.6% and 9.0% for hexane, acetone, water and methanol extracts, respectively. The phenolic content in the acetone extract was found to be increased from 3.7 for the control sample to 3.8 mg/g for the 16 kGy radiation-processed sample. No significant change was observed for the phenolic content of the methanolic extract, while the aqueous extract showed a decrease at dose levels of 12 and 16 kGy. In the control samples, the DPPH radical-scavenging activity was 79.4%, 79.1% and 92.0% for water, acetone and methanol extracts, respectively, at 5 mg/ml concentration. Gamma irradiation enhanced the scavenging activity in acetone and methanol extracts by 10.6% and 5.4%, respectively, at 16 kGy. In summary, gamma irradiation increased the extraction yield and total phenolic content, as well as enhancing the free radical-scavenging activity. In addition, the type of solvent used for extraction also affected the impact of irradiation on antioxidant activity and total phenolic content of N. sativa seed.     

Antioxidant and free radical-scavenging activities of seeds and agri-wastes of some varieties of soybean (Glycine max)

In order to find antioxidant potential, seeds of 30 varieties of Glycine max were studied for their total phenolic contents (TPC), flavonoids and antioxidant activity (AOA). The seed extracts showed wide variation of TPC from 6.4 to 81.7 mg GAE/g, flavonoids 3.5 to 44.6 mg QE/g and AOA 7.5% to 74.7%. Free radical-scavenging activity (FRSA), assayed by DPPH in terms of IC₅₀ (inhibitory concentration), ranged from 0.14 to 0.80 mg/ml, EC₅₀ (efficiency concentration) from 6.1 to 34.8 mg/mg DPPH, ARP (anti-radical power) 2.9 to 16.4 and reducing power from 1.9 to 4.7 ASE/ml. Variety Kalitur showed highest the FRSA followed by Alankar and Hara soya, as evident from their low IC₅₀, EC₅₀ and high ARP values. Alankar, Kalitur NRC-37, PK-472, VLS-47, Hara soya varieties were with comparatively higher TPC (52.7–81.7 mg GAE/g), AOA (50.5–74.7%) and showed better inhibition of peroxide formation assayed through ammonium thiocyanate and egg yolk, non-site-specific and site-specific inhibition of hydroxyl radical induced deoxyribose degradation and ferrous ion-chelating capacity than did the other varieties. Seed extracts of these varieties and leaves of Kalitur showed significant protection against DNA damage caused by free radicals. The agri-wastes of some promising varieties, e.g. Alankar, Kalitur, NRC-37 and PK-472, showed TPC ranging from 27.4 to 167 mg GAE/g, total flavanoids from 10.4 to 63.8 mg QE/g and AOA from 26.5% to 84.7% and their values were highest in the leaves, followed by pod pericarp and twigs. Out of all the varieties studied, leaves of Alankar and Kalitur varieties were more potent free radical-scavengers than were seeds, pod pericarp or twigs. The specific phenolic compositions and their quantifications were performed by HPLC and MS/MS, which showed that the seeds of Kalitur were higher in genistin (127 μg/g), seeds and leaves of Alankar in diadzin (113 μg/g) and gallic acid (87.2 μg/g), respectively. The present studies may be of importance in varietal improvement, nutraceuticals, bio-pharmaceuticals and utilization of agri-wastes as possible cost-effective natural antioxidants.     

Improvement of functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle proteins hydrolyzed by Bacillus mojavensis A21 proteases

Functional properties and antioxidant activities of cuttlefish (Sepia officinalis) muscle protein hydrolysates, with different degrees of hydrolysis (DH from 7.3% to 18.8%), obtained by treatment with Bacillus mojavensis A21 alkaline proteases were investigated. Protein contents for all freeze-dried cuttlefish muscle protein hydrolysates (CMPHs) ranged from 80% to 86%. For the functional properties, hydrolysis by A21 proteases increased (p < 0.05) protein solubility to above 78% over a wide pH range (2.0–11.0). However, the interfacial activities (emulsion activity index, emulsion stability index, foaming capacity and foaming stability) decreased with the increase of the DH. All CMPHs exhibited significant metal chelating activity and DPPH free radical-scavenging activity, and inhibited linoleic acid peroxidation. Antioxidant properties of protein hydrolysates increased with protein hydrolysis and the highest activities were obtained at DH of 16%. The IC₅₀ values for DPPH radical-scavenging and metal chelating activities were found to be 0.52 ± 0.01 mg/ml and 0.67 ± 0.13 mg/ml. The obtained results suggested that functional properties and antioxidant activities of cuttlefish muscle protein hydrolysates were influenced by the degree of hydrolysis.The composition of amino acids of undigested and hydrolyzed proteins was determined. CMPHs have a high percentage of essential amino acids such as arginine, lysine, histidine and leucine. They have a high nutritional value and could be used as supplement to poorly balanced dietary proteins.     

Antioxidant properties of fungal chitosan from shiitake stipes

Fungal chitosan B or C was prepared by alkaline N-deacetylation of crude chitin B or C for 60, 90 and 120 min, which was obtained from air-dried shiitake stipes and its antioxidant properties studied. Chitosan showed antioxidant activities of 61.6-82.4% at 1 mg/ml and showed reducing powers of 0.42-0.57 at 10 mg/ml. At 10 mg/ml, scavenging abilities of chitosan B60 and C60 on 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals were 28.4-31.3% whereas those of chitosan B90, B120, C90 and C120 were 44.5-53.5%. With regard to the scavenging ability on hydroxyl radicals at 0.1 mg/ml, chitosan B60 and C60 were 61.9% and 63.6%, chitosan B90 and C90 were 68.3% and 69.9% and chitosan B120 and C120 were 77.7% and 77.2%, respectively. At 1.0 mg/ml, chelating abilities of chitosan B60 and C60 on ferrous ions were 88.7-90.3% whereas those of the rest chitosan were 97.8-103%. EC₅₀ values of antioxidant activity were below 1 mg/ml whereas those of reducing powers and scavenging abilities on DPPH radicals were 7.69-16.3 mg/ml. EC₅₀ values of scavenging abilities on hydroxyl radicals were below 0.1 mg/ml whereas those of chelating abilities on ferrous ions were 0.58-0.69 mg/ml.     

Effect of different brewing methods on antioxidant properties of steaming green tea

The extracts were prepared from cold or hot brewed steaming green tea at different concentrations (2, 6, and 10%), its antioxidant properties studied and potential antioxidant components determined. The yields of hot water extracts (17.49–28.27%) were significantly higher than those of cold water extracts (11.72–14.70%). EC₅₀ values in antioxidant activity determined by the conjugated diene method and reducing power were 2.19–3.10 and 0.22–0.28 mg/ml, respectively. EC₅₀ values in scavenging ability on 1,1-diphenyl-2-picrylhydrazyl (DPPH) and hydroxyl radicals were 29.45–43.80 and 2.88–3.22 mg/ml, respectively. EC₅₀ values in chelating ability on ferrous ions were 6.45–13.51 mg/ml. Contents of total phenols were 221.71–330.22 mg/g whereas those of total catechins in cold and hot water extracts were 135.05–193.14 and 161.57–195.05 mg/g, respectively. Based on the results obtained, hot water extracts were more effective in antioxidant activity and reducing power. However, cold water extracts were more effective in scavenging ability on DPPH and hydroxyl radicals, and chelating ability on ferrous ions. Summarily, the cold brewing method would be a new alternative way to make a tea.     

The effects of different inorganic salts,buffer systems,and desalting of Varthemia crude water extract on DPPH radical scavenging activity

The DPPH radical-scavenging activity of 25 inorganic salts, two buffer systems, and crude water extract of aerial parts of Varthemia (Varthemia iphionoides) before and after resins purification were investigated. Eight of the 25 inorganic salts tested quenched the DPPH radical colour. Na₂S₂O₃ and FeCl₂ showed markedly high DPPH colour-quenching activity, with inhibition of 65.3% and 47.7% respectively, at a concentration of 10 μg/ml. Four salts slightly increased the intensity of DPPH radical colour. The rest of tested salts, acetate buffer, and phosphate buffer at a concentration less than 0.1 mM did not affect DPPH radical colour. The DPPH radical-scavenging activity of BHT and catechol was considerably affected by the concentration of phosphate buffer (pH 7.0), and by acetate buffer (pH 5.0) at concentrations more than 0.01 mM in the case of BHT only. The DPPH radical-scavenging activity of a crude water extract of aerial parts of Varthemia iphionoides was much higher than that of an extract desalted by cation-exchange resin, indicating that iron ions apparently elevated the DPPH radical-scavenging activity of the extract. Therefore, desalting of plant extracts is important in order to obtain the true value of DPPH radical-scavenging activity.     

Compositions, antioxidant and antimicrobial activities of Helichrysum (Asteraceae) species collected from Turkey

The methanolic extracts of 16 Helichrysum species were investigated for their in vitro antioxidant, radical scavenging and antimicrobial activities. All the extracts showed strong antioxidant and radical scavenging activity. The highest total antioxidant capacity as ascorbic acid equivalents (AAE) of 194.64 mg/g dry extract was obtained for Helichrysum noeanum in the phosphomolybdenum assay. The highest IC₅₀ value (7.95 μg/ml) was observed for the extract of Helichrysum stoechas subsp. barellieri in the DPPH assay. The total phenolic contents of the extracts ranged from 66.74 to 160.63 mg gallic acid equivalents (GAE)/g dry extract. The major component present in the extracts was identified as chlorogenic acid followed by apigenin-7-glucoside and apigenin by HPLC analysis. All the extracts showed significant antimicrobial activity against microorganisms containing 13 bacteria and two yeasts in the agar diffusion method.     

The in vitro and in vivo antioxidant properties of seabuckthorn (Hippophae rhamnoides L.) seed oil

The antioxidant capacity of seabuckthorn (Hippophae rhamnoides L.) seed oil was investigated with a number of established in vitro assays and in an in vivo study of carbon tetrachloride (CCl₄)-induced oxidative stress in mice. The results showed that DPPH radical scavenging activity, ferrous ion chelating activity, reducing power and inhibition of lipid peroxidation activity all increased with increasing concentrations of seabuckthorn seed oil. Moreover, the EC₅₀ values of seabuckthorn seed oil from the hydrogen peroxide, superoxide radical, hydroxyl radical scavenging assays were 2.63, 2.16 and 0.77 mg/ml, respectively. In the in vivo study, seabuckthorn seed oil inhibited the toxicity of CCl₄, as seen from the significantly increased activities of the antioxidant enzymes superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The GSH content in the liver was also increased, whereas hepatic malondialdehyde was reduced. Taken together, these results clearly indicate that seabuckthorn seed oil has significant potential as a natural antioxidant agent.     

Pectin methylesterase in Citrus bergamia R.: purification, biochemical characterisation and sequence of the exon related to the enzyme active site

Three forms of pectin methylesterase (PME) were purified, from bergamot fruit (Citrus bergamia R.), to homogeneity by ion-exchange and affinity chromatography. The isoforms, named PME I, PME II and PME III, according their elution order on a heparin–sepharose column, were characterized for their relative molecular mass, activity kinetic parameters and thermostability. The molecular mass was estimated to be 42 kDa for the three forms, and the apparent K_m values for citrus pectin were 0.9 mg/ml for PME I and 0.5 mg/ml for PME II and PME III. The optimum pH values lie within the range 6.5–9.0, depending on salt concentration. Thermal behaviours of the three PME isoforms were studied in a temperature range from 65 °C to 80 °C with the less abundant PME I isoform showing a higher heat resistance. Moreover, the complete exon 2 sequence of PME gene was acquired (GenBank accession no. DQ458770) using a PCR-based approach on well-known Citrus genomic DNA present in the NCBI database.     

Biological potential of extracts of the wild edible Basidiomycete mushroom Grifola frondosa

Partially purified polysaccharides (FP) and hot alkali extract (FNa) obtained from fruiting bodies of the wild basidiomycete Grifola frondosa were examined for their antimicrobial, antioxidant and cytotoxic activity. The structural properties of FP and FNa samples were investigated by FT-IR and high resolution 1H- and 13C-NMR spectroscopy. From a group of various G − and G + bacteria the antibacterial effects were highest against the G + B. cereus. FNa was the better antioxidant shown by the lower EC₅₀ values of DPPH scavenging ability, ferric-reducing antioxidant power and ferrous ion-chelating ability. Ferric-reducing antioxidant power and ferrous ion-chelating ability were mostly linked to total polysaccharides, total- and β-glucan content, as well as total protein content. Both extracts displayed a moderate dose dependent antiproliferative action towards malignant human breast cancer MDA-MB-453, cervical adenocarcinoma HeLa and myelogenous leukemia K562 cells not observed in the non cancer derived MRC-5 fibroblasts. The highest effect was found in HeLa cells for FP extract. The mean diameter of Ca-alginate bead loading FP was 960.7 μm while the mean diameter of beads encapsulating FNa extract was 1051.7 μm.     

Free radical-scavenging capacity and inhibitory activity on rat erythrocyte hemolysis of feruloyl oligosaccharides from wheat bran insoluble dietary fiber

Water-soluble feruloyl oligosaccharides, which were released from wheat bran insoluble dietary fiber treated with xylanase from Bacillus subtilis and further purified with Amberlite XAD-2, were evaluated for their 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity and their capacity in inhibition of rat erythrocyte hemolysis mediated by 2,2′-azobis-2-amidinopropane dihydrochloride (AAPH) under in vitro conditions. The EC₅₀ value of the water-soluble feruloyl oligosaccharides against the DPPH radical was 0.52 mg/ml. The oxidative hemolysis of rat erythrocytes induced by AAPH was suppressed by the water-soluble feruloyl oligosaccharides in a concentration- and time-dependent manner. The water-soluble feruloyl oligosaccharides, at a concentration of 4 mg/ml, showed 91.7% of inhibition of rat erythrocytes hemolysis, and the erythrocyte hemolysis could be retarded for more than 120 min. These data indicated that the water-soluble feruloyl oligosaccharides from wheat bran insoluble dietary fiber might have potential as natural antioxidants.     

In vitro determination of antioxidant activity of proteins from jellyfish Rhopilema esculentum

In this study, the antioxidant activity of proteins isolated from jellyfish, Rhopilema esculentum Kishinouye (R. esculentum), was determined by various antioxidant assays, including superoxide anion radical-scavenging, hydroxyl radical-scavenging, total antioxidant activity, reducing power and metal chelating activity. Butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), α-tocopherol, vitamin C and mannitol were used as standards in those various antioxidant activities. The crude protein (CP) and the protein fractions isolated by Sephadex chromatography, first peak (FP) and second peak (SP), had very low reductive power and metal chelating abilities compared to EDTA, but they showed strong scavenging effects on the superoxide anion radical, hydroxyl radical and varying total antioxidant activity. FP and SP exhibited stronger scavenging effects on the superoxide anion radical than BHA, BHT or α-tocopherol. The EC₅₀ values of FP and SP were 6.12 and 0.88 μg/ml, respectively, while values EC₅₀ of BHA, BHT and α-tocopherol were 31, 61 and 88 μg/ml, respectively. CP, FP and SP showed far higher hydroxyl radical-scavenging activities than did vitamin C or mannitol. The EC₅₀ values of CP, FP and SP were 48.76, 45.42 and 1.52 μg/ml, but EC₅₀ values of vitamin C and mannitol were 1907 and 4536 μg/ml, respectively. In a β-carotene–linoleate system, SP and CP showed antioxidant activity, but lower than BHA. Of the three samples, SP had the strongest antioxidant activity. So, SP may have a use as a possible supplement in the food and pharmaceutical industries.     

Antioxidant activities of chestnut nut of Castanea sativa Mill. (cultivar ‘Judia’) as function of origin ecosystem

The antioxidant properties of different ecotypes of chestnut nut (cv. Judia) were studied. Total phenolics and flavonoids were also determinated. Total phenolics amount ranged from 9.6 mg/g of GAE (hottest ecotype, Murça) to 19.4 mg/g of GAE (coldest ecotype, Valpaços). Gallic and ellagic acid were the predominant compounds and Valpaços had the highest values while, Murça had the lowest ones. The antioxidant capacity of ethanolic extracts were evaluated through several biochemical essays: ABTS (2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid)) and DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity, FRAP (ferric reducing/antioxidant power) and inhibition of oxidative haemolysis in erythrocytes. In order to evaluate the antioxidant efficiency of each ecotype, the EC₅₀ values were calculated. Once again Valpaços revealed the best antioxidant properties, presenting much lower EC₅₀ values. Climatic conditions influence seems to be a limiting factor for production of phenolic compounds and consequently for the antioxidant properties of chestnut nuts.     

Antioxidant properties of methanolic extracts from Agaricus blazei with various doses of γ-irradiation

Agaricus blazei Murrill (Agariaceae) was irradiated with gamma rays at doses of 2.5, 5, 10, 15 and 20 kGy and the antioxidant properties of its methanolic extracts were studied. At 7.5 and 10.0 mg/ml, antioxidant activities of methanolic extracts from 2.5 to 20 kGy γ-irradiated A. blazei were significantly higher than those of methanolic extract from the nonirradiated control. At 0.5-7.5 mg/ml, reducing powers of methanolic extracts from A. blazei with 2.5, 10, 15 and 20 kGy of irradiation and without irradiation were comparable. All methanolic extracts showed excellent scavenging abilities of 95.2-100.7% against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals at 0.5 mg/ml. With regard to the scavenging ability against hydroxyl radicals, unirradiated and γ-irradiated A. blazei were comparable. Total phenols were the major naturally occurring antioxidant components found in the range of 18.77-21.48 mg/g. All EC₅₀ values were below 10 mg/ml, except values in reducing power, scavenging ability against DPPH radicals and chelating ability against ferrous ions were below 1 mg/ml. That indicates the unirradiated and irradiated A. blazei were good in antioxidant properties. Summarily, up to 20 kGy of irradiation did not remarkably affect the amounts of total antioxidant components in A. blazei.     

Purification and identification of novel antioxidant peptides from enzymatic hydrolysates of sardinelle (Sardinellaaurita) by-products proteins

In order to utilise sardinelle (Sardinellaaurita) protein by-products, which is normally discarded as industrial waste in the process of fish manufacturing, heads and viscera proteins were hydrolysed by different proteases to obtain antioxidative peptides. All hydrolysates showed different degrees of hydrolysis and varying degrees of antioxidant activities. Hydrolysate generated with crude enzyme extract from sardine (Sardinapilchardus) displayed high antioxidant activity, and the higher DPPH radical-scavenging activity (87 ± 2.1% at 2 mg/ml) was obtained with a degree of hydrolysis of 6%. This hydrolysate was fractionated by size exclusion chromatography on a Sephadex G-25 into eight major fractions (P₁–P₈). Fraction P₄, which exhibited the highest DPPH scavenging activity, was then fractionated by reversed-phase high performance liquid chromatography (RP-HPLC). Seven antioxidant peptides were isolated. The molecular masses and amino acids sequences of the purified peptides were determined using ESI-MS and ESI-MS/MS, respectively. Their structures were identified as Leu-His-Tyr, Leu-Ala-Arg-Leu, Gly-Gly-Glu, Gly-Ala-His, Gly-Ala-Trp-Ala, Pro-His-Tyr-Leu and Gly-Ala-Leu-Ala-Ala-His. The first peptide displayed the highest DPPH radical-scavenging activity (63 ± 1.57%; at 150 μg/ml) among these peptides.