Free radical scavenger and antioxidant capacity correlation of alpha-tocopherol and Trolox measured by three in vitro methodologies

The objective of this study was to correlate the free radical scavenging and antioxidant activity of two known substances (Trolox and alpha-tocopherol), using three in vitro methods (linoleic acid emulsion, brain homogenate and 2,2-diphenyl-1-picryl-hydrazyl [DPPH]). At steady state, alpha-tocopherol showed a greater inhibition of spontaneous oxidation of brain homogenate (59.42%+/-1.91) than Trolox (38.50%+/-2.38), while the latter showed a better antioxidant activity performance regarding inhibition of linoleic acid peroxidation (100% versus 84.02%+/-1.98) and free radical scavenging activity (93.56%+/-5.71 versus 66.72%+/-6.28). When the IC50 value was used as a parameter, alpha-tocopherol presented greater antioxidant activity than Trolox evaluated in brain homogenate and DPPH, without a significant difference when using linoleic acid emulsion. Both compounds showed the same antioxidant efficiency measured by DPPH kinetics (0.37 mM). Antioxidant activity significantly changed according to the substrate, the parameter adopted to compare the substances in the same method and the form used to express antioxidant concentration.     

The antioxidant and radical scavenging activities of black pepper (Piper nigrum) seeds

Water and ethanol crude extracts from black pepper (Piper nigrum) were investigated for their antioxidant and radical scavenging activities in six different assay, namely, total antioxidant activity, reducing power, 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities. Both water extract (WEBP) and ethanol extract (EEBP) of black pepper exhibited strong total antioxidant activity. The 75 microg/ml concentration of WEBP and EEBP showed 95.5% and 93.3% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, at the same concentration, standard antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and alpha-tocopherol exhibited 92.1%, 95.0%, and 70.4% inhibition on peroxidation of linoleic acid emulsion, respectively. Also, total phenolic content in both WEBP and EEBP were determined as gallic acid equivalents. The total phenolics content of water and ethanol extracts were determined by the Folin-Ciocalteu procedure and 54.3 and 42.8 microg gallic acid equivalent of phenols was detected in 1 mg WEBP and EEBP.     

The antioxidant and radical scavenging activities of black pepper (Piper nigrum) seeds

Water and ethanol crude extracts from black pepper (Piper nigrum) were investigated for their antioxidant and radical scavenging activities in six different assay, namely, total antioxidant activity, reducing power, 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities. Both water extract (WEBP) and ethanol extract (EEBP) of black pepper exhibited strong total antioxidant activity. The 75?µg/ml concentration of WEBP and EEBP showed 95.5% and 93.3% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, at the same concentration, standard antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and α-tocopherol exhibited 92.1%, 95.0%, and 70.4% inhibition on peroxidation of linoleic acid emulsion, respectively. Also, total phenolic content in both WEBP and EEBP were determined as gallic acid equivalents. The total phenolics content of water and ethanol extracts were determined by the Folin-Ciocalteu procedure and 54.3 and 42.8?µg gallic acid equivalent of phenols was detected in 1?mg WEBP and EEBP.     

Corni fructus scavenges hydroxy radicals and decreases oxidative stress in endothelial cells

Corni fructus has been used as a tonic, analgesic, and diuretic in Korean herbal medicine. The present investigation was undertaken to evaluate the antioxidative effect of corni fructus and its capacity to protect cells against oxidative damage. The radical scavenging activity of corni fructus extracts was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), and the antioxidant activity was determined by measuring the peroxide value in a linoleic acid emulsion system. In addition, human umbilical vein endothelial cells (HUVECs) were treated with corni fructus extracts and incubated with H(2)O(2) to investigate protection against apoptosis induction. Both ethanol and water extracts of corni fructus produced higher DPPH radical scavenging activity than hexane, chloroform, and ethyl acetate extracts. Strong antioxidative activities of both water and ethanol extracts were observed in an emulsion system containing linoleic acid and phosphate buffer. The incubation of HUVECs with the addition of ethanol extract significantly decreased H(2)O(2)-initiated damage of endothelial cells, but the water extract did not. The pretreatment with ethanol extract, but not with water extract, significantly decreased apoptotic damage of the H(2)O(2)-treated HUVECs and kept the morphological normality. This study demonstrates that corni fructus is a potent antioxidant substance, and suggests that further investigation is needed to characterize the difference between extract types and to identify its antioxidant compounds.     

Evaluation of antioxidant properties of a new compound, pyrogallol-phloroglucinol-6,6'-bieckol isolated from brown algae, Ecklonia cava

In this study, antioxidant and free radical scavenging activities of the natural antioxidative compound, pyrogallol-phloroglucinol-6,6''-bieckol (PPB) isolated from brown algae, Ecklonia cava was assessed in vitro by measuring the radical scavenging activities (DPPH, alkyl, hydroxyl, and superoxide) using electron spin resonance (ESR) spectrometry, intracellular reactive oxygen species (ROS) scavenging activity, and DNA damage assay. According to the results of these experiments, the scavenging activity PPB against difference radicals was in the following order: DPPH, alkyl, hydroxyl, and superoxide radicals (IC₅₀; 0.90, 2.54, 62.93 and 109.05 µM). The antioxidant activities of PPB were higher than that of the commercial antioxidant, ascorbic acid. Furthermore, PPB effectively inhibited DNA damage induced by H₂O₂. These results suggest that the natural antioxidative compound, PPB, can be used by the natural food industry.     

Caffeic acid phenethyl ester (CAPE): correlation of structure and antioxidant properties

Caffeic acid phenethyl ester (CAPE), a plant polyphenolic concentrated in honeybee propolis, has been found to be biologically active in a variety of pathways. The aim of this study was to determine the antioxidant activity of CAPE using different methods such as total antioxidant activity by the thiocyanate method, 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid radicals, 1,1-diphenyl-2-picryl-hydrazyl free radicals, N,N-dimethyl-p-phenylenediamine dihydrochloride radicals and superoxide anion radicals scavenging activities, reducing power and ferrous ions (Fe(2+)) chelating activities. CAPE showed 97.9% inhibition on lipid peroxidation of linoleic acid emulsion. On the other hand, butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol and trolox indicated an inhibition of 87.3, 97.6, 75.3 and 90.3% on peroxidation in the same system, respectively.     

蛋清蛋白酶解物抗氧化与血管紧张素转换酶抑制活性的研究

目的研究三种蛋白酶的蛋清蛋白酶解物(EWPH)的抗氧化及血管紧张素转换酶(ACE)抑制活性。方法分别采用Fenton体系、邻苯三酚自氧化体系和亚油酸自氧化体系测定胰蛋白酶、木瓜蛋白酶及alcalase碱性蛋白酶的EWPH清除羟自由基、超氧阴离子及抑制脂质过氧化的能力。利用高效液相色谱法测定ACE抑制活性。超滤分离上述三种蛋白酶的EWPH,检测各组分的抗氧化和ACE抑制活性。结果抗氧化活性最强的是木瓜蛋白酶的EWPH,在浓度10mg/ml时,对羟自由基和超氧阴离子清除率分别为53.14%和27.87%,在浓度0.8mg/ml时,对亚油酸自氧化的抑制率73.72%(D7)。ACE抑制效果最明显的是胰蛋白酶的EWPH,其IC50值为0.985mg/ml。低分子量(Mw3ku)组分酶解物的活性优于高分子量(Mw3ku)组分。结论三种蛋白酶的EWPHs均具有抗氧化和ACE抑制活性,但两种活性之间无明显的相关,且小分子组分的活性最强。     

Antioxidant activity of methanolic extract of emblica fruit (Phyllanthus emblica L.) from six regions in China

The phenolic contents of methanolic extracts of emblica (Phyllanthus emblica L.) fruit from six regions in China were measured in this work. The antioxidant activities of these extracts were also evaluated. Total phenolic content was ranged from 81.5 to 120.9 mg gallic acid equivalents (GAE)/g and the flavonoid content was varied from 20.3 to 38.7 mg quecetin equivalents (QE)/g, while proanthocyanidin content was ranged from 3.7 to 18.7 mg catechin equivalents (CE)/g. Among all the methanolic extracts analyzed, the Huizhou sample exhibited a significantly higher phenolic content than other samples (P<0.05). The antioxidant activities were evaluated by in vitro experiments using scavenging assays of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, hydroxyl radicals, and superoxide anion radicals, chelating ability of ferrous ion, reducing power, and inhibition capability of Fe (II)-induced lipid peroxidation, respectively. The Huizhou sample was found to have the strongest antioxidant activities in scavenging DPPH radicals, superoxide anion radicals, and had the highest reducing power, while the Chuxiong sample showed the best performance in chelating iron and inhibiting lipid peroxidation. Furthermore, the Chuxiong sample exhibited a stronger inhibition activity of the hydroxyl radicals compared with other samples. The high correlation coefficient was existed between the phenolic content and superoxide anion radical scavenging activity, but no significant correlation was found between the former and hydroxyl radical scavenging activity. Methanolic extracts of emblica fruit from some selected regions exhibited stronger antioxidant activities compared to those of the commercial compounds (quercetin and BHA). It might be considered as a potential plant source of antioxidants.     

Free Radical Scavenging Activity of Scoparia dulcis Extract

We studied the scavenging capabilities of an extract of Scoparia dulcis (a cosmopolitan weed widespread in Laos and Vietnam) for 1-diphenyl-2-picrylhydrazyl and measured hemoglobin-catalyzed linoleic acid peroxidation with an oxygen electrode. Our results demonstrated strong antioxidant activity corresponding to mitigation of the generation of hydroxyl radicals, a possible rationale for the observed therapeutic effects of this weed.     

Antioxidative effects of glycoprotein isolated from Solanum nigrum L

Glycoprotein from Solanum nigrum L. (SNL glycoprotein) was isolated and tested for antioxidative effects on oxygen free radicals using a 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the SNL glycoprotein are optimal in acidic pH and up to 60 degrees C. However, it has minimal activities in the presence of EDTA, although such activities are not dependent on M(2+) ions (Ca(2+), Mn(2+), and Mg(2+)) in the presence of EDTA. Interestingly, when SNL glycoprotein was treated with deactivation agents (pronase E and NaIO(4)), the DPPH radical scavenging activity was decreased compared with the SNL glycoprotein treatment alone. The antioxidative effects of SNL glycoprotein on superoxide anion and hydroxyl radical under optimal conditions revealed that SNL glycoprotein has remarkable scavenging effects on both radicals, but exhibited slightly higher scavenging effects on superoxide anion generated by the enzymatic hypoxanthine/xanthine oxidase system than on hydroxyl radicals generated by the Fenton reaction. However, SNL glycoprotein was more effective against hydroxyl radials in cell cultures (NIH/3T3). Consequently, 20 microg/mL SNL glycoprotein has a scavenging ability against superoxide anion corresponding to that of ascorbic acid. On the other hand, its hydroxyl radical scavenging activity corresponds to 0.1 microg/mL catalase. From these results, we suggest that SNL glycoprotein has potent antioxidative potential.     

Synthesis and antioxidative activity of 6-hydroxypyridoxine

An attempt to synthesize 6-hydroxypyridoxine (OPN), hydroxylation on C-6 of pyridoxine (PN) by hydroxyl radical (OH*). was conducted. Application of two well-known OH* generating reactions, i.e. the Fenton reaction and the Fe2+-EDTA/ascorbate reaction, were unsuccessful, as large amounts of by-products were formed. Although generation of OH* by autoxidation of ascorbic acid in the absence of metal ions was slow, by-products were formed in small quantities, and OPN was easily obtained in colorless crystals. Its structure was confirmed by spectral analyses. OPN was comparable to polyphenols such as (+)-catechin, rutin and gallic acid in the antioxidative activity against linoleic acid peroxidation, and was an effective DPPH radical scavenger, though the DPPH radical-scavenging activity of OPN was somewhat lower than that of the polyphenols. PN was relatively inactive under the conditions used here, indicating that the introduction of a hydroxyl group on C-6 of PN greatly enhanced both activities.     

Evaluation of in vitro antioxidant activity of Sida rhombifolia (L.) ssp. retusa (L.)

Sida rhombifolia (L.) ssp. retusa (L.) is widely used in Ayurvedic medicine for the treatment of fever as well as a diuretic. The comparative antioxidant potentials of ethanol extract of roots, stems, leaves, and whole plant were studied. Estimation of total polyphenolic content and high-performance thin-layer chromatography profile were determined. Further inhibition of oxygen-derived free radicals, viz., assays for free radical scavenging, reducing power, superoxide anion scavenging, nitric oxide scavenging, and anti-lipid peroxidation, were performed. All the antioxidant activities were compared with standard antioxidants such as butylated hydroxyanisole and alpha-tocopherol acetate. Extracts were found to be good scavengers of the 1,1-diphenyl-2-picrylhydrazyl radical in the order root > leaves > whole plant > stem with 50% inhibitory concentrations of 546.1, 852.8, 983.8, and 1,222.5 microg/mL, respectively. All extracts of this plant showed effective free radical scavenging activity, reducing power, and superoxide scavenging activity. Only root extract inhibited lipid peroxidation in rat liver and brain homogenate. All these antioxidant properties were concentration dependent. In addition, total polyphenolic contents of all the extracts were determined as gallic acid equivalents. The highest antioxidant activity was observed in root extract. The results obtained from the current study indicate that S. rhombifolia ssp. retusa is a potential source of natural antioxidants.     

Radical scavenging, antibacterial, and antiproliferative activities of Melissa officinalis L. extracts

The aromatic herb Melissa officinalis L. can be used as an easily accessible source of natural antioxidants and as a possible food supplement and as a phytochemical. Radical scavenging, antibacterial, and antiproliferative activities of petroleum ether, chloroform, ethyl acetate, n-butanol, and water extracts of M. officinalis L. extracts were investigated. The results of antioxidative activity, obtained by electron spin resonance spectroscopy, confirmed that investigated extracts suppressed the formation of 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and lipid peroxyl radicals in all investigated systems in a dose-dependent manner. The maximum DPPH and hydroxyl radical scavenging activities (SA(DPPH) = SA(OH) = 100%) were achieved in the presence of n-butanol extract at concentrations of 0.4 mg/mL and 0.5 mg/mL, respectively. The highest lipid peroxyl scavenging activity (93.20%) was observed at a higher concentration (5 mg/mL) of n-butanol extract in the lipid peroxidation system. The most effective antibacterial activities were expressed by petroleum ether and ethyl acetate extracts on Sarcina lutea. Chloroform extract showed the strongest antiproliferative effect with 50% inhibitory concentration values of 0.09 mg/mL and 0.10 mg/mL for HeLa and MCF-7 cell lines, respectively. The present study demonstrated the high phenolic content and radical scavenging, antibacterial, and antiproliferative activities of extracts of M. officinalis L. originating from Serbia.     

Antioxidative activity of hen egg ovalbumin hydrolysates

To evaluate the antioxidative activity of the hydrolysates of ovalbumin, the antioxidative activities of the enzymatic extracts were evaluated using three different methodologies scavenging assays such as superoxide anion, hydroxyl radical, and inhibitory oxidation of linoleic acid in vitro, and the activities of SOD, GSH-PX, CAT and the level of MDA were determined in serum and liver of aged mice induced by G-gal. The results showed that the hydrolysates had a distinctly inhibitory action to superoxide anion made by alkaline pyrogallic acid, HO. produced by Fenton reaction, the oxidation of linoleic acid in linoleic acid autoxidation system, and presented a positive correlation. The inhibition capacity of hydrolysates against superoxide anion and HO. were more than 45% and 56% respectively at the concentration 5 mg/mL. And the hydrolysates could significantly (p< 0.01) prevented the activities of SOD, GSH-Px, and CAT against reducing and all three concentrations could significantly (p< 0.01) decrease the MDA contents in the serum and liver of aged mice induced by G-gal. The antioxidative activity of high concentration was similar to that of control group.     

Antioxidant Activities and Total Phenolic Content of Aqueous Extract of Pleurotus ostreatus (Cultivated Oyster Mushroom)

Pleurotus ostreatus better known as oyster mushroom is widely cultivated and consumed as food in Malaysia. The present study aims to assess the antioxidative potential and total phenolic content of P. ostreatus aqueous extract. The antioxidant activities were evaluated against DPPH and ABTS radical-scavenging activity, ferric-reducing antioxidant power (FRAP) and β-carotene-linoleate bleaching assay, and the Folin-Ciocalteu method for total phenolic content (TPC). The DPPH and ABTS radical-scavenging activity was found to be 63.20% and 87.29% respectively; antioxidant activity using FRAP at 1.45 mM FE/100g and β-carotenelinoleate bleaching assay was 83.51%, while the TPC was found to be 798.55 mg GAE/100g. These antioxidant activities were compared to synthetic antioxidant, BHA and ascorbic acid. Ascorbic acid showed highest scavenging effects on DPPH and ABTS radical, followed by P. ostreatus and BHA (at maximum safety limit). The ferric reducing power of P. ostreatus was significantly higher than BHA and ascorbic acid. The antioxidant activity as assessed in β-carotene-linoleate bleaching assay was found to be higher in BHA compared to P. ostreatus. The aqueous extract of P. ostreatus was found to respond differently in antioxidant assays. The antioxidative activity of the aqueous extract of P. ostreatus correlated with its total phenolic content. Generally, the antioxidant activities of P. ostreatus' aqueous extract are comparable to that of BHA and ascorbic acid to a certain extent.     

Caffeic acid phenethyl ester (CAPE): correlation of structure and antioxidant properties

Caffeic acid phenethyl ester (CAPE), a plant polyphenolic concentrated in honeybee propolis, has been found to be biologically active in a variety of pathways. The aim of this study was to determine the antioxidant activity of CAPE using different methods such as total antioxidant activity by the thiocyanate method, 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid radicals, 1,1-diphenyl-2-picryl-hydrazyl free radicals, N,N-dimethyl-p-phenylenediamine dihydrochloride radicals and superoxide anion radicals scavenging activities, reducing power and ferrous ions (Fe²⁺) chelating activities. CAPE showed 97.9% inhibition on lipid peroxidation of linoleic acid emulsion. On the other hand, butylated hydroxyanisole, butylated hydroxytoluene, α-tocopherol and trolox indicated an inhibition of 87.3, 97.6, 75.3 and 90.3% on peroxidation in the same system, respectively.     

Antioxidant Activity of Anthocyanin Extract from Purple Black Rice

Antioxidant activity was studied for anthocyanins extracted from purple black rice (PBR) by a 3% aqueous trifluoroacetic acid solution (TFA), as well as for anthocyanins extracted from blueberry (Bluetta, high bush type). Capillary zone electrophoresis revealed that the PBR extract contained almost exclusively a single anthocyanin, which was identified as cyanidin 3-O-beta-D-glucoside (Cy 3-Glc) after purification by polyvinylpyrrolidone column chromatography. In contrast, 11 anthocyanins were identified in the blueberry extract. PBR extract showed slightly weaker superoxide scavenging and crocin bleaching activities than blueberry extract did. Both PBR and blueberry extracts, however, showed 10 to 25 times stronger activity than the same concentration of Trolox used as a reference antioxidant. It was further noted that the purified Cy 3-Glc from PBR extract retained approximately 74% of the antioxidant activity (both crocin bleaching and superoxide scavenging) observed in the original TFA extract. The hydroxyl radical scavenging activity of both extracts was several times weaker than that of the same concentration of Trolox, although the PBR extract showed approximately two times stronger activity than blueberry extract did. The hydroxyl radical scavenging activity of the purified Cy 3-Glc from PBR, however, decreased to approximately 20% of that of the original PBR extract. These results indicate that the anthocyanin Cy 3-Glc contributes to the antioxidant activity of PBR through its strong superoxide radical but not hydroxyl radical scavenging activity.     

Radical scavenging-linked antioxidant activities of commonly used herbs and spices in Korea

Herbs and spices not only variety and racy flavour to Korean foods, they also are the richest source for antioxidant power. The present study evaluates the radical scavenging-linked antioxidant activities of hot water extracts from commonly used herbs and spices in Korea. 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical and superoxide anion scavenging activities of bay extract were 39.5% and 22.1%, respectively. The hydroxyl radical scavenging activity was in order of dill (50.0%) > bay (31.3%) > garlic (27.9%) > white pepper and black pepper (15.1-15.3%) > onion (10.1%) extracts. Bay extract had the highest total phenolic content (17.86?μg CE/g). High correlation coefficients were found between the total phenol content and DPPH radical scavenging activity (R?=?0.9162). These results indicate that herbs and spices had high antioxidant activity that is partly due to the phenolic compounds and provide basic data for further development of processed food products.     

Study on the antioxidant activity of tea flowers (Camellia sinensis)

Major chemical compounds in different extracts from tea flowers (Camellia sinensis) were analyzed. Distilled water or 70% ethanol extracts were then fractionated with chloroform, ethyl acetate and n-butanol, respectively. Each extract fraction was tested its scavenging activities on 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydroxyl free radicals. The results showed that ethyl acetate fraction of ethanol-extract of tea flower (EEA) exhibited the highest quenching activity to hydroxyl radicals (SC50 11.6 mug/ml), followed by ethanol-extract (EE) of tea flower (SC50 19.7 microg/ml). Same tea flower extract showed big different scavenging activities on different free radicals. EEA quenched 80% of hydroxyl radicals generated by Fenton's reaction, however, only 40% of DPPH radical was scavenged in the Fe (II)-H2O2 -luminol system. The contents of flavones, polyphenols and catechins in EE and EEA fractions were higher than those in other fractions. We suggest that the stronger scavenging abilities to free radicals might be due to polyphenols, EGCG, ECG and flavones. However, the water extracts of tea flower and their fractions showed lower antioxidant activity for their inhibitory effect on hydroxyl radicals and DPPH radicals.     

Radical scavenging activity of flavonoids from Trollius chinensis Bunge

Objective

Flos trollii is considered as functional tea, as well as a traditional medicinal herb, in China. In this study, total phenolic and flavonoid contents of Flos trollii were determined by a colorimetric method. The antioxidative potential of the hydroalcoholic extract of Flos trollii (FTE, extracted by alcohol:water, 80:20) was also evaluated by various antioxidant assays.

Methods

Chemiluminescence technique was used to determine the radical scavenging activities of FTE toward different reactive oxygen species, including superoxide anion (O_-2·), hydroxyl radical (OH·), lipid-derived radicals (R·), and singlet oxygen (¹O₂).

Results

FTE could effectively scavenge O_-2·, OH·, R·, and ¹O₂ at an efficient concentration (EC₅₀) of 46, 5.64, 5.19, and 3.97 mg/mL, respectively. Moreover, the radical scavenging activities of FTE were higher than those of ascorbic acid. Further, FTE had higher 1,1 diphenyl-2-picrylhydrazyl radical (DPPH) radical-scavenging activity with EC₅₀ 44 mg/mL, compared with butylated hydroxytoluenesynthetic antioxidant with EC₅₀ 52 mg/mL.

Conclusion

All results indicated that FTE is a powerful antioxidant, deserving of better utilization of the extracted Flos trollii as antioxidants.