Enzyme-based online monitoring and measurement of antioxidant activity using an optical oxygen sensor coupled to an HPLC system

It is estimated that up to 50 % of the adult population take antioxidant products on a daily basis to promote their health status. Strangely, despite the well-recognized importance of antioxidants, currently there is no international standard index for labeling owing to the lack of standardized methods for antioxidant measurement in complex products. Here, an online high-performance liquid chromatography (HPLC)-based method to detect and measure the total antioxidant capacity of antioxidant samples is presented. In this approach, complex samples containing antioxidants are separated by the HPLC system, which is further coupled to an antioxidant measuring system consisting of an optical oxygen sensor, laccase, and tetramethoxy azobismethylene quinone (TMAMQ). The antioxidants, separated via HPLC, reduce TMAMQ to syringaldazine, which is then reoxidized by laccase while simultaneously consuming O₂. The amount of consumed oxygen is directly proportional to the concentration of antioxidants and is measured by the optical oxygen sensor. The sensor is fabricated by coating a glass capillary with an oxygen-sensitive thin layer made of platinum(II) meso-tetra(4-fluorophenyl)tetrabenzoporphyrin and polystyrene, which makes real-time analysis possible (t ₉₀?=?1.1 s in solution). Four selected antioxidants (3 mM), namely, catechin, ferulic acid, naringenin (used as a control), and Trolox, representing flavonol, hydrocinnamic acid, flavanone, and vitamin E, respectively, were injected into the online antioxidant monitoring system, separated, and then mixed with the TMAMQ/laccase solution, which resulted in oxygen consumption. This study shows that, with the use of such a system, the antioxidant activity of individual antioxidant molecules in a sample and their contribution to the total antioxidant activity of the sample can be correctly assigned.     

Benzophenones and flavonoids from Hypericum maculatum and their antioxidant activities

The occurrence of three known benzophenones, namely annulatophenonoside, acetylannulatophenonoside and annulatophenone as well as a flavonol O-glycoside guajaverin in the aerial parts of Hypericum maculatum Crantz was established. In addition, hyperoside, isoquercitrin and miquelianin were isolated from this plant, as well. Radical scavenging and antioxidant activities of the isolated compounds were examined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) free radicals, ferric reducing antioxidant power (FRAP) assay and inhibition of lipid peroxidation in linoleic acid system by the ferric thiocyanate method. Isoquercitrin demonstrates the highest DPPH radical scavenging (96.6?±?0.3%), FRAP (23.8?±?0.2 Trolox equivalent, TE?mol?1) and antioxidant activity in linoleic acid system. Guajaverin and acetylannulatophenonoside show significantly strong ABTS radical scavenging activity (93.9?±?0.4% and 93.4?±?0.6%, respectively), which is comparable to that of ascorbic acid (96.2?±?0.4%).     

Seasonal variation in total phenolic and flavonoid contents and DPPH scavenging activity of Bellis perennis L. flowers

Variations in total phenolic and flavonoid contents as well as antioxidant activity of Bellis perennis (common daisy) flowers were investigated. The flowers were collected monthly (from March to October, i.e., during the usual flowering season of the plant) at three localities in three different years. Total flavonoids were determined spectrophotometrically by two methods: by formation of a complex with aluminium chloride after acidic hydrolysis of flower extracts (method 1) and by reaction with boric and oxalic acids in extracts without their modification (method 2). Total phenolics were determined spectrophotometrically using the Folin-Ciocalteu reagent. The antioxidant activity was determined spectrophotometrically by a 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. The contents of flavonoids varied from 0.31 to 0.44 mg quercetin equivalent/100 mg dry weight (method 1) and from 1.37 to 2.20 mg pigenin-7-glucoside equivalent/100 mg dry weight (method 2). Total phenolics ranged from 2.81 to 3.57 mg gallic acid equivalent/100 mg dry weight. The antioxidant activity expressed as IC(50) values varied from 66.03 to 89.27 μg/mL; it is about 50, 30, 20, and 10 times lower as compared with quercetin, ascorbic acid, Trolox?, and butylhydroxytoluene, respectively, and about five times higher in comparison with apigenin-7-glucoside. There is a significant correlation between antioxidant activity and total phenolics. No correlation between total flavonoid contents and antioxidant activity was observed. Contents of phenolics and flavonoids as well as antioxidant activity of daisy flowers vary to a relatively small extent during the year and are not dependant on the time of collection. Thus, the flowers possess comparable quality as to these characteristics over the whole flowering season of Bellis perennis. Effects of environmental factors on the amounts of secondary metabolites in plants are also discussed.     

Synthesis of New Phenolic Derivatives of Quinazolin-4(3H)-One as Potential Antioxidant Agents—In Vitro Evaluation and Quantum Studies

Considering the important damage caused by the reactive oxygen (ROS) and nitrogen (RNS) species in the human organism, the need for new therapeutic agents, with superior efficacy to the known natural and synthetic antioxidants, is crucial. Quinazolin-4-ones are known for their wide range of biological activities, and phenolic compounds display an important antioxidant effect. Linking the two active pharmacophores may lead to an increase of the antioxidant activity. Therefore, we synthesized four series of new hybrid molecules bearing the quinazolin-4-one and phenol scaffolds. Their antioxidant potential was evaluated in vitro, considering different possible mechanisms of action: hydrogen atom transfer, ability to donate electrons and metal ions chelation. Theoretical quantum and thermodynamical calculations were also performed. Some compounds, especially the ortho diphenolic ones, exerted a stronger antioxidant effect than ascorbic acid and Trolox.     

Phytochemical and antioxidant properties of some Cassia species

In this study, the antioxidant activity of aqueous and ethanol extracts of four plants from the genus Cassia were evaluated by various antioxidant assays, including ferric reducing antioxidant power (FRAP), DPPH free radical scavenging, metal chelating activity, phosphomolybdenum reducing power, hydrogen peroxide radical scavenging, hydroxyl radical scavenging, deoxyribose degradation and β-carotene bleaching assay. The various antioxidant activities were compared to standard antioxidant such as ascorbic acid. All the extracts showed antioxidant activity in the tested methods. Among the four species, Cassia auriculata has been found to possess highest activity in most of the tested models. In addition to the antioxidant activity, the total phenolics and flavonoids were measured in the extracts. The ethanolic extract exhibited highest phenolics and flavonoid contents and had also shown potent antioxidant activity in comparison to the aqueous extracts. The possible antioxidant mechanism of the ethanol extract can be due to its hydrogen or electron donating and direct free radical scavenging properties. Hence, the ethanol extract represents a source of potential antioxidants that could be used in pharmaceutical industries.     

4种黄酮小分子对DPPH自由基的清除作用及构效关系研究

通过紫外可见光谱测定了4种黄酮小分子芦丁、牡荆素、山奈素、金丝桃苷对DPPH自由基的清除率、稳定性及半抑制浓度(IC50),并以常用的天然抗氧化剂抗坏血酸作为对照,考察了其抗氧化效果,探讨了黄酮类化合物的抗氧化性与结构的关系。结果表明：不同的抗氧化剂清除DPPH自由基达到平衡的时间不同,芦丁所需时间最长。4种黄酮小分子及抗坏血酸均对DPPH自由基有清除效果,并存在一定的量效关系。对DPPH自由基的清除能力从大到小依次为金丝桃苷、抗坏血酸、芦丁、山奈素、牡荆素。结构分析表明,B环邻二酚羟基是黄酮类化合物抗氧化所必需的基团,其羟甲基化及A环羟基糖苷化不利于黄酮类化合物的抗氧化活性。而C环3-OH的糖苷化对抗氧化活性有利,且单糖苷优于双糖苷。     

Antioxidant phenolic compounds of cassava (Manihot esculenta) from Hainan

An activity-directed fractionation and purification process was used to isolate antioxidant components from cassava stems produced in Hainan. The ethyl acetate and n-butanol fractions showed greater DPPH˙and ABTS?+ scavenging activities than other fractions. The ethyl acetate fraction was subjected to column chromatography, to yield ten phenolic compounds: Coniferaldehyde (1), isovanillin (2), 6-deoxyjacareubin (3), scopoletin (4), syringaldehyde (5), pinoresinol (6), p-coumaric acid (7), ficusol (8), balanophonin (9) and ethamivan (10), which possess significant antioxidant activities. The relative order of DPPH? scavenging capacity for these compounds was ascorbic acid (reference) > 6 > 1 > 8 > 10 > 9 > 3 > 4 > 7 > 5 > 2, and that of ABTS?+ scavenging capacity was 5 > 7 > 1 > 10 > 4 > 6 > 8 > 2 > Trolox (reference compound) > 3 > 9. The results showed that these phenolic compounds contributed to the antioxidant activity of cassava.     

Antioxidant activity of papaya seed extracts

The antioxidant activities of the ethanol, petroleum ether, ethyl acetate, n-butanol and water extract fractions from the seeds of papaya were evaluated in this study. The ethyl acetate fraction showed the strongest DPPH and hydroxyl free radical-scavenging activities, and its activities were stronger than those of ascorbic acid and sodium benzoate, respectively. The n-butanol fraction demonstrated the greatest ABTS? radicals scavenging activity. The ethyl acetate fraction and the n-butanol fraction not only showed higher antioxidant activities than the petroleum ether fraction, water fraction and ethanol fraction, but also showed higher superoxide anion and hydrogen peroxide radicals scavenging activities than those of the other extract fractions. The high amount of total phenolics and total flavonoids in the ethyl acetate and n-butanol fractions contributed to their antioxidant activities. The ethyl acetate fraction was subjected to column chromatography, to yield two phenolic compounds, p-hydroxybenzoic acid and vanillic acid, which possessed significant antioxidant activities. Therefore, the seeds of papaya and these compounds might be used as natural antioxidants.     

Polyphenol composition and antioxidant activity of selected medicinal herbs

The aim of this study was to characterize aqueous and alcoholic extracts [30%, 50% and 70% (w/v)] obtained from medicinal herbs (Calendula officinalis, Hypericum perforatum, Galium verum, and Origanum vulgare) used in traditional medicine from our country. Samples were examined for total and individual content of phenolics and antioxidant activities. The highest content of total polyphenols (9.9 ± 0.02 mg gallic acid equivalents (GAE) L^–1 extract) and antioxidant activities expressed as Trolox Equivalent Antioxidant Capacity [307.51 TEAC mmol g^–1DW by the ABTS (2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) method and 20.90 TEAC mmol g^–1 DW by the DPPH (1,1-diphenyl-2-picrylhydrazyl) method] was found in Origanum vulgare (50%) extract. Polyphenolic compounds were quantified using RP-HPLC.     

Antioxidant and antifungal activity of different extracts obtained from aerial parts of Inula crithmoides L.

The total phenolic content, antioxidant and antifungal activities of three Inula crithmoides extracts (n-hexane, methylene chloride and MeOH) were investigated. The methanolic extract showed the highest total phenolic content. In the DPPH assay, the methanolic and hexane extracts exhibited the highest DPPH-radical scavenging activity; in the 5-lipoxygenase assay, the hexane extract showed greater inhibitory effect with an IC₅₀ similar to that of Trolox and ascorbic acid. The antifungal activity of the methanolic extract revealed a higher activity against Phytophtora cryptogea and Alternaria solani.     

In vitro antioxidant activities of the methanol extract and its different solvent fractions obtained from the fruit pericarp of Terminalia bellerica

Terminalia bellerica has been used as a traditional medicine in a variety of ailments including anaemia, asthma, cancer, inflammation, rheumatism and hypertension. In this study, the free radical scavenging and antioxidant activities of methanol extract (ME) and its different solvent fractions (namely hexane (HE), ethyl acetate (EA), butanol (BL) and water (WA)) of the T. bellerica fruit pericarp were evaluated and compared with standard antioxidant compounds like gallic acid (GA), catechin and ascorbic acid. Among the different fractions tested, the EA fraction exhibited higher antioxidant and radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH), superoxide and hydroxyl radicals than the other fractions, which may be attributed to its higher phenolic and flavonoid content, since a linear relation was observed between the phenolic content and the antioxidant parameters. The HPTLC analysis of the EA fraction revealed that it mainly contains GA and ferulic acid (FA) as major phenolics, and the higher antioxidant activities of EA fraction may be due to the presence of these compounds.     

Evaluation of the antioxidant properties of litchi fruit phenolics in relation to pericarp browning prevention

Phenolics were extracted from litchi fruit pericarp (LFP) tissues, purified and their antioxidant properties analyzed. LFP phenolics strongly inhibited linoleic acid oxidation and exhibited a dose-dependent free-radical scavenging activity against alpha,alpha-diphenyl-beta-picrylhydrazyl (DPPH*) and hydroxyl radicals and superoxide anions. The degradation of deoxyribose by hydroxyl radicals was inhibited by phenolics acting mainly as iron ion chelators, rather than by directly scavenging the radicals. Phenolics from litchi fruit pericarp were found to display similar reducing power activity as ascorbic acid. The effect of phenolic compound treatment on pericarp browning and electrolyte leakage of litchi fruit was also evaluated and it was observed that application of exogenous litchi phenolics to harvested litchi fruit significantly prevented pericarp browning and delayed increases in electrolyte leakage. These results suggest that litchi pericarp tissue phenolics could be beneficial in scavenging free radicals, maintaining membrane integrity and, thereby inhibiting pericarp browning of litchi fruit.     

Antioxidant compounds from propolis collected in Anhui, China

The antioxidant activities of the chloroform, ethyl acetate and n-butanol extract fractions from propolis collected in Anhui, China were evaluated in this study. The ethyl acetate fraction contained the highest amount of total phenolics and total flavonoids, and showed the greatest 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical scavenging capacities and Ferric Reducing/Antioxidant Power (FRAP). The antioxidant activity of twenty-two compounds isolated from the ethyl acetate fraction was also evaluated using the above-mentioned three assays. The results indicated that phenolics contributed to the antioxidant activity of propolis collected in Anhui, China. Therefore, propolis collected in Anhui, China and its phenolics might be used as a natural antioxidant.     

Microwave-assisted extraction of phenolic antioxidants from potato peels

A response surface method was used to optimize the microwave-assisted extraction parameters such as extraction time (t) (min), solvent (methanol) concentration (S) (v/v) and microwave power level (MP) for extraction of antioxidants from potato peels. Max. total phenolics content of 3.94 mg g?1 dry weight (dw) was obtained at S of 67.33%, t of 15 min and a MP of 14.67%. For ascorbic acid (1.44 mg g?1 dw), caffeic acid (1.33 mg g?1 dw), ferulic acid (0.50 mg g?1 dw) max contents were obtained at S of 100%, t of 15 min, and MP of 10%, while the max chlorogenic acid content (1.35 mg g?1 dw) was obtained at S of 100%, t of 5 min, and MP of 10%. The radical scavenging activity of the extract was evaluated by using the DPPH assay and optimum antioxidant activity was obtained at S of 100%, t of 5 min, and MP of 10%.     

Use of DPPH⋅|DPPH Redox Couple for Biamperometric Determination of Antioxidant Activity

A novel electrochemical method for selective determination of antioxidant activity based on 2,2‐diphenyl‐1‐picrylhydrazyl | 2,2‐diphenyl‐1‐picrylhydrazin (DPPH?|DPPH) redox couple and biamperometric technique is proposed. Two identical glassy carbon disk electrodes were mounted in a classic electrochemical cell and the tested working potential difference was between 50 and 200 mV. The DPPH?|DPPH redox couple exhibited a high degree of reversibility. Selectivity of detector was tested by different antioxidant compounds in ethanol‐phosphate buffer solution, pH 7.40. The results of antioxidant activity of pure antioxidant compounds and of actual samples of beverages, expressed as Trolox equivalents and determined by proposed biamperometric and spectroscopic measurements, were in good correlation (R=0.9959). The detection limit for Trolox established by the applied biamperometry was 0.05 μM while the resulting current was linear up to 25 μM of Trolox.     

Antioxidant activities of extracts and fractions from Eupatorium lindleyanum DC

The antioxidant activities of water extract (WE), ethanol extract (EE), residue water extract (RWE) and petroleum ether (PF), ethyl acetate (EF), n-BuOH (BF) and water (WF) fractions of the ethanol extract from Eupatorium Lindley DC were investigated for the first time. Total phenolics content, DPPH radical scavenging activities, superoxide radical scavenging activities, total reduction capability, and ferrous ions chelating activities were determined for all the extracts and fractions. The results showed that all the extracts and fractions exhibited antioxidant activities with different magnitudes of potency. Among all the samples, WE and RWE exhibited the best antioxidant capacities, the BF also exhibited high antioxidant abilities in all tests except for the metal chelating activity, while the other extracts and fractions were relatively weak antioxidants. The BF had the highest total phenolics contents in all extracts and fractions, and the WE and RWE were found to be rich in tannins. Furthermore, the content of total phenolics showed good correlation with DPPH radical scavenging activity, superoxide anion radical scavenging activity, and the reducing power. Phenolic composition of all the extracts and fractions was identified and quantified by HPLC. The results indicate that the extracts of E. Lindley DC might be a useful potential source of natural antioxidant ingredients.     

Essential oil composition and antioxidant activity of Stachys sylvatica L. (Lamiaceae) from different wild populations in Kosovo

Leaves and inflorescences of Stachys sylvatica L. (Lamiaceae) were collected from three different wild populations in Kosovo to study the natural variation of the chemical composition of essential oils, total flavonoids, total phenolics and the antioxidant activity. Essential oils were obtained by steam distillation and analysed by GC-FID and GC-MS, whereas total flavonoids, total phenolics and antioxidant activities were determined by spectrophotometric methods. Yields of essential oils ranged from 0.001% to 0.007% (v per dry weight). Twenty-eight volatile constituents were identified. The main constituents were α-pinene, β-pinene and germacrene-D. Total phenolics ranged from 39.3 to 70.8?mg?g?1 dry mass, whereas total flavonoid content ranged from 30.44 to 70.63?mg?g?1 dm. The antioxidant activity, as measured by the DPPH method, exhibited a rather high degree of activity ranging from 25.5% to 57.2%, whereas the FRAP antioxidant activity showed a lower variability and ranged from 93 to 133.4?mg?g?1 dm.     

Chemical composition and antioxidant activity of the essential oil and various extracts of Haplophyllum robustum Bge

This study was designed to examine the chemical composition and in vitro antioxidant activity of the essential oil and various extracts of Haplophyllum robustum. GC-MS analysis of the oil resulted in the identification of 30 compounds, representing 99.2% of the oil; 1,8-cineole (38.1%), myrcene (10.7%), α-pinene (8.5%), 4-terpineol (7.0%) and sabinene (6.1%) were the main components. The antioxidant potential of the oil and extracts was evaluated using three separate methods: inhibition of the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH), β-carotene-linoleic acid assay and reducing power systems. The essential oil of the plant was able to reduce the stable free radical DPPH with an IC(50) of 72.0?±?1.2?μg?mL(-1) (the IC(50) for ascorbic acid was determined as 5.8?±?0.4?μg?mL(-1)). The essential oil also showed strong reducing power. The level of total phenolics was highest in the essential oil (179.5?±?2.1?μg?mg(-1)).     

Aqueous extracts of the edible Gracilaria tenuistipitata are protective against H₂O₂-induced DNA damage, growth inhibition, and cell cycle arrest

Potential antioxidant properties of an aqueous extract of the edible red seaweed Gracilaria tenuistipitata (AEGT) against oxidative DNA damage were evaluated. The AEGT revealed several antioxidant molecules, including phenolics, flavonoids and ascorbic acid. In a cell-free assay, the extract exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity that significantly reduced H?O?-induced plasmid DNA breaks in a dose-response manner (P < 0.001). The AEGT also suppressed H?O?-induced oxidative DNA damage in H1299 cells by reducing the percentage of damaged DNA in a dose-response manner (P < 0.001) as measured by a modified alkaline comet-nuclear extract (comet-NE) assay. The MTT assay results showed that AEGT confers significant protection against H?O?-induced cytotoxicity and that AEGT itself is not cytotoxic (P < 0.001). Moreover, H?O?-induced cell cycle G2/M arrest was significantly released when cells were co-treated with different concentrations of AEGT (P < 0.001). Taken together, these findings suggest that edible red algae Gracilaria water extract can prevent H?O?-induced oxidative DNA damage and its related cellular responses.     

Antioxidant activity of aqueous methanol extracts of Protaetia brevitarsis Lewis (Coleoptera: Scarabaedia) at different growth stages

In this study, we evaluate the antioxidant properties of the various extracts of Protaetia brevitarsis Lewis (Coleoptera: Scarabaedia) at different growth stages. The antioxidant activities of six different extracts from larvae, pupae and imago were measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) and singlet oxygen (1O? ). The larval methanol extracts (LME) and imago methanol extracts (IME) displayed the greatest effect in DPPH and ABTS radical scavenging assay, but the activity of water extracts was weaker in the all tested assays. However, LME and IME could be compared to ascorbic acid in 1O? quenching ability (the effective concentrations of 50% 1O? quenching: EC?? 0.174, 0.149 and 0.177?mg?mL?1, respectively). The antioxidant ability of the extracts to scavenge free radicals could significantly change the contents of gallic acid equivalent, an important factor based on the value of R2. The results suggest that our study may contribute to the development of new bioactive products with potential applications to reduce oxidative stress as well as play a vital role in protecting insect organisms against oxidative damage caused by undesirable conditions.