Enrichment of Hazelnut Oil with Several Polyphenols: An Alternative Approach to A New Functional Food.

Hazelnut oil has been examined according to its oxidative stability and antioxidant activity. The oil sample has been treated with gallic acid, ascorbic acid, catechin, vanillic acid, p-coumaric acid and rutin. Stability of the pure and treated oils against the oxidation has been assessed via Rancimat by detecting the protection factor. The quality parameters of the oil samples were compared depending on their antioxidant activity. D-Optimal design of Response Surface Method has been applied to optimize the enrichment conditions of hazelnut oil with several polyphenols. Principal component analysis has been applied to comprehend the relationship between the groups and their quality parameters. Depending on the analysis of variance test, the most important parameter (at p < 0.0001) affecting the relevant system has been found polyphenol type with respect to stability and antioxidant capacity. Gallic acid has enhanced the stability of hazelnut oil against oxidation ~3 times over that of pure sample. The maximum yields of protection factor, antioxidant activity and dissolved polyphenol level have been 2.738, 46.14% and 259.424 ppm under the optimum conditions (300 ppm gallic acid).

Optimizing the extraction of polyphenols from Sideritis montana L. using response surface methodology.

Sideritis montana L. endemic of Turkey was screened for its polyphenols content and antioxidant activity. Factor analysis and experimental design have been applied to understand the structure of the separation process, to determine the effective parameters, and to accomplish the performance improvement. Face-centred composite design (FCD) of response surface methodology (RSM) was applied to evaluate the influences of solvent concentration, solvent amount, extraction time, and stirring speed of homogenizer-assisted extraction (HAE) as well as to model and to optimize the HAE. Quadratic models were highly significant (p < 0.0001) for the responses studied with high coefficients of determination (R2) of 0.9440, 0.9415 and 0.9521. The result of the study suggests that 15.02 mL of 22.69% EtOH solution (v/v), 70.16 s, and 9524.52 rpm of mixing speed are the optimal conditions to obtain the highest yield of total polyphenols (TPC) and flavonoids (TFC), and the best antioxidant activity (AA). Rosmarinic acid was identified as the most abundant component.

Identification and Determination of Phenolics in Lamiaceae Species by UPLC-DAD-ESI-MS/MS.

This study reports the phenolic profile screening of aromatic Lamiaceae species such as marjoram (Origanum majorana L.), lavender (Lavandula officinalis) and pennyroyal (Mentha pulegium L.) using a novel and validated ultra performance liquid chromatography method coupled with DAD diode array detector and tandem mass spectrometry (MS/MS) in negative mode of electrospray ionization. Identification and quantification of phenolics in these plant extracts has been realized within 12 min. This method showed good precision (percentage relative standard deviation; RSD% 0.54-2.72 for intra-day, 1.71-4.64 for inter-day), reproducibility (percentage recovery, REC% 92.0-109.0) and linearity (r = 0.9988-0.9999). Limits of detection ranged from 0.02 to 18.2 ng/mL. The extraction of plants was performed using microwave-assisted extraction technique and 60% (v/v) aqueous methanol solvent medium was selected as suitable solvent because of maximum extraction efficiency. Total antioxidant capacity, total phenolic content and free radical scavenging activity of these plant extracts were tested and the results correlated well among each other. According to the Folin assay, phenolic contents of Origanum majorana L., Mentha pulegium L. and Lavandula officinalis were calculated as 119 ± 3.4, 85.1 ± 2.8 and 57.8 ± 2.1 mg GAE/g dry matter, respectively.

Optimization of microwave-assisted extraction of polyphenols from herbal teas and evaluation of their in vitro hypochlorous acid scavenging activity.

Hypochlorous acid (HOCl) is an important reactive oxygen species (ROS) and non-radical and is taking part in physiological processes concerned with the defense of the organism, but there has been limited information regarding its scavenging by polyphenols. This study was designed to examine the HOCl scavenging activity of several polyphenols and microwave-assisted extracts of herbal teas. HOCl scavenging activity has usually been determined spectrophotometrically by a KI/taurine assay at 350 nm. Because some polyphenols (i.e., apigenin and chrysin) have a strong ultraviolet (UV) absorption in this range, their HOCl scavenging activity was alternatively determined without interference using resorcinol (1,3-dihydroxybenzene) as a fluorogenic probe. In the present assay, HOCl induces the chlorination of resorcinol into its non-fluorescent products. Polyphenols as HOCl scavengers inhibit the chlorination of the probe by this species. Thus, the 25% inhibitive concentration (IC25) value of polyphenols was determined using the relative increase in fluorescence intensity of the resorcinol probe. The HOCl scavenging activities of the test compounds decreased in the order: epigallocatechin gallate > quercetin > gallic acid > rutin > catechin > kaempferol. The present study revealed that epigallocatechin gallate (IC25 = 0.1 µM) was the most effective scavenging agent. In addition to polyphenols, four herbal teas were evaluated for their HOCl activity using the resorcinol method. The proposed spectrofluorometric method was practical, rapid, and less open to interferences by absorbing substances in the range of 200-420 nm. The results hint to the possibility of polyphenols having beneficial effects in diseases, such as atherosclerosis, in which HOCl plays a pathogenic role.

Novel oxime based flavanone, naringin-oxime: synthesis, characterization and screening for antioxidant activity.

Recent interest in polyphenolic antioxidants due to their involvement in health benefits has led to the investigation of new polyphenolic compounds with enhanced antioxidant activity. Naringin (4',5,7-trihydroxyflavanone-7-ß-l-rhamnoglucoside-(1,2)-α-d-glucopyranoside) is one of the major flavanones in citrus and grapefruit. The present study aimed to synthesize naringin oxime from naringin and to evaluate its antioxidant and anticancer potential using in vitro assay system. The structure of the synthesized compound, naringin oxime, was elucidated by FT-IR, (1)H NMR, elemental analysis and UV-vis spectroscopy. Antioxidant capacity of naringin oxime, as measured by the cupric reducing antioxidant capacity (CUPRAC) method, was found to be higher than that of the parent compound naringin. Other parameters of antioxidant activity (scavenging effects on OH, O2(-), and H2O2) of naringin and naringin oxime were also determined.

Identification and anti-oxidant capacity determination of phenolics and their glycosides in elderflower by on-line HPLC-CUPRAC method.

INTRODUCTION: Development and application of an on-line cupric reducing anti-oxidant capacity (CUPRAC) assay coupled with HPLC for separation and on-line determination of phenolic anti-oxidants in elderflower (Sambucus nigra L.) extracts for their anti-oxidant capacity are significant for evaluating health-beneficial effects. Moreover, this work aimed to assay certain flavonoid glycosides of elderflower that could not be identified/quantified by other similar on-line HPLC methods (i.e. 2,2-diphenyl-1-picrylhdrazyl and 2, 2'-azino-bis-3-ethylbenzothiazoline-6-sulphonic acid). OBJECTIVE: To identify anti-oxidant constituents in elderflower by HPLC and to evaluate their individual anti-oxidant capacities by on-line HPLC-CUPRAC assay with a post-column derivatisation system. METHODS: The separation and UV detection of polyphenols were performed on a C18 -column using gradient elution with two different mobile phase solutions, that is acetonitrile and 1% glacial acetic acid, with detection at 340 nm. The HPLC-separated anti-oxidant polyphenols in column effluent react with copper(II)-neocuproine in a reaction-coil to reduce the latter to copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. RESULTS: The detection limits of tested compounds at 450 nm after post-column derivatisation were compared with those of at 340 nm UV-detection without derivatisation. LOD values (µg/mL) of quercetin and its glycosides at 450 nm were lower than those of UV detection at 340 nm. This method was applied successfully to elderflower extract. The flavonol glycosides of quercetin and kaempferol bound to several sugar components (glucose, rhamnose, galactose and rutinose) were identified in the sample. CONCLUSION: The on-line HPLC-CUPRAC method was advantageous over on-line ABTS and DPPH methods for measuring the flavonoid glycosides of elderflower.

Novel pro-oxidant activity assay for polyphenols, vitamins C and E using a modified CUPRAC method.

In this study, a direct assay, a modified CUPRAC (Cupric Ion Reducing Antioxidant Capacity) method, is developed to determine transition metal ion (Cu(II))-catalyzed pro-oxidant activity of polyphenolic compounds, vitamins C and E, and herbal samples in the presence of proteins containing thiol groups. Since transition metal ion-catalyzed pro-oxidant activity of phenolics is usually initiated with the reduction of the metal to lower oxidation states (as a prerequisite of Fenton-type reactions), this method involves the reduction of copper(II) ions to copper(I) by polyphenolic compounds (simultaneously giving rise to reactive species), binding of the formed Cu(I) to egg white protein -SH groups, and liberation of copper(I)-neocuproine (Cu(I)-Nc) chelate (showing maximum absorbance at 450 nm) by treating the incubation product with a neocuproine-ammonium acetate mixture. The proposed method is validated against atomic absorption spectrometric (AAS) determination of protein-bound copper and protein carbonyl assay of oxidative stress. The proposed assay is faster and more specific than the carbonyl assay, and uses low-cost reagents and equipment. Pro-oxidant activity (i.e. proportional to absorbance) varies linearly over a relatively wide range with concentration, as opposed to the reciprocal correlations (i.e. linear regression of 1/(pro-oxidant activity) versus 1/concentration) of other similar assays. The pro-oxidant activity order of the tested antioxidant compounds in terms of 'Quercetin Equivalent Pro-oxidant Activity' (QREPA) coefficients is: gallic acid > epicatechin > quercetin ≈ catechin > α-tocopherol > rosmarinic acid > trolox > caffeic acid > ascorbic acid.

Development of a silver nanoparticle-based method for the antioxidant capacity measurement of polyphenols.

A sensitive colorimetric method for the detection of polyphenols (i.e., flavonoids, simple phenolic, and hydroxycinnamic acids) was proposed in this research based on the reduction of Ag(+) ions by polyphenols in the presence of citrate-stabilized silver seeds. The color of the stable suspension was controlled by varying the concentration of trisodium citrate, silver nitrate, and silver seeds. The reduction of Ag(+) to spherical silver nanoparticles (SNPs) by polyphenols in the presence of trisodium citrate and silver seeds produced a very intense surface plasmon resonance (SPR) absorption band of SNPs at 423 nm. The plasmon absorbance of SNPs allows the quantitative spectrophotometric detection of the polyphenols, and the developed method gave a linear response over a wide concentration range of standard polyphenolic compounds. In contrast to other reported NP-based antioxidant assays, it was established in this work that growth but not nucleation of SNPs gave a linear concentration-dependent response. The trolox equivalent antioxidant capacity (TEAC) values of various (hydrophilic and lipophilic) antioxidants using the developed method were comparable to those of the CUPRAC assay. Common food ingredients like oxalate, citrate, fruit acids, amino acids, and reducing sugars did not interfere with the proposed sensing method. This assay was validated through linearity, additivity, precision and recovery, demonstrating that the assay is reliable and robust. The developed method was used to screen total antioxidant capacity (TAC) of some commercial fruit juices and herbal teas without preliminary treatment, and showed a promising potential for the preparation of antioxidant inventories of a wide range of food plants.

Determination of antioxidants by a novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) assay with post-column detection.

A novel on-line HPLC-cupric reducing antioxidant capacity (CUPRAC) method was developed for the selective determination of polyphenols (flavonoids, simple phenolic and hydroxycinnamic acids) in complex plant matrices. The method combines chromatographic separation, constituent analysis, and post-column identification of antioxidants in plant extracts. The separation of polyphenols was performed on a C18 column using gradient elution with two different mobile phase solutions, i.e., MeOH and 0.2% o-phosphoric acid. The HPLC-separated antioxidant polyphenols in the extracts react with copper(II)-neocuproine (Cu(II)-Nc) reagent in a post-column reaction coil to form a derivative. The reagent is reduced by antioxidants to the copper(I)-neocuproine (Cu(I)-Nc) chelate having maximum absorption at 450 nm. The negative peaks of antioxidant constituents were monitored by measuring the increase in absorbance due to Cu(I)-Nc. The detection limits of polyphenols at 450 nm (in the range of 0.17-3.46 microM) after post-column derivatization were comparable to those at 280 nm UV detection without derivatization. The developed method was successfully applied to the identification of antioxidant compounds in crude extracts of Camellia sinensis, Origanum marjorana and Mentha. The method is rapid, inexpensive, versatile, non-laborious, uses stable reagents, and enables the on-line qualitative and quantitative estimation of antioxidant constituents of complex plant samples.

Solvent effects on the antioxidant capacity of lipophilic and hydrophilic antioxidants measured by CUPRAC, ABTS/persulphate and FRAP methods.

Antioxidants are health beneficial compounds that can protect cells and macromolecules (e.g., fats, lipids, proteins, and DNA) from the damage of reactive oxygen species (ROS). Solvent effect is a crucial parameter on the chemical behaviour of antioxidant compounds but there has been limited information regarding its role on antioxidant capacity and its assays. Therefore, the present study was undertaken to investigate the total antioxidant capacity (TAC) of some certain lipophilic and hydrophilic antioxidants, measured in different solvent media such as ethanol (EtOH) (100%), methanol (MeOH) (100%), methanol/water (4:1, v/v), methanol/water (1:1, v/v), dichloromethane (DCM)/EtOH (9:1, v/v). The cupric reducing antioxidant capacity (CUPRAC) values of selected antioxidants were experimentally reported in this work as trolox equivalent antioxidant capacity (TEAC), and compared to those found by reference TAC assays, i.e., 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)/persulphate (ABTS/persulphate) and ferric reducing antioxidant power (FRAP) methods. The TAC values of synthetic mixtures of antioxidants were experimentally measured as trolox equivalents and compared to those theoretically found by making use of the principle of additivity of absorbances assuming no chemical interaction between the mixture constituents. Possible synergistic (e.g., BHT and BHA in DCM/EtOH) or antagonistic behaviours of these synthetic mixtures were investigated in relation to solvent selection.

Hydroxyl radical detection with a salicylate probe using modified CUPRAC spectrophotometry and HPLC.

Reactive oxygen species (ROS) may attack biological macromolecules giving rise to oxidative stress-originated diseases, so it is important to establish efficient methods to screen hydroxyl radical scavengers for antioxidant therapy. Since *OH is very short-lived, secondary products resulting from *OH attack to various probes are measured. As a low-cost measurement technique, we used a salicylate probe for detecting hydroxyl radicals generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide. The produced hydroxyl radicals attacked both the probe and the water-soluble antioxidants in 37 degrees C-incubated solutions for 2 h. The CUPRAC (cupric ion reducing antioxidant capacity) assay absorbance of the ethylacetate extract due to the reduction of Cu(II)-neocuproine reagent by the hydroxylated probe decreased in the presence of *OH scavengers, the difference being proportional to the scavenging ability of the tested compound. Attack by *OH radicals upon salicylate produced 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, and 2,5-dihydroxybenzoate as major products. HPLC separation combined with CUPRAC spectrophotometry was used to identify and quantify hydroxylated salicylate derivatives in the presence of synthetic water-soluble antioxidants and green tea infusion. The developed spectrophotometric method for *OH detection was validated with HPLC, i.e., the concentrations of dihydroxybenzoates produced by radical attack from the probe were determined by HPLC, and the sum of (concentrationxabsorptivity) products of these components approximately agreed with the experimentally found CUPRAC absorbances, confirming the validity of Beer's law for the selected system. Statistical comparison of the results found with the proposed methodology and HPLC was made with two-way ANOVA (analysis of variance) test. Under optimal conditions, about 53% of the probe (salicylate) was converted into dihydroxybenzoate isomers in the absence of *OH scavengers, and these isomers were more specific markers of hydroxyl radicals than the non-specific malondialdehyde end-product of the TBARS test. Thus, the more costly and less speedy HPLC method could advantageously be substituted with the proposed spectrophotometric assay of *OH detection, which was also of much higher yield than the TBARS colorimetric assay.

The cupric ion reducing antioxidant capacity and polyphenolic content of some herbal teas.

The total antioxidant capacity of the aqueous extracts of some endemic herbs-prepared as infusions by steeping these herbs in hot water--was assayed with bis(neocuproine)copper(II) chloride, also known as the cupric ion reducing antioxidant capacity (CUPRAC) reagent, which was easily accessible, rapid, stable and responsive to both hydrophilic and lipophilic antioxidants. The highest antioxidant capacities of some herbal teas available in the Turkish market were observed for scarlet pimpernel (Anagallis arvensis), sweet basil (Ocimum basilicum), green tea (Camellia sinensis) and lemon balm (Melissa officinalis), in this order (1.63, 1.18, 1.07, and 0.99 mmol trolox equivalent (TR)/g, respectively). For infusions prepared from ready-to-use tea bags, the CUPRAC values were highest for Ceylon blended ordinary tea (4.41), green tea with lemon (1.61), English breakfast ordinary tea (1.26) and green tea (0.94), all of which were manufactured types of C. sinensis. Following the strongest antioxidant herbs with capacities close to or slightly exceeding 1.0 mmol TR/g, sage, thyme, coriander, coltsfoot, blackberry and immortelle (Helichrysum) exhibited capacities around 0.5 mmol TR/g. The correlation of the Folin total phenolic content of herbal teas with their CUPRAC and ABTS (2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) total antioxidant capacities gave linear curves with correlation coefficients of 0.966 and 0.936, respectively, showing that the CUPRAC assay results better correlated with total phenolic content of herbal teas. Absorbance versus concentration data at different dilutions and upon standard additions of model antioxidant compounds (trolox and quercetin) to herbal tea infusions showed that the absorbances (at 450 nm of the CUPRAC method) due to different antioxidant compounds in herbal tea infusions were additive; that is, the tested antioxidants did not chemically interact to cause apparent deviations from Beer's law.