Biochemical and functional properties of indigenous Australian herbal infusions

The phytochemical profile, organic acid content, minerals, various antioxidant assays and consumers acceptability of indigenous Australian herbal infusions namely gulban (Melaleuca citrolens), anise myrtle (Syzygium anisatum), and lemon myrtle (Backhousia citriodora) were compared with a commercial green tea (Camellia sinensis). Total phenolic content and catechin derivatives were higher in green tea as compared to indigenous herbal infusions (P < 0.05). Phytochemical profiles showed high levels of caffeine in green tea, but, it was not found in herbal infusions (P < 0.05). Australian indigenous herbal infusions were a good source of calcium and magnesium compared to green tea (P < 0.05). Oxalic acid was higher in green tea, whereas gulban and anise myrtle infusions were rich in citric acid (P < 0.05). Antioxidant activities of green tea and gulban herbal infusions were comparable (P ≥ 0.05). Overall liking scores were higher for herbal infusions compared to green tea (P < 0.05). Indigenous Australian herbal infusions particularly gulban has a potential to become a successful commercial herbal beverage

Reversible Keap1 inhibitors are preferential pharmacological tools to modulate cellular mitophagy

Mitophagy orchestrates the autophagic degradation of dysfunctional mitochondria preventing their pathological accumulation and contributing to cellular homeostasis. We previously identified a novel chemical tool (hereafter referred to as PMI), which drives mitochondria into autophagy without collapsing their membrane potential (ΔΨm). PMI is an inhibitor of the protein-protein interaction (PPI) between the transcription factor Nrf2 and its negative regulator, Keap1 and is able to up-regulate the expression of autophagy-associated proteins, including p62/SQSTM1. Here we show that PMI promotes mitochondrial respiration, leading to a superoxide-dependent activation of mitophagy. Structurally distinct Keap1-Nrf2 PPI inhibitors promote mitochondrial turnover, while covalent Keap1 modifiers, including sulforaphane (SFN) and dimethyl fumarate (DMF), are unable to induce a similar response. Additionally, we demonstrate that SFN reverses the effects of PMI in co-treated cells by reducing the accumulation of p62 in mitochondria and subsequently limiting their autophagic degradation. This study highlights the unique features of Keap1-Nrf2 PPI inhibitors as inducers of mitophagy and their potential as pharmacological agents for the treatment of pathological conditions characterized by impaired mitochondrial quality control

Modeling and optimizing microwave-assisted extraction of antioxidant compounds from marigold (Calendula offieinalis L.) using response surface methodology

Calendula officinalis L. is a commercially important plant that finds application in the treatment of various diseases in traditional medicine. The total antioxidant capacity, radical scavenging activity, and total phenolic content of marigold extracts were investigated by Folin, CUPRAC, and DPPH assays, respectively. The optimum operating conditions of microwave-assisted extraction (MAE) including temperature, extraction time, solvent-to-solid ratio, and solvent concentration were ascertained by employing response surface methodology (RSM). The solvent (ethanol) concentration was the most significant operating factor among all responses of MAE. At the optimum extraction conditions, the maximum yield of total phenolic content, total antioxidant capacity, and radical scavenging activity obtained experimentally were very close to their predicted values, thus showing the suitability of the model used and the success of RSM in optimizing the extraction conditions. Chromatographic analysis of marigold extract was performed by UPLC-PDA-ESI-MS/MS system and chlorogenic acid was the main component (1742.50 +/- 42.23 mu g/g DS)

A Novel Spectrofluorometric Probe for the Determination of Peroxynitrite Anion Scavenging Activity of Biothiols and Amino Acids

In this study, a novel fluorometric method for the determination of peroxynitrite anion (ONOO-) scavenging (PAS) activity of amino acids and biothiols, which can mostly trap peroxynitrite in vivo, is described. This assay is based on the conversion of a gentisic acid probe to its non-fluorescent oxidation products with ONOO-. The attenuation of the fluorescence intensity (PI) of the probe upon peroxynitrite attack is diminished with antioxidants, the difference in FI being related to the PAS activity of the antioxidants. The IC50 (50% inhibitive concentration) values of biothiols, amino acids and tissue homogenates were estimated, in comparison with the reference Pyrogallol Red (PR) bleaching method. PR is the most suitable and frequently used dye to determine PAS activity, but is relatively insensitive. The developed fluorometric assay is highly sensitive to allow determinations of the PAS activity of amino acids

A novel colorimetric sensor for measuring hydroperoxide content and peroxyl radical scavenging activity using starch-stabilized gold nanoparticles

A novel colorimetric nanosensor was developed for evaluating peroxyl radical scavenging activity of phenolic antioxidants and for the detection of hydroperoxides formed during AAPH-induced oxidation of linoleic acid emulsions. Starch was used as a green reduction/stabilization agent for gold nanoparticles (AuNPs) synthesis in alkaline medium. When tert-butyl hydroperoxide (ten-BHP) was incubated with an excess of iodide ions in a 37 degrees C water bath for 90 min, triodide (I-3(-)) was formed in an amount equivalent to tert-BHP concentration. Upon the addition of starch-stabilized gold nanoparticles (ss-AuNPs) solution to the incubation mixture, triiodide ions were rapidly adsorbed on the surface of AuNPs and caused their aggregation. A concomitant red shift (from 525 nm to 563 nm) of surface plasmon resonance (SPR) absorption of the nanoparticles was observed, absorbance linearly increasing with aqueous tert-BHP concentration. The method provided an LOD of 39 mu M for tert-BHP, and was validated through linearity, precision and accuracy. The concentration of hydroperoxides estimated in linoleic acid peroxidation correlated well with those found by the reference ferric thiocyanate assay. Peroxyl radical scavenger antioxidants decreased the red-shifted SPR absorption of aggregated ss-AuNPs, thereby enabling an indirect estimation of antioxidant activity. This AuNPs-based colorimetric sensor is the first of its kind to directly determine peroxyl radical scavenging activity of polyphenols. The half-maximal inhibitive concentrations (IC50) of selected antioxidant compounds were calculated by utilizing the decrease in absorbance with increasing concentration of scavengers, and compared to those of classical oxygen radical absorbance capacity (ORAC) assay. The proposed nanosensor was superior over FL-based ORAC in determining the peroxyl radical scavenging activity of the lipophilic antioxidant alpha-tocopherol. The percentage scavenging of real samples such as green tea infusion and synthetic serum were determined. The proposed assay can be used for estimating the peroxyl scavenging of various food and biological samples in terms of its low cost, ease of use and compatibility

Novel spectroscopic sensor for the hydroxyl radical scavenging activity measurement of biological samples

A novel spectroscopic sensor was developed and validated for hydroxyl radical scavenging (HRS) activity estimation using terephthalate (TP) as probe. This sensor was designed by electrostatic immobilization of the chromogenic oxidizing agent of the CUPric Reducing Antioxidant Capacity (CUPRAC) method, Cu(II)-Neocuproine (Cu(II)-Nc) complex, on a Nation cation-exchange membrane, and the spectrophotometric assay developed in aqueous-alcoholic solutions was integrated to the CUPRAC sensor. Hydroxyl radicals ((OH)-O-center dot) generated from an equivalent mixture of Fe(II)+EDTA with hydrogen peroxide attacked both the probe and the (OH)-O-center dot scavengers in 37 degrees C-incubated solutions for 1/2 h. The HRS activity was measured using the decrease in CUPRAC absorbance at 450 nm - arising from the reduction of Cu(II)-Nc reagent to the Cu(I)-neocuproine chelate - of the hydroxylated probe (TP) undergoing radical attack in the presence of (OH)-O-center dot scavengers. The HRS activity was evaluated as the second-order rate constants of biologically active compounds for (OH)-O-center dot scavenging and also as the percentage scavenging of a measured compound or sample relative to a reference compound. Using this reaction, a kinetic approach was adopted to assess the HRS activity of amino acids, plasma- and thiol-antioxidants. This assay, applicable to small molecule antioxidants and tissue homogenates, proved to be efficient for serine and albumin for which the widely used TBARS (thiobarbituric acid-reactive substances) test is nonresponsive. Under optimal conditions, about half of the probe (TP) was converted into 2-hydroxyterephthalate (hTP), and this monohydroxylated derivative, being the only product of hydroxylation, was a more specific marker of (OH)-O-center dot than the non-specific malondialdehyde end-product of the TBARS test. The sensor gave a linear response to scavenger concentration in the competition kinetic equation. (C) 2012 Elsevier B.V. All rights reserved

tert-Butylhydroquinone as a Spectroscopic Probe for the Superoxide Radical Scavenging Activity Assay of Biological Samples

As a more convenient and less costly alternative to electron spin resonance (ESR) and nonspecific nitroblue tetrazolium (NET) and cytochrome c assays of superoxide radical (SR, O-2(center dot-)) detection, a novel probe, tert-butylhydroquinone (TBHQ), is introduced for SR nonenzymatically generated in the phenazine methosulfate-beta-nicotinamide adenine dinucleotide (PMS-NADH) system. SR attacks both TBHQ and SR scavengers incubated in solution for 30 min where scavengers compete with TBHQ for the O-2(center dot-) produced. TBHQ but not its O-2(center dot-) product, tert-butyl-1,4-benzoquinone (TBBQ), is responsive to the CUPRAC (cupric reducing antioxidant capacity) spectrophotometric assay. The CUPRAC absorbance of the ethyl acetate extract of the incubation solution arising from the reduction of Cu(II)-neocuproine reagent by the remaining TBHQ was higher in the presence of O-2(center dot-) scavengers (due to less conversion to TBBQ), the difference being correlated to the SR scavenging activity (SRSA) of the analytes. With the use of this reaction, a kinetic approach was adopted to assess the SRSA of amino acids, vitamins, and plasma and thiol antioxidants. This assay, applicable to small-molecule antioxidants and tissue homogenates, proved to be efficient for cysteine, uric acid, and bilirubin, for which the widely used NBT test is nonresponsive. Thus, conventional problems of NBT assay arising from formazan insolubility and direct reduction of NBT by tested scavengers were overcome

Resorcinol as a Spectrofluorometric Probe for the Hypochlorous Acid Scavenging Activity Assay of Biological Samples

A novel spectrofluorometric method was developed and validated for hypochlorous acid (Hod) scavenging activity estimation using resorcinol, which is a highly sensitive and chemically stable fluorogenic probe. This assay is based on the chlorination of resorcinol to its nonfluorescent products in the presence of HOCl. HOCl reacts with both resorcinol and HOCl scavengers incubated in solution for 10 min, where scavengers compete with resorcinol for the HOCl. Thus, the relative increase in fluorescence intensity of intact resorcinol is proportional to the antioxidative activity of HOCl scavengers. Using this reaction, a kinetic approach was adopted to assess the HOCl scavenging activity of amino acids, vitamins, and plasma and thiol antioxidants. This assay, which is applicable to small molecule antioxidants and tissue homogenates, proved to be efficient for thiol-type antioxidants for which the widely used 5-thio-2-nitrobenzoic acid (TNB) test is not accurately responsive. Thus, conventional problems of the TNB assay arising from the reactivity of thiol-type scavengers to produce extra TNB by direct reduction of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were overcome. Moreover, unlike enzymatic assays (e g, elastase), there is no confusion as to whether the putative scavenger actually reacts with HOCl or inhibits the enzyme

A novel hypobromous acid scavenging activity assay using p-cresol as a spectrofluorometric probe

In this study, a novel spectrofluorometric assay based on p-cresol (4-methyl phenol) probe is developed for the measurement of HOBr scavenging activity. It is the first study involving the use of a p-cresol probe for the determination of the HOBr scavenging activity of biothiols. While the p-cresol probe (lambda(ex) = 260 nm, lambda(em) = 305 nm) has fluorescence characteristics, its brominated derivatives emerging at the end of the oxidation reaction with HOBr do not show fluorescence. The initial fluorescence intensity of the p-cresol probe is decreased in the presence of the brominating agent, HOBr, and this decrease is lower in the presence of HOBr scavenging antioxidants. The scavenging activities of biothiols tested with respect to the developed method decrease in the following order: penicillamine > N-acetyl cysteine > L-glutathione (reduced) > cysteamine > homocysteine > glutathione ethyl ester > cysteine > 1,4-dithiothreitol > lipoic acid > methionine. Penicillamine (IC50 = 10.12 mu M) was the most effective HOBr-scavenger among the tested biothiols. The results obtained with the developed method for biothiols and some pharmaceutical samples were statistically compared (using ANOVA) to those found by the reference methods (KI/taurine and UPLC). The advantage of the proposed method over the KI/taurine assay was demonstrated