The present paper aims at reviewing and commenting on the analytical methods applied to antioxidant and antioxidant capacity assessment in plant-derived products. Aspects related to oxidative stress, reactive oxidative species' influence on key biomolecules, and antioxidant benefits and modalities of action are discussed. Also, the oxidant-antioxidant balance is critically discussed. The conventional and nonconventional extraction procedures applied prior to analysis are also presented, as the extraction step is of pivotal importance for isolation and concentration of the compound(s) of interest before analysis. Then, the chromatographic, spectrometric, and electrochemical methods for antioxidant and antioxidant capacity determination in plant-derived products are detailed with respect to their principles, characteristics, and specific applications. Peculiarities related to the matrix characteristics and other factors influencing the method's performances are discussed. Health benefits of plants and derived products are described, as indicated in the original source. Finally, critical and conclusive aspects are given when it comes to the choice of a particular extraction procedure and detection method, which should consider the nature of the sample, prevalent antioxidant/antioxidant class, and the mechanism underlying each technique. Advantages and disadvantages are discussed for each method.  In the course of molecular oxygen stepwise reduction, a series of reactive oxygenated species occur –3. Reactor HClO) –7.Free radicals generated in aerobic metabolism are involved in a series of regulatory processes such as cell proliferation, apoptosis, and gene expression. When generated in excess, free radicals can counteract the defense capability of the antioxidant system, impairing the essential biomolecules in the cell by oxidizing membrane lipids, cell proteins, carbohydrates, DNA, and enzymes. Oxidative stress results in cytotoxic compounds occurrence  and alters the oxidant-antioxidant balance (redox homeostasis) that characterizes normal cell functioning –4.α- and β-dicarbonyls may also result during this glycolaldehyde autooxidation [•, as one of the most active radical species, and HO2∙ attack lipid substrates (L-H), yielding the corresponding lipid radicals L•. The attack on polyunsaturated fatty acids by singlet oxygen can yield lipid peroxides [With respect to alteration in the protein structure, amino acid oxidation, free radical-induced cleavage, and cross-linking due to reaction with lipid peroxidation products may occur . In nuclxidation . Peroxideroxides , 13.In recent studies, it has been repeatedly asserted that oxidative stress not only is not limited to free radical-induced damage on biomolecules but also involves perturbation of cellular redox status, which has been described as “a disruption in redox signaling and control”; hence the antioxidant system implies more than mere free radical capture –17.Oxidative stress-induced pathology includes cancer , 19, car Initiation:LH is the lipid substrate, R• is the initiating oxidizing radical, and L• is the allyl radical endowed with high reactivity. Propagation:So, during this step, the lipid peroxyl radicals LOO• act as chain carriers, further oxidizing the lipid substrate and generating lipid hydroperoxides (LOOH), which can decompose into alcohols, aldehydes, alkyl formates, ketones, hydrocarbons, and radicals such as lipid alkoxyl radical LO• [ Branching:The decay of lipid hydroperoxides often takes place in the presence of transition metal ions, generating lipid peroxyl and lipid alkoxyl radicals:Termination implies the combination of radicals to form nonradical chemical species:The antioxidant action mechanism cannot be understood without describing the model lipid peroxidation in cell membranes or foodstuffs, a radical mechanism that these biomolecules undergo, with initiation, propagation, and chain termination stages, which is promoted by heat, light, and ionizing radiation or by metal ions or metalloproteins –31. Initical LO• . BranchiAntioxidants can act as chain breakers, scavenging chain initiating radicals like hydroxyl, alkoxyl, or peroxyl, quenching singlet oxygen, decomposing hydroperoxides, and chelating prooxidative metal ions , 33. EpiChain breaking antioxidants able to scavenge radical species are called primary antioxidants. Secondary antioxidants are singlet oxygen quenchers, peroxide decomposers that yield nonradical species, oxidative enzyme  inhibitors, UV radiation absorbers, or compounds that act by metal chelating –40. endogenous or exogenous.Natural antioxidants constitute the essential part in the cell's defense mechanisms and they can beEndogenous antioxidants can be nonenzymatic, such as glutathione, alpha-lipoic acid, coenzyme Q, ferritin, uric acid, bilirubin, metallothionein, l-carnitine, melatonin, albumin, and antioxidant enzyme cofactors, or enzymatic, such as superoxide dismutase, catalase, glutathione peroxidases, thioredoxins, and peroxiredoxins. Peroxiredoxins regulate cytokine-induced peroxide levels and mediate cell signal transduction [sduction .Enzymatic antioxidants at their turn are grouped within the primary and secondary defence systems. The primary defence is formed by three crucial enzymes capable of preventing the occurrence or neutralizing free radicals: glutathione peroxidase, which donates two electrons that reduce peroxides, catalase that decomposes hydrogen peroxide into water and molecular oxygen, and superoxide dismutase that turns superoxide anions into hydrogen peroxide , 41. The exogenous, diet-sourced antioxidants [Apart from the endogenous, enzymatic, and nonenzymatic antioxidants previously discussed, there are alsooxidants , 43, repoxidants –47.The intake of antioxidants from diet is always meant to counterpart the organism's antioxidant defense. Enzymic natural antioxidants in food  can be inactivated during processing.Particularly plant-sourced low-molecular weight antioxidants such as glutathione and ascorbate are synthesized within the chloroplast stroma and the cytosol in the presence of reduced coenzyme molecules (NADPH) acting as the final electron source . These lAlthough review papers have been previously published on antioxidant activity in plants, the present paper provides a novel way of gathering and also critically and comparatively presenting these aspects. The section devoted to critical and conclusive aspects provides the reader with an original discussion over extraction techniques and their comparison, as well as methods' performances (in a way that has not been systematized until now), with the following aspects concerned: sample, mechanism underlying the method, working parameters, and detection.Extraction techniques aim not only at extracting the active biocompounds from the plant sample but also at imparting selectivity and optimizing sensitivity of the applied analytical methodology due to the increase of the concentration of the compound of interest. The biocompound is more easily detected and separated from other matrix components, and the assay becomes independent on the variable matrix characteristics .Classical extraction techniques are based on the extractive potential of various solvents, using heating or mixing. The main shortcomings of conventional extraction are long extraction times, the need for high purity expensive solvents, evaporation of solvents in significant amounts, reduced selectivity, and, finally, the thermal decomposition in the case of thermolabile substances . These pAn important goal is represented by high extraction efficiency and efficacy. Efficiency was defined as the yield of extraction, whereas efficacy represents the potential to induce bioactivity and the ability to produce an effect. Therefore, a selection of the most appropriate extraction method is required in each case, as it was proven that various techniques applied on the same plant material employing the same solvent can lead to different extraction efficiencies. Moreover, it has been confirmed that the most convenient method in this regard requires standardization to attain reproducibility .Soxhlet extraction was first applied only for lipid extraction, but its use has been extended for extracting active principles. The solvent is heated, vaporized, and condensed and extracts the interest compound(s) by contact with the sample-containing thimble. When the solvent in the extraction chamber reaches the overflow level, the solution in the thimble-holder is aspirated by a siphon and returns in the distillation flask. Significant extraction yields can be reached, with a small solvent amount. It can be applied in batch at small scale, but it can be converted into a continuous extraction set-up on medium or large scale [ge scale .Maceration is applied to obtain essential oils and bioactive compounds. The plant material is ground to improve the surface area. The solvent is then added and allowed to stand at ambient temperature for several days, and the mixture is subject to frequent stirring until dissolution. The damped material is then pressed and then the liquid is purified by filtration or decantation [antation , 56.Hydrodistillation  is applied to the extraction of bioactive compounds and essential oils from plants, generally prior to dehydration, and does not imply the use of organic solvents [solvents . Hot watsolvents . Hydrodisolvents , 56.Infusions are prepared by shortly macerating the raw plant material with either cold or boiling water. It is often mentioned that concentrated infusions are the result of a modified percolation or maceration procedure [rocedure .Percolation is a recognized procedure applied for the preparation of tinctures and fluid extracts and makes use of a cone-shaped vessel opened at both ends (percolator). The solid material is moistened with an adequate amount of the appropriate solvent (menstruum) and left for about 4 h. Solvent amount is necessary, until the percolate represents about three-quarters of the quantity corresponding to the final product. The marc is then pressed and the eliminated liquid is added to the percolate. Solvent is again added to get the required volume, and the liquid mixture is clarified by filtration or by decanting [ecanting . decoction process, the crude plant material is subject to boiling in an appropriate water amount, for a well-defined period, followed by cooling and then straining or filtering. This approach is adequate for the extraction of hydrosoluble, thermostable components, being popular for obtaining Ayurvedic extracts [In theextracts .Cold pressing or expression consists in pressing or grinding fruits or seeds by using a press. Oil release is possible due to crushing or breaking of essential oil glands in the peel. Olive, peanut, sunflower, and citrus oils are obtained through cold pressing, which results in preserving flavor, aroma, and nutritional value.Aqueous Alcoholic Extraction by Fermentation. The formed ethanol enables extraction of the active principles from the material and also contributes to preserving the product's qualities. In Ayurveda, this method is not standardized, but, with progresses in the fermentation technology, standardization would be of use for obtaining herbal drug extracts [extracts .Vortex apparatus is commonly used to mix the interest plant sample with the dilutant. It is applied for dissolution, namely, in aqueous environment and polar solvents, of samples of plants to yield a fluid and homogeneous solution subject to analysis. As in the case of other techniques like shaking or sonication, it can be followed by centrifugation, with use of the supernatant.Supercritical Fluid Extraction (SFE). Critical point was defined as the temperature and pressure above which distinction between gas and liquid phases does not exist [2 use does not imply high costs. It operates at room temperature, so it is adequate for thermosensitive compounds; smaller samples can be extracted compared with conventional solvent extraction. It is characterized by facility of coupling with chromatographic procedures and reutilization of SCF [ot exist . In supeot exist . The advot exist . CO2 usen of SCF , 56. DisSolid Phase Microextraction (SPME). SPME employs a sorbent, which usually coats the surface of small fibers, for the isolation and concentration of target compounds from the sample and is applied to quantitative assay of analytes  in aqueous or gaseous phase.Microwave-Assisted Extraction (MAE). Microwaves interact with the dipoles of polar and polarizable matrixes [matrixes , 62. As matrixes , 63. Thematrixes . When thmatrixes . Separatmatrixes , enhancematrixes , 66.Ultrasound-Assisted Extraction (UAE). Ultrasound waves with frequencies comprised between 20 kHz and 100 MHz induce compression and expansion as they pass through the extractable plant matrix, producing cavitation. The energy produced can promote the conversion of kinetic energy into thermal one, inducing heating of the bubble contents. In solid plant samples, ultrasounds enable compound leaching from the plant materials [aterials . The mecaterials . The phyaterials , 70. Rapaterials . Neverthaterials .Pulsed-Electric Field (PEF) Extraction. Living cells are suspended in an electric field, and an applied potential crosses the membrane. The electric potential induces molecule separation according to the molecular charge. At values greater than 1 V for the transmembrane potential, the electrostatic repulsion between the charged molecules results in pore generation in the membrane and produces dramatic permeability increase, and yield is optimized [ptimized , 72. Theptimized . Pulsed-ptimized . It can ptimized .Enzymatic Treatment. Enzymes used are cellulase, α-amylase, and pectinase, which act by breaking the cellular wall, with subsequent hydrolysis of the structural polysaccharides and lipids [d lipids , 77. Enzd lipids . Enzyme d lipids . Enzyme-d lipids . The enzd lipids . In enzyd lipids .Pressurized Liquid Extraction (PLE). PLE implies exerting an elevated pressure to the remaining liquid solvent above the boiling point. High pressure values favor the extraction process, which is easily prone to automation. Pressurized liquid extraction benefits much shorter extraction times and lower solvent requirements, when compared to conventional Soxhlet extraction. At elevated temperatures and pressures, the extraction performances are improved by the increased analyte solubility and mass transfer rate, as well as by the diminished viscosity and low surface tension of solvents [solvents , 83.The investigation of performant analytical methods aiming to assess the antioxidant capacity in plants and plant extracts remains a constant goal and a series of classifications have been proposed. Antioxidant measurement techniques were classified as methods based on the inhibition of low-density lipoprotein oxidation estimation and the ones relying on the quantification of the free radical scavenging capacity .• scavenging, the phosphomolybdenum method, and beta-carotene linoleate bleaching are applied in vitro, while the lipid peroxidase, catalase, and glutathione peroxidase activity assays are techniques used in vivo [Considering the mechanism underlying the antioxidant–oxidant reaction, the methods were also divided in hydrogen atom transfer (HAT) and single electron transfer (SET) techniques. HAT-based methods measure the capacity of an antioxidant to trap free radicals by hydrogen donation, while SET methods rely on the one electron transfer reductive ability of an antioxidant compound versus a radical species . ORAC, T in vivo . The anaThe main chemical processes underlying antioxidant activity assay (a–d) and lipid oxidation status evaluation (e) are detailed in In  Bergia suffruticosa  proved antiradical activity by bleaching DPPH•. This free radical scavenging activity was assigned to the high tannin and phenolic amounts [• assay allowed for the swift detection of the antioxidant potential of nine out of ten tested polyphenols (except for apigenin 7-O-glucoside), present in five analysed plant species: Hypericum perforatum L., Matricaria recutita L., Achillea millefolium L., Thymus vulgaris L., and Salvia officinalis L. By LC–MS, the presence of compounds previously identified by TLC was confirmed. Four other compounds (caffeic acid and apigenin in St. John wort and apigenin and apigenin 7-O-glucoside in sage) have been identified. Their presence was not revealed by TLC and it has been stated that their low level in the plant samples could be the reason [Thin layer chromatograms of the methanolic extract of amounts . A recene reason .Sonneratia caseolaris (astringent and antiseptic) extracts were tested for their antioxidant composition: column chromatography with a Diaion HP-20 column and successive elution with methanol and acetone was first applied. The chlorophyll-free eluate was separated into 5 fractions by C18 column chromatography, with methanol and acetone for elution. The methanol-eluted fraction containing DPPH positive spots was then applied to a silica gel presqualene column with n-hexane-acetone–methanol eluents, resulting in eight fractions. The first compound was obtained after precipitation from the fraction corresponding to the n-hexane-acetone 1 : 1 eluate. One acetone-eluted fraction also yielded a precipitate, which after washing with methanol resulted in the second compound. The structures of the isolated compounds were assessed by one-dimensional and two-dimensional NMR and mass spectroscopy. Moreover, both showed positive (discolored) spots with a reddish purple background on the thin layer chromatogram, using a 0.02% (w/v) methanolic solution of DPPH as spray reagent. Luteolin and luteolin-7-O-β-glucoside were identified as the two bioactive antioxidant and anti-inflammatory compounds [ompounds . Plantago lanceolata. The best eluent composition was determined: in first step of development, the mobile phase contained hexane, diisopropyl ether, and formic acid 90% (6.0 : 4.0 : 0.5) v/v. In the second and third steps, a mixture of hexane, diisopropyl ether, dichloromethane, formic acid 90%, and propan-2-ol (6.0 : 4.0 : 2.0 : 1.0 : 0.1) v/v was employed. The application of this HPTLC technique with area measurements at 320 nm led to a caffeic acid amount equal to 99.3 μg/g of dried plant, with RSD of 3.19% [High performance thin layer chromatography combined with densitometry was applied for caffeic acid quantitation inof 3.19% . Scoparia dulcis, known for many health benefits [HPTLC  was alsobenefits   Gas Chromatography. The composition of various extracts of Merremia borneensis was assessed by GC-MS, showing the presence of flavonoids, terpenoids, alkaloids, and glycosides in the analysed organic crude extracts [ Datura metel was performed in crude extracts by GC/MS, revealing abundancy of high-molecular weight components such as polyphenols, flavonoids, triterpenoids, and hydrocarbons. The phenolic level was expressed as gallic acid equivalents, with chloroform having the best extractive potential, followed by methanol, butanol, ethyl acetate, and hexane. It has been concluded that the chloroform crude extract had the highest phenolics amount and its potential as antibiotic has been stated [extracts . The quan stated . Ajuga bracteosa and Lavandula dentata obtained by hydrodistillation were analysed by GC and GC/MS. 47 and 48 biocomponents were identified for the two analysed plants, respectively. The oils contained high amounts of oxygenated monoterpenes (34 to 51%). Borneol (20.8%) and hexadecanoic acid (16.0%) were the major compounds present in the oil of A. bracteosa, which also contained aliphatic acids (30.3%). Camphor (12.4%), trans-pinocarveol (7.5%), and β-eudesmol (7.1%) were prevalent in Lavandula dentata oil. The antioxidant activity of the oil extracts was confirmed by DPPH• scavenging assay [Essential oils from the aerial parts ofng assay .(2) Liquid Chromatography. The rapid and resolution-high determination of six bioactive flavonoids present in the pericarp of Citri reticulata has been performed by liquid chromatography/electrospray ionization coupled with mass spectrometry. The chromatographic system used a C18 column and a 0.1% formic acid/acetonitrile mobile phase with a gradient elution. Naringin, hesperidin, nobiletin, 3,5,6,7,8,3′,4′-heptamethoxyflavone, tangeritin, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone were assessed by the above-mentioned chromatographic technique and were also investigated for their antiproliferative activities by Cell Counting Kit-8 Assay. In the cultivars analysed, hesperidin presented the highest content, ranging from 50.137 to 100.525 mg/g. The levels of nobiletin, tangeritin, and 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone were higher in the peel of Citrus reticulata “Chachi” than in other cultivars. With respect to the antiproliferative activity against A549 and HepG2 cells, 5-hydroxy-6,7,8,3′,4′-pentamethoxyflavone has been proven to be the most effective [Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots, and leaves of dill (Anethum graveolens) extracts, relying on the antioxidant compounds' specific oxidation. It has been confirmed that the method is characterized by sensitivity and simplicity of detection, since no additional instrumentation (reagent pump or secondary detector) is necessary. In comparison to the results obtained using reversed-phase chromatographic separation with online postcolumn DPPH scavenging detection, HPLC-ED provided much richer chromatographic profiling of celery leaves extracts. At elevated electrooxidation potential values higher than 700 mV, compounds that are electroactive contribute to HPLC-ED detection but are missed in the postcolumn DPPH scavenging [Chromatography followed by electrochemical detection proved its viability in the assessment of onion . Nevertheless, it was stated that in this case the correlation between the antioxidant activity of analysed extracts and their phenolic composition is very difficult to be described by statistical tools. It was assumed that this difficulty may result not only from the fact that total phenolics do not include all the antioxidants but also from the synergism and structure interaction among the antioxidants, which does not always involve concentration influence. For instance, samples such as Artemisia arborescens and Oudneya africana, with close concentration values of total phenolics, exhibited varying antioxidant activity. On the whole, the antioxidant activity and flavonoids concentration did not correlate significantly in comparison to hydroxycinnamic acids and hydroxybenzoic acids. Artemisia campestris was assessed as the most powerful inhibitor of radical-induced red blood cells hemolysis, more active than caffeic acid, more than three times more active than ascorbic acid, and two times more active than α-tocopherol. The UV spectra were obtained in the range of 220–600 nm and the amounts of phenolics in the extracts were assessed from the calibration curves developed at the absorption maxima of each phenolic class [Eleven Algerian medicinal plants were subject to analysis for their antioxidant capacity and phenolic profile. The HPLC results revealed that the hydroxycinnamic acid derivatives were the predominant phenolics of the extracts endowed with best antioxidant activity . For all extracts, the carnosol content ranged from 5.4 to 25.5 mg/g, and the carnosic acid level ranged from 3.8 to 115.8 mg/g [Methanolic extracts of the leaves of5.8 mg/g .The phenolic ingredients in samples of 24 cereal grains were analysed by HPLC, relying on the peak area of maximum absorption wavelength. The chromatographic setup was comprised of a C18 column, a mobile phase with an elution gradient between solution A (acetic acid-water and methanol) and solution B (methanol and acetic acid-water solution), and a photodiode array detector. Gallic acid, kaempferol, quercetin, galangin, and cyanidin 3-glucoside were found in high amounts in these cereals .μg/g dry weight) was estimated by comparing the peak areas (at 254 nm) of the samples with that of standards, proving the prevalence of caffeic acid: 500.0 μg/g dry weight [HPLC was also applied along with LC-MS for the estimation of polyphenolic compounds from bitter cumin. The amount of phenolic compounds  were excreted and assessed in urine after ingestion. One prevalent monoglucuronide form was detected in urine in masses 10-fold higher than the other two monoglucuronide forms. It was assessed that anthocyanins from strawberries present a linear dose response over ranges of 15–60 mmol. The 24 h urinary recoveries were much more elevated than those reported for most of the other anthocyanins and it has been concluded that pelargonidin-based anthocyanins may be more efficiently absorbed than other anthocyanins . Ocimum basilicum. In the extracts obtained from harvested samples, rutin (665.052 mg/100 g dried plant) and caftaric acid (1595.322 mg/100 g dried plant) were determined in the largest amount. Commercial samples contained hydroxycinnamic acid derivates, dihydroxybenzoic acid, flavonols, and flavonoid glycosides [18 phenolic compounds have been analysed by HPLC-MS in harvested and commercial 50% methanolic extracts ofycosides . Salvia maxima and Salvia verde was carried out by HPLC. Methanol was employed for the extraction of the Salvia samples; then filtration (on a 0.45 mm PTFE filter) was performed before injection in the LC-DAD-ESI/MS setup. The mobile phase was comprised of 0.1% (v/v) formic acid and acetonitrile, with the application of linear gradient. The content of phenolics in the analysed samples was assessed through interpolation of the peak area using the calibration curve developed per reference to the rosmarinic acid peak and retention time. The results obtained, as rosmarinic acid equivalent content, ranged from 103 ± 2 µg/g fresh material for S. maxima to 174 ± 2 µg/g fresh material for Salvia verde, with a limit of detection of 3.4 × 10−7 mol L−1 [The determination of rosmarinic acid content of mol L−1 . Andrographis paniculata and in dietary supplements containing this plant. This Ayurveda plant is used for healing purposes  led to the successful detection of antioxidant purine alkaloids  and indole alkaloids  inoses see , hence t−1 monosodium phosphate, with gradient elution. BHA content ranged between 20.1 μg g−1 in rapeseed oil and 55.9 μg g−1 in sesame oil. BHT was only found in blend oil at a level of 21.4 μg g−1. TBHQ amount ranged between 25.4 μg g−1 in rapeseed oil and 47.2 μg g−1 in corn oil [The analysis of phenolic synthetic antioxidants BHA, BHT, and TBHQ in edible oils was carried out by HPLC with UV-VIS detection at 280 nm on the basis of peak area ratios. The mobile phase was composed of methanol and 0.01 mol Lcorn oil . Reversed-Phase High Performance Liquid Chromatography with an UV-VIS multiwavelength detection. The separation was performed on a Hypersil ODS C18 column at ambient temperature. The mobile phase comprised acetonitrile and water with 0.2% H2SO4. The flow rate was established at 0.5 mL/min and gradient elution was applied. Rosmarinic acid was the main phenolic acid found in the unripe seeds. Then, p-coumaric acid was confirmed as the prevalent phenolic in half ripe and full ripe seeds [ Artemisia capillaris extracts proved that the main compounds imparting antioxidant capacity were chlorogenic acid, 3,5-dicaffeoylquinic acid, and 3,4-dicaffeoylquinic acid [A number of 19 phenolic compounds were determined by HPLC, during the ripening of cumin seeds. The phenolic compounds were analysed bype seeds . HPLC annic acid . Spathodea campanulata revealed antioxidant potential of this traditionally used plant against malaria and inflammation due to the presence of bioactive compounds such as verminoside (10.33%) and 1-O-(E)-caffeoyl-beta-gentiobiose (6.58%) [µmol/g [ Bambusa textilis McClure indicated active antiradical fractions, as presented in The HPLC profile of methanolic extracts of (6.58%) . Glucosi [µmol/g . HPLC ch Acacia confusa bark extracts was determined by free radical scavenging against DPPH. The total phenolic content was assessed according to the Folin-Ciocalteu method, using gallic acid as a standard. The scavenging activity exhibited against the DPPH free radical diminished in the following order: 3,4,5-trihydroxybenzoic acid = 3,4-dihydroxybenzoic acid = 3,4-dihydroxybenzoic acid ethyl ester > 4-hydroxy-3-methoxybenzoic acid > 3-hydroxy-4-methoxybenzoic acid > 4-hydroxybenzoic acid = benzoic acid. It has been stipulated that this trend is due to the presence of catechol moieties in 3,4,5-trihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, and 3,4-dihydroxybenzoic acid ethyl ester, which impart antioxidant activity [The antioxidant activity ofactivity . Lycium species were subject to sequential extraction with petroleum ether, ethyl acetate, methanol, n-butanol, and water in a Soxhlet extractor. All the extracts were analysed for their scavenging potential towards the free DPPH• radical by in vitro method. The composition of each extract was also studied for the Folin-Ciocalteu reactive species. It was stressed out that the butanol extracts of both species (Lycium barbarum and Lycium ruthenicum) were endowed with the highest scavenging potential . A linear relationship (correlation) was established between the total phenol content  and the radical scavenging potential [Fruits ofotential . Ocimum basilicum showed that the scavenging effect against the DPPH radical was proportional to the phenolic content, to which flavonoids and caffeic acid derivatives contribute. The DPPH scavenging activity proper to harvested samples  was greater than the one obtained for commercial ones (12.05 and 11.24%), considering the one of BHT as 94.77% [A research dedicated to the antioxidant composition investigation and antioxidant capacity determination ins 94.77% .Allium cepa), parsley (Petroselinum crispum) roots and leaves, celery (Apium graveolens) roots and leaves, and leaves of dill (Anethum graveolens), were subject to analysis for the total phenolic amount and the antioxidant activity assessed by DPPH scavenging. The celery leaves exhibited the highest total phenolic content, namely, 1637.1 mg gallic acid equivalents/100 g, and the highest radical scavenging activity against DPPH [Six spice plant samples, namely, onion  and highest flavonoid level. Hence, it has been asserted that acetone is an efficient solvent for antioxidant extraction. It has been also suggested that the substances with best antioxidant activity in Clusiaceae fruits possess intermediate polarity [In another study, the antioxidant activity ofpolarity .50). The obtained average values of EC50 were 20.1 μL for red and 98.4 μL for white dry wines. The highest EC50 of red dry wines, 26.9 μL (illustrating the lowest antioxidant capacity), was inferior to the one proper to white wines with the most reduced antioxidant capacity, 56.4 μL. It was inferred that, regarding DPPH radical scavenging, red wines are around 5 times stronger than white wines, despite the absence of statistically significant differences between the grape varieties studied, as well as among different wine regions [The antioxidant activity of 52 wine samples was assessed spectrophotometrically and expressed as the amount of wine able to engender 50% decolorization of the DPPH radical solution per reference to the control  were assessed in the seeds of cumin at different ripening stages. At full ripening stage, for which the highest level of total phenolics was determined (17.74 and 25.15 mg GAE/g DW), the antioxidant capacity also attained its peak, with the smallest values of IC50, 6.24 and 42.16 μg/mL, respectively, for maceration and Soxhlet methods applied for extraction [The total phenolic amount and antioxidant potential expressed by the ICtraction .Citrus grandis Osbeck). The antioxidant and antibacterial activity of both crude extracts and isolated compounds were determined using DPPH scavenging and paper disc diffusion method. The 100% methanol extract was steeped in water at different pH values and subject to partitioning with ethyl acetate yielding basic, acidic, neutral, and phenolic fractions. The neutral fraction revealed the highest antioxidant potential and antibacterial efficacy [Another study was performed to assess the antioxidant and antimicrobial potential of methanol (100 and 80%) aqueous extracts of pumelo fruits albedo  were tested. Methanol extracts of Artemisia capillaris herba possessed the highest phenolic content and were endowed with the strongest antioxidant power, when compared to the other solvent extracts; namely, the scavenging potential of three extracts exhibited against the DPPH radical varied as follows: ethyl acetate extracts < acetone extracts < methanol extracts, corresponding to the inhibition percentages of 35.2, 57.1 and 91.1% at a dose of 200 ppm, respectively. The scavenging potential of 100 ppm methanol extract (90.8%) equaled the one proper to 100 ppm BHT (90.5%) and was very close to the one of α-tocopherol (92.3%). The methanolic extract also exhibited the strongest antioxidant activity in the β-carotene bleaching system [The antioxidant activities ofg system . Sonchus asper crude extracts was assessed by DPPH radical scavenging. Methanol extract showed the highest scavenging potential  followed by chloroform, ethyl acetate, and hexane extracts, as revealed in The antioxidant potential of nonpolar  and polar (methanol) Bergia suffruticosa, bone, and sore healer. The whole plant extracts proved dose-increasing scavenging activities versus DPPH• and superoxide, with EC50 values of 13.1 µg and 139.4 µg, respectively. The Fe3+ to Fe2+ reducing ability was also proven to be dose-dependent, reaching a maximum for 300 µg extract [The antiradicalic activity against DPPH and the superoxide anion scavenging activity (in a riboflavin-light-nitro blue tetrazolium chloride system) were determined in the case of methanolic extracts of extract .β carotene equivalents/100 g). Raisins possessed the highest total phenolic concentration  and proanthocyanidins . Figs presented the highest flavonoid  and anthocyanin (5.9 mg/100 g) amount. The antioxidant activities were also assessed. The apricot aqueous extract had the best reducing power, Agen prune presented the highest antioxidant activity furnished by the phosphomolybdenum method, and the raisin extract in ethanol showed the best DPPH• quenching capacity [In another paper, the antioxidant profiling and antioxidant activities of dried fruits have been assessed, namely, prunes, apricots, raisins, and figs . The higcapacity . chile ancho extract exhibits a lower antioxidant capacity when compared to rosemary and both BHT and BHA. In the case of rosemary, due to the preponderance of ethanol in the extraction mixture, the polyphenol amount increases, being the most elevated for an ethanol : water ratio of 75 : 25. The rosemary extract is rich in rosmarinic acid, carnosic acid, and carnosol. In the case of chile ancho, the maximum extracted polyphenol amount is obtained for an ethanol : water ratio of 50 : 50. The chile ancho extract contains flavonoids , carotenoids, ascorbic acid, and capsaicinoids [A study based on ABTS scavenging ability proves that rosemary extract exhibits an antioxidant capacity equivalent to the one of BHA and superior to the one proven by BHT. Theicinoids . Vitis vinifera extracts were followed under different storage conditions. The total phenolic content was determined by Folin-Ciocalteu method and the antioxidant capacity by the scavenging ability versus the ABTS cation radical. The extract proved stable for up to one year at storage in darkness as a hydroalcoholic solution at 4°C or as a freeze-dried powder at 25°C. The total phenolic content was found constant at different pH values  for up to 400 days, while the antioxidant capacity diminished at pH values greater than 5.0. The thermal treatment  neither decreased nor increased the ABTS radical cation-scavenging activity. Nevertheless, it was found that the total phenolic content increased after heating at all pH values tested [The phenolic content and antioxidant activity ofs tested . Anthemis arvensis and Artemisia campestris had the highest phenolics amounts  and the most enhanced antioxidant power assessed by ABTS•+ decolorization . They also promoted an enhanced delay of free radical-induced red blood cells hemolysis, even when compared to caffeic acid, the reference antioxidant endowed with the most effective inhibition capacity [Another study was dedicated to the assessment of the antioxidant potency of phenolic substances from wild Algerian medicinal plants by chemical and biological methods.µmol Fe(II)/g DW. Black rice exhibited the highest FRAP value, followed by organic black rice, purple rice, and organic black millet. With respect to the fat-soluble fractions, the FRAP values ranged between 4.27 ± 0.19 and 21.91 ± 1.27 µmol Fe(II)/g, with red rice, buckwheat, organic black rice, and brown rice exhibiting the most elevated FRAP values. The TEAC values (relying on the ability of antioxidants to scavenge ABTS•+) ranged between 0.18 ± 0.01 and 25.28 ± 1.07 µmol Trolox/g DW for the water-soluble fractions, with black rice, organic black rice, buckwheat, and red glutinous rice exhibiting the highest values. For fat-soluble fractions, the TEAC values varied from 0.06 ± 0.04 to 5.22 ± 0.29 µmol Trolox/g. The antioxidant capacities of cereals showed a significant correlation between the FRAP value and the TEAC values. A strong correlation between antioxidant capacity and total phenolic content  was also obtained, indicating that phenolic compounds mainly contribute to the antioxidant capacities of these cereals [Both lipophilic and hydrophilic components of 24 cereal grains from China were spectrophotometrically assessed. For water-soluble fractions of the analysed grains, the FRAP values varied from 0.87 ± 0.08 to 114.69 ± 2.15  cereals . Melissae folium infusions exhibited a ferric reducing antioxidant power greater than 20 mmole Fe(II)/L and a phenol antioxidant coefficient greater than 3. The DPPH radical scavenging ability of Melissae folium phenolics was close to that of catechin. With respect to ABTS radical cation scavenging, Melissae folium phenolics exhibited superior efficacy in comparison to Trolox and vitamin C [The total antioxidant capacity and phenolic content were assessed for 70 medicinal plant infusions.itamin C .Sidastrum micranthum (A. St.-Hil.) Fryxell, Wissadula periplocifolia (L.) C. Presl, Sida rhombifolia (L.) E. H. L., and Herissantia crispa L. (Brizicky)) were investigated for the total phenolic content, DPPH radical scavenging activity, and Trolox equivalent antioxidant capacity. The antioxidant activity of the crude extract, aqueous and organic phases and isolated flavonoids, kaempferol 3,7-di-O-α-L-rhamnopyranoside (lespedin), and kaempferol 3-O-β-D-(6′′-E-p-coumaroyl) glucopyranoside (tiliroside) was assessed. A firm correlation was noticed between total polyphenol content and antioxidant activity of the crude extract of Sidastrum micranthum and Wissadula periplocifolia; this was not the case for Sida rhombifolia and Herissantia crispa. The ethyl acetate phase exhibited the best total phenolics content, as well as antioxidant capacity in DPPH and TEAC assays, followed by the chloroform phase. To lespedin, present in the ethyl acetate phase of W. periplocifolia and H. crispa, no significant antioxidant activity has been ascribed ; tiliroside, isolated from W. periplocifolia, H. crispa, and S. micranthum, had small IC50 (1.63 ± 0.86 mg/mL), proving better antioxidant capacity as provided by TEAC method [Species belonging to Malvaceae family  stembark were assessed by ABTS, DPPH, and ORAC assays. The antioxidant properties that contribute to the bioactivity of the plant extract were mainly imparted by ismailin [The antioxidant properties ofismailin .•, ABTS•+ scavenging, and β-carotene bleaching proved strong correlation with the total polyphenols assessed by Folin-Ciocalteu assay. It has been concluded that proteins do not significantly contribute to the samples' antioxidant activity and that buckwheat followed by quinoa and amaranth are the most proper as cereal substitutes [During the investigation of the antioxidant potentials of some cereals and pseudocereals, polyphenol dry matter extracts from seeds of buckwheat, rice, soybean, amaranth, and quinoa (obtained with 1.2 M HCl in 50% methanol/water) showed better inhibition of lipid peroxidation than the ones extracted with 50% methanol/water and were close to the antioxidant activity of BHT at concentration of 0.2 mg/mL. The antioxidant activities of seed extracts determined by DPPHstitutes .A series of foods usually consumed in Italy were analysed for their antioxidant capacity, by TEAC, TRAP , and FRAP. Among vegetables, spinach had the highest antioxidant capacity in the TEAC and FRAP assays, followed by peppers, while asparagus had the greatest antioxidant capacity in the TRAP assay. Among fruits, berries  possessed the highest antioxidant capacity in all assays. With respect to beverages, coffee had the greatest total antioxidant activity regardless of the technique, followed by citrus juices. As for oils, soybean oil had the highest antioxidant capacity, followed by extra virgin olive oil, whereas peanut oil proved less effective . in vitro antioxidant activity of wines has been investigated by a series of determinations such as ORAC, ABTS, DPPH, and DMPD quenching. Also, the total phenolic index was assessed for the 41 samples subject to analysis. Red wines necessitated solid phase extraction to discriminate three main fractions. ABTS, DPPH, and ORAC provided the same reactivity ranking of the analysed fractions: fraction 2 (flavan-3-ol and anthocyanins) showed the best activity followed by fractions 1 (phenolic acids) and 3 (flavonols). Nevertheless, a much reduced reactivity of fraction 2 components (anthocyanins and flavanols) towards DMPD∙+ was noticed which is consistent with the lower correlation with the total phenolic index and the smaller difference (red versus white wines) in comparison to the other methods' results [The results . n-butanol extracts, along with that of seven flavonol glycosides isolated from Dorycnium hirsutum aerial parts, was assessed using the DPPH method and the lipoxygenase assay. With respect to the inhibition activity towards the DPPH radical, kaempferol 3-O-α-L-rhamnopyranoside possessed the highest antioxidant activity . The lipoxygenase assay has been also performed, in the presence of linoleic acid and commercial lipoxygenase at pH = 6.80, and the hydroperoxide generation was monitored at 235 nm. It was proven that kaempferol 3-(4′′-O-acetyl)-O-α-L-rhamnopyranoside-7-O-α-L-rhamnopyranoside exhibited the best antioxidant protection activity in the lipid peroxidation system. The butanolic extract proved less active than the ethyl acetate residue, which confirmed the higher flavonoid level assessed in the latter [The antioxidant activity of the ethyl acetate ande latter . Lantana camara obtained from different plant parts. The absorbance diminution of the DPPH solution was measured at 517 nm, and the XO inhibition assay was based on uric acid production estimated according to the absorbance increase at 290 nm. Allopurinol (XO inhibitor) was used as positive control. The DPPH assay showed that the leaf extract had the best antioxidant activity, and the highest phenolic content was also found in the leaves: 245.50 ± 3.54 mg gallic acid/g [The scavenging properties of the DPPH radical and the xanthine oxidase inhibition activity were determined for methanol extracts ofc acid/g . in vitro antioxidant capacities of methanolic extracts (80%) prepared from Cornus mas L., Diospyros kaki L., and Laurocerasus officinalis Roem were tested by a series of recognized methods: DPPH, superoxide radical scavenging, FRAP, CUPRAC, metal-chelating capacity, β-carotene bleaching test in a linoleic acid emulsion system, and TEAC [ Diospyros kaki yielded the best results, except for the β-carotene bleaching assay. Also, good correlation was obtained between the phenolic profile and antioxidant activity. No metal-chelating activity was shown [Theand TEAC . For theand TEAC , 186. Thand TEAC . Diospyrµg amounts, trapping effectively DPPH•, lipid peroxyl, hydroxyl, and superoxide anion radicals. Superoxide anions were generated in the samples containing nitroblue tetrazolium, nicotinamide adenine dinucleotide-reduced, and phenazine methosulphate. The absorbance taken at 560 nm decreased in the presence of cumin extracts, pointing towards superoxide anion scavenging activity. The measurement of lipid peroxidation activity in rat liver microsomes was performed incubating microsomal protein with ferrous sulphate and ascorbic acid, yielding malonyl dialdehyde, which led to the assessment of TBARS in the presence of thiobarbituric acid with readings taken at 535 nm. The presence of cumin antioxidants led to the decrease of microsomal lipid peroxidation, assessed in the presence of soybean lipoxygenase and linoleic acid. The absorbance due to lipid hydroperoxides was measured at 234 nm. Cumin phenolics decreased lipid peroxidation and proved radical scavenging ability in Fenton OH-initiated DNA damage [The isolation and characterization of bioactive polyphenolic compounds from bitter cumin  were performed, by various spectrophotometric determinations, to assess their scavenging activities. The bitter cumin seed extract showed enhanced antioxidant activity at A damage .2O2-induced erythrocyte membrane lipid peroxidation. Human erythrocyte superoxide dismutase and catalase activities, after in vitro incubation with the extracts, were also tested in order to check altered enzymatic efficiency. Rosmarinus officinalis samples were collected from three different locations, namely, Canakkale , Izmir , and Mersin , on four different intervals as follows: December 2003 , March , June , and September 2004 . TEAC values ranged between 11.7 and 5.3 mmol Trolox/kg FW, with Canakkale–March samples having the highest value (11.7 mmol/kg). With respect to the H2O2-forced human erythrocyte membrane lipid peroxidation test, most of rosemary extracts acted efficiently except for C-S2, I-S1, IS2, I-S3, and I-S4. I-S2 and I-S3 even exhibited prooxidant activity and caused an increase of MDA. The M series together with C-S4 have been proven to be the most active antioxidants in the in vitro test. M-S4 extract showed closest effect to the three reference antioxidants: BHT, vitamin C/vitamin E mixture, and quercetin. All extracts caused significant increases in SOD activity, except C-S1, I-S1, I-S4, and M-S4. M series extracts did not affect CAT activity, while C and I series significantly decreased CAT activity.Twelve methanolic extracts from rosemary leaves harvested from different locations of Turkey at four different times of the year were analysed for their radical scavenging capacities and antioxidant activities by applying different techniques: DPPH radical scavenging activity, Trolox equivalent antioxidant capacity, and reversing H−1), and decreased in December, attaining a minimum in March (0.4 mg g−1). In the Canakkale samples, the rosmarinic acid level raised progressively from December to September. As in the case of rosmarinic acid, the levels of carnosol peaked in September and then diminished stepwise, attaining a minimum in March. For carnosic acid, seasonal differences were not as significant as for rosmarinic acid and carnosol: all September samples possessed higher levels of carnosic acid. Mersin samples exhibited the highest and lowest carnosic acid levels in September and March, respectively [All extracts proved high ability to quench the free radicals (DPPH and ABTS) and to inhibit malonyl dialdehyde formation. On the whole, the obtained results revealed that the plants harvested in September possess higher levels of active constituents and superior antioxidant activities in comparison to those harvested in other year seasons. Plants harvested from the Izmir region had lower total phenol and active constituent levels and hence smaller antioxidant activity. These differences were assigned to the various compositions in bioactive compounds characterizing the plants harvested from different locations and in various year seasons: the rosmarinic acid contents in the Izmir and Mersin samples increased in summer, with a maximum in September (30.4 mg gectively .−1) led to the development of multivariate models for quantifying total phenolics. The reference experimental results and those predicted by the partial least squares regression model obtained for the 26 samples ranged between 27.28 and 44.55 mg g−1 [111 samples of yerba mate from three  regions of the Brazilian state of Parana were subject to analysis of the total phenolic content (TPC) by near infrared spectroscopy. Multivariate calibration models were developed to assess the TPC from the NIR spectra using partial least squares regression. Namely, the characteristic spectral signal of phenolic groups  was investigated to assess the appropriateness as topical formulation against free radicals-induced damage. Horseradish peroxidase catalyses luminol oxidation to 3-aminophthalate by H extract .The antioxidant capacity of dry extracts of green tea, black tea, rosemary, and coffee, acerola and açaì, was assessed voltammetrically at a glassy carbon electrode. Methanol proved better efficacy in extracting antioxidant principles. The antioxidant capacities given by anodic area on the voltammogram and expressed as mg ascorbic acid equivalents ranked as follows: green tea > black tea > rosemary > arabica coffee > herb tea > acerola > quality tea > açaì .A cyclic voltammetric study of the electrooxidation of phenolic compounds present in wine revealed a pivotal influence of the structure on the propensity to undergo oxidation. Compounds with an orthodiphenol group  and morin possess the greatest antioxidant potential and are oxidized at low potentials at about 400 mV. Ferulic acid, trans-resveratrol, and malvin as well as vanillic and p-coumaric acids that have a solitary phenol moiety in many cases close to a methoxy function are less electrooxidizable. Anthocyanins determine the existence of a peak at 650 mV. It was also assessed that phenolics that imparted a first peak in the 370–470 mV range on the voltammogram can be secondly oxidized at around 800 mV: catechin and epicatechin by reason of the meta-diphenol groups on the A-ring, quercetin owing to the -OH group on the C-ring, and gallic acid due to the third -OH group placed next to the orthodiphenol moiety oxidized during the first wave .280) for amounts from 50 to 2400 mg L−1 gallic acid equivalents [A voltammetric electronic tongue system composed of an array of modified graphite-epoxy composites and a gold microelectrode was applied in the qualitative and quantitative analysis of polyphenols in wine. Samples were analysed by cyclic voltammetry and did not necessitate sample pretreatment. The analytical responses were processed by discrete wavelet transform in order to compress and extract the essential features of the voltammetric analytical responses. External test subset samples results correlated well with the ones furnished by the Folin-Ciocalteu assay and UV absorbance polyphenol index  and some of the most significant anions  did not interfere also up to a 100-fold increase in their concentrations [BHA, BHT, and TBHQ in edible oil samples were assessed by first derivative voltammetry at glassy carbon electrode modified with gold nanoparticles. First derivative pretreatment was applied to the linear sweep voltammetric signals, aiming at minimizing noise influence. The values of the peak potentials of 0.273, 0.502, and 0.622 V allowed discrimination between TBHQ, BHA, and BHT with detection limits as low as 0.039, 0.080, and 0.079 trations .Sesamol and lignans contents in sesame, tahina, and halva were assessed by polarography and stripping voltammetry. Differential pulse polarography used a capillary hanging mercury drop electrode. In cathodic stripping, sesamol reacts forming a reduced derivative, which is oxidized, yielding a cyclic voltammetric peak. The cathodic stripping voltammetric assessment involved preconcentration (when sesamol accumulates to the electrode-surface at −1650 mV) and scanning . Using these electrochemical methods, sesamol was assessed at levels of 0.26–0.32 mg/100 g oil in three varieties of sesame, 10.98–12.33 mg/100 g oil in tahina, and 4.97–9.12 mg/100 g oil in halva .−1, in the 0–800 mV potential range and for an optimal pulse amplitude of 10 mV. The antioxidant activity coefficient was assessed considering O2 reduction current in the presence of added antioxidants and the limiting O2 current in the absence of antioxidants. To check reversibility and electrooxidation mechanism, cyclic voltammetric assay was performed for the tested flavonoids  in the potential range of 0–1000 mV, at 50 mV s−1. All flavonoids presented reversible peaks in the range of 300 mV–400 mV. The intensity of the CV oxidation peak  increases with the dihydroquercetin concentration. The peak intensity also depended linearly on the square root of the scan rate, so it was concluded that the oxidation of this flavonoid was limited by mass transfer. Both the easiness of electron transfer reflected by the redox potentials (in CV) and the antioxidant activity (assessed by DPV) correlated with the following trend of increase: rutin–dihydroquercetin–catechin–quercetin [The antioxidant activity of flavonoids  was voltammetrically assessed at a glassy carbon working electrode, and the measured redox potentials were correlated to the antioxidant activity results. Differential pulse measurements relied on molecular oxygen cathodic reduction, at 50 mV suercetin .• radicals. The electrooxidation of the left unimpaired adenine bases can give an oxidation product able to catalyse NADH oxidation [μM, calculated as per reference to ascorbic acid standard [A DNA-modified carbon paste voltammetric biosensor functioned on the basis of DNA layer oxidative insults, induced by Fenton OHxidation , 227. Anxidation . Ascorbistandard .Differential pulse voltammetry a Pt working electrode allowed for ascorbic acid quantitation in fruit juices, based on its electrooxidation, as presented in Square wave voltammetry at disposable screen-printed carbon electrodes was developed for the polyphenol antioxidant screening in freshly squeezed blackcurrant and strawberry juices from various cultivars and at different maturity stages. Prior to the electrochemical assessment, the anthocyanins and nonanthocyanins were separated by solid phase extraction. It was proven that the charges passing to 500 and 1000 mV correlated well with the antioxidant activity, as well as with anthocyanin and ascorbate levels. The use of disposable screen-printed sensors was able to surpass the shortcoming of electrode deactivation due to fouling by the polymeric film formed through coupling of phenoxyl radicals during electrooxidation. Nevertheless, it was asserted that phenolics other than anthocyanins, with low formal oxidation potentials, should be quantified on the basis of sensor cumulative responses at 300 mV. Blackcurrant juices possessed high oxidation peaks at low potentials (<400 mV) and so enhanced antioxidant capacities .−1 and 990.25 mg kg−1 [The phenolic content of extra virgin olive oils was assessed by an array of 12 voltammetric electrodes: five lanthanide bis-phthalocyanines-based sensors, six polypyrrole-based sensors, and one unmodified carbon paste electrode. Apart from the peaks related to phthalocyanine, a peak due to the redox process associated with the polyphenolic fraction was also present as a shoulder in the domain of 300–500 mV (assigned as first peak). The oxidation of lanthanide bis-phthalocyanines complexes occurs at 0.55 V in KCl electrolyte and at 0.66 V in the oil extract, so the antioxidant features of polyphenols render the oxidation of phthalocyanine more difficult. The electrocatalytic efficacy of phthalocyanines enables a more facile oxidation of phenols: for the extract with the highest polyphenol content, when using a bare carbon paste electrode, the peak assigned to polyphenols appeared at 0.68 V and at 0.5 V when using a praseodymium bis-phthalocyanine electrode. The polyphenol content of the extracts ranged between 403.06 mg kg mg kg−1 .Square wave voltammetry and cyclic voltammetry at different pH values was performed to investigate the antioxidant capacity of cashew nut shell liquid components, such as cardol, cardanol, and tert-butylcardanol which were characterized by lower oxidation potentials in comparison to BHT (Epa = 0.989 V). Cardol possessed the smallest Epa (0.665 V) and hence best antioxidant potential among the tested compounds, followed by tert-butylcardanol (Epa = 0.682 V) and cardanol (Epa = 0.989 V). A linear shift of the peak potential to more negative values with the pH increase was noted for all antioxidant compounds. Increasing the pH of the electrolyte resulted in a nonlinear decrease of the peak currents, so the highest values were obtained at pH = 2.0 .−8 to 5 ×10−7 M with a detection limit of 4 × 10−9 M in acetate buffer pH = 4.0. By operating in the square wave voltammetric mode, this technique proved viable in the determination of caffeic acid in the herbs of Plantago lanceolata. The caffeic acid amount obtained by the developed voltammetric method was 107.4 µg/g of dried plant with RSD of 2.95% [Adsorptive stripping voltammetry has been applied for the determination of caffeic acid at a Pb film electrode. The working electrode was obtained in situ on a glassy carbon basis. The analyte accumulates by adsorption on the lead film electrode and is subsequently electrooxidized during the stripping step. The analytical signal depended linearly on caffeic acid concentration in the range of 1 × 10of 2.95% .Citrus paradisi, antioxidant, antiallergic, and anticarcinogenic) peel and juice has been performed by capillary electrophoresis with electrochemical detection. Hydrodynamic voltammetric measurements aimed at optimizing operational parameters. An applied potential of +0.95 V (versus SCE) was chosen for an optimum signal to noise ratio and a pH of the running buffer of 9.0, because at this value a rapid and efficient separation was obtained between the five flavonoids. 60 mM was the best buffer concentration, with higher values negatively influencing the detection limit. Hesperidin, naringin, hesperidin, naringenin, rutin, and ascorbic acid were separated and quantified in grapefruit juice by capillary electrophoresis with electrochemical detection, comparing the migration times with those of the standards. Hesperidin, naringin, and ascorbic acid were assessed in grapefruit peel extract, with good peak repeatabilities and recoveries [The determination of flavonoids and ascorbic acid in grapefruit , ranged from 97.8 ± 8.2 µg/g fresh material for Salvia maxima to 162.2 ± 11.3 µg/g fresh material for Salvia verde, with a limit of detection of 4.2 × 10−7 M. The biosensor retains more than 85% of its initial analytical response up to 90 days. The Michaelis-Menten kinetic apparent constant of 8.3 × 10−6 M revealed the enzyme affinity for the substrate [A laccase-based biosensor aimed at polyphenols determination fromubstrate .µA µM−1 and a 0.89 µM detection limit. The polyphenol content of green tea samples, as mg catechin, ranged from 19 ± 1.45 to 98 ± 3.86 [A tyrosinase-based biosensor was prepared by enzyme immobilization on single wall carbon nanotubes screen-printed electrodes modified with iron(II) phthalocyanine. The electrochemical behavior of the biosensor was studied with optimization of the parameters: the cyclic voltammograms in catechin solution do not present peaks related to phthalocyanine but exhibit only the peak related to the reduction of the o-quinone that resulted from tyrosinase-catalysed catechin oxidation. This peak points to the retaining of the catalytic activity of tyrosinase after immobilization. The maximum analytical response was obtained at 0.15 V, pH = 7.0, in phosphate buffer solution. The amperometric signal at increasing catechin concentrations was registered, with a sensitivity of 0.937 8 ± 3.86 .A superoxide dismutase-based biosensor allowed for the amperometric antioxidant activity determination in aromatic herbs, olives, and fruits. Superoxide dismutase was immobilized between a cellulose acetate membrane and a dialysis membrane. Enzymic xanthine oxidation yields superoxide radical anion and the disproportionation of the latter by superoxide dismutase results in molecular oxygen and hydrogen peroxide occurrence :(6)xanth2O2 oxidation at the Pt anode and 650 mV. As the electroactive superoxide anion radicals are trapped by antioxidants in the sample, the amperometric signal diminishes. The highest antioxidant capacity was exhibited by sage as herb and by medlar, among analysed fruits [Finally, the electrochemical response correlatable to the superoxide radical concentration is imparted by Hd fruits .3+/Fe2+, I2/I−, Fe(CN)63−/Fe(CN)64−) is present. The analyte reacts with the indicating redox couple: the selectivity of the technique depending on the specificity of the reaction involving the oxidized form of the redox pair and the antioxidant [This method relies on recording at a small potential difference, the current intensity between two identical working electrodes, found in a solution where a reversible redox couple  decreasing its concentration and generating DPPH (reduced form). The DPPH• reduction at one electrode gives rise to a cathodic current proportional with the concentration of the radical form, whereas the oxidation of DPPH at the other electrode yields an anodic current proportional with the molecular form concentration. When employed working conditions are as such, for the radical form concentration to be smaller than the one proper to the molecular form, cathodic current is limited by the lower concentration of DPPH• radical in the indicating mixture. DPPH•/DPPH biamperometry was employed in the analysis of tea, wine, and coffee using glassy carbon electrodes [•+/ABTS biamperometry enables analysis of juices, tea, and wine [μM Trolox [Particularly in DPPHectrodes . ABTS•+/and wine , as wellM Trolox .Quercus sp.) extracts were subject to ceric reducing antioxidant capacity (CRAC) analysis at a boron-doped diamond film electrode, relying on Ce4+/Ce3+ redox couple. Chronoamperometric determinations enabled quantification of the decrease of Ce4+ concentration, which was caused by its reduction by antioxidants present in the sample. The following variation of the antioxidant activity of analysed extracts was observed: oak (Quercus sp.) (1.73) > cabreuva-vermelha  (1.05) > cabreuva (Myrocarpus frondosus) (0.90) > imbuia (Octea porosa) (0.71) > pequi (Caryocar brasiliense) (0.31) [Samples of Brazilian woods and oak  (0.31) .4+ concentration after its reaction with antioxidants. The following trend of variation of antioxidant capacities resulted from this comparative investigation, which relied on chronoamperometric measurements: tannic acid > quercetin > rutin > gallic acid ≈ catechin > ascorbic acid > butylated hydroxyanisole > Trolox. The results were consistent with those furnished by the applied conventional FRAP assay [The ceric reducing antioxidant capacity assay was also exploited given its direct electron transfer facility for determining the antioxidant capacity of eight antioxidant compounds. The developed technique was based on observing the decrease of CeAP assay .2/I− employed as oxidizing agent. The biamperometric detection of the amount of iodine consumed allowed for the assessment of vitamin C with a linear range of analytical response comprised between 5 × 10−5 and 5 × 10−4 M: a 1.08% RSD  and high throughput of 60 samples h−1 [Vitamin C assessment relied on the oxidation of the analyte at acidic pH by Iples h−1 .The analytical signal represented by the potential change is the result of the variation of an ionic species concentration. A flow injection potentiometric method was developed to rapidly and reproducibly evaluate the antioxidative ability characteristic for several aqueous plant extracts. This potentiometric technique relies on recording the potential shift in the ferricyanide/ferrocyanide mediator system in the presence of antioxidants, which react with the oxidized form of the redox pair, modifying the concentration ratio between the oxidized and reduced forms. The developed potentiometric method used as detector a Pt electrode transducer based on logarithmic dependence, which provided the antioxidant activity of several hydrosoluble antioxidants . The total antioxidant activity of aqueous fruit extracts was comprised between 0.066 ± 0.002 ascorbic acid equivalents (for lemon) and 0.490 ± 0.001 (for orange). The tea infusions' values ranged between 3.60 ± 0.1 ascorbic acid equivalents for Dolche vita and 18.0 ± 0.2 for Sweet osman .−5 and 1.0 × 10−3 M in model solutions. The study also assessed the contribution of ascorbic acid to the total antioxidant capacity of aqueous extracts of hips, hop cones, and lemon juice, namely, 26.0, 0.16, and 15%, respectively [A linear potentiometric response versus ascorbic acid at an iodine-modified platinum electrode was obtained between 1.0 × 10ectively .In As previously discussed in this review, the first step is represented by the choice of the adequate extraction method and solvent, and here the nature of the sample and contained active principles, as well as the analytical technology subsequently applied, should be considered. Detailed discussions devoted to the choice of the proper solvent as well as to the extraction methodology are given in specific cases. M. pubescens were extracted by maceration employing comparatively solvents with various polarities. It has been revealed that the use of aqueous methanol (50%), aqueous ethanol (50%), and aqueous acetone (50%) imparted the highest antioxidant capacity values. The aqueous methanol (50%) and the aqueous ethanol (50%) extracts possessed the highest polyphenol content [ Ouratea lucens and Acomastylis rossii, namely, conventional sonication (shaker) bath and homogenizer method, revealed a better performance of the latter. Sonication efficacy depends on the cell wall disruption during grinding, and the better homogenizer performances were attributed to efficacious cell wall breakdown [Ethanol, methanol, and water, due to their polarity, favor the extraction of polar substances such as phenolics and flavonoids. Owing to its low toxicity and being an organic solvent, ethanol is employed with high frequency. Methanol toxicity hinders somehow its use. Nonpolar solvents like ether, as well as solvents endowed with low polarity , are proper for particular cases and have limited availability . Phenoli content . The difreakdown .• scavenging potential was not increased. So, in this case, by applying Soxhlet extraction, compounds other than antioxidants can take part in the extraction [Three genotypes of horseradish roots were subject to extraction employing conventional solvent, as well as Soxhlet extraction. The solvents endowed with the most potent extractive power were ethanol and ethanol/water mixture. The total phenolic content obtained in Soxhlet extracts proved superior to the one obtained by conventional solvent extraction. Nevertheless, the DPPHtraction . Soxhlettraction .•. Its efficacy has been proven to be solvent dependent, with best performances being obtained with acetone : water [Vortex extraction led to superior performances when compared to sonication and shaking, for phenolics extraction from oregano leaves, as proved by the results of total phenolic content assay and scavenging activity determination by DPPH : water .Quercus infectoria extract) revealed that the supercritical CO2 extraction resulted in lower extraction yield in comparison to Soxhlet conventional technique, though the antioxidant capacity and selectivity with respect to total phenolics given by supercritical CO2 extraction has been proven to be higher than the one imparted by Soxhlet extraction [The comparative analysis of both conventional and nonconventional extraction techniques applied on the same plant material  were applied to the aerial roots of solvent . in vitro AlCl3 treatment, and UV-C radiation [Grape resveratrol extraction performances were investigated by liquid-liquid extraction, solid-liquid extraction, pressurized liquid extraction-solid phase extraction, and supercritical carbon dioxide extraction and in all situations the yield can be improved by postharvest ultrasonication, fungal pathogens,adiation .The health benefits of plant-derived products have been already stated: the lipid oxidation delaying ability led to preventing the occurrence of mutagenic and carcinogenic lipid peroxides and aldehydes. Moreover, spices and herbs have been used for years for flavour, aroma, and color preserving . Dried fWith respect to the analytical methodology, the development of experimental conditions of the working protocols (that should be tuned to the nature of the sample/target compound(s)) and also the interpretation of the results should be carefully considered. A plethora of analytical methods were applied to total antioxidant capacity in plant extracts, yet difficulties may arise when it comes to choosing the most adequate method. It was stressed out that conditions, nature of substrate, and concentration of analysed antioxidants should be as close as possible to those encountered in food or biological media . So, theIn the case of the radical scavenging antioxidants, the antioxidant-radical reactivity (both rate constant and stoichiometry), antioxidant localization, mobility in reaction media, stability of the antioxidant-derived radical, interrelation with other antioxidants, and metabolism should be measured to thoroughly understand their dynamic action as antioxidants. For instance, vitamin E has been proven to be an efficacious radical scavenger, but it was asserted that it possesses weak activity against lipid peroxidation by lipoxygenase. Carotenoids are not efficient in radical trapping but they inhibit single oxygen-induced oxidation . In the The choice of the appropriate method should be grounded on the biomolecules targeted during the oxidation process . A viable antioxidant protocol necessitates the quantification of more than one property relevant to either foods or biological systems. Antioxidant standardization is required to diminish the discrepancies that may result from only one technique applied to antioxidant assessment .A comparative discussion of the detection mechanism should consider the following aspects. Gas chromatography allows separation and determination of a precise amount of certain antioxidants in different media. HPLC as well as gas chromatography coupled to various detectors also results in detecting individual, particular antioxidant compounds . These methods require qualification; they are laborious but benefit from most accurate and efficacious separation and quantitation , 251. HP in vitro methods, their frequency of application decreases in the order: DPPH scavenging > hydroxyl radical scavenging > superoxide dismutase activity > beta-carotene linoleate bleaching. Recently, reliable results have been obtained by combining in vitro method (DPPH assay) with high performance liquid chromatography. Such a DPPH-HPLC online assay especially for natural-sourced antioxidants requires minimum sample preparation [ in vivo techniques, the lipid peroxidase assay is the most frequently used, followed by catalase and glutathione peroxidase [ in vivo antioxidant capacity assay uses biological media [ in vivo versus in vitro is of vital importance in these types of assays, as the antioxidant capacity of plants and phytocompounds is influenced by various parameters in vivo, such as gut absorption, metabolism, bioavailability, and the presence of other antioxidants or transition metal ions [ in vitro assays should be complemented by in vivo efficacy testing [ in vivo, the mechanisms of action should be subject to analysis in vitro to avoid discrepancies that may occur when validating in vitro confirmed methods in vivo [With respect to the methods relying on optical detection, the antioxidant activity is exerted by various mechanisms, so the results furnished by a technique that relies solely on one mechanism may not take account on the actual antioxidant activity value. There are assays referring to various oxygenated/nitrogenated species, methods appliable to both lipophilic and hydrophilic antioxidants, and techniques relying on either hydrogen transfer or single electron transfer . It has paration . Moreoveparation . With reroxidase , 251. Thal media , so the al media , 257. Thtal ions , and scr testing . Moreove in vivo .When it comes to the chemiluminescent assay, the major shortcoming is represented by light emission from other sources , 251. Th in vitro studies cannot be extrapolated to in vivo effects, and the clinical trials testing benefits of dietary antioxidants may result in mixed results. Moreover, it was mentioned that ORAC values expressed as Trolox equivalents give account on both the inhibition time and the extent of oxidation inhibition. Novel versions of the ORAC assay have been developed, which use other substrates as reference , which makes data comparison not an easy task. These considerations led to ORAC withdrawal from the online catalog of United States Department of Agriculture [ in vitro antioxidant properties remains useful, as far as the benefits are also related to what happens outside human body.Nevertheless, in ORAC assay, only the antioxidant activity against peroxyl radicals is measured, disregarding the other reactive oxygenated species. It has been also asserted that antioxidant molecules present in foodstuffs exert numerous functions, some not related to the capacity to trap free radicals. Also, it has been considered that the values of antioxidant capacity do not give account on all the effects of particular bioactive principles . The valiculture . So, if The type of the prevalent antioxidant class present or the reference antioxidant chosen plays important roles with respect to the correlation of results obtained by different spectrometric methods: in the case of guava fruit methanol extract, the ABTS, DPPH, FRAP, and ORAC methods gave close results. The antioxidant activity of methanol extract exhibited good correlation with ascorbic acid or total phenolics, with best correlation being shown by FRAP results with both ascorbic acid and total phenolic content. On the other hand, ABTS, DPPH, and FRAP results for methanolic extracts were negatively correlated with total carotenoids. The antioxidant capacity of dichloromethane extract (that took account on lipophilic antioxidants) was low, compared to antioxidant activity of methanol extract. So, hydrophilic ascorbic acid and phenolics have been proven to be the main contributors to antioxidant capacity of guava fruit . In Foli•, ABTS•+, and FRAP and total polyphenol content assays as follows: positive correlation between TPC-DPPH• (0.8277), TPC-ABTS•+ (0.8835), and TPC-FRAP (0.9153) [ the antioxidant capacity values reported to different standard antioxidants: the ABTS•+ antioxidant capacities of basil in 57% ethanol extract were 47.27 ± 2.16 mg Trolox equivalents/g DW or 31.17 ± 1.42 mg ascorbic acid equivalents/g DW (white holy basil) and 65.86 ± 5.51 mg Trolox equivalents/g DW or 43.43 ± 3.63 mg ascorbic acid equivalents/g DW (red holy basil). The DPPH• values of 57% ethanol extract of basil were also expressed as per reference to different standards: 5.41 ± 0.04 mg Trolox equivalents/g DW or 4.59 ± 0.03 mg ascorbic equivalents/g DW (white holy basil) and 6.23 ± 0.19 mg Trolox equivalents/g DW or 5.28 ± 0.16 mg ascorbic equivalents/g DW (red holy basil) [•+ assay were explained by the fact that compounds which have ABTS•+ scavenging activity may not be endowed with DPPH• scavenging potential [•+ scavenging reaction may exert a higher antioxidant capacity than the antioxidants initially present in the reaction medium and can react with ABTS•+ [Several correlations have been reported between the results obtained at the application of DPPH(0.9153) . Also, cy basil) . The higotential . Moreoveh ABTS•+ . (Ilex paraguariensis) ethanol extracts evaluated by DPPH• method were expressed as ascorbic acid equivalents or Trolox equivalents , trying to facilitate a comparative assessment: 12.8–23.1 g Trolox equivalents % dry matter and 9.1–16.4 g ascorbic acid equivalents % dry matter [The results of total antioxidant capacity assay of yerba matey matter .• photometry, the absorbance variations can be subject to more inaccuracy, whereas in voltammetry deviations in the peak potential value smaller than ±3 mV were noticed. Therefore, in photocolorimetry, at low antioxidant capacity values, the results may be prone to a greater uncertainty [Rapidity, lower cost, simpler instrumentation, and oxidation potential value of each particular sample component evaluated with the same accuracy  are reported advantages of electroanalysis versus spectrophotometry. It was asserted that, in DPPHertainty . MoreoveQ500 value as analytical signal does not give account on less oxidizable components, such as phenolics with more elevated oxidation potentials  [−9 mol/L in acetate buffer, pH = 4.0 [Nevertheless, in the case of samples rich in various phenolic classes and possessing different electrooxidation potentials ,  800 mV) , 269. St 800 mV) . ExcellepH = 4.0 . AmperompH = 4.0 . BiamperpH = 4.0 , 271. ThpH = 4.0 .Employing minimum two analytical techniques (relying on different mechanisms) and applying three sample dilutions are generally recommended , 274. HeResearches focusing on free radicals, plant extracts, plant-derived antioxidants in foodstuffs, and biological media should be accompanied by validation of biological markers meant to define the efficacy of antioxidant compounds in diet . The com


Bilateria) ranging from flies to whales. A rigorously vetted sample set containing 265 specimens from 64 species was assembled. Bacterial lineages were characterized by 16S rRNA gene sequencing. Previously published samples were also compared, allowing analysis of over 1,098 samples in total. A restricted number of bacterial phyla was found to account for the great majority of gut colonists. Gut microbial composition was associated with host phylogeny and diet. We identified numerous gut bacterial 16S rRNA gene sequences that diverged deeply from previously studied taxa, identifying opportunities to discover new bacterial types. The number of bacterial lineages per gut sample was positively associated with animal mass, paralleling known species-area relationships from island biogeography and implicating body size as a determinant of community stability and niche complexity. Samples from larger animals harbored greater numbers of anaerobic communities, specifying a mechanism for generating more-complex microbial environments. Predictions for species/abundance relationships from models of neutral colonization did not match the data set, pointing to alternative mechanisms such as selection of specific colonists by environmental niche. Taken together, the data suggest that niche complexity increases with gut size and that niche selection forces dominate gut community construction.Classical ecology provides principles for construction and function of biological communities, but to what extent these apply to the animal-associated microbiota is just beginning to be assessed. Here, we investigated the influence of several well-known ecological principles on animal-associated microbiota by characterizing gut microbial specimens from bilaterally symmetrical animals ( The intestinal microbiome of animals is essential for health, contributing to digestion of foods, proper immune development, inhibition of pathogen colonization, and catabolism of xenobiotic compounds. How these communities assemble and persist is just beginning to be investigated. Here we interrogated a set of gut samples from a wide range of animals to investigate the roles of selection and random processes in microbial community construction. We show that the numbers of bacterial species increased with the weight of host organisms, paralleling findings from studies of island biogeography. Communities in larger organisms tended to be more anaerobic, suggesting one mechanism for niche diversification. Nonselective processes enable specific predictions for community structure, but our samples did not match the predictions of the neutral model. Thus, these findings highlight the importance of niche selection in community construction and suggest mechanisms of niche diversification. Classical ecology provides extensive theories for investigating community dynamics that are beginning to be applied to sequence-based studies of microbes. For example, species-area relationships  have beeAnother ecological framework, Hubbell’s neutral model, makes specific predictions for neutral colonization processes that can then be compared to data to interrogate assembly mechanisms. Hubbell’s neutral theory models construction of a population via migration from a source population using information on birth and death rates, migration rates, and abundances while assuming all organisms are equally fit colonists . NeutralBilateria—the bilaterally symmetrical clade of animals that includes protostomes and deuterostomes. We focused on intestinal bacterial communities to access a consistent sample type and to interrogate an organ system relevant to human and veterinary medicine. In all, we sampled gut contents from 10 classes of animals, representing 64 species. We purified DNA from feces or dissected gut tissue from species that ranged in size from bedbug (0.009 g) to right whale , spanning almost 109 g in body size. In this analysis, we assumed that overall organismic size is a viable surrogate for gut size, though it is clearly an approximation  and western lowland  and eastern lowland  gorilla, as well as the bonobo , in sub-Saharan Africa. Field sites are labeled by a two-letter code, with circles, squares, and hexagons identifying locations where fecal samples were collected from chimpanzee species, gorilla species, and both groups of species, respectively. Triangles denote ape rescue centers, and ovals indicate bonobo field sites. CC, center for chimp conservation (sanctuary); GA, Gashaka; MT, Minta; DG, Diang; SY, Sanaga-Yong (sanctuary); BQ, Belgique; GT, Goalougo triangle; UB, Ubangi; LG, Lingunda; KR, Kokolopori; LK, Lui-Kotal; IK, Ikela; TL2, Tshuapa-Lomami-Lualaba; BI, Babingi; PA, Parisi; OP, Opienge; KE, Kasese; TC, Tchimpounga (sanctuary); GM, Gombe national park; LU, Lubutu; UG, Ugalla. The Chimfunshi (sanctuary) site (CF) in Zambia is not indicated. Download FIG S1, PDF file, 1 MB.(A) Taxonomic breakdown of bilaterian host samples reported in this study. (B) Summary of the average numbers of reads per sample for various sample types. Four samples  with 0 reads are not shown. Naming of control samples is as follows: dissection, washes of instruments used prior to dissection; extraction, mock purification of DNA-free water; negControl, extractions with no added sample; posControl, synthetic DNAs used as methodological positive controls as described in reference Copyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We expected that bacteria in little-studied bilaterian species would be disproportionately difficult to assign to genera and species, so bacterial lineages were characterized as operational taxonomic units (OTUs) based on sequence and not as phylogenetic assignments based on database references. Sequence reads were condensed into clusters of 97% identity using UCLUST . Phyloge10.1128/mBio.00319-18.8Table S2 Table S2, XLSX file, 8.4 MB.OTUs in bilaterian samples and negative controls. Download Copyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the We also collected additional data from previously published studies on the microbiome of insects , birds , primate17–Mammalia were dominated by Firmicutes and Bacteroides, as has been reported in many studies. Proteobacteria and Actinobacteria were present to lesser extents. Birds showed high levels of colonization with Firmicutes and Tenericutes. Fish and sharks, in contrast, consistently showed high levels of colonization with levels of Proteobacteria. Among invertebrates, colonization by Proteobacteria typically predominated.10.1128/mBio.00319-18.2FIG S2 FIG S2, PDF file, 2.8 MB.(A) Summary of the abundances and diversity of a gut microbiome data set from myrmecophagous mammals . (B) SumCopyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria, Tenericutes, Spirochaetes, Fusobacteria, Cyanobacteria, and TM7 . Related patterns could be seen in the published data sets analyzed in We began to assess community structure by asking what proportion of the full sample was comprised of the most predominant OTU . As is dGiven these survey data, we could also compare levels of oxygen utilization by gut bacterial communities. Literature data were used to annotate the oxygen utilization and sensitivity of each bacterial species; values were first pooled for bacteria in a community and then for individuals within a host species, to generate an overall value for each host species  and B. A10.1128/mBio.00319-18.3FIG S3 FIG S3, PDF file, 0.3 MB.(A) Proportions of inferred oxygen utilization averaged across samples from gut bacterial communities for each species. (B) Correlation between the ratio of the reads assigned and the ratio of aerobes to obligate anaerobes and host organismic weight. Points indicate the average for each species and are colored by host phylogenetic class; the line indicates a linear regression of the ratio of aerobes to obligate anaerobes on log host weight, and shading indicates the 95% confidence interval for the regression. Download Copyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Wolbachia, a Gram-negative Alphaproteobacteria species of the Rickettsiaceae family. A previous sequence survey identified Wolbachia in multiple insect species  for divergent OTUs is shown. Divergent OTUs from the inferred phylogenetic tree were selected by finding any OTU whose ancestry contained a single branch length of at least 10% nucleotide difference. This resulted in a set of 669 OTUs . Read counts for 254 divergent OTUs were aggregated into 85 taxonomic assignments from Greengenes. OTUs with taxonomic assignments are labeled with their assigned phylum (indicated by "p_") concatenated to their most specific phylogenetic assignment . The remaining 415 divergent OTUs had no taxonomic assignment in the Greengenes database and are labeled “Unassigned” (concatenated to an arbitrary identifier). Taxonomic assignments were ranked by number of distinct host species in which they were detected, and the top 24 are shown as columns in the heat map, with aggregate counts of all the remaining divergent OTUs indicated in the far right column. Download Copyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Cetacea, Artiodactyla, Langomorpha, and Rodentia. Primates, extensively represented in this data set, formed a broad cluster, with specific subclusters classified by species. Members of Insecta formed a particularly tight group, with subclusters for Orthoptera, Diptera, Hymenoptera, and Coleoptera.We next compared bilaterian gut community structures on a phylogenetic scaffold using UniFrac. In this method, two samples are compared by arranging their OTU sequences on a common tree and quantifying how much of the branch length is unique to each sample. 10.1128/mBio.00319-18.5FIG S5 FIG S5, PDF file, 0.2 MB.(A) t-SNE plot of unweighted UniFrac species centroids. A two-dimensional representation of unweighted UniFrac distances between species centroids was generated using t-distributed stochastic neighbor embedding (t-SNE). Colors indicate host phylogenetic class as described for Copyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P < 10−6 for phylum, class, order, family, genus, and species). In another analytic approach, evolutionary times of divergence among all host species were obtained from TimeTree (P < 10−6), indicating a strong association.We used several approaches to measure this clustering with host taxonomy. Permutational multivariate analysis of variance (PERMANOVA) testing showed that gut microbial communities clustered with host taxonomy for all host taxonomic levels queried . Even after accounting for phylum identity, the clustering was still highly significant (PERMANOVA P < 10−6). Samples from Chondrichthyes, carnivorous sharks and rays, clustered (PERMANOVA P < 10−6), as did samples from Carnivora . Among the previously published whale samples (Firmicutes (P = 0.0002) from the carnivorous toothed whales which were dominated by other phyla (P < 10−6).Samples also partitioned by diet, although diet was commonly correlated with host phylogeny and difficult to disentangle in our data. Samples clustered by diet type  at a high level of significance  –3. Here –Modeling log bacterial richness (expected number of rarefied OTUs) as a linear function of log weight yielded a significant positive slope of 0.054 , indicating greater richness in the larger guts . This suP < 10−6), suggesting that the larger guts may be more anaerobic in the luminal space and that this may result in new niche creation.Assessment of oxygen utilization by bacterial communities showed a correlation with host body weight. Each bacterial lineage was analyzed where possible for its status as an anaerobe or aerobe, and the results were pooled for the bacterial lineages within a host individual and then for host individuals within a species . The ratEcological theory describing colonization of islands in the absence of deterministic factors—the neutral model—predicts the shape of rank abundance curves, allowing our data to be interrogated to assess agreement with this prediction. According to this model, colonization is a function of immigration of individuals from a source population, together with their birth and death rates, and all potential colonists are equal. Lack of agreement between predicted and observed rank-abundance curves allows rejection of the neutral model, pointing instead to alternative theories such as selection of specific colonists by environmental niches.Several approaches were taken to assess the influence of sequencing error on our conclusions. Chimera filtering was not applied to the data initially due to the difficulty of identifying chimeras involving pairs of previously unstudied 16S rRNA gene sequences. To test for possible confounding by chimeras, we applied chimera filtering using ChimeraSlayer and USEARCH to our gut microbiome samples , 30. ChiTo test for possible effects of sequencing error, we denoised our data set using the filtering program DADA2 . DADA2 a10.1128/mBio.00319-18.6FIG S6 FIG S6, PDF file, 1 MB.(A) Fit of models to rank-abundance curves after denoising of sequence reads with DADA2. The abundances of DADA2 sequence variants from samples were assessed for their fits to community models  (columns) using Akaike information content (AIC) and the AIC averaged within each species (rows). Within each row, the best-fitting models  appear red, with the color code showing the difference for each model from the minimum AIC. White coloring indicates a model convergence failure. (B to E) Empirical rank-abundance curves and comparisons to model best fits are shown for single samples from human (B), right whale (C), tiger shark (D), and cricket (E). (B) Comparison of animal weight to the number of DADA2 filtered sequence variants. Points indicate the average animal weights and the expected number of DADA2 filtered sequence variants after rarefaction to 500 reads for each species. Points are colored by host phylogenetic class as described for Copyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Here we sought to test the hypothesis that the bilaterian gut microbiota is assembled by selective forces, resulting in communities diverging from the predictions of neutral colonization. For this, we assembled a broad sample set, ranging from small insects to baleen whales. Although myriad factors affect microbial community structure in each organism, our survey establishes several generalizations. The structure of gut microbial communities commonly partitioned with host taxonomy, indicating that gut microbiome structure can be preserved over long evolutionary time scales. Species-area analysis showed an increase in species richness with gut size, supporting the ideas that bacterial populations in larger guts are less likely to become extinct due to chance fluctuations and that larger guts may have more types of environmental niches. Consistent with these findings, predictions from quantitative models based on ecological neutral colonization did not match most samples, supporting an alternative model in which selection drives gut microbial community assembly. We also report numerous detections of highly divergent bacterial taxa, which can help target future studies to characterize global bacterial diversity.9 bacterial cells to undetectable numbers  processes in colonization of the bilaterian gut. The profiles of species-abundance curves display long tails —represen35-Numerous further theories have attempted to explain the shapes of species-abundance curves . UnfortuWolbachia in invertebrates  and associations with humans.This study had several limitations. The use of 16S rRNA gene tags provides a tractable window on community structure for a sample set that includes tissue specimens, but additional studies using shotgun metagenomic sequencing could yield richer (though quite complex) data. Although a large number of specimens and species were studied, our sampling scheme was opportunistic and the coverage of In summary, these data suggest that neutral assembly models cannot explain the structure of the bilaterian gut microbiome and that niche selection is a likely driver. Larger animals harbor richer bacterial communities, potentially a consequence of the presence of more-diverse niches in gut, including an increase in anaerobic habitat, and greater resistance to stochastic extinction due to larger sizes of microbial populations. This work provides baseline data for understanding the structure and dynamics of the global bilaterian microbiome and provides a point of departure for additional studies. Going forward, the data and analytic methods presented here may be useful in assessing colonization mechanisms in human disease states , 44 and Sources of samples are listed in DNA from all samples was isolated in a sterile class II laminar flow hood. Due to differences in starting material, DNA was purified using three methods. First, DNA was isolated from fecal samples using a Mo Bio PowerSoil HTP 96 DNA isolation kit . Sample inputs weighed between 0.0085 g (gerbil pellet) and 1.6408 g (gut contents of the large intestine of a sandbar shark). Samples were incubated for 10 min at 70°C and homogenized for 20 min with a Qiagen TissueLyser II instrument, and DNA was purified per the manufacturer’s protocol. Second, to reduce background, DNA from low-biomass samples  was purified using a Qiagen DNeasy UltraClean microbial kit , in single tubes, per the protocol of the manufacturer, including the recommended bead-beating step. Finally, DNA from whale feces was isolated with a PSP Stool DNA Plus kit . Samples were homogenized in PSP stool DNA stabilization buffer using the TissueLyser II instrument and incubated at 95°C, and DNA was purified per the manufacturer’s protocol. All DNA was stored at −20°C.16S rRNA gene copies were quantified by the amplification of the V1V2 region of the 16S rRNA gene by quantitative PCR (qPCR). Dilutions (1:4) of each DNA sample were used in reaction mixtures  and measured in triplicate. Primer and probe sequences and amplification conditions are described in 10.1128/mBio.00319-18.9Table S3 Table S3, XLSX file, 0.01 MB.Sequences of DNA oligonucleotides used in this study. Download Copyright © 2018 Sherrill-Mix et al.2018Sherrill-Mix et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Taq, 5 µl of the forward primer (2 µM), and 5 µl of the reverse primer (2 µM). PCRs were prepared in a PCR clean room using an EpMotion 5075 liquid handling workstation  and run in quadruplicate on an Applied Biosystems GeneAmp PCR 9700 system  under the following cycling conditions: initial denaturation at 95°C for 5 min; 30 cycles of denaturation at 95°C for 30 s, annealing at 56°C for 30 s, and extension at 72°C for 90 s; and a final extension at 72°C for 8 min. Reaction replicates were pooled into libraries and purified using Agencourt AMPure XP beads  per the manufacturer’s protocol. The purified PCR products were then pooled and sequenced using an Illumina MiSeq platform. qPCR quantification of 16S rRNA gene copies was used to ensure that a minimum of 1,000 16S rRNA gene copies were input into each reaction. To pass our quality filter, samples were also required to contain at least 1,000 Illumina sequence reads.For each sample, the V1V2 region of the bacterial 16S rRNA gene was amplified with Golay bar-coded universal primers 27F and 338R  41, 46,,46, 47. Sequence data was processed using QIIME 1.9.1 with default parameters . OTUs wehttps://www.ncbi.nlm.nih.gov/Traces/study/?acc=SRP115877).Sequences from this study are available at the NCBI SRA (