A Rapid HPLC-UV Protocol Coupled to Chemometric Analysis for the Determination of the Major Phenolic Constituents and Tocopherol Content in Almonds and the Discrimination of the Geographical Origin

Reversed phase-high-pressure liquid chromatographic methodologies equipped with UV detector (RP-HPLC-UV) were developed for the determination of phenolic compounds and tocopherols in almonds. Nineteen samples of Texas almonds originating from USA and Greece were analyzed and 7 phenolic acids, 7 flavonoids, and tocopherols (−α, −β + γ) were determined. The analytical methodologies were validated and presented excellent linearity (r² > 0.99), high recoveries over the range between 83.1 (syringic acid) to 95.5% (ferulic acid) for within-day assay (n = 6), and between 90.2 (diosmin) to 103.4% (rosmarinic acid) for between-day assay (n = 3 × 3), for phenolic compounds, and between 95.1 and 100.4% for within-day assay (n = 6), and between 93.2–96.2% for between-day assay (n = 3 × 3) for tocopherols. The analytes were further quantified, and the results were analyzed by principal component analysis (PCA), and agglomerative hierarchical clustering (AHC) to investigate potential differences between the bioactive content of almonds and the geographical origin. A decision tree (DT) was developed for the prediction of the geographical origin of almonds proposing a characteristic marker with a concentration threshold, proving to be a promising and reliable tool for the guarantee of the authenticity of the almonds.     

Toward the prediction of the activity of antioxidants: Experimental and theoretical study of the gas-phase acidities of flavonoids

The relative gas-phase acidities were determined for eight flavonoids, applying the kinetic method, by means of electrospray-ion trap mass spectrometry. The experimental acidity order, myricetin > luteolin > quercetin > (+/-)-taxifolin > kaempferol > apigenin > (+)-catechin > (+/-)-naringenin shows good agreement with the order obtained by theoretical calculations at the B3LYP/6-311 + G(2d,2p)//HF/6-31G(d) level. Moreover, these calculations provide the gas-phase acidities of the different OH groups for each flavonoid. The calculated acidity values (Delta(ac)H), corresponding to the most favorable deprotonation, cover a narrow range, 314.8-330.1 kcal/mol, but the experimental method is sensitive enough to differentiate the acidity of the various flavonoids. For all the flavones and the flavanol, catechin, the 4'-hydroxyl group is the most favored deprotonation site whereas for the flavanones studied, taxifolin and naringenin, the most acidic site is the 7-hydroxyl group. On the other hand, the 5-hydroxyl, in flavones and naringenin, and the 3-hydroxyl, in taxifolin and catechin, are always the less acidic positions. The acidity pattern observed for this family of compounds mainly depends on the following structural features: The ortho-catechol group, the 2,3 double bond and the 4-keto group.     

Influence of extraction methods on stability of flavonoids

The LC-MS/MS was applied for the determination of flavonoids' stability under four types of solvent extraction methods (reflux heating, sonication, maceration and microwave) from maize samples. The 11 flavonoids belong to different groups: flavonols (kaempferol, myricetin, rhamnetin, quercetin, rutin), flavanones (naringenin, naringin, hesperedin), flavones (apigenin, luteolin), isoflavones (genistein) were studied. The effect of the degradation of flavonoids depended on extraction mode and chemical structure. The smallest decomposition was observed by heated reflux extraction procedure within 30 min in water bath and by microwave assisted extraction under 160 W during 1 min. The decomposition for flavonoids depends on number of substituents in flavonoid molecule. The most unstable compound (recovery below 50%) in tested condition was myricetin. The higher number of hydroxyl groups promote degradation of flavonoids, whereas sugar moiety and methoxyl groups protect flavonoids of degradation during microwave and ultrasonic assisted extraction.     

Composition and content of seed flavonoids in forage and grain legume crops

Flavonoid content and composition in 124 accessions of forage and grain legumes was assessed by means of HPLC-DAD and HPLC-ESI-MS analyses of seed extracts. Excluding soybean, only 7 out of 77 grain legume accessions exhibited a total flavonoid content higher than 0.1 mg/g fresh seed weight (mean content 0.33 +/- 0.31 mg/ g fresh seed weight); while in 38 out of 47 forage legume, the mean flavonoid content was 1.49 +/- 0.89 mg/g fresh seed weight. On the basis of their higher seed flavonoid content and agronomic relevance, two grain and five forage legume accessions were selected for further HPLC-ESI-MS analyses aimed to identify bioactive compounds. Chemical analysis of the selected accessions permitted the identification of several flavonoids with pharmaceutical properties such as vitexin in Vigna angularis and Trigonella foenum-graecum, quercetin in all Trifolium spp and Medicago sativa, taxifolin in Trifolium pratense and Trifolium alexandrinum, myricetin in Trifolium repens, kaempferol in Phaseolus vulgaris and T. repens and luteolin in M. sativa and T. alexandrinum. Since much of literature information is regarding flavonoid content and composition of the green part of legume species, the obtained results contributed to fill the gap in knowledge about flavonoid content at the seed level.     

Observation of apigenin anionic clusters in the gas phase

The ubiquity and favorable medicinal properties of flavonoids make essential the determination of flavonoid levels in various matrices. While developing a liquid chromatography/tandem mass spectrometry method for the analysis of the flavonoid, apigenin, anionic oligomers and nitrate- and chloride-bound clusters of this compound were observed. Tandem mass spectrometry of these oligomers and cluster ions showed the cleavage of apigenin molecules from the precursor. The observation of these cluster ions shows the possibility of post-column derivatization techniques to enhance specificity in analysis. Copyright 2000 John Wiley & Sons, Ltd.     

高效液相色谱-串联质谱法同时测定银杏保健茶中的16种黄酮类功效成分

建立了银杏保健茶中16种黄酮类物质的液相色谱-串联质谱(LC-MS/MS)测定方法。16种黄酮成分分别为儿茶素、牡荆素、葛根素、大豆苷元、水飞蓟宾、槲皮素、木犀草素、芹菜素、柚皮素、橙皮素二氢查尔酮、山柰酚、橙皮素、异鼠李素、黄芩素、川陈皮素、桔皮素。实验优化了液相色谱条件和质谱参数。采用C₁₈柱分离,流动相为乙腈-水(含0.1%甲酸)梯度洗脱,流速0.25 mL/min,以电喷雾离子源正离子多反应监测(MRM)模式进行MS/MS检测。16种黄酮类物质在各自的线性范围内具有良好的线性关系,相关系数大于0.996,低、中、高3个添加水平的平均回收率在70.9%~100.0%之间,相对标准偏差小于10%。通过检测发现实际样品中9种黄酮物质含量较高,分别是:山柰酚、槲皮素、橙皮素、牡荆素、木犀草素、儿茶素、芹菜素、柚皮素、异鼠李素,占总量的99.6%,此9种物质可作为银杏保健茶的质量控制指标。本法简便、快速、准确可靠,可用于控制银杏保健茶的质量。     

Optimization of Micellar LC Conditions for the Flavonoid Separation

The chromatographic conditions for the separation of a complex set of flavonoids (aglycones and glycosides) by micellar liquid chromatography with spectrophotometric detection were optimized. A good separation for all analytes was obtained and satisfactory peak shapes were achieved by isocratic elution with Ultrasphere ODS column (250 mm × 4.6 mm, 5 μm). The optimal mobile phase range for flavonoids separation is: SDS concentration between 0.014 and 0.018 mol L^?1 and 1-propanol volume fraction between 2.2 and 4.5% (v/v) in a diluted (1:5) phosphate buffer solution pH 6.86. The flavonoids (robinin, rutin, hyperoside, quercitrin, liquroside, luteolin-7O-glucoside, apigenin-7O-glucoside, isosalipurposide, myricetin, fisetin, luteolin, apigenin, quercetin and caempferol) were successfully separated within 40 min with isocratic elution. The developed method is an alternative to reversed-phase LC in the assay of flavonoids in plants, plant extracts and plant extract containing drugs.     

Simultaneous determination of polyphenols and major purine alkaloids in Greek Sideritis species, herbal extracts, green tea, black tea, and coffee by high-performance liquid chromatography-diode array detection

Herein, a high-performance liquid chromatography-diode array detection method has been developed for the simultaneous determination of 15 phenolic antioxidants: flavan-3-ols, (-)-epigallocatechin, (+)-catechin, (-)-epigallocatechin gallate, (-)-epicatechin, (-)-epicatechin gallate, (-)-gallocatechin, a phenolic acid (gallic acid), a hydroxycinnamic acid (chlorogenic acid), flavones (apigenin), flavonols (kaempferol, quercetin, and myricetin), and purine alkaloids (caffeine theophylline, theobromine) in different herb extracts, tea, and coffee varieties. The developed method was validated and successfully applied in order to determine the polyphenolic content to estimate the antioxidant activity of the Sideritis species commonly known as Greek mountain tea. To the best of our knowledge, this is the first report on the quantitative determination of catechins and other polyphenols in Greek mountain tea. Acidic hydrolysis was necessary for the simultaneous determination of the aglycones of the target analytes. According to our results, chlorogenic acid, myricetin, apigenin, catechin, and epicatechin gallate are found in the Sideritis species.     

Flavonoid profile,antioxidant and antiglycation properties of Retama sphaerocarpa fruits extracts

Retama sphaerocarpa occurs in the Mediterranean area of North-east Africa and in the Iberian Peninsula, and grows on a variety of soil types and climatic conditions. Used in Algerian folk medicine, it is a valuable species for revegetation and soil restoration. The aim of this study is to evaluate flavonoid composition and antioxidant and antiglycation properties of methanolic and aqueous extracts from R. sphaerocarpa fruits. HPLC-PDA/ESI-MS was used to identify/quantify flavonoid content. Antioxidant activity was evaluated by Folin–Ciocalteu, ORAC, FRAP, TEAC, and DPPH assays, and antiglycation capability by glucose/fructose-BSA assay. Results showed that fruits contain isoflavones (daidzein and genistein derivatives) and flavonols (apigenin, isorhamnetin, kaempferol and quercetin derivatives), and extracts (especially the methanolic one, richer in flavonoids) possess good in vitro antioxidant and antiglycation properties. These findings evidence that R. sphaerocarpa fruits are a source of valuable phytochemicals, with potential applications in the field of phytopharmaceuticals and in food industry.     

Towards Adsorptive Enrichment of Flavonoids from Honey Using h-BN Monolayer

Density functional theory (DFT) was used to explore the possibility of h-BN monolayer acting as an adsorbent for the flavonoids. Four flavonoids named apigenin, kaempferol, myricetin, and quercetin as well as glucose (Glu) were selected as representatives of honey. DFT and ab initio molecular dynamics simulation results show that the four flavonoids interact with the h-BN monolayer much stronger than the Glu does in both vacuum and solutions, indicating a good adsorptive selectivity of the flavonoids over Glu. The interaction of the flavonoids and the Glu with water as well as the solvation energy of the flavonoids in water, methanol and ethanol was obtained using both the PBE-D and B3LYP-D functionals. It is shown that the h-BN monolayer can provide high selective adsorption of the flavonoids from bee honey and ethanol can be used as an elution solvent to recover the adsorbed flavonoids.     

Extraction and determination of bioactive flavonoids from Abelmoschus manihot (Linn.) Medicus flowers using deep eutectic solvents coupled with high‐performance liquid chromatography

A highly efficient and ecofriendly extraction method using deep eutectic solvents was developed to extract bioactive flavonoids from Abelmoschus manihot (Linn.) Medicus flowers. First, a series of deep eutectic solvents using choline chloride as hydrogen bond acceptor with different hydrogen bond donors was successfully synthesized. Then, the types of deep eutectic solvents and the extraction conditions for bioactive flavonoids (hyperoside, isoquercitrin, and myricetin) were optimized based on the flavonoids extraction efficiencies. The optimized deep eutectic solvent for hyperoside and isoquercitrin extraction was composed of choline chloride and acetic acid with a molar ratio of 1:2. The optimized deep eutectic solvent for myricetin extraction was composed of one mole of choline chloride and two moles of methacrylic acid. The optimal extraction conditions were set as: solid to solvent ratio, 35:1 (mg/mL); extraction time, 30 min; extraction temperature, 30°C. Qualitative and quantitative analysis were performed using ultra high performance liquid chromatography with tandem mass spectrometry and high‐performance liquid chromatography. And the extraction efficiencies of hyperoside, isoquercitrin, and myricetin under optimal extraction conditions were calculated as 11.57, 5.64, and 1.11 mg/g, much higher than those extracted by traditional extraction solvents. Therefore, the prepared deep eutectic solvents can be selected as alternative solvent to extract bioactive flavonoids.     

Palynological origin, phenolic content, and antioxidant properties of honeybee-collected pollen from Bahia, Brazil

The aim of this study was to determine the palynological origin, phenolic and flavonoid content, and antioxidant properties of twenty-five samples of bee pollen harvested during a nine-month period (February-November) from the Canavieiras municipality (northeastern Brazil). Of the 25 samples analyzed, only two (February 01 and 02) were heterofloral. The predominant pollens in the samples analyzed during that month were: Cecropia, Eucalyptus, Elaeis, Mimosa pudica, Eupatorium, and Scoparia. Ethyl acetate fractions were analyzed by HPLC-DAD. The flavonoids isoquercetin, myricetin, tricetin, quercetin, luteolin, selagin, kaempferol, and isorhamnetin were detected. The flavonoid present in all 22 samples was isolated and identified as isorhamnetin 3-O-b-neohesperidoside. The total phenolic contents determined using the Folin-Ciocalteu reagent ranged from 41.5 to 213.2 mg GAE/g. Antioxidant activities based on the 1,1-diphenyl-2-picryl hydrazyl (DPPH), 2,2-azinobis 3-ethylbenzothiozoline-6-sulfonic acid (ABTS), and Fe2+ ion chelating activity assays were observed for all extracts, and correlated with the total phenolic content.     

Comparative Studies of Selected Criteria Enabling Optimization of the Extraction of Polar Biologically Active Compounds from Alfalfa with Supercritical Carbon Dioxide

The aim of this research was to provide crucial and useful data about the selection of the optimization criteria of supercritical carbon dioxide extraction of alfalfa at a quarter-technical plant. The correlation between more general output, including total phenolics and flavonoids content, and a more specified composition of polar constituents was extensively studied. In all alfalfa extracts, polar bioactive constituents were analyzed by both spectrometric (general output) and chromatographic (detailed output) analyses. Eight specific phenolic acids and nine flavonoids were determined. The most dominant were salicylic acid (221.41 µg g⁻¹), ferulic acid (119.73 µg g⁻¹), quercetin (2.23 µg g⁻¹), and apigenin (2.60 µg g⁻¹). For all seventeen analyzed compounds, response surface methodology and analysis of variance were used to provide the optimal conditions of supercritical fluid extraction for each individual constituent. The obtained data have shown that eight of those compounds have a similar range of optimal process parameters, being significantly analogous for optimization based on total flavonoid content.     

Determination of flavonoid aglycones in several food samples by mixed micellar electrokinetic chromatography

The application of mixed micellar electrokinetic chromatography to the separation of ten flavonoid aglycones (catechin, epicatechin, naringenin, morin, fisetin, quercetin, kaempferol, galangin, apigenin, and chrysin) belonging to four different classes (flavanols, flavanones, flavonols, and flavones), and expected to be prominent in commonly consumed foods, has been developed. A micellar system composed of 25 mM SDS and 25 mM sodium cholate buffered at pH 7.0 provided a simultaneous separation of all compounds in less than 20 min. The procedure could be easily adapted to the determination of some flavonoids from each of these classes in real complex samples (propolis, Ginkgo biloba, etc.). The LODs of these compounds were in the range of 1.2-4 microg/mL, and the peak area and migration time repeatabilities were below 6.0 and 3.1%, respectively.     

Direct and simultaneous determination of representative byproducts in a lignocellulosic hydrolysate of corn stover via gas chromatography-mass spectrometry with a Deans switch

Pretreatment is one of the most important steps in producing fuel ethanol from lignocellulosic biomass. Simple, fast and accurate quantification of byproducts in lignocellulosic hydrolysates is critical to optimize the pretreatment procedures, but still a challenge. In this paper, a new GC-MS (SIM) method based on a Deans switch has been developed for the determination of byproducts in a corn stover hydrolysate. The Deans switch was incorporated into a hardware system that facilitated the direct aqueous injection (DAI) on GC-MS system. Simultaneous chromatographic separation and quantification of 18 byproducts including four aliphatic acids, five furan derivatives, four phenolic compounds and five others were achieved within 45 min. The detection limits of the presented method for various byproducts were in the range of 0.007-0.832 mg/L. The within-day and between-day precisions of the method were less than 6.0% (RSD, n=6). The accuracy of the method was confirmed with recoveries of 86-128%. A lignocellulosic hydrolysate sample of corn stover was successfully analyzed using this method, with aliphatic acids and furan derivatives accounting for 89.15% of the selected total byproducts.     

Antioxidant activities and quali-quantitative analysis of different Smallanthus sonchifolius [(Poepp. and Endl.) H. Robinson] landrace extracts

Five landraces of Smallanthus sonchifolius [(Poepp. and Endl.) H. Robinson], known as yacon, were investigated in total phenolic content, antioxidant activity and chemical composition of ethanol extracts (EEs) and decoction extracts (DEs). The results demonstrated that DEs are rich in phenolic acids as caffeic acid, while the EEs show an higher amount of flavonoids, as luteolin 3′,7-O-diglucoside and luteolin 7-O-glucoside. These flavonoid glycosides were identified for the first time in yacon extracts, together with apigenin and luteolin. The phytochemical profile explains the different antioxidant activities shown in our study. The landraces PER6-DE and PER4-DE showed the highest radical-scavenging activity and reducing power related to their polyphenolic contents. Results also show that yacon can be considered an important source of bioactive compounds with significant differences among the analysed landraces.     

Spectrophotometric determination of total procyanidins in wine vinegars

A widely-used method for the spectrophotometric determination of procyanidins in wines has been adapted to wine vinegar samples. Reagent concentrations have been established and the analytical method tested for possible matrix effects. The recovery of catechin was approximately 98% and the limit of reliable measurement was 0.48 mg l(-1). The within-day and between-day precisions were evaluated and according to the two-tailed F-test the precisions were statistically equivalent. Application to wine vinegars obtained by traditional and quick acetification methods showed differences in concentration between the two groups.     

Comparing the Affinities of Flavonoid Isomers with Protein by Fluorescence Spectroscopy

Abstract

Dietary flavonoids can be detected in plasma as protein‐bound conjugates. Flavonoids–protein interaction is expected to modulate the bioavailability of flavonoids. In this work, the binding flavonoid isomers (galangin, baicalein, apigenin, and genistein; MW=270.25) and B‐ring hydroxylation flavonols (galangin, kaempferol, quercetin, and myricetin, which share the same structure on the A and C rings but have 0, 1, 2, and 3 moieties of ‐OH on the B‐ring, respectively) to protein were investigated by fluorescence quenching method. The apparent binding constants (K _a ) of were flavonoid isomers determined as: flavones (10⁶–10⁷ L mol^?1)>isoflavone≈flavonol (10⁵ L mol^?1). For B‐ring hydroxylation flavonols, the binding affinity increased with increasing number of hydroxyl groups on the B‐ring. The binding constants (K _a ) were determined as follows: myricetin>quercetin>kaempferol>galangin.     

Nonaqueous capillary electrophoretic separation of polyphenolic compounds in wine using coated capillaries at high pH in methanol

Nonaqueous capillary electrophoretic separation of a group of flavonoids (quercetin, myricetin, catechin, epicatechin) and resveratrol in wine was investigated in methanol at high pH. Malonate background electrolyte (pH* 13.5, ionic strength I = 14.2 mmol/L) provided highly repeatable separations of the analytes. Tests of untreated and coated (poly(glycidylmethacrylate-co-N-vinylpyrrolidone)) capillaries showed the analysis to be faster (6.5 min vs. 25 min) and the repeatability better in the coated capillaries. The coating procedure was simple and highly repeatable and the coating was stable during 40-45 runs. Determination of the last migrating peaks (epicatechin, resveratrol and catechin) was achieved merely by evaporating the wine samples and reconstituting the residue in methanol. For determination of the first migrating peaks (quercetin and myricetin) the samples were submitted to solid-phase extraction in C8 cartridges.     

Photoprotective and Antigenotoxic Effects of the Flavonoids Apigenin,Naringenin and Pinocembrin

This work evaluated the photoprotective and antigenotoxic effects against ultraviolet B (UVB) radiation of flavonoid compounds apigenin, naringenin and pinocembrin. The photoprotective efficacy of these compounds was estimated using in vitro photoprotection indices, and the antigenotoxicity against UVB radiation was evaluated using the SOS chromotest and an enzymatic (proteinase K/T4 endonuclease V enzyme) comet assay in UV‐treated Escherichia coli and human (HEK‐293) cells, respectively. Naringenin and pinocembrin showed maximum UV‐absorption peak in UVC and UVB zones, while apigenin showed UV‐absorption capability from UVC to UVA range. These compounds acted as UV filters reducing UV‐induced genotoxicity, both in bacteria and in human cells. The enzymatic comet assay resulted highly sensitive for detection of UVB‐induced DNA damage in HEK‐293 cells. In this work, the photoprotective potential of these flavonoids was widely discussed.