Antithrombotic activity of fractions and components obtained from raspberry leaves (Rubus chingii)

The 70% ethanol fraction from an aqueous extract of raspberry leaves was shown to be the most antithrombotic fraction in in vitro and in vivo tests. The total flavonoids and phenolics in this fraction were 0.286 g/g and 0.518 g/g by colorimetry. Six compounds, including salicylic acid, kaempferol, quercetin, tiliroside, quercetin 3-O-β-d-glucopyranoside and kaempferol 3-O-β-d-glucopyranoside, were isolated from the active fraction. Among them, kaempferol, quercetin and tiliroside obviously delayed plasma recalcification time (PRT) in blood.     

Determination of iodate in table salt by transient isotachophoresis–capillary zone electrophoresis

A transient isotachophoresis–capillary zone electrophoresis (tITP–CZE) method was first time developed for determining iodate in table salt. Sensitivity enhancement was accomplished by coupling on-capillary tITP with CZE. The interference of sample matrix was overcome by using electrophoretic buffer containing high concentration of sodium chloride. The optimal terminating electrolyte for tITP was 1500 mmol/L phosphate and the separation buffer of capillary zone electrophoresis was 10 g/L sodium chloride (pH 8.0) containing 20 mmol/L cetyltrimethylammonium chloride (CTAC). Calibration graphs based on peak height and peak area showed good linearity. Use of chromate as the internal standard significantly improved the precision of the quantitative results. The wavelength of UV detection for iodate was set at 218 nm with the detection limits of 3.5 μg/L for iodate. The quantitative results of iodate in table salt measured by the tITP–CZE method were compared with those measured by the redox titration and there was no significant difference between the two means. The method developed was sensitive, fast and simple.     

A rapid quantitative determination of phenolic acids in Brassica oleracea by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis method to quantitatively determine the phenolic acid contents in brassica vegetables is described. Phenolic compounds were extracted from broccoli, broccolini, Brussels sprouts, cabbage and cauliflower and the main hydroxycinnamic acids (sinapic, ferulic, p-coumaric and caffeic acids) were isolated by solid phase extraction with C18 cartridges. Using an optimised method, the four analytes were separated in less than 7 min in a 50 μm × 60 cm capillary with a 15 mM borate buffer (pH = 9.13) and a separation voltage of 30 kV at 30 °C. A linear relationship was observed for the method (r = 0.9997–0.9999) with detection limits ranging from 1.1 to 2.3 mg/kg of vegetables for the analytes. This method demonstrated good reproducibility with coefficients of variation of less than 5% for peak area and less than 1% for migration time (n = 7). The method was successfully applied to quantitatively determine the phenolic acid contents in a range of brassica vegetables and the results were in good agreement when compared to those from high performance liquid chromatography analysis.     

Determination of red wine flavonoids by HPLC and effect of aging

A new method for simultaneous determination of 10 flavonols and 2 flavones by high performance liquid chromatography was developed in this paper. The identified compounds contained quercetin, kaempferol, myricetin, rhamnetin, isorhamnetin, quercetrin, rutin, morin, galangin, fisetin, apigenin and luteolin. The chromatographic separation of these flavonoids was performed in a single run by using the mobile phase gradient elution of acetonitrile–methanol–water mixture (1% tetrahydrofuran, THF) at 20 °C, with the flow rate at 1.0 ml/min and the detection wavelength at 360 nm. With direct injection of wine samples, seven red wine samples, differing in their origin of producing places and time, were analyzed for flavonoids content by this method. The results showed the presence of myricetin, luteolin, quercetin, kaempferol, isorhamnetin and galanin. Additionally, the changes of flavonoids in red wines stored in the three types of oak barrels with aging time were investigated, which indicated that the component of flavonoids in red wine is related to wine aging greatly. These provide a substantial basis for the further research on control of flavonoids during winemaking.     

Study of flavonoids in aqueous spinach extract using positive electrospray ionisation tandem quadrupole mass spectrometry

The presence of three flavonols (quercetin, kaempferol, myricetin) and two flavones (apigenin, luteolin) were investigated in the extract of fresh spinach leaves. Aqueous spinach extracts were prepared with a leaf/water ratio of 1:2 at 80 °C for 30 min stirring. Ferric ammonium sulphate method was used for measuring total polyphenols in the extracts and expressed as catechins and tannic acid equivalents. The flavonoids glycosides in the extract were hydrolysed to their aglycons with 1.2 M HCl in boiling 50% water methanol solution. The resulting aglycons were identified and quantified by a C18 reverse phase high-performance liquid chromatography (RP-HPLC). Furthermore, the results were confirmed by HPLC coupled to an electrospray ionisation tandem triple-quadrupole mass spectrometer ESI-MS performing low-energy collision induced dissociation (CID-MS/MS) in the collision cell (HPLC-ESI-MS/MS). Analyses were made in the multiple reaction monitory (MRM) mode. Results showed that total polyphenols contents in fresh spinach leaves were 270 mg kg⁻¹ and 390 mg kg⁻¹ as tannic acid and catechin equivalents, respectively, in which, major flavonoids aglycons were apigenin (170 mg kg⁻¹), quercetin (50 mg kg⁻¹) and kaempferol (30 mg kg⁻¹).     

Large-Volume Sample Staking of Rice Polyphenols Prior to Their Determination by Non-aqueous Capillary Electrophoresis

A rapid non-aqueous capillary electrophoretic (NACE) method for the separation of (?)-epicatechin (EPI), (+)-catechin (?CAT), kaempferol (KAE), quercetin (QUR), naringanin (NAR), ferulic acid (FA), and p-coumaric acid (p-CA) has been developed and applied to the determination of these compounds in different rice varieties. All seven compounds were separated on capillary of 50 μm?×?68 cm (60-cm effective length) using 20 mmol L^?1 borate buffer of pH 9.0 and 5 % acetonitrile in methanol. Large-volume sample stacking (LVSS) technique was optimized and used to preconcentrate non-detectable polyphenols of white polished rice. Rice extracts were concentrated on-line by LVSS prior to separation by non-aqueous capillary electrophoresis. An improvement of 10–55 times in detectability was achieved with injection at 50 mbar for 30 s followed by voltage inversion (?20 kV) for 5 s. Linear calibration range of 1–300 μg L^?1 and 0.01–60 μg L^?1 was observed for NACE and NACE-LVSS method respectively, with the detection limit of 0.33–2.0 and 0.006–0.19 μg L^?1. Good reproducibility with standard deviations of less than 5 % was achieved. Polyphenol contents of different rice varieties were determined using developed method.     

Application of response surface methodology to optimise extraction of flavonoids from fructus sophorae

Response surface methodology (RSM) based on a central composite design (CCD) was applied to optimise the extraction conditions for flavonoids from fructus sophorae with advantages in terms of resisting flavonoids during the whole process and maximising of extraction yield. Three aglycon forms of the flavonoids, namely, quercetin, kaempferol and isorhamnetin were quantified by high-performance liquid chromatography with ultraviolet detection (HPLC-UV) to estimate extraction yield. The combined effects of independent variables were studied and the optimal extraction conditions were obtained as ethanol concentration, 74.47%; solid–liquid ratio, 17.99 ml/g; temperature, 89.13 °C; and extraction time, 2.10 h. The reliability of the method was confirmed by recovery experiments, performed under optimal conditions. Recoveries indicated that flavonoids resisted the extraction conditions. The experimental extraction yield under optimal conditions was found to be 10.459%, which was well matched with the predicted values of 10.461%.     

Chemical characterization and antioxidant evaluation of muscadine grape pomace extract

Concentrated muscadine pomace extract was chromatographically analyzed for its individual phenolic, flavonoid, and anthocyanidin compounds. This extract was also characterized regarding its total phenolic content (TPC), total flavonoid content (TFC), antioxidative activities in terms of scavenging DPPH free radicals, reducing ferric, and chelating Fe²⁺. The TPC of this product was 34.1 ± 1.8 mg of gallic acid equivalents(GAE)/g of extract, and TFC was 3.0 ± 0.3 mg of quercetin equivalents/g of extract. Some phenolic compounds including ellagic acid, gallic acid, (−)-epicatechin, (−)-epigallocatechin, catechin, myricetin, quercetin, and kaempferol were identified. Some anthocyanidins including delphinidin, cyanidin, petunidin, peonidin, and malvidin were also identified in the extract by using a combination of retention and spectral properties on a reverse-phase HPLC–PDA. In addition, 3,3′,4,4′5,5′-hexahydroxystilbene-a resveratrol analogue present in the extract was identified for the first time by LC–MS. The results from this study demonstrate that the muscadine pomace extract is rich of natural antioxidants such as phenolics, flavonoids and anthocyanins, and possesses strong antioxidant properties. Besides, the developed methods can be used for routine quality control of the muscadine products for manufacturing efficiency and consistency.     

Antioxidant activity and phenolic compounds of fractions from Portulaca oleracea L.

Portulaca oleracea fractions obtained from the crude extract of the plant by reversed-phase separation were investigated for their antioxidant activities and phenolic compounds content. Five fractions were classified based on optical absorption between the wavelength range of 200–400 nm. Fraction 3 displayed higher absorption than the other fractions. The total amount of quantified phenolics in this fraction was found to be quite high compared to that of crude extract. Chlorogenic, caffeic, p-coumaric, ferulic and rosmarinic acids were the free phenolic acids, and quercetin and kaempferol were the free flavonoids detected in Fraction 3. IC₅₀ value of crude extract was found to be 511.8 μg/ml whereas Fraction 3 exhibited an IC₅₀ value of 154.1 μg/ml. TEAC of Fraction 3 was found to be almost four times higher than that of the crude extract. Fraction 3 was also found to show the highest lipid peroxidation inhibiting capacity, determined by TBARS assay.     

HPLC-DAD-ESIMS analysis of phenolic compounds in bayberries (Myrica rubra Sieb. et Zucc.)

Qualitative analysis of the ethyl acetate extracts from three bayberry cultivars, Xiangshan, Biqi and Dongkui, was performed by means of a hyphenated technique of HPLC coupled to photodiode array detection and electrospray ionization mass spectrometry (HPLC-DAD-ESIMS). Three phenolic compounds were identified (gallic acid, protocatechuic acid, and quercetin 3-glucoside) and seven others (two myricetin hexoside and two myricetin deoxyhexoside derivatives; quercetin hexoside and quercetin deoxyhexoside derivatives; kaempferol hexoside derivative) partially identified. Quantification of phenolic compounds was performed by HPLC-DAD, which revealed that gallic acid (2.6–7.0 mg/kg FW) was the major phenolic acid in all analysed cultivars. Myricetin glycosides (71.1 mg/kg FW) were the major flavonol glycosides in cultivar Xiangshan and quercetin glycosides (117.8 mg/kg FW) were the major ones in cultivar Biqi. Cultivar Dongkui had medium contents of quercetin glycosides (48.0 mg/kg FW) and myricetin glycosides (53.2 mg/kg FW). Kaempferol glyosides (3.1–4.6 mg/kg FW) were the lowest contents of flavonol glycosides in the assayed bayberries. These results are relevant not only from a nutritional point of view, but also in the control of color stability and haze formation during bayberry juice processing and storage.     

The effect of solar radiation on the flavonol content in broccoli inflorescence

The effect of solar radiation on the quercetin and kaempferol contents in the inflorescence of three broccoli cultivars (‘Lord’, ‘Marathon’ and ‘Fiesta’) was investigated from 1999 to 2001. Great differences in the contents of both flavonols, dependent on growing time and cultivar, were found. Quercetin and kaempferol contents varied from 14.3 to 81.0 mg kg⁻¹ f.w. and from 35.9 to 213 mg kg⁻¹ f.w., respectively. Inflorescences of the cultivar ‘Lord’ were characterised by the highest mean content of quercetin and those of cultivar ‘Fiesta’ of kaempferol. The contents of both flavonols were highly positively correlated with total solar radiation in the period from planting to the harvest of broccoli inflorescences.     

Monitoring the enrichment of virgin olive oil with natural antioxidants by using a new capillary electrophoresis method

The enrichment of virgin olive oil (VOO) with natural antioxidants contained in various herbs (rosemary, thyme and oregano) was studied. Three different enrichment procedures were used for the solid–liquid extraction of antioxidants present in the herbs to VOO. One involved simply bringing the herbs into contact with the VOO for 190 days; another keeping the herb–VOO mixture under stirring at room temperature (25 °C) for 11 days; and the third stirring at temperatures above room level (35–40 °C). The efficiency of each procedure was assessed by using a reproducible, efficient, reliable analytical capillary zone electrophoresis (CZE) method to separate and determine selected phenolic compounds (rosmarinic and caffeic acid) in the oil. Prior to electrophoretic separation, the studied antioxidants were isolated from the VOO matrix by using an optimised preconcentration procedure based on solid phase extraction (SPE). The CZE method was optimised and validated.     

Hollow fibre-based liquid phase microextraction combined with high-performance liquid chromatography for the analysis of flavonoids in Echinophora platyloba DC. and Mentha piperita

A simple, inexpensive and efficient three phase hollow fibre liquid phase microextraction (HF-LPME) technique combined with HPLC was used for the simultaneous determination of flavonoids in Echinophora platyloba DC. and Mentha piperita. Different factors affecting the HF-LPME procedure were investigated and optimised. The optimised extraction conditions were as follows: 1-octanol as an organic solvent, pH_donor = 2, pH_acceptor = 9.75, stirring rate of 1000 rpm, extraction time of 80 min, without addition of salt. Under these conditions, the enrichment factors ranged between 146 and 311. The values of intra and inter-day relative standard deviations (RSD) were in the range of 3.18–6.00% and 7.25–11.00%, respectively. The limits of detection (LODs) ranged between 0.5 and 7.0 ng mL⁻¹. Among the investigated flavonoids quercetin was found in E. platyloba DC. and luteolin was found in M. piperita. Concentration of quercetin and luteolin was 0.015 and 0.025 mg g⁻¹ respectively.     

Determination of capsaicin and dihydrocapsaicin in Capsicum anuum and related products by capillary electrophoresis with a mixed surfactant system

An easy, rapid, sensitive, and cheap capillary electrophoresis (CE) method based a mixed surfactant system formed by sodium dodecyl sulphate (SDS) and polyoxyethylene sorbitan monolaurate (Tween 20) as modifier in the buffer was reported. Quantitative analysis of capsaicin and dihydrocapsaicin in Capsicum anuum, pepper sauce and porous capsicum plaster was demonstrated. After conducting a series of optimisations, baseline separation was obtained for the analytes within 5 min under the optimum conditions (15 mM sodium tetraborate–0.05% (v/v) Tween 20–2.2 mM SDS buffer (pH 10.1), 20 kV voltage, 214 nm UV detection). The method resulted in excellent linearity, with r² of regression equation of 0.9994 and 0.9996 for capsaicin and dihydrocapsaicin, respectively. Recoveries were in the range 90–107% and 92–109% for capsaicin and dihydrocapsaicin, respectively.     

Flavonoid pattern of sage (Salvia officinalis L.) unifloral honey

The aim of the present paper was to determine the flavonoids in monofloral sage (Salvia officinalis L.) honey which is characteristic and specific for the area of Croatian coast and islands. For that purpose 38 sage honey samples from two production seasons were analysed. After specific pollen content determination, and analyses of selected physicochemical parameters which confirmed that samples are in compliance with national and international regulations and can be regarded as unifloral sage honeys, flavonoid fraction was isolated and analysed using RP-HPLC/DAD method. The HPLC analysis showed that all examined sage honey samples contain quercetin (3,3′,4′,5,7-pentahydroxyflavone), luteolin (3′,4′,5,7-tetrahydroxyflavone), kaempferol (3,4′,5,7-tetrahydroxyflavone), apigenin (4′,5,7-trihydroxyflavone), chrysin (5,7-dihydroxyflavone) and galangin (3,5,7-trihydroxyflavone), as well as p-coumaric (trans-4-hydroxycinnamic acid) and caffeic acid (3,4-dihydroxycinnamic acid). Total amount of identified flavonoids varied from 109.4 μg/100 g of honey to 589.9 μg/100 g of honey, with the average of 288.5 μg/100 g of honey. All analysed honey samples showed common and specific flavonoid profile which could be the basis for differentiating sage from other monofloral honeys.     

Effect of hydrothermal treatment on the antioxidant properties of broccoli (Brassica oleracea var. botrytis italica) florets

In the extracts of fresh raw and frozen broccoli, caffeic, ferulic, sinapinic acids and kaempferol were identified. Boiling reduced the amounts of caffeic acid and kaempferol in both samples. The concentration of polyphenols was 2.69 mg/g fresh mass and 0.96 mg/fresh mass in fresh raw broccoli and frozen raw broccoli, respectively. Boiling significantly decreased the amounts of phenolic compounds in fresh broccoli (1.58 mg/g of fresh mass). In the case of frozen broccoli, boiling increased the concentration of polyphenols by 38%. Fresh broccoli extract neutralized free radicals by 19.87%. Boiling significantly reduced its antiradical activity (to 15.06%). Samples of frozen broccoli had a 27.06% antiradical ability. Boiling did not change the antiradical activity in frozen broccoli case. Hydrothermal processing significantly influenced on the ability of the extracts to inhibit the decolorization of β-carotene emulsion. The extract of fresh broccoli had a higher activity when uncooked. Boiling seemed to increase this activity in the case of frozen broccoli samples. The results of the studies on the ability of broccoli extracts to inhibit linoleic acid autooxidation were ambiguous and depended on the method applied. No correlation was found between the content of phenolic compounds and the antioxidant activity of the extracts, regardless of the experimental variant and technique used.     

Determination of glucosinolates and phenolic compounds in rocket salad by HPLC-DAD–MS: Evaluation of Eruca sativa Mill. and Diplotaxis tenuifolia L. genetic resources

The glucosinolate and phenolic profiles of 37 rocket salad accessions (32 Eruca sativa and 5 Diplotaxis tenuifolia) were obtained by liquid chromatography–mass spectrometry. Eleven desulpho-glucosinolates (DS-GLSs) were isolated and the glucosinolate profiles did not differ between the two species. Total DS-GLS content, expressed as sinigrin equivalents (SE) revealed a certain variability, ranging from 0.76 to 2.46 g kg⁻¹ d.w. but, again, the quantitative analysis did not discriminates Eruca from Diplotaxis. The polyphenol evaluation by HPLC-DAD–MS allowed the identification of two different classes of compounds in the two rocket salad species. Qualitative differences were observed between the polyphenol profiles at specific level: quercetin derivatives were the main phenolics of Diplotaxis, whereas kaempferol derivatives characterised Eruca samples. The contents of total flavonoids determined as rutin equivalents (RE) ranged from 4.68 to 31.39 g kg⁻¹ d.w. Kaempferol-3,4′-diglucoside (71.4–82.2%) and isorhamnetin-3,4′-di-glucoside (7.8–18.4%) were always isolated as first and second more abundant phenolic compounds in Eruca samples. No marker phenolic compounds were isolated in Diplotaxis samples.