Application of capillary electrophoresis to study phenolic profiles of honeybee-collected pollen

Honeybee-collected pollen is promoted as a health food with a wide range of nutritional and therapeutic properties. A high-performance capillary electrophoresis with amperometric detection method has been developed for the simultaneous determination of bioactive ingredients in 10 samples of honeybee-collected pollen in this work. Under the optimum conditions, 13 phenolic components can be well-separated or nearly baseline-separated (apigenin and vanillic acid peaks) within 29 min at the separation voltage of 14 kV in a 50 mM borax running buffer (pH 9.0), and adequate extraction was obtained with ethanol for the determination of the above 13 compounds. Recovery (94.1-104.0%), repeatability of the peak current (<5.4%), and detection limits (6.9 x 10(-7)-6.4 x 10(-9) g mL(-1)) for the method were evaluated. This procedure was successfully used for the analysis and comparison of the phenolic content of honeybee-collected pollen samples originating from different floral origins based on their electropherograms or "phenolic profiles".     

Evaluation of antioxidant activity of some natural polyphenolic compounds using the Briggs-Rauscher reaction method

A new method based on the inhibitory effects of antioxidants on the oscillations of the hydrogen peroxide, acidic iodate, malonic acid, and Mn(II)-catalyzed system (known as the Briggs-Rauscher reaction), was used for the evaluation of antioxidative capacity. With this method, which works near the pH of the fluids in the stomach (pH approximately 2), a group of natural compounds present in fruits and vegetables or in medicinal plants assumed to have antioxidant capacity, was tested successfully. The aim of the present study is to evaluate the antioxidative properties of some active principles contained in vegetables and aromatic plants, namely, cynarin (from Cynara scolymus), rosmarinic acid (from Rosmarinus officinalis), echinacoside (from Echinacea species), puerarin (from Pueraria lobata), and oleuropein (from Olea europea). Also studied with the Briggs-Rauscher reaction method was the antioxidant activity of cyanidin 3-O-beta-glucopyranoside (from Citrus aurantium) in order to compare the results with those obtained by other methods. The conclusions on the dependency of the antioxidative activity on the pH of the testing system are given.     

Identification of two polyphenolic compounds with antioxidant activities in longan pericarp tissues

Longan fruits contain a significant amount of polyphenols. In the present study, polyphenols were extracted from longan pericarp tissues, and then two representative polyphenols were separated and purified by polyamide column chromatography, Sephadex LH-20 column chromatography, and silica gel column chromatography. On the basis of 1H NMR, 13C NMR, and electrospray ionization mass spectrometric (ESI-MS) data, the two compounds were identified as 4-O-methylgallic acid and (-)-epicatechin, respectively. In terms of reaction with longan polyphenol oxidase (PPO), (-)-epicatechin was further identified as the PPO substrate that caused longan fruit to brown. The results of antioxidant activity showed that 4-O-methylgallic acid had higher reducing power and 2,2-diphenyl-1-picrylhydrazyl- (DPPH-), hydroxyl radical-, and superoxide radical-scavenging activities than (-)-epicatechin.     

Identification and quantification of polyphenolic compounds in Longan (Euphoria longana Lam.) fruit

Regular consumption of fruit and vegetables is associated with a lower risk of some chronic diseases including various forms of cancer and cardiovascular diseases. The health-promoting potential of these foods may be due, in part, to the phytochemical bioactive compounds present in the plants. Fruit of Euphoria longana Lam. (longan) are consumed throughout Asia and are a major crop in Thailand. In the present study phytochemicals were extracted with 70% methanol from peel, pulp, and seed tissues of longan fruit, and the major components were identified as gallic acid, corilagin (an ellagitannin), and ellagic acid. A high-through-put reversed phase HPLC method was developed to determine the content of these three compounds in different parts of the longan fruit and among different cultivars. The analyses showed that there was a large variation in the contents of gallic acid, corilagin, and ellagic acid in different plant tissues and cultivars. Seed contained the highest levels of the three phenolics, and pulp contained the lowest. Among commercial cultivars, Biewkiew and Edor contained the highest levels of gallic and ellagic acid while Srichompoo contained the highest content of corilagin. These three cultivars may be used in directed breeding and cultivation programs and to develop concentrated longan seed extracts to promote good health. Utilization of this byproduct material will support the use of thousands of tons of waste longan seeds after the production of canned longan pulp.     

Inhibitory effect of blueberry polyphenolic compounds on oleic acid-induced hepatic steatosis in vitro

Nonalcoholic fatty liver disease (NAFLD) is one of the most common chronic liver diseases worldwide and is closely associated with metabolic syndromes, such as obesity, diabetes, and insulin resistance. Nonalcoholic fatty liver (NAFL), also called simple steatosis, is the initial phase of NAFLD, which is accompanied the characteristic pathological overaccumulation of lipids without inflammation. To prevent NAFLD from reaching the NAFL stage through dietary therapy, in the present work, wild Chinese blueberries (Vacciniun spp.) were selected for their well-known benefits in inhibiting metabolic syndrome. After being purified from wild Chinese blueberries, polyphenol-rich extracts were subsequently separated into three fractions, namely, anthocyanin-rich fraction, phenolic acid-rich fraction, and ethyl acetate extract. The inhibition of oleic acid (OA)-induced triglyceride (TG) deposition in HepG 2 cells was referred to as the potential activity of preventing NAFL. Biochemical indicators, such as cytotoxicity, TG level, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), and intracellular reactive oxygen species, were used to evaluate the analogous pathological stage of NAFLD. The results show that OA ≤ 1.0 mM exhibits a dose-dependent induction of TG accumulation, and no inflammation was observed based on the changes in ALT and AST levels. Therefore, 1.0 mM OA was used to simulate an in vitro fatty liver. Blueberry polyphenol-rich extract efficiently inhibited OA-induced TG accumulation in HepG2 cells, and the phenolic acid-rich fraction performed efficiently. Seven phenolic acids were subsequently identified using a high-performance liquid chromatography assay, and the main types were caffeic, chlorogenic, ferulic, p-coumaric, and cinnamic acids. These phenolic acid standards also displayed good efficiency in inhibiting TG accumulation in HepG2 cells. These results imply that wild Chinese blueberries have a potential preventive effect on NAFLD in its early stage, and phenolic acids are the most efficient component.     

Antioxidant and chemopreventive properties of polyphenolic compounds derived from Greek legume plant extracts

Recently, phytochemical compounds present in legumes have gained a lot of interest because they are considered to be possible chemopreventive agents. In the present study, 14 polyphenolic compounds were extracted and identified from two unique varieties of Leguminosae family plants cultivated in Greece and screened for their antioxidant and chemopreventive properties. Ten polyphenolic fractions, which are mainly mixtures of two compounds and five pure flavonoids, were isolated from the methanolic extracts of aerial plant parts of Vicia faba and Lotus edulis (Leguminosae), respectively. All of these fractions exhibited significant DPPH(*) radical scavenging capacity. Furthermore, they exerted significant protective activity against free radical-induced DNA damage. This activity was more potent against ROO(*) radical-induced DNA damage than against that induced by OH(*) radicals. Finally, they exhibited significant ability to inhibit the activity of the topoisomerase I enzyme. These results imply that the polyphenolic compounds identified in the fractions were responsible of the observed properties of the fractions and the initial extracts and indicate different mechanisms by which these phenolic compounds may act as chemopreventive agents.     

Modelling start of oak pollen season in different climatic zones in Spain

Quercus pollen and meteorological data for several years from eight sites in Spain have been statistically analysed to select the threshold temperature and calculate the mean heat accumulation for predicting the Quercus pollination start in different climatic areas. The growing degree days method, which assumes the daily temperature varies as a sine wave, was used for heat accumulation calculations. Threshold temperatures between 4 and 12 °C were chosen using linear regression equations forced through the origin and their root mean square error (RMSE) of predicted against the observed dates for each observation site. Above the threshold, the average growing degree days (up to 1999) for the studied years was taken as the predictor value. Results showed a relationship between the selected threshold and elevation and a stronger and statistically significant correlation between threshold and yearly mean temperature, for each site. Regression analysis indicated that the selected threshold and the calculated heat accumulation were optimum for most of the localities. The validity of the results was tested using the meteorological data for the year 2000 as independent variable and this confirmed that there were only a few days difference between the predicted and observed day of the first pollen release for most of the studied localities.     

Antioxidant and anti-inflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds

The antioxidant, anti-inflammatory, and cytotoxic activities of water and ethanol extracts of 14 Chinese medicinal plants were investigated and also their total phenolics and flavonoid contents measured. The antioxidant activity was evaluated in a biological assay using Saccharomyces cerevisiae , whereas the radical scavenging activity was measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH) method. Total phenolics and flavonoid contents were estimated by Folin-Ciocalteu and aluminum chloride methods, respectively. The anti-inflammatory activities of the plant extracts were determined by measuring the inhibition of production of nitric oxide (NO) and TNF-α in LPS and IFN-γ activated RAW 264.7 macrophages. Their cytotoxic activities against macrophages were determined by Alamar Blue assay. Four plants, namely, Scutellaria baicalensis , Taxillus chinensis , Rheum officinale , and Sophora japonica , showed significant antioxidant activity in both yeast model and also free radical scavenging methods. The ethanol extract of S. japonica showed highest levels of phenolics and flavonoids (91.33 GAE mg/g and 151.86 QE mg/g, respectively). A positive linear correlation between antioxidant activity and the total phenolics and flavonoid contents indicates that these compounds are likely to be the main antioxidants contributing to the observed activities. Five plant extracts (S. baicalensis, T. chinensis, S. japonica, Mahonia fortunei , and Sophora flavescens ) exhibited significant anti-inflammatory activity by in vitro inhibition of the production of NO and TNF-α with low IC(50) values. These findings suggest that some of the medicinal herbs studied in this paper are good sources of antioxidants.     

Control of pitch pine seed germination and initial growth exerted by leaf litters and polyphenolic compounds

We investigated the influence of three concentrations of water extracts of three leaf litter species (pitch pine, huckleberry and white oak) and a mixture of all litters on the germination of pitch pine seeds and initial seedling growth in a microcosm experiment. All three plant species are important components of the pine barrens ecosystems in New Jersey, where it has been seen that pine seedling recruitment occurs only after stand replacing fire or in disturbed sites, where surface organic soil horizons and leaf litter have been removed. Leaf litter extracts did not influence seed germination, but significantly reduced seedling growth at high concentrations. There was little difference between the leaf litter species in growth suppression. As charcoal is a natural residue on the forest floor following fire, its influence on growth suppression was examined; it has been shown to immobilize polyphenols. Charcoal removed the inhibitory effect of leaf litter extracts and allowed the fertilizer effect of nutrients leached from the leaves to enhance seedling growth, particularly at the higher concentration of litter extract used. Responses to litter extracts were compared to four pure phenolic compounds, catchecol, p-coumaric acid, p-hydroxybenzoic acid and tannic acid. None of these compounds suppressed pine seedling growth, suggesting that these phenolics are not allelopathic to pine seedlings. The results are discussed in the context of fire as a driving factor in these oligotrophic and seasonally dry ecosystems and the interactions between nutrient supply and allelopathic chemistry of different leaf litters.     

The use of micromorphology in soil pollen analysis: The interpretation of the pollen content of slope deposits in Galicia, Spain

Humic horizons of soil profiles often contain pollen and spores. Comparison of the sequence of pollen associations of soil profiles with pollen zones of reference diagrams offers palyno-ecological information, relevant for reconstruction of landscape ecological development. In palynological studies of semiterrestrial and aquatic deposits, pollen is considered to be part of the sediment and anaerobic conditions in water-saturated deposits promote its conservation. This is not true for pollen in soils. Aerobic conditions in drained soils result in bio-oxidation of unprotected pollen grains. Interpretation of soil pollen associations requires knowledge of the processes of infiltration and conservation of pollen grains in the soil material. Micromorphology is an important technique to investigate these processes. This is demonstrated in a case study of polycyclic slope deposits in Galicia (Spain). The buried cambisols, in pleistocene solifluction deposits, contain post-sedimentary infiltrated pollen. The pollen associations reflect successive phases in soil ecological development. The holocene colluvial deposits contain syn-sedimentary incorporated pollen. The pollen associations reflect environmental conditions during the deposition of colluvial layers.     

Determination of water-soluble polyphenolic compounds in commercial herbal teas from Lamiaceae: peppermint, melissa, and sage

Chromatographic techniques (HPLC and HPTLC) were used for qualitative and quantitative determination of eriocitrin, luteolin 7-O-rutinoside, luteolin 7-O-beta-glucuronide, lithospermic acid, rosmarinic acid, and methyl rosmarinate together with other known compounds in commercial herbal teas from the Lamiaceae family: peppermint leaf (Menthae piperitae folium), melissa leaf (Melissae folium), and sage leaf (Salviae officinalis folium). Contents of analyzed compounds in infusions, the most popular forms, were established using a C18 column with acetonitrile-water-formic acid as a mobile phase. The HPLC method was validated for linearity, precision, and accuracy. Luteolin 7-O-beta-glucuronide and lithospermic acid were identified as new Mentha x piperita compounds. The investigated herbal teas delivered polyphenols in high amounts, up to 182.2 mg for the infusion of one peppermint tea bag.     

New polyphenolic compounds with xanthylium skeletons formed through reaction between (+)-catechin and glyoxylic acid

The reaction between (+)-catechin and glyoxylic acid was studied in a model solution system. The major (+)-catechin carboxymethine-linked dimer was isolated and shown to proceed to new polyphenolic compounds exhibiting absorption maxima around 440 and 460 nm. Three yellow pigments were obtained by incubation of the 8-8 colorless isomer. One was the previously reported xanthylium compound NJ2 with a maximum at 440 nm. The other two, showing absorption maxima at 460 nm, were obtained separately by incubation of the colorless dimer in hydroethanolic or methanolic medium. Structural elucidation of these two new yellow pigments was achieved by means of MS and 1D and 2D NMR techniques and showed that they were, respectively, ethyl and methyl esters of NJ2. The fact that these compounds were not obtained when NJ2 was incubated in hydromethanolic or ethanolic medium showed that esterification took place before the formation of the xanthylium chromophores. The detection of the esterified colorless compounds and the corresponding xanthene intermediates confirmed the postulated mechanism. New pigments exhibiting a strong absorption at 560 nm were also observed.     

Volatile compounds in samples of cork and also produced by selected fungi

The production of volatile compounds by microbial communities of cork samples taken during the cork manufacturing process was investigated. The majority of volatiles were found in samples collected at two stages: resting after the first boiling and nontreated cork disks. Volatile profiles produced by microbiota in both stages are similar. The releasable volatile compounds and 2,4,6-trichloroanisole (TCA) produced in cork-based culture medium by five isolated fungal species in pure and mixed cultures were also analyzed by gas chromatography coupled with mass spectrometry (GC-MS).The results showed that 1-octen-3-ol and esters of fatty acids (medium chain length C8-C20) were the main volatile compounds produced by either pure fungal species or their mixture. Apparently, Penicillium glabrum is the main contributor to the overall volatile composition observed in the mixed culture. The production of releasable TCA on cork cannot be attributed to any of the assayed fungal isolates.     

Changes in the content of bioactive polyphenolic compounds of olive mill wastewater by the action of exogenous enzymes

The aim behind the present research is to develop an enzymatic treatment for olive mill wastewater (OMW) to release high amounts of simple phenolics having high antioxidant value. OMW was hydrolyzed by a mixed enzyme preparation rich in β-glucosidase produced by Aspergillus niger . This research shows that A. niger β-glucosidase played a major role in the release of simple phenolic compounds from OMW. These compounds were recovered by ethyl acetate extraction and identified by HPLC and LC-MS. The main identified phenolic compound is hydroxytyrosol. The results of enzymatic hydrolysis of OMW under optimum conditions indicated a maximum hydroxytyrosol concentration of 2.9 g L(-1) compared to 0.015 g L(-1) contained in the control (test without added enzyme). The above results prove that OMW is a potential substrate for producing hydroxytyrosol through enzymatic hydrolysis of its glycosides.     

Evaluation of solid-phase microextraction for the isotopic analysis of volatile compounds produced during fermentation by lactic acid bacteria

The use of solid-phase microextraction (SPME) coupled with isotope ratio mass spectrometry (IRMS) for the analysis of flavor compounds produced by lactic acid bacteria has been evaluated using both liquid and headspace sampling modes. Initially, it was necessary to optimize the conditions for the SPME extraction of flavors-diacetyl and acetoin-in standard aqueous solutions. The effects of salt, headspace versus liquid sampling, and coating phase were tested. Second, the suitability of the coupling of SPME and gas chromatography-combustion interface-IRMS (GC-C-IRMS) for the determination of delta(13)C values was assessed. It is shown that neither the analyte concentration nor the period of fiber exposure has an effect on the delta(13)C values. Finally, having verified that there are no matrix effects from the fermentation medium, it is reported for the first time that flavor compounds can be extracted directly from culture supernatant by SPME and their delta(13)C values can be obtained by GC-C-IRMS.     

Characterization and quantitation of polyphenolic compounds in bark, kernel, leaves, and peel of mango (Mangifera indica L.)

The contents of secondary plant substances in solvent extracts of various byproducts (barks, kernels, peels, and old and young leaves) in a range of Brazilian mango cultivars were identified and quantitated. The results show that the profiles of secondary plant substances such as xanthone C-glycosides, gallotannins, and benzophenones in different byproducts vary greatly but are fairly consistent across cultivars. The free radical scavenging activity of the solvent extracts was evaluated using a high-performance liquid chromatography-based hypoxanthine/xanthine oxidase assay and revealed dose-dependent antioxidant capacity in all extracts. Four (mangiferin, penta- O-galloyl-glucoside gallic acid, and methyl gallate) of the major phenolic compounds detected were also evaluated in additional in vitro bioassay systems such as oxygen radical absorbance capacity, 2,2-diphenyl-1-picrylhydrazyl, and ferric reducing ability of plasma. Mangiferin in particular, detected at high concentrations in young leaves (Coite = 172 g/kg), in bark (Momika = 107 g/kg), and in old leaves (Itamaraka = 94 g/kg), shows an exceptionally strong antioxidant capacity.     

Volatile compounds produced from monosodium glutamate in common food cooking

A mixture of soybean oil and/or water, sugar and/or monosodium glutamate (MSG) was water-boiled or oil-heated at 100, 120, 140, 160, or 170 degrees C, respectively, and volatile compounds produced were isolated by solid-phase microextraction and then identified by GC and GC/MS. Oxidative thermal degradation products of fatty acids (OTDPFA) were the major volatile compounds detected for all water-boiled samples. When MSG and sugar were heated together, 2, 5-dimethyl pyrazine and methyl pyrazine were also detected at or after 160 min heating. Water added in soybean oil increased OTDPFA production. In oil-heated samples of soybean oil alone, soybean oil with MSG, and soybean oil with sugar, OTDPFA were also the major compounds found. The samples containing MSG also produced 2-pyrrolidone, and the samples containing sugar also produced furfural and 5-hydroxy methyl furfural. The samples containing soybean oil, sugar, and MSG produced 23 pyrazines, OTDPFA, and gamma-butyrolactone as the major volatile compounds. The contents of OTDPFA were lower in MSG plus sugar and MSG-added samples, and it is postulated that antioxidant activities were produced in cooking.     

Quantitative characterization of flavonoid compounds in Rooibos tea (Aspalathus linearis) by LC-UV/DAD

Rooibos tea originates from the leaves and stems of the indigenous South African plant Aspalathus linearis. It has gained much attention for clinical purposes in the case of nervous tension, allergies (dermatitis), and various indigestive problems. Recently, antioxidative activity was also attributed to the tea on the basis of its flavonoid content. Therefore, an HPLC method using a C(18) reversed phase column was developed for the assay of 10 flavonoids in aqueous and methanolic infusions. Main compounds determined were the dihydrochalcone aspalthin, rutin, and orientin, and their content was in the range of 1.0 to 1.3 mg/g. The identity of detected flavonoids was confirmed by comparing their retention times and UV and MS spectra with those of corresponding standards. In addition, the MS analysis showed evidence of the presence of other compounds such as nothofagin, dihydroisoorientin, and dihydroorientin.     

不同镍盐对果树花粉萌发及花粉管生长的影响

以桃和杏不同品种为试材,研究不同镍（Ni2+）盐（氯化镍、硝酸镍、硫酸镍）处理对果树花粉萌发和花粉管生长的影响。结果表明,较低浓度的Ni2+盐可以明显促进果树花粉萌发和花粉管的伸长生长;浓度较高时则明显产生抑制作用,且3种Ni2+盐表现规律一致。桃品种花粉萌发的适宜浓度为Ni2+ 4～6 mol/L;杏品种花粉萌发的适宜浓度为2 mol/L。其作用效应能力大小因Ni2+盐种类、浓度和果树品种而异。表明镍肥在果树上应用是有益的。     

A comparative study of flavonoid compounds, vitamin C, and antioxidant properties of baby leaf Brassicaceae species

A comparative study of antioxidant compounds, flavonoids and vitamin C, and also antioxidant activity was carried out in four species of Brassicaceae vegetables used for salads: watercress ( Nasturtium officinale R. Br.), mizuna [ Brassica rapa L. subsp. nipposinica (L.H. Bailey) Haneltand], wild rocket [ Diplotaxis tenuifolia (L.) DC.], and salad rocket [ Eruca vesicaria (L.) Cav.]. The characterization of individual phenolic compounds by HPLC-DAD-MS/MS-ESI in watercress and mizuna completes the polyphenol study previously reported for wild rocket and salad rocket. The qualitative study of flavonoids in watercress leaves showed a characteristic glycosylation pattern with rhamnose at the 7 position. Isorhamnetin 3,7-di- O-glucoside was identified in mizuna leaves and may be considered a chemotaxonomical marker in some B. rapa subspecies. Brassicaceae species showed differences in the quantitative study of flavonoids, and the highest content was detected in watercress leaves. Watercress and wild rocket leaves had the highest content of vitamin C. The antioxidant activity evaluated by different methods (ABTS, DPPH, and FRAP assays) showed a high correlation level with the content of polyphenols and vitamin C. In conclusion, the Brassicaceae leaves studied, watercress, mizuna, wild rocket, and salad rocket, presented a large variability in the composition and content of antioxidant compounds. These baby leaf species are good dietary sources of antioxidants with an important variability of bioactive compounds.