Supercritical fluid extraction and fractionation of different preprocessed rosemary plants

Two-step supercritical fluid extraction of rosemary leaves at selected conditions of pressure and temperature is proposed to divide the oleoresin into two fractions with different antioxidant activities and essential oil compositions. Rosemary leaves obtained from different sources have been extracted and evaluated in terms of antioxidant activity and essential oil yield and composition. Also, a new device is proposed to improve the performance of the technique in terms of sample collection after SFE.     

Anti-inflammatory effects of supercritical carbon dioxide extract and its isolated carnosic acid from Rosmarinus officinalis leaves

Rosemary (Rosmarinus officinalis) leaves possess a variety of bioactivities. Previous studies have shown that the extract of rosemary leaves from supercritical fluid extraction inhibits the expression of inflammatory mediators with apparent dose-dependent responses. In this study, three different extraction conditions (5000 psi at 40, 60, and 80 °C) of supercritical carbon dioxide (SC-CO(2)) toward the extraction of antioxidants from rosemary were investigated. Furthermore, simultaneous comparison of the anti-inflammatory properties between rosemary extract prepared from SC-CO(2) under optimal conditions (5,000 psi and 80 °C) and its purified carnosic acid (CA) using lipopolysaccharide (LPS)-treated murine RAW 264.7 macrophage cells was also presented. Results showed that the yield of 3.92% and total phenolics of 213.5 mg/g extract obtained from the most effective extraction conditions showed a high inhibitory effect on lipid peroxidation (IC(50) 33.4 μg/mL). Both the SC-CO(2) extract and CA markedly suppressed the LPS-induced production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α), as well as the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), phosphorylated inhibitor-kappaB (P-IκB), and nuclear factor-kappaB (NF-κB)/p65 in a dose-dependent manner. The five major compounds of verbenone, cirsimaritin, salvigenin, carnosol, and CA existing in the SC-CO(2) extract were isolated by semipreparative HPLC and identified by HPLC-MS/MS analysis. CA was the most abundant recorded compound and the most important photochemical with an anti-inflammatory effect with an IC(50) of 22.5 μM or 7.47 μg/mL presented to the best inhibitory activity on NO production better than that of the 14.50 μg/mL dosage prepared from the SC-CO(2) extract. Nevertheless, the effective inhibition of LPS-induced NF-κB signaling in RAW 264.7 cells from the SC-CO(2) extract extends the potential application of nutraceutical formulation for the prevention of inflammatory diseases.     

Polyphenolic transmission to Segureno lamb meat from ewes' diet supplemented with the distillate from rosemary (Rosmarinus officinalis) leaves

The aim of the present work is to study whether the introduction of rosemary plant byproduct, from plant steam distillation, in daily Segurena sheep feeding allows the transfer of active antioxidant components to lamb meat, without detriment to the animal productivity. For this, 36 Segurena ewes were assigned randomly to three homogeneous groups. One group was fed a basal diet as a control and the diet of the other two groups was modified by substituting 10 or 20% of the control diet (respectively) with distilled rosemary leaves. Chromatographic analysis allowed the identification of 11 polyphenolic components previously identified in the rosemary and basal diet pellets, respectively. Among them, rosmarinic acid, carnosol, and carnosic acid were the phenolic components that had a significantly increased presence ( P < 0.05) in the lamb meat from sheep mothers fed this aromatic herb, when compared to the control group. The incorporation of this byproduct into the animal diet favored the antioxidant capacity of these lamb meat samples. Fresh meat produced on rosemary had higher total ferric reducing antioxidant power (FRAP) ( P < 0.05), greater ability to reduce ABTS*+, and lower IC50 (DPPH*) ( P < 0.05) values when compared to the control group. Because no statistically significant differences were detected among the results obtained from the lamb meat belonging to the different ewe groups fed rosemary leaf extract (10 or 20%), it can be concluded that the incorporation of distilled rosemary leaves at a rate of 10% of the ewes' diet should be enough to improve the lamb meat antioxidant status.     

Pressurized fluid extraction of bioactive compounds from Phormidium species

In the search for new functional ingredients with potential use in the food industry, extracts of unknown species of microalgae, such as Phormidium species have been studied. Three solvents of different polarities (i.e., hexane, ethanol, and water) have been used to obtain pressurized liquid extracts with different compositions. Moreover, extractions were performed at four different extraction temperatures (50, 100, 150, and 200 degrees C) with 20 min as extraction time. Antioxidant activity of the extracts has been measured by the TEAC assay. In general, hexane and ethanol extracts showed a higher antioxidant capacity that was mainly attributed to carotenoid compounds, as the TEAC value trend seems to be similar to the carotenoid content of the extracts. On the other hand, the high antioxidant activity of the 200 degrees C water extracts is likely related to the presence of Maillard reaction compounds produced by thermal degradation of the sample. beta-Carotene, lutein, violaxanthin, and neoxanthin were identified in 150 degrees C ethanol extracts. Four different microbial species ( Escherichia coli, Staphylococcus aureus, Candida albicans, and Aspergillus niger) were used to screen the potential antimicrobial activity of the Phormidium sp. extracts. The most sensitive microorganism was the yeast, C. albicans, whereas the fungus, A. niger, was the most resistant. In general, no drastic differences were found for solvents and temperatures tested, showing a very diverse nature of the compounds responsible for the antimicrobial activity of these microalgae. In ethanol extracts, antimicrobial activity could be mainly attributed to the presence of terpenes (i.e., beta-ionone, neophytadiene) and fatty acids (i.e., palmitoleic and linoleic acids) in the samples. Toxicity studies carried out with the extracts evaluated in the present work showed a cellular toxicity lower than those of other cyanobacteria such as Spirulina plantensis.     

Isolation of antioxidant compounds from orange juice by using countercurrent supercritical fluid extraction (CC-SFE).

Antioxidants from orange juice are isolated by the use of countercurrent supercritical fluid extraction (CC-SFE) and characterized by reversed-phase liquid chromatography (RPLC) coupled to mass spectrometry (MS) and diode-array detection (DAD). A pilot-scale SFE plant equipped with a packed column has been employed for countercurrent extraction and fractionation of raw orange juice with carbon dioxide. Several experiments have been performed in order to study the effect of the countercurrent conditions on the content of antioxidative compounds. In this study, the main variable that has been considered is the solvent-to-feed ratio (S/F) because it plays an essential role in the extraction efficiency. The values tested covered a wide range of sample and solvent (CO(2)) flow rates. In each experimental run, two different extracted fractions and the residual nonextracted juice were obtained and characterized. Different flavonoids have been identified in the fractions obtained after CC-SFE. The possibility of using this process for antioxidant compounds enrichment is discussed.     

Castanea sativa Mill. leaves as new sources of natural antioxidant: an electronic spin resonance study

The antioxidant potential of Castanea sativa Mill. leaf (sweet chestnut) was explored as a new source of active extracts. The capacity of the different fractions issued from aqueous, methanol, and ethyl acetate extracts to inhibit the stable free radical 2,2-diphenyl-1-pycryl-hydrazyl, superoxide anion, and hydroxyl radical was measured by electronic spin resonance. Their scavenging potential was analyzed versus their amount of phenolic compounds. Among the active fractions, the most effective one was A6, an ethyl acetate fraction, which contained a high level of total phenolic compounds (29.1 g/100 g). Thus, a different extraction procedure was performed to concentrate the active compounds of A6 in the new C. sativa leaf extract (CSLE). Compared to reference antioxidants (quercetin and vitamin E) and standard extracts (Pycnogenol, from French Pinus maritima bark, and grape marc extract), it was observed that A6 and CSLE have high antioxidant potentials, equivalent to at least those of reference compounds.     

Flavanol content and antioxidant activity in winery byproducts

Proanthocyanidins, particularly those coming from wine and grape products, have became of interest to nutritionists. Particular attention is currently being paid to the exploitation of this kind of grape byproducts for obtaining bio-active phenolic compounds with potential application as food antioxidants and preventive agents against cancer and other diseases. In this work, the flavanol composition of various winery byproducts submitted to different degrees of industrial exploitation has been studied and their antioxidant activity determined using two different methods (TBARS and TEAC) to evaluate their interest as suitable sources for the preparation of flavanol-rich antioxidant extracts. All the byproducts studied were still good flavanol sources no matter their exploitation degree. An important conclusion was that dried grape seeds, obtained as an end byproduct after the color extraction and alcohol distillation of the wine pomace, still kept important flavanol concentrations and significant antioxidant activity, even if they were submitted to high temperatures. These byproducts can be considered a cheap source for the extraction of antioxidant flavanols, which can be used as dietary supplements or in the production of phytochemicals.     

亚临界丙烷萃取米糠油及其微量活性组分动力学模型

为了揭示亚临界流体萃取米糠油及其微量活性组分的动力学规律,该文以亚临界丙烷为萃取介质,研究了米糠油、γ-谷维素及α-生育酚等活性组分在不同萃取温度和时间的萃取率,并基于 Baümler 模型对目标物的萃取率进行模型拟合,建立相应的动力学模型并验证。结果表明,亚临界萃取过程中目标物的相对萃取率随时间增长递增,且在初始阶段（洗涤过程）的增长速率快于第二阶段（扩散过程）的增长速率;同时,升高萃取温度可同时提高目标产物的扩散系数和相对萃取率;基于Baümler模型可较好拟合亚临界丙烷萃取米糠油、γ-谷维素和α-生育酚的动力学过程（R2>0.95）,所得动力学模型具有较好的预测准确性。由Arrhenius方程可计算出米糠油、α-生育酚和γ-谷维素的亚临界萃取反应活化能,其中米糠油的活化能最低（5.23 kJ/mol）,α-生育酚次之（7.05 kJ/mol）,γ-谷维素最高（9.11 kJ/mol）,表明γ-谷维素最难萃取且对温度依赖程度最高;根据拟合所得动力学模型,对米糠油提取率进行预测,结果表明,该模型对米糠油萃取率的预测准确率达95.8%,而对γ-谷维素和α-生育酚萃取率的预测准确度稍低,分别达94.7%和94.4%,对三者均具有很好的预测准确性。总之,基于 Baümler 模型可以较好地建立米糠油及其有益伴随物的亚临界萃取动力学模型,从而较为准确的预测米糠油及其微量活性组分的萃取率,对米糠油的亚临界萃取工艺具有一定的理论指导意义。     

Optimization of microwave-assisted extraction for the characterization of olive leaf phenolic compounds by using HPLC-ESI-TOF-MS/IT-MS(2)

In the present work, a simple and rapid method for the extraction of phenolic compounds from olive leaves, using microwave-assisted extraction (MAE) technique, has been developed. The experimental variables that affect the MAE process, such as the solvent type and composition, microwave temperature, and extraction time, were optimized using a univariate method. The obtained extracts were analyzed by using high-performance liquid chromatography (HPLC) coupled to electrospray time-of-flight mass spectrometry (ESI-TOF-MS) and electrospray ion trap tandem mass spectrometry (ESI-IT-MS(2)) to prove the MAE extraction efficiency. The optimal MAE conditions were methanol:water (80:20, v/v) as extracting solvent, at a temperature equal to 80 °C for 6 min. Under these conditions, several phenolic compounds could be characterized by HPLC-ESI-MS/MS(2). As compared to the conventional method, MAE can be used as an alternative extraction method for the characterization of phenolic compounds from olive leaves due to its efficiency and speed.     

Antioxidant properties of various solvent extracts of total phenolic constituents from three different agroclimatic origins of drumstick tree (Moringa oleifera Lam.) leaves

Water, aqueous methanol, and aqueous ethanol extracts of freeze-dried leaves of Moringa oleifera Lam. from different agroclimatic regions were examined for radical scavenging capacities and antioxidant activities. All leaf extracts were capable of scavenging peroxyl and superoxyl radicals. Similar scavenging activities for different solvent extracts of each collection were found for the stable 1,1-diphenyl 2-picrylhydrazyl (DPPH(*)) radical. Among the three different moringa samples, both methanol and ethanol extracts of Indian origins showed the highest antioxidant activities, 65.1 and 66.8%, respectively, in the beta-carotene-linoleic acid system. Nonetheless, increasing concentration of all the extracts had significantly (P < 0.05) increased reducing power, which may in part be responsible for their antioxidant activity. The major bioactive compounds of phenolics were found to be flavonoid groups such as quercetin and kaempferol. On the basis of the results obtained, moringa leaves are found to be a potential source of natural antioxidants due to their marked antioxidant activity. This is the first report on the antioxidant properties of the extracts from freeze-dried moringa leaves. Overall, both methanol (80%) and ethanol (70%) were found to be the best solvents for the extraction of antioxidant compounds from moringa leaves.     

Extraction of tricyclazole from soil and sediment with subcritical water

The use of subcritical water to extract tricyclazole from soils and sediments was examined. Extraction efficiency and kinetics were determined as a function of temperature, sample age, sample matrix, sample size, and flow rate. Extraction temperature was the most influential experimental factor affecting extraction efficiency and kinetics, with increasing temperature (up to 150 degrees C) yielding faster and higher efficiency extractions. Higher extraction temperatures were also important for quantitative recovery of tricyclazole from aged samples. Extraction at 50 degrees C yielded 97% recoveries from samples aged 1 day but only 30% recoveries for samples aged 202 days, whereas extraction at 150 degrees C yielded recoveries of 85-100% that were independent of incubation time and sample matrix, with the exception of one sediment that contained a large amount of organic matter. Sample extracts from subcritical water extraction were generally a pale yellow color, contrasted with a dark brown color from organic solvent extractions of the same matrixes. Less sample cleanup was therefore required prior to analysis, with the total time for the extraction and analysis of a single sample being approximately 2 h. Subcritical water extraction is an effective technique for the rapid and quantitative extraction of tricyclazole from soils and sediments.     

Total oxidant scavenging capacity of Euterpe oleracea Mart. (açaí) seeds and identification of their polyphenolic compounds

The antioxidant capacity of methanol and ethanol seed extracts from Euterpe oleracea Mart. (a?aí) against the reactive oxygen species (ROS) peroxyl radicals, peroxynitrite, and hydroxyl radicals was studied with the total oxidant scavenging capacity (TOSC) assay in a modified and automated version. Cold methanol digestion was the most efficient extraction method with respect to the antioxidant capacity. The extracts exhibit good antioxidant capacity against peroxyl radicals, similar to the capacity of the pulp. The antioxidant capacity against peroxynitrite and hydroxyl radicals is even higher. The main antioxidants identified by HPLC-MS and HPLC-CEAD are five different procyanidins (di- through pentamers); furthermore, protocatechuic acid and epicatechin were identified as minor compounds. Determination of TOSC values of HPLC seed extract fractions indicates that the procyanidins contribute substantially to the overall antioxidant capacity. In addition, however, other compounds that have not yet been identified are responsible for a large part of the observed antioxidant capacity.     

Relevance of carnosic acid concentrations to the selection of rosemary, Rosmarinus officinalis (L.), accessions for optimization of antioxidant yield

Methods were developed to identify and select accessions of rosemary, Rosmarinus officinalis (L.), producing optimum antioxidant activity. Extracts from 12 different rosemary accessions, using three solvents of varying polarity, were assayed for their antioxidant activity, and their major antioxidant compounds were identified and quantified by high-performance liquid chromatography (HPLC). Carnosic acid concentrations were correlated with (i) the free radical scavenging activity of these extracts, as measured by the 2,2-diphenyl-1-picrylhydrazyl assay (adjusted R(2) = 77.3%) and (ii) their inhibition of linoleic acid oxidation, as measured by the beta-carotene assay (adjusted R(2) = 44.1%). The correlation was broadly confirmed by the production of volatile aldehydes as measured by the hexanal assay. The variation of carnosic acid concentrations in extracts of 29 accessions, grown in field trials at three sites in England, was determined.     

Extraction of organic compounds from airborne particulate matter

The extraction of organic compounds from airborne particulate matter, obtained by glass fiber filtration, has been studied with different solvents and has been followed as a function of time. For many compounds, almost quantitative extraction was obtained after S h. Polyaromatic hydrocarbons and polar compounds often yielded higher extraction efficiencies with methanol than with benzene. The gas chromatographic mass spectrometric determination of these compounds is somewhat simplified by a separation of the sample into neutral, acidic and basic fractions. The efficiency of this procedure was tested on a mixture of compounds representative for the actual aerosol composition. It was concluded that the basic fraction yielded poor results. The techniques for these measurements included quantitative gas chromatography with electronic integration for standard mixtures and quantitative mass chromatography for natural sample extracts.     

Identification and quantification of antioxidant components of honeys from various floral sources

Little is known about the individual components of honey that are responsible for its antioxidant activity. The present study was carried out to characterize the phenolics and other antioxidants present in honeys from seven floral sources. Chromatograms of the phenolic nonpolar fraction of the honeys indicated that most honeys have similar but quantitatively different phenolic profiles. Many of the flavonoids and phenolic acids identified have been previously described as potent antioxidants. A linear correlation between phenolic content and ORAC activity was demonstrated (R(2) = 0.963, p < 0.0001). Honeys were separated by solid-phase extraction into four fractions for sugar removal and separation based on solubility to identify the relative contribution of each fraction to the antioxidant activity of honey. Antioxidant analysis of the different honey fractions suggested that the water-soluble fraction contained most of the antioxidant components. Specific water-soluble antioxidant components were quantified, including protein; gluconic acid; ascorbic acid; hydroxymethylfuraldehyde; and the combined activities of the enzymes glucose oxidase, catalase and peroxidase. Of these components, a significant correlation could be established only between protein content and ORAC activity (R(2) = 0.674, p = 0.024). In general, the antioxidant capacity of honey appeared to be a result of the combined activity of a wide range of compounds including phenolics, peptides, organic acids, enzymes, Maillard reaction products, and possibly other minor components. The phenolic compounds contributed significantly to the antioxidant capacity of honey but were not solely responsible for it.     

Combined use of supercritical fluid extraction, micellar electrokinetic chromatography, and reverse phase high performance liquid chromatography for the analysis of antioxidants from rosemary (Rosmarinus officinalis L.)

Antioxidants from rosemary were determined by the combined use of supercritical fluid extraction (SFE) prior to reverse-phase high-performance liquid chromatography (RP-HPLC) or micellar electrokinetic chromatography (MEKC). The separation of antioxidants found in the SFE fractions was achieved by using a new MEKC method and a published HPLC procedure, both with diode array detection. The characterization of the different antioxidants was further done by HPLC-mass spectrometry. Advantages and drawbacks of HPLC and MEKC for analyzing the antioxidants found in the different extracts are discussed. From the results it is concluded that HPLC renders higher peak area and is better in its reproducibility than MEKC; both techniques provide similar analysis time reproducibility. The main advantage of MEKC is its much higher separation speed, which is demonstrated to be useful for the quick adjustment of SFE conditions, allowing rosemary fractions of higher antioxidative power to be obtained quickly. Moreover, the possibilities of this approach for following the degradation of antioxidants are discussed.     

New alternative method of benzo[a]pyrene extractionfrom soils and its approbation in soil under technogenic pressure

Purpose

The optimization of benzo[a]pyrene extraction conditions by subcritical water extraction method from soils is the purpose of the research. The optimal conditions for benzo[a]pyrene recovery are 30-min extraction by water in a special steel cartridge at 250 °C and 100 atm.

Materials and methods

Studies were conducted on the soils of monitoring plots subjected to Novocherkassk Power Station emissions. Monitoring plots were established at different distances from the Novocherkassk Power Station (NPS; 1.0–20.0 km).

Results and discussion

It was shown that the use of water in subcritical state as a solvent for benzo[a]pyrene extraction from soil allows to avoid large volumes of organic solvents and to decrease the time of sample preparation. It is shown that the maximum benzo[a]pyrene maintenance was observed in soils of the monitoring plots located most close (to 5 km) to a pollution source in the area of the prevailing direction of a wind rose. Dynamics of pollutant accumulation in soils depend on number of Novocherkassk state district power station emissions.

Conclusions

The method of benzo[a]pyrene subcritical water extraction from soil was developed and approbated during long-term monitoring researches of technogenic polluted territories. The optimum conditions for benzo[a]pyrene extraction from soil have been determined: the soil is treating by subcritical water at 250 °C and 100 atm of pressure for 30 min. Trends in the accumulation of benzo[a]pyrene in soil zones of the thermal power plant influence have been researched over a 5-year period of monitoring observations by subcritical water extraction method. Benzo[a]pyrene accumulation in soils depends on the technogenic emissions to the atmosphere from Novocherkassk power station and on the soil physical and chemical properties.

     

Multielemental speciation analysis of fungi porcini (Boletus edulis) mushroom by size exclusion liquid chromatography with sequential on-line UV-ICP-MS detection

An analytical methodology to determine the molecular weight (MW) distribution patterns of several elements among different compounds present in commonly consumed edible mushrooms is presented in this work. A hyphenated technique based on size exclusion liquid chromatography (SEC) coupled on-line to UV and inductively coupled plasma mass spectrometry (ICP-MS) detection was used. The association of the elements to high and low MW fractions was confirmed with sequential detection by UV and ICP-MS. Separation of the fractions was performed by injecting a 100 microL sample volume to a Superdex 75 column. The effect of different mobile phases on the separation was evaluated. Additionally, three different extraction conditions including 0.05 mol L(-1) NaOH, 0.05 mol L(-1) HCl, and hot water at 60 degrees C were applied to extract the elemental species from the mushroom samples. Significant differences were observed in the chromatograms depending on the extraction conditions utilized. Optimization of the experimental variables involved in the SEC-UV-ICP-MS coupling was carried out. The method was applied to investigate the fractionation patterns of Bi, Co, Cu, Fe, I, Mo, Ni, Se, and Zn in fungi porcini (Boletus edulis) mushroom. The results obtained in this work indicate an important association of most of the elements to high MW fractions.     

Optimization of the extraction of antioxidants from Dunaliella salina microalga by pressurized liquids

In this work, extraction of antioxidant compounds from Dunaliella salina microalga is optimized by combining pressurized liquid extraction (PLE) and experimental design (three-level factorial design) with three different solvents (hexane, ethanol, and water). Two main factors were considered, the extraction temperature (40, 100, and 160 degrees C) and the extraction time (5, 17.5, and 30 min). As response variables, the extraction yield (percent dry weight/initial weight) and the antioxidant activity of the extracts (determined using the TEAC method) were used. The parameters of the model were estimated by multiple linear regression. Results showed that the extraction temperature was the factor having the strongest influence (positive) on the two response variables. The best yields were obtained with ethanol at the higher extraction temperature and time tested. Besides, although hexane extracts provided the best antioxidant activity, ethanol extracts were also very active. The chemical characterization of ethanol extracts was carried out using HPLC-DAD, and attempts have been made to correlate their chemical composition with the antioxidant activity measured. Results pointed out that the extracts contained, besides all-trans-beta-carotene and isomers, several different minor carotenoids that seemed to make a contribution to the antioxidant activity of the extracts.     

Total Phenolics and Antioxidant Activity of Water and Ethanolic Extracts from Distillers Dried Grains with Solubles With or Without Microwave Irradiation

The purpose of this study was to determine the efficacy of extracting phenolic compounds with antioxidant activity from distillers' dried grains with solubles (DDGS) with water, 50% aqueous ethanol, and absolute ethanol, using microwave irradiation or a water bath at various temperatures. DDGS was extracted for 15 min with each solvent while heating at 23, 50, 100, and 150°C by microwave irradiation or in a water bath at 23, 50, and 100°C. Phenolic content of extracts increased with increasing temperature to a maximum of 12.02 mg/g in DDGS extracts that were microwave irradiated in water or with 50% aqueous ethanol at 150°C. Antioxidant activity range was 1.49–6.53 μmol of Trolox equivalents/g of DDGS. Highest antioxidant activities were obtained from 50% aqueous ethanol extracts at all temperatures, and water extracts that were heated at 100 and 150°C. These data indicate that DDGS extracts with high phenolic content and antioxidant activity can be obtained from DDGS, particularly with the use of water or 50% ethanol and high temperature (100 or 150°C). This may be valuable to ethanol manufacturers, livestock producers, and food and nutraceutical companies.