New possibilities for the valorization of olive oil by-products

In this contribution, the capabilities of pressurized liquid extraction (PLE) using food-grade solvents, such as water and ethanol, to obtain antioxidant extracts rich on polyphenolic compounds from olive leaves are studied. Different extraction conditions were tested, and the PLE obtained extracts were characterized in vitro according to their antioxidant capacity (using the DPPH radical scavenging and the TEAC assays) and total phenols amounts. The most active extracts were obtained with hot pressurized water at 200 °C (EC(50) 18.6 μg/mL) and liquid ethanol at 150 °C (EC(50) 27.4 μg/mL), attaining at these conditions high extraction yields, around 40 and 30%, respectively. The particular phenolic composition of the obtained extracts was characterized by LC-ESI-MS. Using this method, 25 different phenolic compounds could be tentatively identified, including phenolic acids, secoiridoids, hydroxycinnamic acid derivatives, flavonols and flavones. Among them, hydroxytyrosol, oleuropein and luteolin-glucoside were the main phenolic antioxidants and were quantified on the extracts together with other minor constituents, by means of a UPLC-MS/MS method. Results showed that using water as extracting agent, the amount of phenolic compounds increased with the extraction temperature, being hydroxytyrosol the main phenolic component on the water PLE olive leaves extracts, reaching up to 8.542 mg/g dried extract. On the other hand, oleuropein was the main component on the extracts obtained with ethanol (6.156-2.819 mg/g extract). Results described in this work demonstrate the good possibilities of using PLE as a useful technique for the valorization of by-products from the olive oil industry, such as olive leaves.     

An efficient microwave assisted extraction of phenolic compounds and antioxidant potential of Ginkgo biloba

Flavonoid glycosides are a significant group of compounds found in Ginkgo biloba leaves, but the long extraction procedures in existing methods are a challenging problem. In this work, a microwave-assisted extraction (MAE) method has been developed for extracting bioactive compounds from G. biloba. Several variables were optimized, such as extracting solvent, microwave power, and extraction time that can potentially affect the extraction yield. The total phenolic content, antioxidant activity (using DPPH, ABTS and FRAP assays) and flavonoid glycosides of different extracts using RP-HPLC were assessed. The antioxidant capacity was found to be highest with MAE using 60% aq. ethanol as extracting solvent and microwave power of 120W for 20 min.     

Evaluation of antioxidant activities of aqueous extracts and fractionation of different parts of Elsholtzia ciliata

The aim of this study was to investigate the antioxidant and free-radical scavenging activity of extract and fractions from various parts of Elsholtzia ciliata. The inflorescences, leaves, stems and roots of E. ciliata were extracted separately and two phenolic component enrichment methods: ethyl acetate-water liquid-liquid extraction and macroporous resin adsorption-desorption, were adopted in this study. The antioxidant activities of water extracts and fractions of E. ciliata were examined using different assay model systems in vitro. The fraction root E (purified by HPD300 macroporous resin) exhibited the highest total phenolics content (497.2 ± 24.9 mg GAE/g), accompanied with the highest antioxidant activity against various antioxidant systems in vitro compared to other fractions. On the basis of the results obtained, E. ciliata extracts can be used potentially as a ready accessible and valuable bioactive source of natural antioxidants.     

Green processes for the extraction of bioactives from Rosemary: Chemical and functional characterization via ultra-performance liquid chromatography-tandem mass spectrometry and in-vitro assays

In this contribution, the performance of three different extraction procedures towards the extraction of antioxidants from rosemary (Rosmarinus officinalis) is presented. Namely, pressurized liquid extraction (PLE), using water and ethanol as solvents, supercritical fluid extraction (SFE), using neat CO₂ and supercritical CO₂ modified with ethanol, as well as a novel extraction process called Water Extraction and Particle formation On-line (WEPO) are directly compared. Different extraction conditions including temperatures, times and pressures have been studied. The produced extracts have been characterized in terms of extraction yield, antioxidant activity (using the DPPH radical scavenging method) and total phenols (using the Folin method). Besides, all the extracts have been chemically characterized using a new quantitative UPLC-MS/MS method. This method allowed the determination of the main antioxidants present in rosemary, including, among others, rosmarinic acid, carnosic acid and carnosol, attaining detection limits as low as 2 ng/mL. The results obtained in this study show that PLE using ethanol at high temperatures (200 °C) was able to produce extracts with high antioxidant activity (EC₅₀ 8.8 μg/mL) and high yield (ca. 40%) while efficiently extracting antioxidants of diverse polarity, among them, carnosic and rosmarinic acids, regarded as the most important antioxidants present in rosemary. Nevertheless, in this work, the ability of the three studied environmentally friendly extraction techniques to obtain bioactives from natural sources is demonstrated.     

Characterization via liquid chromatography coupled to diode array detector and tandem mass spectrometry of supercritical fluid antioxidant extracts of Spirulina platensis microalga

Spirulina platensis microalga has been extracted on a pilot scale plant using supercritical fluid extraction (SFE) under various extraction conditions. The extraction yield and the antioxidant activity of the extracts were evaluated in order to select those extracts with both the highest antioxidant capacity and a good extraction yield. These extracts were characterized using LC coupled to diode array detection (DAD) and LC coupled to mass spectrometry (MS) with two different interfaces, atmospheric pressure chemical ionization (APCI) and electrospray (ESI) which allowed us to perform tandem MS by using an ion trap analyzer. The best extraction conditions were as follows: CO2 with 10% of modifier (ethanol) as extraction solvent, 55 degrees C (extraction temperature) and 220 bar (extraction pressure). Fractionation was achieved by cascade depressurization providing two extracts with different activity and chemical composition. Several compounds have been identified in the extracts, corresponding to different carotenoids previously identified in Spirulina platensis microalga along with chlorophyll a and some degradation products. Also, the structure of some phenolic compounds could be tentatively identified. The antioxidant activity of the extracts could be attributed to some of the above mentioned compounds.     

Separation and characterization of antioxidants from Spirulina platensis microalga combining pressurized liquid extraction, TLC, and HPLC-DAD

A new procedure has been developed to separate and characterize antioxidant compounds from Spirulina platensis microalga based on the combination of pressurized liquid extraction (PLE) and different chromatographic procedures, such as TLC, at preparative scale, and HPLC with a diode array detector (DAD). Different solvents were tested for PLE extraction of antioxidants from S. platensis microalga. An optimized PLE process using ethanol (generally recognized as safe, GRAS) as extraction solvent has been obtained that provides natural extracts with high yields and good antioxidant properties. TLC analysis of this ethanolic extract obtained at 115 degrees C for 15 min was carried out and the silica layer was stained with a DPPH (diphenyl-pycril-hydrazyl) radical solution to determine the antioxidant activity of different chromatographic bands. Next, these colored bands were collected for their subsequent analysis by HPLC-DAD, revealing that the compounds with the most important antioxidant activity present in Spirulina extracts were carotenoids, as well as phenolic compounds and degradation products of chlorophylls.     

Antioxidant capacity and antibacterial activity from Annona cherimola phytochemicals by ultrasound-assisted extraction and its comparison to conventional methods

In recent years, the food, pharmacy, and cosmetic industries have focused on the search of natural compounds with antimicrobial and antioxidant properties; commonly, these compounds are obtained from Kingdom plantae. The aim of the present work is comparing antibacterial and antioxidant capacity of Annona cherimola Mill leaves, using different extraction methods. The ultrasound assisted extraction technique (UAE) was compared with conventional techniques: Soxhlet and maceration. Water and ethanol were used as solvents for leaves extractions performed with these three methods. The main acetogenins reported in Annona cherimola Mill and Annona muricata L. species were simulated using the functional hybrid B3LYP and to confirm its presence, analysis of the compound composition was performed using FT-IR, UV–Vis and HPLC. Total phenolics (TP) and flavonoids (TF) were determined by spectroscopy techniques and novel Differential Pulse Voltammetry (DPV) electrochemical technique. Total Antioxidant Capacity (TAC) of the extracts was measured, using the DPPH, FRAP and CUPRAC techniques. The highest antioxidant content was found in the Soxhlet water extracts; even so, the UAE technique presented an attractive alternative due to considerable reduction in extraction time, which was greater than 99%, and possible selectivity in compounds extraction. Finally, antibacterial activity of the extracts was evaluated, obtaining the best results against gram-positive bacteria using UAE water extract. In this way, the UAE technique presents an excellent extraction option due to the considerable reduction in time and energy, as well as the increase in antibacterial activity.     

Evaluation of influence of selected metal cations on antioxidant activity of extracts from savory (<Emphasis Type="Italic">Satureja hortensis</Emphasis>)

The analysis of the antioxidant activity of ethanol, methanol, acetone and aqueous extracts from the dried leaves and stems of savory is presented. The culinary herb used to study was procured from ecological agriculture in the Podlasie region of Poland. The antioxidant properties of extracts were calculated using the 1,1-diphenyl-2-picryl-hydrazyl radical (DPPH) and ferric-reducing antioxidant power (FRAP) (expressed as mg Trolox per g of dry mass) methods. In addition, the total phenolics contents of the herbal extracts were determined using the Folin-Ciocalteu reagent. The antioxidative activity of extracts as dependent on the type of solvent used for the extraction and concentration of savory in extracts is discussed. The influence of the concentration of different metal ion solutions on the anti-radicals’ properties of savory extracts was evaluated.     

Combining stir bar sorptive extraction and MEKC for the determination of polynuclear aromatic hydrocarbons in environmental and biological matrices

In this work, stir bar sorptive extraction and liquid desorption was combined with MEKC and diode-array detection (SBSE-LD-MEKC-DAD) for the determination of polynuclear aromatic hydrocarbons (PAHs) in aqueous medium, using biphenyl, fluorene, anthracene, phenanthrene, fluoranthene and pyrene as model compounds. MEKC-DAD conditions and parameters affecting SBSE-LD efficiency are fully discussed. Assays performed on aqueous samples spiked at trace levels, yielded recoveries ranging from 55.5 +/- 6.1% (pyrene) to 70.7 +/- 7.1% (anthracene), under optimized experimental conditions. The methodology proved to be nearly described by the octanol-water partition coefficients (K(PDMS/W) approximately K(O/W)). The analytical performance showed good precision (<12.0%), suitable detection limits (2-11 microg/L) and convenient linear dynamic ranges (r(2)>0.99) from 5 to 25 microg/L for anthracene and 25 to 125 microg/L for the remaining compounds. The application of the proposed methodology to environmental water, sediments and fish bile matrices demonstrated good selectivity and accuracy. SBSE-LD combined with MEKC-DAD was shown to be an easy, reliable and robustness methodology, as well as a good analytical alternative to monitor environmental priority pollutants.     

HPLC and GC-MS determination of bioactive compounds in microwave obtained extracts of three varieties of Labisia pumila Benth

Microwave extraction of phytochemicals from medicinal plant materials has generated tremendous research interest and shown great potential. This research highlights the importance of microwave extraction in the analysis of flavonoids, isoflavonoid and phenolics and the antioxidant properties of extracts from three varieties of the Malaysian medicinal herb, Labisia pumila Benth. High and fast extraction performance ability, equal or higher extraction efficiencies than other methods, and the need for small samples and reagent volumes are some of the attractive features of this new promising microwave assisted extraction (MAE) technique. The aims of the present research were to determine the foliar phenolics and flavonoids contents of extracts of three varieties of L. pumila obtained by a microwave extraction method while flavonoid, isoflavonoid and phenolic compounds were analyzed using RP-HPLC. Furthermore, the antioxidant activities were measured by the DPPH and FRAP methods and finally, the chemical composition of the crude methanolic extracts of the leaves of all three varieties were analyzed by GS-MS.     

Chemical composition of bioactive pressurized extracts of Romanian aromatic plants

In this contribution, pressurized liquid extraction (PLE) has been employed to isolate bioactive compounds from three native Romanian plants, oregano (Origanum vulgare), tarragon (Artemisia dracunculus) and wild thyme (Thymus serpyllum). Different PLE conditions have been tested including extraction with water, ethanol and their mixtures in a wide range of extraction temperatures (50-200°C), and the antioxidant capacity of the extracts was measured using different assays (DPPH radical scavenging, TEAC assay and Folin-Ciocalteau assay to measure total phenols). Moreover, a complete chemical characterization by using LC-MS/MS was carried out to be able to correlate the bioactivity with the particular chemical composition of each extract and plant. The use of PLE with water as a solvent at the highest temperature tested (200°C) always provided the highest extraction yields for the three studied plants, being maximum for oregano (>60%). Besides, oregano's pressurized water extracts at lower temperatures (50°C) presented the highest content on total phenols (184.9 mg gallic acid/g extract) and the best antioxidant activities (EC(50) 6.98 μg/ml). In general, oregano extracts were the most active, followed by wild thyme extracts. The antioxidant capacity measured by DPPH assay was highly correlated with the amount of total phenols. Moreover, the use of a LC-MS/MS method allowed the identification of 30 different phenolic compounds in the different extracts, including phenolic acids, flavones, flavanones and flavonols, which have an important influence on the total antioxidant capacity of the different extracts.     

Liquid chromatographic-mass spectrometric analysis of supercritical-fluid extracts of rosemary plants

A two-step supercritical fluid extraction process of rosemary leaves, on a pilot plant scale, is proposed to divide the oleoresin into two fractions with different antioxidant activities and essential oil composition. Rosemary leaves were extracted by using different conditions of pressure and temperature as well as different conditions for fractionation of the extracts. Conditions can be tuned to selectively extract one antioxidant fraction with almost no residual aroma. In the present investigation, the antioxidant fraction was exhaustively studied in terms of antioxidant activity measurements as well as of chemical composition. An LC–MS method was adapted to perform the analysis and identification of the compounds responsible for the antioxidant activity of the extracts. Different extraction and fractionation conditions were studied in order to correlate the process conditions with the antioxidant activities obtained.     

Improved method for the in vitro assessment of antioxidant activity of plant extracts by headspace solid-phase microextraction and gas chromatography-electron capture detection

The simultaneous monitoring of malondialdehyde, pentanal and hexanal, final products of lipid peroxidation is reported, using a headspace solid-phase microextraction (HS-SPME) technique with on-fibre derivatisation. The aldehydes are extracted and subjected to on-sorbent derivatisation into stable hydrazones with 2,4,6-trichlorophenylhydrazine (TCPH) and analyzed. The degree of inhibition of oxidation is performed by monitoring the chlorinated hydrazones after thermal desorption, by gas chromatography-electron capture detection. The procedure was employed to evaluate in vitro the antioxidant activity of Hypericum perforatum L. extracts and of the well-known antioxidant vitamin E following induction of oxidation of sunflower oil, as a model lipid system. Prior to the measurement of antioxidant activity, the optimal process conditions, i.e. headspace volume, temperature, agitation, extraction/derivatisation time and desorption time and temperature were properly established. Aqueous extracts of H. perforatum L. exhibited the highest antioxidative effect. The method is shown to be promising for screening purposes for antioxidant substances and natural extracts.     

Optimization of Extraction Conditions for Flavonoid Composition and Antioxidant Activity of Radix Scutellariae

Microwave, ultrasonic, and reflux extraction were compared to provide an optimized method for flavonoids from Radix Scutellariae including the antioxidant activity of the extracts. The antioxidant activities of the extracts were evaluated by 2,2-diphenyl-1-picrylhydrazyl and oxygen radical absorbance capacity assays. Baicalin, wogonoside, baicalein, wogonin, and oroxylin A were quantified using ultra-high performance liquid chromatography. For microwave extraction, with a ten minutes treatment, the extraction efficiencies of the analytes were higher than treatments by refluxing for 180 minutes and sonication for sixty minutes. In addition, the antioxidant activity of the microwave extracts was slightly higher than the extracts obtained by the other methods. Microwave extraction conditions were further optimized by a single-factor experiment and the optimum conditions were 50 percent ethanol–water (volume by volume), an extraction temperature of 100 degrees celsius, an extraction time of ten minutes, and a sample to solvent ratio of 1:50. This study combines extraction, phytochemistry, and bioactivity to screen the most efficient extraction procedure for flavonoids from Radix Scutellariae.     

HPTLC and ATR/FTIR Characterization of Antioxidants in Different Rosemary Extracts

The effect of spontaneous fermentation by lactic acid bacteria on the extraction yield of bioactive compounds and antioxidant activity from rosemary leaf extracts was investigated using high-performance thin-layer chromatography (HPTLC). Brining and spontaneous fermentation with lactic acid bacteria more than doubled extraction of polyphenolics and antioxidants from the rosemary leaves. The results show that lactic acid fermentation enhances antioxidant activity in extracts by increasing the total phenolic content but does not increase extraction of phytosterols. Increased extraction of phenolic oxidants during fermentation assisted extraction, results from the in situ generated natural eutectic solvent from the plant sample. ATR-FTIR spectra from the bioactive bands suggests that this increased antioxidant activity is associated with increased extraction of rosmarinic acid, depolymerised lignin, abietane diterpenoids and 15-hydroxy-7-oxodehydroabietic acid.     

Bioactive compounds, RP-HPLC analysis of phenolics, and antioxidant activity of some Portuguese shrub species extracts

In the ecosystem of Serra Da Estrela, some plant species have the potential to be used as raw material for extraction of bioactive products. The goal of this work was to determine the phenolic, flavonoid, tannin and alkaloid contents of the methanolic extracts of some shrubs (Echinospartum ibericum, Pterospartum tridentatum, Juniperus communis, Ruscus aculeatus, Rubus ulmifolius, Hakea sericea, Cytisus multiflorus, Crataegus monogyna, Erica arborea and Ipomoea acuminata), and then to correlate the phenolic compounds and flavonoids with the antioxidant activity of each extract. The Folin-Ciocalteu's method was used for the determination of total phenols, and tannins were then precipitated with polyvinylpolypyrrolidone (PVPP); a colorimetric method with aluminum chloride was used for the determination of flavonoids, and a Dragendorff's reagent method was used for total alkaloid estimation. The 2,2-diphenyl-1-picrylhydrazyl (DPPH) and beta-carotene bleaching tests were used to assess the antioxidant activity of extracts. The identification of phenolic compounds present in extracts was performed using RP-HPLC. A positive linear correlation between antioxidant activity index and total phenolic content of methanolic extracts was observed. The RP-HPLC procedure showed that the most common compounds were ferulic and ellagic acids and quercetin. Most of the studied shrubs have significant antioxidant properties that are probably due to the existence of phenolic compounds in the extracts. It is noteworthy to emphasize that for Echinospartum ibericum, Hakea sericea and Ipomoea acuminata, to the best of our knowledge, no phytochemical studies have been undertaken nor their use in traditional medicine been described.     

Evaluation of eco-friendly compounds in the manufacturing of antibacterial papers

The papers have a porous structure which can be a suitable medium for the growth of bacteria. Therefore, in the sanitary papers, creation of a suitable antibacterial property is necessary. In this way, the plant extracts were prepared using solid–liquid extraction method from the Rosmarinus officinalis, Olea europaea, Mentha spicata and Punica granatum. Relatively, the total phenol and flavonoid contents were determined according to colorimetric method. Their antioxidant activity was evaluated using DPPH method. The extracts were sprayed on the surface of the handsheets, and then their antibacterial activity was investigated. The results showed that extracts from the P. granatum has the highest phenol (5.82%) and antioxidant activity (71.13%), while the extract from the R. officinalis had the highest flavonoid (4.23%) and antibacterial activity (90.43%). Hence, it can be concluded that these two extracts could be the suitable combinations to create desirable antibacterial properties in the manufacture of papers with sanitary purposes.     

Selective Extraction of Sinapic Acid Derivatives from Mustard Seed Meal by Acting on pH: Toward a High Antioxidant Activity Rich Extract

The aim of this paper is to study the effect of the pH on the extraction of sinapic acid and its derivatives from mustard seed meal. Solutions of acidic pH (pH 2), basic pH (pH 12) and distilled water (uncontrolled pH ~ 4.5) were tested at different percentages of ethanol. The maximum extraction yield for sinapic acid (13.22 µmol/g of dry matter (DM)) was obtained with a buffered aqueous solution at pH 12. For ethyl sinapate, the maximum extraction yield reached 9.81 µmol/g _DM with 70% ethanol/buffered aqueous solution at pH 12. The maximum extraction yield of sinapine (15.73 µmol/g _DM) was achieved with 70% ethanol/buffered aqueous solution at pH 2. The antioxidant activity of each extract was assessed by DPPH assay; the results indicated that the extracts obtained at pH 12 and at low ethanol percentages (<50%) exhibit a higher antioxidant activity than extracts obtained at acidic conditions. Maximum antioxidant activity was reached at pH 12 with buffer solution (11.37 mg of Trolox Equivalent/g _DM), which confirms that sinapic acid-rich fractions exhibit a higher antioxidant activity. Thus, to obtain rich antioxidant extracts, it is suggested to promote the presence of sinapic acid in the extracts.     

Effect of extraction method on phenolic content and antioxidant activity of mistletoe extracts from Viscum album subsp. abietis

The total content of polyphenols and flavonoids determined in the same plant and their corresponding antioxidant activities may vary widely, depending on the extraction conditions applied. This study was conducted to optimise the extraction conditions of phenolics and flavonoids from the mistletoe plant. Various extraction methods, i.e. ultrasound-assisted extraction technology, maceration, maceration with stirring, accelerated solvent extraction (ASE), and extraction under reflux were evaluated for their percentage extraction of polyphenols (TPC) and flavonoids (TFC) from Viscum album subsp. abietis. In addition, the anti-radical activity of extracts was analysed using the 2,2-diphenyl-1-picrylhydrazyl method. The effects of temperature, solvent type, and concentration on the phenolic extraction efficiency and antioxidant activity were studied using chemometric and statistical methods. The results showed that the extracts of V. album subsp. abietis contained large amounts of polyphenols and flavonoids (up to 57.673 mg g^?1 and 9.955 mg g^?1 of dry extract, respectively) and exhibited potent antioxidant activity, hence representing promising sources of powerful antioxidants. Due to its high extraction efficiency and considerable saving of time and solvent, ASE was more effective than the other extraction techniques. Extracts prepared with water-polar solvent mixtures displayed the highest TPC, TFC, and antioxidant activity, while organic polar solvents were the least efficient extractants.     

Coupling of capillary electrophoresis with reaction detection for the on-line evaluation of radical scavenging activity of analytes

The main task of this work was to create a rapid, simple and less material and time consuming method involving capillary electrophoretic separation in order to separate analytes and evaluate antioxidant activity within a single analytical run. Several interfaces were developed and used to couple CE to the reaction detector. The method developed enables simultaneous electrophoretic separation and evaluation of antioxidant activity of each separated compound in the mixture. The analysis was performed using DPPH (2,2-diphenyl-1-picrylhydrazyl) as synthetic radical reagent. The on-line capillary electrophoresis-reaction (antioxidant activity) detection method can be used for a rapid evaluation of individual antioxidants in complex mixtures, particularly extracts of natural products. Possibility to evaluate radical scavenging activity of extracts of natural products components is demonstrated on an example of aqueous propolis extract. Four phenolic acids where separated and their radical scavenging activity was on-line evaluated.