Antioxidant activity of hydro distillation water residues from Rosmarinus officinalis L. leaves determined by DPPH assays

Rosemary (Rosmarinus officinalis L.) is a perennial herb with an intensive aromatic flavor. Its most important chemical constituents are essential oils (e.g., 1,8-cineole and camphor) and antioxidants (e.g., carnosic acid and rosmarinic acid). The common methods to extract the essential oil of rosemary are steam or hydro distillation. The aim of this work is to investigate the residual antioxidants after hydro distillation, especially rosmarinic acid and carnosic acid. For this purpose, the hydro distillation water residues were analyzed by HPLC-UV. Moreover, the influence of the extraction duration on the concentration of the antioxidants was investigated. Also, the residual amount of these compounds in the leaves was examined. The total antioxidant activity of the extracts and of the pure compounds was determined by DPPH assays. It is shown that after 2.5 h of hydro distillation the amount of rosmarinic acid and the antioxidant activity in the water residue reaches a maximum value. In addition, the yield and the quality of the essential oil were investigated to draw a comparison between steam and hydro distillations of Moroccan rosemary leaves.     

Capillary electrophoretic separation of phenolic diterpenes from rosemary

The major phenolic diterpenes responsible for the antioxidant properties of rosemary extracts, namely carnosol and carnosic acid, were separated by capillary zone electrophoresis (CZE) using a 56 cm long uncoated fused-silica capillary and a 50 mM disodium tetraborate buffer of pH 10.1. The effect of the buffer type, pH and concentration, and the capillary length on the separation, was studied. Carnosol and carnosic acid were identified in the electrophoregrams of rosemary extracts through their migration times and UV spectra obtained by CZE analysis of pure compounds isolated from a rosemary extract by HPLC fractionation. The CZE method had good reproducibility (relative standard deviation less than 5%) and was applied to compare the contents of carnosol and carnosic acid in solid and oil-dispersed commercial extracts of rosemary and in rosemary leaves. The separation of carnosol and carnosic acid was accomplished in less than 11 min.     

Antioxidant capacity of abietanes from Sphacele salviae

Carnosol 1, rosmanol 2, carnosic acid 3 and 20-deoxocarnosol 4, the main phenolic abietanes present in aerial parts of the Chilean medicinal plant Sphacele salviae were tested for antioxidant activity of measuring the decay of the radical cation diphenyl-picrylhydrazyl (DPPH). All compounds displayed higher antioxidant levels than BHT under the same conditions and carnosic acid was more efficient than vitamin E.     

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.     

Direct determination of carnosic acid in a new active packaging based on natural extract of rosemary

A new antioxidant film is being developed that incorporates a natural extract of rosemary and is intended for contact with food. The rosemary extract has been screened and carnosic acid and carnosol have been determined as the major antioxidant components (6.96% and 0.88%, respectively) that are responsible for the antioxidant properties of the whole extract. Thus, a fast method for the direct determination of carnosic acid in the packaging material, in order to evaluate the antioxidant capacity of the new active plastic, has been developed and optimized. The method consists of extraction from the plastic with methanol, followed by anion exchange solid-phase extraction and final analysis by UPLC–MS. Using this process, the recovery of carnosic acid is about 99%. The complete analytical performance of the method developed here is also assessed. The analytical features of the method, such as the relative standard deviation, reproducibility, repeatability, linear range, and detection and quantification limits, are shown. This method can be subsequently modified to monitor other active components in different packages, and it constitutes a crucial step forward in research into new and improved commercial antioxidant packages.     

Isolation and identification of radical scavengers in olive tree (Olea europaea) wood

Several extracts of Olea europaea wood (Picual olive cultivar) were obtained with solvents of different polarity and their antioxidant activities determined. The active compounds were detected in fractions of an ethyl acetate extract using HPLC with on-line radical scavenging detection. After applying different separation techniques, hydroxytyrosol, tyrosol, cycloolivil, 7-deoxyloganic acid, oleuropein and ligustroside were isolated and characterized. Hydroxytyrosol showed a higher activity than the natural antioxidant rosmarinic acid in scavenging the DPPH model radical. Cycloolivil and oleuropein showed stronger activities than the synthetic antioxidant BHT against the same radical. Ligustroside, tyrosol and 7-deoxyloganic acid showed little activity. The latter compound has not been previously identified in the genus Olea.     

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.     

A Study on the In Vitro Antioxidant Activity of Juniper (Juniperus communis L.) Fruit Extracts

Abstract

This study aimed at evaluating the in vitro antioxidant activity of water and ethanol extracts of juniper (Juniperus communis L., Family Cupressaceae) fruit. The antioxidant properties of both Juniper extracts were studied using different antioxidant assays, including reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities. Both the water and the ethanol extracts exhibited strong total antioxidant activity. The concentrations of 20, 40, and 60 µg/mL of water and ethanol extracts of juniper fruit showed 75%, 88%, 93%, 73%, 84%, and 92% inhibition on peroxidation of linoleic acid emulsion, respectively. On the other hand, 60 µg/mL of standard antioxidant such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and α‐tocopherol exhibited 96, 96, and 61 inhibitions on peroxidation of linoleic acid emulsion, respectively. However, both extracts of juniper had effective reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activities at these same concentrations (20, 40, and 60 µg/mL). Those various antioxidant activities were compared to BHA, BHT, and α‐tocopherol as standard antioxidants. In addition, total phenolic compounds in both aqueous and ethanolic juniper extracts were determined as gallic acid equivalents. Accordingly, these results indicate that juniper has in vitro antioxidant properties and these may be major reasons for the inhibition of lipid peroxidation properties.     

Fast separation and determination of carnosic acid and rosmarinic acid in different rosemary (Rosmarinus officinalis) extracts by capillary zone electrophoresis with ultra violet-diode array detection

Summary Carnosol, carnosic acid, rosmarinic acid and other not identified phenolic compounds were separated by capillary zone electrophoresis (CZE) using a 40-cm long capillary and a 20 mM tetraborate buffer (pH 9.0), within 3 min. A UV-diode array detector was employed to collect spectra of phenolic compounds. The effect of some separation parameters on peak resolution and migration time of phenolic species present in a refined rosemary extract was studied. The repeatability of the method was also investigated: the intraday relative standard deviation on total peak area was less than 4%, while the intraday relative standard deviation on migration time was less than 0.6%. Moreover the CZE method showed good sensitivity (0.0007 μg mL¹ for carnosic acid and rosmarinic acid). Carnosic acid and rosmarinic acid have been quantified in different commercial extracts of rosemary. Finally, the optimized method was also applied to evaluate the recovery of these two compounds when different organic solvents were employed during the extraction procedure.     

Pressurized liquid extraction-capillary electrophoresis-mass spectrometry for the analysis of polar antioxidants in rosemary extracts

A method based on capillary electrophoresis-electrospray-mass spectrometry (CE-ESI-MS) was developed to qualitatively characterize natural antioxidants from rosemary (Rosmarinus officinalis L.) in different fractions obtained by pressurized liquid extraction (PLE) using subcritical water. The parameters of CE-ESI-MS were adjusted allowing the separation and characterization of different compounds from rosemary in the PLE fractions. These parameters for CE are kind, pH and concentration of the separation buffer, parameters for ESI-MS are dry gas temperature and flow, nebulizing gas pressure, and make-up flow. The following analytical conditions were found most favorable: aqueous CE buffer (40 mM ammonium acetate/ammonium hydroxide, pH 9); sheath liquid containing 2-propanol-water (60:40, v/v) and 0.1% (v/v) triethylamine at a flow rate of 0.24 mL/h; drying gas flow rate equal to 7 L/min at 350 degrees C, nebulizing gas pressure of 13.8 kPa (2 psi), using a compound stability of 50%. Different antioxidant compounds (e.g., rosmarinic acid and carnosic acid) could be detected in the rosemary extracts by CE-ESI-MS without any additional treatment, enabling the determination of variations in the extract composition caused by the different PLE conditions (i.e., 60 and 100 degrees C). The results provide complementary information to HPLC analysis.     

Radical scavenging activity and antioxidant capacity of bay leaf extracts

Bay leaves (BL) (Laurus nobilis L., Family: Lauraceae) are traditionally used orally to treat the symptoms of gastrointestinal problems, such as epigastric bloating, impaired digestion, eructation, and flatulence. In this study, lyophilized extracts (both water and ethanol) of BL were studied for their antioxidant properties. The antioxidant activity, reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities were evaluated to determine the total antioxidant capacity of both BL extracts. Both extracts exhibited strong total antioxidant activity in linoleic acid emulsion. Concentrations of 20, 40, and 60 μg ml^?1 showed 84.9, 95.7, 96.8, and 94.2, 97.7, and 98.6% inhibition of lipid peroxidation of linoleic acid emulsion, for water and ethanol extracts, respectively. On the other hand, 60 μg ml^?1 of the standard antioxidants butylated hydroxyianisole (BHA), butylated hydroxytoluene (BHT), and α-tocopherol exhibited 96.6, 99.1, and 76.9% inhibition of lipid peroxidation in linoleic acid emulsion, respectively. In addition, the both BL extracts had effective reducing power, DPPH? free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging and metal chelating activities at 20, 40, and 60 μg ml^?1. The total amount of phenolic compounds in each BL extract was determined as gallic acid equivalents.     

An Improved HPLC Post-Column Methodology for the Identification of Free Radical Scavenging Phytochemicals in Complex Mixtures

The importance of natural antioxidants principally relates to their health-promoting properties. The discovery of new sources of established compounds or more potent compounds is a costly exercise and any technique capable of aiding this procedure would be highly significant. An approach combining chromatographic separation, component analysis and post-column identification of free radical scavenging has been reported. However, its effectiveness is dependent upon various factors and the number of samples analyzed without operator intervention is restricted. A more applicable technique using a 10% to 100% methanol with 2% acetic acid mobile phase and a citric acid-sodium citrate buffered methanolic 1,1-diphenyl-2-picrylhydrazyl free radical reagent is presented, whereby free radical scavengers can be detected within crude extracts of variable polarity without special considerations. As an illustration, acetone, 70% (aq.) methanol and acetonitrile-soluble extracts were prepared from sage (Salvia officinalis L.) for extraction of polar and non-polar radical scavengers and analyzed. Radical scavenging components were identified as carnosic, caffeic and rosmarinic acids and luteolin-7-O-glycoside. Others radical scavengers were tentatively identified as benzoic and hydroxycinnamic acid derivatives, flavonoids and diterpenoids. Through the application of this technique, carnosic acid and rosmarinic acid were identified as the principal free radical scavengers.     

Antioxidant activity of two wild edible mushrooms (Morchella vulgaris and Morchella esculanta) from North Turkey

The ethanol extracts of Morchella vulgaris (EEMV) and Morchella esculanta (EEME) were analysed for their antioxidant activities in different systems including reducing power, free radical scavenging, superoxide anion radical scavenging, total antioxidant activity, and metal chelating activity. EEMV and EEME had similar reducing power, free radical scavenging, superoxide anion radical scavenging, hydrogen peroxide scavenging, and metal chelating activity at concentrations of 50, 100, and 150 microg/mL. These various antioxidant activities were compared to standard antioxidants such as butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), and alpha-tocopherol. The percent inhibition of different concentrations of EEMV on peroxidation in the linoleic acid system was 85 and 87 % respectively, which was greater than that of 100 and 250 microg/mL of alpha-tocopherol (50 and 77%, respectively) and similar to 250 microg/mL of BHA (85, 87%, respectively). The percent inhibition of different concentrations of EEME on peroxidation in the linoleic acid system was 80 and 87 % respectively, which was greater than that of 100 and 250 microg/mL of alpha-tocopherol (50, 77%) and similar to 250 microg/mL BHA (87%). On the other hand, the percent inhibition of 100 and 250 microg/mL of BHT was 97 and 99%, respectively. In addition, the total phenolic compounds in EEMV and EEME were determined as gallic acid equivalents.     

Rancimat and PDSC accelerated techniques for evaluation of oxidative stability of soybean oil with plant extracts

Taking into account the importance of natural antioxidants in the preservation of oils and fats, the present study evaluated the antioxidant action of five plant extracts in the control of soybean oil stability, by means of the accelerated techniques Rancimat and PDSC. These plants are rosemary (Rosmarinus officinalis L.), chamomile (Matricaria recutita L.), coriander (Coriandrum sativum L.), fennel (Foeniculum vulgare), and senna (Cassia angustifolia Vahl). The plant extracts and also the synthetic antioxidant BHT were added to the samples of crude soybean oil at the concentration of 1,000 mg kg^?1. The values of total phenolic contents ranged from 8.7 ± 0.4 to 63.0 ± 2.3 mg GAE g^?1 extract and a strong positive correlation was observed between the total phenolic contents and the overall antioxidant activity of the plant extracts. Such high values indicate a good protection of the analyzed soybean oil, moreover for the Rosemary extract that was superior to the remaining extracts. In the Rancimat technique the rosemary extract was more effective than the synthetic BHT antioxidant. The OIT values of Rosemary extract and the BHT antioxidant were equivalent, and the former, showed the highest phenolic contents among the extracts, for all the performed tests, confirming that it is a powerful natural source of antioxidants.     

Obtaining Bioactive Compounds from the Coffee Husk (Coffea arabica L.) Using Different Extraction Methods

Coffee husks (Coffea arabica L.) are characterized by exhibiting secondary metabolites such as phenolic compounds, which can be used as raw material for obtaining bioactive compounds of interest in food. The objective of this study is to evaluate different methods for obtaining the raw material and extracting solutions of bioactive compounds from coffee husks. Water bath and ultrasound-assisted extraction methods were used, using water (100%) or ethanol (100%) or a mixture of both (1:1) as extracting solutions and the form of the raw material was in natura and dehydrated. The extracts were evaluated by their antioxidant potential using DPPH radicals, ABTS, and iron reduction (ferric reducing antioxidant power (FRAP)), and later total phenolic compounds, total flavonoids, and condensed tannins were quantified the phenolic majority compounds were identified. It was verified that the mixture of water and ethanol (1:1) showed better extraction capacity of the compounds with antioxidant activity and that both conventional (water bath) or unconventional (ultrasound) methods showed satisfactory results. Finally, a satisfactory amount of bioactive compounds was observed in evaluating the chemical composition (total phenolic compounds, total flavonoids, condensed tannins, as well as the analysis of the phenolic profile) of these extracts. Corroborating with the results of the antioxidant activities, the best extracting solution was generally the water and ethanol mixture (1:1) using a dehydrated husk and water bath as the best method, presenting higher levels of the bioactive compounds in question, with an emphasis on chlorogenic acid. Thus, it can be concluded that the use of coffee husk as raw material to obtain extracts of bioactive compounds is promising. Last, the conventional method (water bath) and the water and ethanol mixture (1:1) stood out among the methods and extracting solutions used for the dehydrated coffee husk.     

Storage method, drying processes and extraction procedures strongly affect the phenolic fraction of rosemary leaves: an HPLC/DAD/MS study

The Rosmarinus officinalis L. is widely known for its numerous applications in the food field but also for the increasing interest in its pharmaceutical properties. Two groups of compounds are mainly responsible for the biological activities of the plant: the volatile fraction and the phenolic constituents. The latter group is mainly constituted by rosmarinic acid, by a flavonoidic fraction and by some diterpenoid compounds structurally derived from the carnosic acid. The aim of our work was to optimize the extractive and analytical procedure for the determination of all the phenolic constituents. Moreover the chemical stability of the main phenols, depending on the storage condition, the different drying procedures and the extraction solvent, have been evaluated.This method allowed to detect up to 29 different constituents at the same time in a relatively short time. The described procedure has the advantage to being able to detect and quantify several classes of compounds, among them numerous minor flavonoids, thus contributing to improving knowledge of the plant.The findings from this study have demonstrated that storing the raw fresh material in the freezer is not appropriate for rosemary, mainly due to the rapid disappearing of the rosmarinic acid during the freezing/thawing process. Regarding the flavonoidic fraction, consistent decrements, were highlighted in the dried samples at room temperature if compared with the fresh leaf. Rosmarinic acid, appeared very sensitive also to mild drying processes. The total diterpenoidic content undergoes to little changes when the leaves are freeze dried or frozen and limited losses are observed working on dried leaves at room temperature. Nevertheless it can be taken in account that this fraction is very sensitive to the water presence during the extraction that favors the conversion of carnosic acid toward it oxidized form carnosol. From our findings, it appear evident that when evaluating the phenolic content in rosemary leaves, several factors, mainly the type of storage, the drying process and the extraction methods, should be carefully taken into account because they can induce partial losses of the antioxidant components.     

Phytochemical composition,in vitro antioxidant activity and antibacterial mechanisms of Neolamarckia cadamba fruits extracts

Aqueous extracts of Neolamarckia cadamba fruits prepared at different maturity stages were used for the analysis of various phytochemicals, and their antioxidant and antibacterial activities were determined. Ripe fruit extract had highest phenolics (3.14 mM GAE/ g fruit extract) with caffeic acid, tannic acid, syringic acid and quercetin as major phenolic compounds. The ripe fruit extract showed lowest IC₅₀ values in DPPH radical scavenging assay (231.33 μg fruit extract/ mL), and highest ABTS radical scavenging activity (111.18 μM TEAC/g). Immature fruit extract showed lowest minimum inhibitory concentration against tested bacteria, and the antibacterial activity was probably due to membrane permeation, as was evident by leakage of genetic material and reduction in propidium iodide uptake by bacterium; and by inhibition of sugar and amino acid uptake. The appreciable amount of phenolic compounds and biological activities in the aqueous extracts of N. cadamba fruits suggests it's potential application as natural preservative.     

Optimization and validation of post-column assay for screening of radical scavengers in herbal raw materials and herbal preparations

On-line method, which combines HPLC distribution and post-column reaction, was designed for the search of individual antioxidants. Optimization of the assay was performed evaluating optimal ABTS(+) radical cation concentration in the reactor, reaction time, impact of flow rate, reaction coil length. HPLC-ABTS assay validation in this work was performed by assessing reference antioxidant negative peak areas in radical scavenging chromatogram. Sample free radical scavenging activity is expressed as trolox equivalent antioxidant capacity (TEAC). Optimized and validated method was applied in detection of compounds possessing free radical scavenging ability in complex mixtures. Antioxidant compounds were studied in perilla (Perilla frutescens (L.) Britton var. crispa f. viridis) herbal raw material and its preparations. The HPLC-separated antioxidant compounds were identified using HPLC-photodiode array coupled to mass spectrometer, using a reference mass for determining accurate masses. Radical scavenging characteristics of rosmarinic acid, which is the dominant phenolic compound in medicinal herbal raw material of perilla and its preparations, were confirmed by the calculated TEAC values. Compounds responsible for antioxidant effect in herbal raw materials and herbal preparations were identified, evaluated and compared.     

An Improved On-Line HPLC-DPPH Method for the Screening of Free Radical Scavenging Compounds in Water Extracts of Lamiaceae Plants

An on-line HPLC-1,1-diphenyl-2-picrylhydrazyl (DPPH*) method has been improved for the detection of polar and nonpolar radical scavenging compounds from complex plant extracts. Eight water extracts were prepared from steam-distilled essential oil-extracted Lamiaceae plants (Origanum vulgare L., O. Onites L., O. Minutiflorum O. Schwartz et P. H. Davis, O. Syriacum L., Satureja cuneifolia Ten., Thymbra spicata L., Coridothymus capitatus (L.) Reichb. f., Majorana hortensis Moench). After the components within each extract had been separated by reverse phase chromatography using 10% to 100% methanol with 2% acetic acid as a mobile phase, analytes capable of scavenging a citric acid-sodium citrate buffered methanolic DPPH* solution were detected by post-column derivatization at 517 nm. The HPLC-DPPH* on-line method was applied to the qualitative and quantitative analysis of these Lamiaceae plant extracts. There was a strong correlation between the scavenging (negative) peak area and the concentration of the radical scavenging reference substances used. The radical scavenging compounds within the extracts were determined as benzoic acid and hydroxycinnamic acid derivatives, flavonoids and diterpenoids according to their retention time and UV spectral data. Rosmarinic acid and carnosic acid were identified as the dominant radical scavengers in these extracts by this method.     

Selective recovery of rosmarinic and carnosic acids from rosemary leaves under ultrasound- and microwave-assisted extraction procedures

Ultrasound-assisted extraction (UAE) and microwave-assisted extraction (MAE) are currently amongst the foremost green techniques for accelerating extraction processes. Several methods for the efficient recovery of the phenolic compounds from rosemary leaves have so far been proposed, but little data are available on the use of UAE and MAE. The aim of this work is to investigate the efficiency and selectivity of these techniques in recovering fractions of specific phenolic compounds from dried rosemary leaves using solvents that are suitable for food applications. UAE has been carried out by means of a probe system equipped with a titanium horn working at 19.5 kHz (140 W). MAE has been performed in a closed multimode reactor under N₂ (20 bar) at 100 °C. All obtained extracts were dissolved in a defined solvent volume and the solutions were directly analyzed using a combination of the HPLC-DAD-MS and TOF techniques. MAE and UAE in ethanol and acetone dramatically increased phenol yield (more than three times) as compared to more traditional solid–liquid extraction processes. In terms of selectivity, remarkably high rosmarinic acid content (6.8% of the dried extract) was obtained in ethanol under ultrasound (US). Even more impressive is the selectivity of UAE in n-hexane which gave the highest carnosic acid content, up to 13% of the dried extract. In conclusion, non-conventional energy sources and, in particular, high-intensity US have proven themselves to be rapid, efficient, and selective techniques for rosemary leaf extraction and provide fractions with high rosmarinic and carnosic acid contents.