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.     

Within-Plant Variation in Rosmarinus officinalis L. Terpenes and Phenols and Their Antimicrobial Activity against the Rosemary Phytopathogens Alternaria alternata and Pseudomonas viridiflava

This study investigated within-plant variability of the main bioactive compounds in rosemary (Rosmarinus officinalis L.). Volatile terpenes, including the enantiomeric distribution of monoterpenes, and phenols were analyzed in young and mature foliar, cortical and xylem tissues. In addition, antimicrobial activity of rosmarinic acid and selected terpenes was evaluated against two rosemary pathogens, Alternaria alternata and Pseudomonas viridiflava. Data showed that total concentration and relative contents of terpenes changed in relation to tissue source and age. Their highest total concentration was observed in the young leaves, followed by mature leaves, cortical and xylem tissues. Rosmarinic acid and carnosic acid contents did not show significant differences between leaf tissues of different ages, while young and mature samples showed variations in the content of four flavonoids. These results are useful for a more targeted harvesting of rosemary plants, in order to produce high-quality essential oils and phenolic extracts. Microbial tests showed that several terpenes and rosmarinic acid significantly inhibited the growth of typical rosemary pathogens. Overall, results on antimicrobial activity suggest the potential application of these natural compounds as biochemical markers in breeding programs aimed to select new chemotypes less susceptible to pathogen attacks, and as eco-friendly chemical alternatives to synthetic pesticides.     

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.     

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.     

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.     

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.     

Liquid chromatograpic-mass spectrometric analysis of phenolics and free radical scavenging activity of rosemary extract from different raw material

The antioxidant activity of rosemary (Rosmarinus officinalis) extract from different raw materials has been studied. Extracts were prepared from wild or drip-irrigated plants, as well as from the by-product resulting from the distillation of the aromatic essential oil. The radical scavenging activity of rosemary extracts was compared with that of antioxidants widely used in food, such as BHT and delta-tocopherol, using an optimization of the method based on the reduction of the radical 2,2-diphenyl-1-picrylhydrazyl (DPPH). The results pointed the excellent antioxidant activity of the crude fresh rosemary extracts, which was almost identical to that of pure delta-tocopherol, and higher than that of BHT; extracts prepared from distilled rosemary showed the lowest activity, although they are also of interest due to the low cost of the raw material. High performance liquid chromatography (HPLC) combined with diode array (DAD) and electrospray (ESI)-ion trap-MS detection was used to separate and identify the compounds present in the rosemary extracts. Rosmarinic acid, carnosic acid and seven of their terpene-type metabolites, and seven flavones were identified. The drying and/or distillation treatments used with the plant material strongly affected the content of the two compounds of higher antioxidant activity: rosmarinic acid and carnosic acid.     

Determination of poly-beta-hydroxybutyric acid in Bacillus thuringiensis by capillary zone electrophoresis with indirect ultraviolet absorbance detection

A new capillary electrophoresis method for determining poly-beta-hydroxybutyric acid (PHB) in Bacillus thuringiensis was established. Poly-beta-hydroxybutyric acid in samples was hydrolyzed by sulphuric acid and neutralized by Ba(OH)2. The content of produced beta-hydroxybutyrate was then determined by capillary zone electrophoresis (CZE) with indirect UV detection at 254 nm. With 5 mM p-hydroxybenzoate and 0.5 mM tetradecyltrimethylammonium bromide (TTAB) at pH 8.0 as carrier electrolyte, beta-hydroxybutyrate can be determined within 6 min. Standard regression equation was made by beta-hydroxybutyrate, and the linear range was 2-1000 microg/ml. The relative standard deviations (RSDs) for migration time and peak area are both less than 1.0%. The detection limit for beta-hydroxybutyrate was 0.2 microg/ml, which is two to three orders of magnitude lower than that of the gas chromatography (GC) method. The capillary electrophoresis method was successfully applied to determine poly-beta-hydroxybutyric acid in fermentation broth and single colony. The added standard recovery was 96%.     

Reversed-polarity capillary zone electrophoretic analysis of usnic acid.

A capillary zone electrophoretic (CZE) method for the determination of usnic acid is described for the first time. Usnic acid is an antibiotic substance from lichens. Due to its low solubility in water, a high content of methanol in CZE buffer is required. Because of the methanol in the buffer, the electroosmotic flow velocity was lower than the electrophoretic mobility of usnic acid. Accordingly, the use of reversed-polarity (with the anode on the detector side of the capillary) was necessary. The optimal buffer composition was 50 mM NaOH, 20 mM acetic acid and 5% water in methanol. The detection limit of UV detector at 290 nm for usnic acid in the injected extract was 3.5 mg/L and the relative standard deviation of the normalized peak area was 3.3% at 250 mg/L.     

Comprehensive two-dimensional liquid chromatography in analysis of Lamiaceae herbs: characterisation and quantification of antioxidant phenolic acids

A fast and effective dynamic sonication assisted ethanol extraction method was developed for extracting phenolic acids from basil, oregano, rosemary, sage, spearmint and thyme of the Lamiaceae family. The results were compared with results obtained by conventional solvent extraction techniques. A comprehensive two-dimensional liquid chromatography (LC x LC) system interfaced to electrospray ionisation time-of-flight (TOF) mass spectrometry was then optimised for analysis and quantification of the herb extracts. The optimised LC x LC system employed a combination of C18 and cyano columns. The relative standard deviations for the retention times were better than 0.05% (rosmarinic acid 0.1%) and those for the peak areas 2-14% (2 mg/l, n=3). Limits of detection were 18-90 ng/ml. The LC x LC-MS method was applied to the quantitative analysis of phenolic acids, and the results were compared with those obtained with conventional LC-MS.     

Separation and identification of photodegradation products of benzoic acid by capillary zone electrophoresis

A capillary zone electrophoresis (CZE) method was developed for separation and identification of photodegradation products of benzoic acid under irradiation at a wavelength of 300 nm. Parameters such as run buffer, applied voltage and injection time were optimized for the separation of benzoic acid and its photodegradation products. Linearity, limit of detection, and repeatability of migration time as well as peak area of the method were examined. Four reaction products, including salicylic acid, 3-hydroxybenzoic acid, 4-hydroxybenzoic acid and 3,4-dihydroxybenzoic acid have been separated and identified by spiking the known compounds into the irradiated samples using the CZE method developed. The confirmation of the reaction products is one of the key steps for proposing the possible reaction mechanisms involved in the photodegradation of benzoic acid.     

Determination of baicalin, chlorogenic acid and caffeic acid in traditional chinese medicinal preparations by capillary zone electrophoresis

Summary A capillary zone electrophoresis (CZE) method was developed for the simultaneous assay of three bioactive components—baicalin, chlorogenic acid and caffeic acid—in seven traditional Chinese medicinal preparations. The analytes were separated successfully within 3.5 min using 10 mM borate buffer (pH8.6). Regression equations revealed linear relationships (correlation coefficients 0.9942–0.9996) between the peak area and concentration of the three analytes. The relative standard deviations of the migration times and the peak areas of the three constituents were 1.12–2.68% and 1.62–5.73%, respectively. Recovery of the three constituents ranged from 89 to 107%. The extraction efficiencies of different extraction solutions are also discussed. The contents of the three components in seven different Chinese medicinal preparations containing Honeysuckle flower and/orScutellariae radix were determined by the CZE method with satisfactory results.     

Peptide nucleic acid and amino acid modified peptide nucleic acid analysis by capillary zone electrophoresis

A rapid, high resolution, and low sample consumption CZE method is developed for peptide nucleic acid (PNA) analysis for the first time. 30% v/v acetonitrile in PNA sample and 20% v/v acetonitrile in 50 mM borax‐boric acid (pH 8.7) as BGE were employed after optimization. The calibration curves were linear for PNA concentration ranging from 1 to 50 μmol/L. LOD and LOQ of PNA were 0.2 and 1.0 μmol/L, respectively. Since the commercially available reagent gives rise to huge PNA peak and an apparent impurity peak, the purity of PNA was evaluated to be about 81.4% by CZE method, obviously lower than the supplier's purity value of 99.9% evaluated by RP–HPLC, and also lower than 94.8% determined with RP–HPLC by our research group. The CZE method takes only 5 min, needs only 90 nL PNA, much less than 20 min and 20 μL PNA in RP–HPLC method. Moreover, the CZE method is applicable for the analysis of glutamic acid modified and lysine modified PNAs, they show different migration time with their corresponding complementary PNAs. Our results show CZE provides a new choice for PNA and modified PNA analysis, also their purity or quality evaluation.     

Application of ionic liquids based microwave-assisted simultaneous extraction of carnosic acid, rosmarinic acid and essential oil from Rosmarinus officinalis

An ionic liquid based microwave-assisted simultaneous extraction and distillation (ILMSED) method has been developed for the effective extraction of carnosic acid (CA), rosmarinic acid (RA) and essential oil (EO) from Rosmarinus officinalis. A series of 1-alkyl-3-methylimidazolium ionic liquids differing in composition of anion and cation were evaluated for extraction yield in this work. The results obtained indicated that the anions and cations of ionic liquids had influences on the extraction of CA and RA, 1.0M 1-octyl-3-methylimidazolium bromide ([C8mim]Br) solution was selected as solvent. In addition, the ILMSED procedures for the three target ingredients were optimized and compared with other conventional extraction techniques. ILMSED gave the best result due to the highest extraction yield within the shortest extraction time for CA and RA. The novel process developed offered advantages in term of yield and selectivity of EO and shorter isolation time (20 min in comparison of 4h of hydrodistillation), and provides a more valuable EO (with high amount of oxygenated compounds). The microstructures and chemical structures of rosemary samples before and after extraction were also investigated. Moreover, the proposed method was validated by the stability, repeatability and recovery experiments. The results indicated that the developed ILMSED method provided a good alternative for the both extraction of non-volatile compounds (CA and RA) and EO from rosemary as well as other herbs.     

Capillary zone electrophoretic determination of phenolic compounds in chess (Bromus inermis L.) plant extracts

A simple CZE method for quantification of phenolic compounds (vanillin, cinnamic, sinapic, chlorogenic, syringic, ferulic, benzoic, p-coumaric, vanillic, p-hydroxybenzoic, rosmarinic, caffeic, gallic and protocatechuic acids) in less than 10 min using 20 mM sodium tetraborate (pH 9.2) with 5% v/v methanol as a BGE and with UV detection at 254 nm is described. The LODs (3 S/N) ranged between 0.02 and 0.12 microg/ mL. Repeatabilities (RSDs) were 0.66-1.8 and 1.56-4.23% for migration times and peak areas (n = 5), respectively. The method was applied to the determination of phenolic compounds in chess (Bromus inermis L.) after Soxhlet extraction and purification of the crude extracts with SPE procedures. The results compared well with those obtained by liquid chromatographic method. B. inermis was found as a suitable model plant containing a broad spectrum of phenolic compounds in easily detectable concentrations and as a potential source of antioxidants.     

Simultaneous determination of seven phenolic acids in three Salvia species by capillary zone electrophoresis with β‐cyclodextrin as modifier

A capillary zone electrophoresis method was developed for the simultaneous determination of seven phenolic acids, including protocatechuic aldehyde ( 1 ), salvianolic acid C ( 2 ), rosmarinic acid ( 3 ), salvianolic acid A ( 4 ), danshensu ( 5 ), salvianolic acid B ( 6 ), and protocatechuic acid ( 7 ), in Danshen and related medicinal plants. A running buffer composed of 20 mM sodium tetraborate adjusted to pH 9.0, and containing 12 mM β‐cyclodextrin as modifier. Baseline separation was achieved within 17 min running at the voltage of 20 kV, temperature of 25°C and detection wavelength of 280 nm. The relative standard deviations of migration time ranged from 0.2 to 0.7% and the peak area ranged from 1.5 to 3.7% for the seven analytes, indicating the good repeatability of the proposed method. The method was extensively validated by evaluating the linearity (R² ≥ 0.9992), limits of detection (0.14–0.36 μg/mL), limits of quantification (0.47–1.19 μg/mL), and recovery (96.0–102.6%). Under the optimum conditions, samples of Danshen and related medicinal plants were analyzed using the developed method with high separation efficiency.     

Capillary electrophoresis for the monitoring of carboxylic acid production by Gluconobacter oxydans

Determination of carboxylic acids in Gluconobacter oxydans fermentations of wheat straw hydrolyzate was carried out. This matrix is of complex composition containing carbohydrates, organic compounds (e.g., amino acids, toxins), and inorganic salts making the analysis challenging even with separation techniques. A method based on capillary electrophoresis with indirect UV detection was developed for the simultaneous quantification of 18 carboxylic acids. The background electrolyte solution of ammonia, 2,3-pyridinedicarboxylic acid, and Ca²⁺ and Mg²⁺ salts, containing myristyltrimethylammonium hydroxide as a dynamic capillary coating reagent, was validated for the robust and repeatable separation of the carboxylic acids. Intraday relative standard deviations in the optimized method were less than 1.6% for migration times and between 1.0% and 5.9% for peak area. Interday relative standard deviations were less than 5.0% for migration times and between 5.7% and 9.3% for peak area. With 11 nl injected, detection limits for the analytes were between 10 and 43 μmol/l. Detection limits ranged from 0.1 to 0.5 pmol at signal-to-noise ratio of 3. The results demonstrated that wheat straw hydrolyzate was a suitable substrate for G. oxydans with a product yield of 45% for the formation of xylonic acid from xylose and 96% for the formation of gluconic acid from glucose.     

Continuous on-line derivatization and selective separation of D-aspartic acid by a capillary electrophoresis system with a continuous sample introduction interface

A rapid and selective method is described for the separation of D-aspartic acid (D-Asp) using a continuous on-line derivatization system coupled to capillary electrophoresis (CE). D-Asp was derivatized using o-phthaldialdehyde/N-acetyl-L-cysteine (OPA/NAC). By on-line derivatization, amino acid enantiomers were automatically and reproducibly converted to the UV-absorbing diastereomer derivatives which were separated by capillary zone electrophoresis (CZE) in the presence of 10 mmol/L beta-cyclodextrin (beta-CD). Under the investigated separation conditions, D-Asp is resolved from L-aspartic acid (L-Asp) and other amino acids in a standard mixture of amino acids. The separation could be achieved within 4 min and the sample throughput rate can reach up to 16 h(-1). The repeatability (defined as relative standard deviation, RSD) was 3.21%, 3.58% with peak area evaluation and 3.72%, 4.03% with peak height evaluation for L-Asp and D-Asp.     

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.     

高效毛细管电泳分离测定水中的乙酸和氯代乙酸

用邻苯二甲酸氢钾(pH=5.4)缓冲液作为电泳的背景电解质,以十六烷基三甲基溴化铵作为电渗流抑制剂,用甲基叔丁基醚作为萃取剂处理水样,建立了一种测定水中的乙酸和氯代乙酸的简单灵敏的毛细管区带电泳(紫外检测)法.测定结果表明,分析物的迁移时间和峰面积的日内相对标准偏差分别低于0.33%和4.45%,日间相对标准偏差分别低...