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.     

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.     

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.     

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.     

Chemical characterization of leaves and calli extracts of Rosmarinus officinalis by UHPLC-MS

Rosmarinus officinalis L. (Lamiaceae) is an aromatic plant widely popular mainly due to its uses in traditional medicine as an anti-inflammatory, diuretic and antimicrobial, as well as in the prevention and treatment of diseases. These biological activities are mainly related to the presence of phenolic and terpenic compounds. This work reports a chemical profile analysis of extracts from leaves and calli of rosemary obtained by both pressurized liquid extraction and maceration. Chemical profiles were determined on calli extracts of 3, 6, 9, and 15 days of culture; chemical characterization and quantification of compounds was carried out using ultrahigh performance liquid chromatography-mass spectrometry. A total of 53 metabolites were identified in callus and 47 compounds in leaf extracts, of which 25 correspond to phenolic compounds, mainly flavonoids and flavones, 13 terpenes that include phenolic terpenes and one diterpenolactone, two glycosides which correspond to 6-O-caffeoyl-β-D-fructofuranosil-(2→1)-α-D-glucopyranoside and primulaverin, an aromatic compound identified as fenantrenone and a growth regulator 12-hydroxy jasmonic acid. These results showed that undifferentiated rosemary cells accumulate the same compounds identified mainly in highly specialized tissues such as leaves. The plant cell culture supply the possibility of developing biotechnological processes to obtain compounds of commercial interest.     

Multivariate optimisation of the microwave-assisted extraction of oleuropein and related biophenols from olive leaves

Microwave assistance is proposed for the first time in order to accelerate the extraction of biophenols from olive leaves. Under optimal working conditions, obtained using a multivariate methodology, complete extraction of the target analytes was achieved in 8 min. The extracts required no clean-up nor concentration prior to injection into a chromatograph–photodiode array detector assembly for individual separation–quantification. The optimal extractant (an 80:20 ethanol–water mixture) was also used in the development of a stirring-based extraction method which required around 24 h for complete extraction of the target compounds. These mixtures can be used as replacements for toxic extractants, with a view to exploiting olive leaves in order to obtain biophenols for human use.     

Fast high performance liquid chromatography and ultraviolet-visible quantification of principal phenolic antioxidants in fresh rosemary

An improved HPLC method is reported for the determination of rosemary's principal phenolic antioxidants, rosmarinic and carnosic acids, providing a fast and simultaneous determination for both of them by using a solid phase column. The analysis was performed with fresh methanolic extractions of Rosmarinus officinalis. To quantify the amount of antioxidants in a fast and reproducible way by means of UV-vis absorption measurements, a spectrophotometric multi-wavelength calibration curve was constructed based on the antioxidant contents obtained with the recently developed HPLC method. This UV-vis methodology can be extended to the determination of other compounds and herbs if the restrictions mentioned in the text are respected.     

Solvent-free microwave-assisted extraction of fluoroquinolones from soil and liquid chromatography-fluorescence determination

Presented hereafter is a novel method entailing solvent free microwave-assisted extraction (MAE) and HPLC equipped with Fluorimetric Detector (HPLC-FD) for the simultaneous determination at μgkg(-1) concentration of eight fluoroquinolone antibiotics (FQs) (Ciprofloxacin, Danofloxacin, Enrofloxacin, Flerofloxacin, Levofloxacin, Marbofloxacin, Norfloxacin and Orbifloxacin) in agricultural soils. The extraction was quantitatively performed, in a single step, by using an aqueous solution containing Mg(II) as complexing agent, thus avoiding consumption of organic solvents. The optimal MAE conditions have been established through a chemometric approach by considering temperature, irradiation time and matrix moisture or solvent, as the most important recognized variables affecting the extraction yield. Satisfying recoveries (69-110%, spikes 0.03-0.5mgkg(-1)) were gained with a single MAE cycle of 20min, at 80°C in 20% (w/v) Mg(NO(3))(2) solution as leaching agent. MAE-SPE recoveries at 10μgkg(-1), concentration near method quantification limits (MQLs), were in the range 60-85%. Good repeatability and within-lab reproducibility were observed (both in the range 1-16%). The applicability of the method to real samples was assessed on natural contaminated soils. Compared to ultrasonic-assisted extraction (UAE), MAE was shown to be highly competitive in terms of extraction efficacy and analysis speed.     

Application of ionic liquids based microwave-assisted extraction of three alkaloids N-nornuciferine, O-nornuciferine, and nuciferine from lotus leaf

The application of ionic liquids based microwave-assisted extraction (ILMAE) was successfully developed for extracting three alkaloids N-nornuciferine, O-nornuciferine, and nuciferine from lotus leaf. Seven kinds of 1-alkyl-3-methylimidazolium with different cations and anions were investigated in this work and 1.0 M 1-hexyl-3-methylimidazolium bromide ([C₆MIM]Br) solution was selected as solvent. In addition, the microwave parameters including irradiation power, extraction time and solid-liquid ratio were optimized. Compared with the regular MAE and conventional heat-reflux extraction (HRE), the proposed approach exhibited higher efficiency (0.9-43.7% enhanced) and shorter extraction time (from 2 h to 2 min), which indicated ILMAE was an efficient, rapid and simple sample preparation technique. Moreover, the proposed method was validated by the linearity, reproducibility, and recovery experiments. Good linearity was observed with the regression coefficients (r²) between 0.9998 and 0.9999. The recoveries of all methods were in the range of 94.6% and 105.5% with RSD lower than 6.6%, which indicated that the proposed method was credible.     

Parallel determination of rosmarinic and caffeic acids by TLC-densitometry

Summary A TLC-densitometric method was introduced for the parallel determination of rosmarinic and caffeic acids. Experiments performed to establish the optimal measurement parameters related to the excitation wave-length, colour stability, linearity of the calibration curves, sensitivity and reproducibility. The application of TLC permits utilization of the fluorescence of both phenols, often without the need for prior purification. This fluorescence measurement makes the detection extremely sensitive. The densitometric quantification allows the routine analysis of many samples in a short time with reasonable accuracy. The practical application of the elaborated method will be demonstrated on 5Salvia species.     

Ultrasound-assisted extraction and determination of tartaric and malic acids from grapes and winemaking by-products

The optimization of an ultrasound-assisted extraction (UAE) method for tartaric and malic acids from grape derived samples is shown. A fractional factorial experimental design allowed for the determination of the effects of seven extraction variables. Relationships between all the variables were examined. By applying graphical analysis, the best extractions conditions were obtained. The most important variables were the extracting liquid and the extraction temperature. Later, a central composite design was applied for optimizing the temperature and the composition of the extracting liquid. The optimized method was applied to grapes and to winemaking by-products. The repeatability of the method was studied and the recovery of tartaric and malic acids was established. Organic acids quantification was done by liquid chromatography (LC) using a post-column buffer and a conductivity detector.     

Application of surfactants and microemulsions to the extraction of pyrene and phenanthrene from soil with three different extraction methods

In the present work, the use of surfactants and oil-in-water (O/W) microemulsions as alternative extractants in accelerated solvent extraction (ASE) for the extraction of polycyclic aromatic hydrocarbons (pyrene and phenanthrene) from soils was investigated. In particular, the effect of each individual component within the microemulsions, i.e., oil phase, surfactant and co-surfactatnt, and extraction conditions on the percentage recovery was systematically studied. When compared to the water and organic solvent, the important findings were that the common surfactant solutions at the concentrations above their critical micelle concentrations (CMC) were shown to enhance the percentage recovery at the lower extraction temperature. Moreover, the highest percentage recovery can be obtained using microemulsion as the extractant. The chemical component within the microemulsions and relative amounts of the oil phase appeared to play a much more significant role in ensuring high percentage recovery. Finally, an overall comparison between the percentage recoveries obtained with ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE) and ASE using organic solvents, surfactants and microemulsions as extractants was exhibited.     

Optimization of a microwave-assisted extraction of secondary metabolites from crustose lichens with quantitative spectrophotodensitometry analysis

A focused and rapid microwave-assisted extraction (MAE) process was carried out and optimized for secondary metabolites from crustose lichens using Taguchi experimental design and quantitative analysis on TLC by a Camag^® spectrophotodensitometer. The procedure was improved by quantitative determination of norstictic acid (NA), a common depsidone isolated from Pertusaria pseudocorallina (Sw.) Arn. Various experimental parameters that can potentially affect the NA extraction yields including extraction time, irradiation power, volume and the percentage of tetrahydrofuran (THF) were optimized. Results suggest that THF percentage and solvent volume were statistically the most significant factors. The optimal conditions were determined as follows: THF level of 100%, solvent volume of 15 mL, microwave power of 100 W and extraction time of 7 min. Compared to the reflux method, MAE showed a drastic reduction of extraction time (7 min vs. 3 h) and solvent consumption (15 mL vs. 30 mL). The NA in total yield was 90% using the two methods. The optimal conditions were applied to other crustose lichens, Aspicilia radiosa, Diploicia canescens and Ochrolechia parella for the extraction of NA, diploicine (DP) and variolaric acid (VA), with 83%, 90% and 95% of recovery, respectively.     

Semisynthesis of miltirone, 1,2-dehydromiltirone,saligerone from carnosic acid and cytotoxities of their derivatives

Miltirone, 1,2-dehydromiltirone, saligerone have been synthesized form carnosic acid. Among them, one step transformation of carnosic acid into miltirone was realized when decarboxylation/aromatization was promoted by Lewis acid. Moreover, 4 derivatives exhibited potent cytotoxic activities against three human cancer lines in vitro.     

水中痕量苯酚的微波萃取气相色谱分析

A method of microwave-assisted extraction and gas chromatography for determination of trace phenol in water was established.The conditions of microwave extraction and derivation were optimized.Acetone-cyclohexane(1∶1) mixture was used as extracting agent for water sample containing phenol and acetic anhydride was employed for the direct acetylation of phenol.Petroleum ether was used as the extracting agent to extract the derivative of phenol.Detection was carried out in GC-FID equipped with DB-17(30 m×0.53 ...     

Optimization of microwave-assisted solvent extraction for volatile organic acids in tobacco and its comparison with conventional extraction methods

In the present study, a new method using microwave-assisted solvent extraction (MASE) technique followed directly GC analysis was developed for the extraction of volatile organic acids (VOAs) in tobacco. The MASE conditions (heating time, volume of extracting solvent and extraction temperature) were optimized by means of an orthogonal array design (OAD) procedure. The results suggested that extractant, temperature and heating time were statistically the most significant factors. The extracts were directly analyzed with capillary GC operating in splitless-injection mode on an Agilent HP-FFAP capillary column. Under optimum operating conditions, MASE showed significantly better recoveries than those obtained by the conventional extraction method (ultrasonic and reflux extraction), ranging from 90.6% to 103.2%. In addition, a drastic reduction of the extraction time (20 min versus 4 h) and solvent consumption (20 mL versus 100 mL) was achieved with an outstanding reproducibility (CV ≤5%).     

Fast determination of chlorogenic acid in tobacco residues using microwave-assisted extraction and capillary zone electrophoresis technique

In this work, for the first time, capillary zone electrophoresis (CZE) technique combined with microwave-assisted extraction (MAE) was developed for the fast quantification of chlorogenic acid (CA) in tobacco residues. CA in tobacco residue samples were extracted by MAE technique, and then analyzed by CZE. As a new sample preparation method for tobacco residues, the MAE procedure is optimized, validated and compared with conventional methods including ultrasonic extraction (USE) and reflux extraction (RE). It is found that MAE gives the best result due to the highest extraction efficiency within shortest extraction time (only 4.0 min). Here, CA is determined by CZE based on the calibration curve of its authentic standard. The method linearity, detection limit, precision and recovery are studied. The results show that the combined MAE and CZE method has a linearity (R² 0.991, 0.003-0.5 mg ml⁻¹), a limit of detection (0.003 mg ml⁻¹), a limit of quantification (0.01 mg ml⁻¹), good precision (R.S.D. = 4.28%) and a finer recovery (89.0%). The proposed method was successfully applied to the analysis of CA in tobacco residue samples. The experiment results have demonstrated that the CZE combined with MAE is a convenient, fast, economical and reliable method for the determination of CA in tobacco residues.     

Extraction of polycyclic aromatic hydrocarbons and organochlorine pesticides from soils: a comparison between Soxhlet extraction, microwave-assisted extraction and accelerated solvent extraction techniques

The methods of simultaneous extraction of polycyclic aromatic hydrocarbons (PAHs) and organochlorine pesticides (OCPs) from soils using Soxhlet extraction, microwave-assisted extraction (MAE) and accelerated solvent extraction (ASE) were established, and the extraction efficiencies using the three methods were systemically compared from procedural blank, limits of detection and quantification, method recovery and reproducibility, method chromatogram and other factors. In addition, soils with different total organic carbon contents were used to test the extraction efficiencies of the three methods. The results showed that the values obtained in this study were comparable with the values reported by other studies. In some respects such as method recovery and reproducibility, there were no significant differences among the three methods for the extraction of PAHs and OCPs. In some respects such as procedural blank and limits of detection and quantification, there were significant differences among the three methods. Overall, ASE had the best extraction efficiency compared to MAE and Soxhlet extraction, and the extraction efficiencies of MAE and Soxhlet extraction were comparable to each other depending on the property such as TOC content of the studied soil. Considering other factors such as solvent consumption and extraction time, ASE and MAE are preferable to Soxhlet extraction.     

Development of a microwave-assisted extraction method for the recovery of bioactive inositols from lettuce (Lactuca sativa) byproducts

A microwave-assisted extraction (MAE) method was developed for the extraction of bioactive inositols (D-chiro- and myo-inositols) from lettuce (Lactuca sativa) leaves as a strategy for the revalorization of these agrofood residues. Gas chromatography-mass spectrometry was selected for the simultaneous determination of inositols and sugars (glucose, fructose, and sucrose) in these samples. A Box–Behnken experimental design was used to maximize the extraction of inositols based on the results of single factor tests. Optimal conditions of the extraction process were as follows: liquid-to-solid ratio of 100:1 v/w, 40°C, 30 min extraction time, 20:80 ethanol:water (v/v), and one extraction cycle. When compared with conventional solid-liquid extraction (SLE), MAE was found to be more effective for the extraction of target bioactive carbohydrates (MAE 5.42 mg/g dry sample versus SLE 4.01 mg/g dry sample). Then, MAE methodology was applied to the extraction of inositols from L. sativa leaves of different varieties (var. longifolia, var. capitata and var. crispa). D-chiro- and myo-inositol contents varied between 0.57–7.15 and 0.83–3.48 mg/g dry sample, respectively. Interfering sugars were removed from the extracts using a biotechnological procedure based on the use of Saccharomyces cerevisiae for 24 h. The developed methodology was a good alternative to classical procedures to obtain extracts enriched in inositols from lettuce residues, which could be of interest for the agrofood industry.     

Application of ionic liquids in the microwave-assisted extraction of polyphenolic compounds from medicinal plants

Ionic liquids (ILs) solutions as solvents were successfully applied in the microwave-assisted extraction (MAE) of polyphenolic compounds from medicinal plants. ILs, its concentration and MAE conditions were investigated in order to extract polyphenolic compounds effectively from Psidium guajava Linn. (P. guajava) leaves and Smilax china (S. china) tubers. The results obtained indicated that the anions and cations of ILs had influences on the extraction of polyphenolic compounds as well as the ILs with electron-rich aromatic π-system enhanced extraction ability. Under the optimized conditions, the extraction yields of the polyphenolic compounds were in the range of 79.5-93.8% with one-step extraction, and meanwhile the recoveries were in the range of 85.2-103% with relative standard deviations (R.S.D.s) lower than 5.6%. Compared to conventional extraction procedures, the results suggested that the proposed method was effective and alternative for the extraction of polyphenolic compounds from medicinal plants. In addition, the extraction mechanisms and the structures of samples before and after extraction were also investigated. ILs solutions as green solvents in the MAE of polyphenolic compounds from medicinal plant samples showed a great promising prospect.