Protein extraction from Phoenix dactylifera L. leaves, a recalcitrant material, for two-dimensional electrophoresis

This work was aimed at optimizing a protein extraction procedure for date palm (Phoenix dactylifera L.) leaves, a highly recalcitrant plant tissue for 2-DE. Five protein extraction protocols based on different protein precipitation agents (TCA/acetone vs. phenol (Ph) methods) and protein resolubilization methods (physical treatments, e.g., sonication, shaking and/or heating) were tested. Ph/SDS extraction with methanol/ammonium acetate precipitation, followed by DOC preincubation and TCA/acetone precipitation and, finally, solubilization by shaking in rehydration solution was found to be the best protein extraction method. We conclude that DOC with TCA/acetone precipitation step eliminates interfering compounds, thus allowing efficient resolubilization of date palm leaf proteins. This method could be appropriate for proteomic studies such as date palm colonization by entomopathogenic fungi.     

Efficient extraction of proteins from recalcitrant plant tissue for subsequent analysis by two‐dimensional gel electrophoresis

Protein extraction for two‐dimensional electrophoresis from tissues of recalcitrant species is quite problematic and challenging due to the low protein content and high abundance of contaminants. Proteomics in Shorea robusta is scarcely conducted due to the lack of a suitable protein preparation procedure. To establish an effective protein extraction protocol suitable for two‐dimensional electrophoresis in Shorea robusta, four procedures (borate buffer/trichloroacetic acid extraction, organic solvent/trichloroacetic acid precipitation, sucrose/Tris/phenol, and organic solvent/phenol/sodium dodecyl sulfate) were evaluated. Following these, proteins were isolated from mature leaves and were analyzed for proteomics, and also for potential contaminants, widely reported to hinder proteomics. The borate buffer/trichloroacetic acid extraction had the lowest protein yield and did not result in any banding even in one‐dimensional electrophoresis. In contrast, organic solvent/phenol/sodium dodecyl sulfate extraction allowed the highest protein yield. Moreover, during proteomics, organic solvent/phenol/sodium dodecyl sulfate extracted protein resolved the maximum number (144) of spots. Further, when proteins were evaluated for contaminants, significant (77–95%) reductions in the nucleic acids, phenol, and sugars were discernible with refinement in extraction procedure. Accumulated data suggested that the organic solvent/phenol/sodium dodecyl sulfate extraction was the most effective protocol for protein isolation for proteomics of Shorea robusta and can be used for plants that have a similar set of contaminants.     

Protein extraction for 2-DE from the lamina of Ecklonia kurome (laminariales): recalcitrant tissue containing high levels of viscous polysaccharides

Extraction of proteins from the tissues of laminarialean algae, i.e. kelp, is difficult due to high levels of nonprotein interfering compounds, mainly viscous polysaccharides. To establish proteomic analysis of kelp species, an ethanol/phenol extraction method was developed and compared to other popular methods. Proteins were extracted with phenol from crude protein powder, obtained by homogenizing the kelp tissues in ice-cold ethanol. The ethanol/phenol method produced high-quality proteins of the highest purity from the lamina of Ecklonia kurome, one of the Japanese dominant laminarialean algae. This method gave well-resolved 1-D SDS-PAGE or 2-DE images with low background and the highest number of bands or spots. In particular, proteins with neutral to basic pI's were efficiently extracted. Furthermore, 27 spots on the 2-DE gel were extensively identified by MALDI-TOF/TOF analysis. To the best of our knowledge, this is the first report of a protocol for protein extraction from kelp tissues that gives satisfactory 2-D protein profiles. It is expected that the protocol can be applied to other algae tissues or other recalcitrant plant tissues containing high levels of nonprotein interfering compounds.     

适于双向电泳分析的苹果叶片蛋白质提取方法

为了探索适用于双向电泳(2-DE)分析的苹果叶片蛋白质提取方法,比较了三氯乙酸(TCA)/丙酮沉淀法、二硫苏糖醇(DTT)/丙酮法、Tris-HCl提取法和改良的Tris-HCl提取法等4种蛋白质提取方法。以7 cm、pH 3～10的线性固相pH梯度(immobilized pH gradient,IPG)胶条作为第一向电泳,以十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)(12.5%的分离胶)作为第二向电泳,对提取物进行2-DE分离,采用银染显色。结果表明,上述4种方法在2-DE图谱上分别得到140,215,181和616个蛋白质点。其中以改良的Tris-HCl提取法得到的蛋白质点数最多,且背景清晰、图谱上没有明显的横纵条纹。为了进一步验证改良的Tris-HCl提取法的有效性,用18 cm、pH 3～10的线性IPG胶条和12.5%的分离胶对提取的苹果叶片蛋白质进行2-DE分离,考马斯亮蓝R-250染色,共检测到455个蛋白质点,其相对分子质量主要分布在14000～66000范围内,图谱背景清晰,再次证明应用该方法制备的样品适用于双向电泳分析,可用于苹果叶片的蛋白质组学分析。     

Ultrasound-assisted extraction of pesticides from olive branches: a multifactorial approach to method development

A rapid and simple method has been developed for the analysis in olive branches of two insecticides currently used in olive pest control, dimethoate and α-cypermethrin. The effects of analytical conditions on pesticide recovery and the optimal extraction conditions were evaluated by means of a factorial design. The use of this chemometric tool in analytical method development allows the identification of the principal and interaction effects of the extraction conditions on the recovery of pesticides. It also gives information about the location of pesticide maximum recovery with minimal experimental investment. Extraction was carried out with an ultrasonic bath and the experimental conditions studied included the volume of extractant, the time of extraction, the number of extraction steps and the sample weight. The sample was further cleaned up using a Florisil solid-phase extraction cartridge. For the overall extraction procedure, recoveries of 99 % for α-cypermethrin and 90 % for dimethoate from the spiked samples were found for 1 g of sample extracted three times with 35 mL hexane, sonicating for 2 min in each step. The complete process including ultrasonic extraction and filtration will not require more than 15–20 min, in contrast with several hours for conventional liquid–solid extraction techniques. The proposed method allows a high sample throughput, as commonly required in monitoring studies.     

Simple and fast determination of phenolic compounds from different varieties of olive oil by nonaqueous capillary electrophoresis with UV‐visible and fluorescence detection

In this article, we proposed very simple procedures to analyze important phenolic compounds in olive oil samples from different olive varieties. A nonaqueous CE method has been employed. The main phenolic alcohols in virgin olive oil (tyrosol and hydroxytyrosol) and some among the most abundant secoiridoid aglycone derivatives (dialdehydic form of decarboxymethyl elenoic acid linked to hydroxytyrosol, an isomer of oleuropein aglycone and the dialdehydic form of decarboxymethyl elenoic acid linked to tyrosol) were determined by a direct injection into the capillary of the olive oil dissolved in 1‐propanol 1:1 v/v. For the determination of compounds present at lower concentrations, a very simple liquid–liquid extraction method with ethanol has been proposed. The extraction was performed using a relationship 5:1 w/v olive oil/ethanol to achieve the necessary preconcentration of the analytes and the ethanolic extracts were directly injected into the capillary to obtain a very important time reduction. Good recoveries were obtained with both the procedures, using an internal standard. Finally, these procedures were applied to the analysis of these compounds in extra virgin olive oil samples from different varieties of olive.     

Development of a selective sorbent for the solid‐phase extraction of terbuthylazine in olive oil samples: A molecular imprinting strategy

Aiming to implement an analytical methodology that is highly selective for the extraction and quantification of terbuthylazine from olive oil, we successfully achieved: (i) the development of a molecularly imprinted polymer by bulk polymerization using terbuthylazine as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as cross‐linker, and dichloromethane as porogen; (ii) characterization of the imprinting material using Fourier transform infrared spectroscopy, thermogravimetric analysis, nitrogen adsorption at 77 K, and scanning electron microscopy; (iii) their molecular recognition for the template molecule using high‐performance liquid chromatography, and (iv) optimization of a solid‐phase extraction procedure using as sorbent the synthesized molecularly imprinted polymer for the selective extraction and clean‐up of terbuthylazine from spiked organic olive oil and further quantification of the pesticide levels by high‐performance liquid chromatography. The suitability of the implemented analytical methodology was demonstrated, as concentrations of terbuthylazine below the tolerated maximum residue limits in the spiked organic olive oil samples could be satisfactorily analyzed with good precision/accuracy with high recovery rates (96%). Overall, the implemented methodology has proven to be reliable and robust and is highly promising in the field of sample preparation, particularly for the isolation/preconcentration of terbuthylazine in complex food samples.     

Application of deep eutectic solvents for the extraction of phenolic compounds from extra-virgin olive oil

A HPLC–DAD/ESI–MS method has been developed and validated for the analysis of the most representative phenolic compounds in extra-virgin olive oil (EVOO) samples using a green extraction approach based on deep eutectic solvents (DESs) at room temperature. We examined ten DESs based on choline chloride and betaine in combination with different hydrogen bond donors comprising six alcohols, two organic acids, and one urea. Five phenolic compounds, belonging to the classes of secoiridoids and phenolic alcohols, were selected for the evaluation of extraction efficiency. A betaine-based DES with glycerol (molar ratio 1:2) was found to be the most effective for extracting phenolic compounds as compared to a conventional solvent. The optimization of the extraction method involved the study of the quantity of water to be added to the DES and evaluation of the sample-to-solvent ratio optimal condition. Thirty percent of water added to DES and sample to solvent ratio 1:1 (w/v) were selected as the best conditions. The chromatographic method was validated by studying LOD, LOQ, intraday and interday retention time precision, and linearity range. Recovery values obtained spiking seed oil sample aliquots with standard compounds at 5 and 100 μg/g concentration were in the range between 75.2% and 98.7%.     

Preparation of an efficient sorbent by washing then pyrolysis of olive wood for simultaneous solid phase extraction of chloro-phenols and nitro-phenols from water

Simultaneous preconcentration of phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol, 2-nitrophenol, 4-nitrophenol, and 2,4-dinitrophenol was improved by using olive wood (OW) washed with ethanol then pyrolyzed at 200 °C as preconcentrating sorbent. Various OW sorbents were prepared by either washing OW (with ethanol, ether, dichloromethane, tetrahydrofuran or n-hexane); or by pyrolysis (at 100, 150, 200, 250 or 300 °C); or by combined pyrolysis/washing. The adsorbents were characterized by elemental analysis, total acidity/basicity, methylene blue relative surface area, point of zero charge, distribution coefficients of the phenols, and sample loading flow rate. It seems that washing and pyrolysis have removed compounds covering the OW pores, which improved the OW porosity and exposed more acidic groups on the OW surface. The pores and the surface acidic groups seem to play major role in phenols sorption. Ethanol-washed OW then pyrolyzed at 200 °C gave the best preconcentration performance towards phenols (300 mg sorbent, 150 mL of the sample (pH 7), and elution with 3 mL of acetonitrile). The method was linear up to 100 μg L⁻¹; limit of quantification: 0.20-0.48 μg L⁻¹. The method could detect phenol and 2,4-dinitrophenol in industrial wastewater with spiked recovery range from 80.2% to 91.4% (±1.1 to 5.5%RSD) for all the phenols.     

Protein extraction for proteome analysis from cacao leaves and meristems, organs infected by Moniliophthora perniciosa, the causal agent of the witches' broom disease

Preparation of high-quality proteins from cacao vegetative organs is difficult due to very high endogenous levels of polysaccharides and polyphenols. In order to establish a routine procedure for the application of proteomic and biochemical analysis to cacao tissues, three new protocols were developed; one for apoplastic washing fluid (AWF) extraction, and two for protein extraction--under denaturing and nondenaturing conditions. The first described method allows a quick and easy collection of AWF--using infiltration-centrifugation procedure--that is representative of its composition in intact leaves according to the smaller symplastic contamination detected by the use of the hexose phosphate isomerase marker. Protein extraction under denaturing conditions for 2-DE was remarkably improved by the combination of chemically and physically modified processes including phenol, SDS dense buffer and sonication steps. With this protocol, high-quality proteins from cacao leaves and meristems were isolated, and for the first time well-resolved 1-DE and 2-DE protein patterns of cacao vegetative organs are shown. It also appears that sonication associated with polysaccharide precipitation using tert-butanol was a crucial step for the nondenaturing protein extraction and subsequent enzymatic activity detection. It is expected that the protocols described here could help to develop high-level proteomic and biochemical studies in cacao also being applicable to other recalcitrant plant tissues.     

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.     

Effective extractants for the extraction of lithium from aqueous solutions containing sodium and potassium compounds

The extraction power of newly obtained pure methoxy-1,3-diketones in diluents and in their mixtures with electron-donating additives during the extraction of lithium from aqueous solutions containing sodium and potassium was investigated. High separation factors were obtained; no appreciable amounts of sodium and potassium were found in the extract after total extraction of the lithium.Institute of Chemical Kinetics and Combustion, Siberian Branch, Russian Academy of Sciences, 630090 Novosibirsk. Translated fromIzvestiya Akademii Nauk, Seriya Khimicheskaya, No. 6, pp. 1304–1310, June, 1992.     

Protein extraction and 2-DE of water- and lipid-soluble proteins from bovine pericardium, a low-cellularity tissue

Bovine pericardium (BP) is an important biomaterial used in the production of glutaraldehyde-fixed heart valves and tissue-engineering applications. The ability to perform proteomic analysis on BP is useful for a range of studies, including investigation of immune rejection after implantation. However, proteomic analysis of fibrous tissues such as BP is challenging due to their relative low-cellularity and abundance of extracellular matrix. A variety of methods for tissue treatment, protein extraction, and fractionation were investigated with the aim of producing high-quality 2-DE gels for both water- and lipid-soluble BP proteins. Extraction of water-soluble proteins with 3-(benzyldimethylammonio)-propanesulfonate followed by n-dodecyl beta-D-maltoside extraction and ethanol precipitation for lipid-soluble proteins provided the best combination of yield, spot number, and resolution on 2-DE gels (Protocol E2). ESI-quadrupole/ion trap or MALDI-TOF/TOF MS protein identifications were performed to confirm bovine origin and appropriate subcellular prefractionation of resolved proteins. Twenty-five unique, predominantly cytoplasmic bovine proteins were identified from the water-soluble fraction. Thirty-two unique, predominantly membrane bovine proteins were identified from the lipid-soluble fraction. These results demonstrated that the final protocol produced high-quality proteomic data from this important tissue for both cytoplasmic and membrane proteins.     

Selective extraction of derivates of p-hydroxy-benzoic acid from plant material by using a molecularly imprinted polymer

Selective SPE of derivates of p-hydroxybenzoic acid (pHBA) from plant extract of Melissa officinalis is presented using a molecularly imprinted polymer (MIP) made with protocatechuic acid (PA) as template molecule. MIP was prepared with acrylamide as functional monomer, ethylene glycol dimethacrylate as crosslinking monomer and ACN as porogen. MIP was evaluated towards six phenolic acids: PA, gallic acid, pHBA, vanillic acid (VA), gentisic acid (GeA) and syringic acid (SyrA), and then steps of molecularly imprinted SPE (MISPE) procedure were optimized. The best specific binding capacity of MIP was obtained for PA in ACN (34.7 microg/g of MIP). Other tested acids were also bound on MIP if they were dissolved in this solvent. ACN was chosen as solvent for sample application. M. officinalis was extracted into methanol/water (4:1, v/v), the extract was then evaporated to dryness and dissolved in ACN before application on MIP. Water and ACN were used as washing solvents and elution of benzoic acids was performed by means of a mixture methanol/acetic acid (9:1, v/v). pHBA, GA, PA and VA were extracted with recoveries of 56.3-82.1% using this MISPE method. GeA was not determined in plant extract.     

Evaluation of vacuum microwave-assisted extraction technique for the extraction of antioxidants from plant samples

In the present work, vacuum microwave-assisted extraction (VMAE) was to perform microwave-assisted extraction in vacuum. Two well-known antioxidants, vitamin C from guava and green pepper, and vitamin E (α-tocopherol and γ-tocopherol) from soybean and tea leaves, which were easy to be oxidized, were chosen as representative target compounds for the evaluation of VMAE. The extraction yields of vitamin C, α-tocopherol and γ-tocopherol in VMAE and those in MAE performed in atmosphere (air-MAE) were compared and the effects of extraction time, extraction temperature and sample matrix were studied. Moreover, the effects of the oxygen and subpressure invacuo were also discussed via performed MAE in N₂ atmosphere (N₂-MAE). The extraction yields of vitamin C, α-tocopherol and γ-tocopherol in VMAE were higher than that in air-MAE, 35% increments of vitamin C from green pepper, 22% increments of α-tocopherol and 47% increments of γ-tocopherol from tea leaves were obtained, respectively. The comparable increased extraction yields of vitamin C, α-tocopherol and γ-tocopherol in N₂-MAE to that in air-MAE confirmed that oxygen in system was the crucial factor for the oxidation of vitamin C and vitamin E, VMAE was beneficial for the extraction of these oxygen-sensitive compounds. In addition, the subpressure invacuo in the VMAE system also showed positive affect on the extraction yields. On the basis of preventing oxidation and improving extraction efficiency of target compounds because of less oxygen and subpressure invacuo in the extraction system, VMAE has good potential for the extraction of oxygen-sensitive and thermosensitive compounds from plant samples.     

Preparation of molecularly imprinted polymers based on magnetic nanoparticles for the selective extraction of protocatechuic acid from plant extracts

In this study, highly selective core–shell molecularly imprinted polymers on the surface of magnetic nanoparticles were prepared using protocatechuic acid as the template molecule. The resulting magnetic molecularly imprinted polymers were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy, X‐ray diffraction, and vibrating sample magnetometry. The binding performances of the prepared materials were evaluated by static and selective adsorption. The binding isotherms were obtained for protocatechuic acid and fitted by the Langmuir isotherm model and Freundlich isotherm model. Furthermore, the resulting materials were used as the solid‐phase extraction materials coupled to high‐performance liquid chromatography for the selective extraction and detection of protocatechuic acid from the extracts of Homalomena occulta and Cynomorium songaricum with the recoveries in the range 86.3–102.2%.     

Evaluation of the suitability of low hazard surfactants for the separation of phenols and carotenoids from red‐flesh orange juice and olive mill wastewater using cloud point extraction

Natural antioxidants derived from plant sources attract considerable scientific interest. While classic extraction methods consume high volumes of toxic organic solvents, cloud point extraction requires surfactant not exceeding 15% of the waste volume. In preliminary tests, the suitability of various low hazard surfactants (Span 20, PEG 400, Tween 80 and 20) was explored for separation of phenols and carotenoids from olive mill wastewater and red‐flesh orange juice. Tween 80 showed the highest recovery and further applied to the next experiments. The most appropriate surfactant concentrations were 5% (for olive mill wastewater) and 7% (for orange juice) as indicated by recovery % and the rest cloud point extraction parameters (analyte concentration, concentration factor, and phase volume ratio). A double step CPE with 5% + 5% of Tween 80 recovered up to 94.4% of the total phenols from olive mill wastewater, while a 7% + 7% of Tween 80 recovered up to 72.4% of the total carotenoids from orange juice. Evaluation of the final effects and extraction efficiency of single and double step cloud point extraction shows that double step scheme seems to be preferable in both cases. Finally, phenols and carotenoids recovered by Tween 80 maintained high antiradical activity (DPPH test).     

Application of a thiourea‐containing task‐specific ionic liquid for the solid‐phase extraction cleanup of lead ions from red lipstick,pine leaves,and water samples

Here, task‐specific ionic liquid solid‐phase extraction is proposed for the first time. In this approach, a thiourea‐functionalized ionic liquid is immobilized on the solid sorbent, multiwalled carbon nanotubes. These modified nanotubes packed into a solid‐phase extraction column are used for the selective extraction and preconcentration of ultra‐trace amounts of lead(II) from aqueous samples prior to electrothermal atomic absorption spectroscopy determination. The thiourea functional groups act as chelating agents for lead ions retaining them and so, give the selectivity to the sorbent. Elution of the retained ions can be performed using an acidic thiourea solution. The effects of experimental parameters including pH of the aqueous solution, type and amount of eluent, and the flow rates of sample and eluent solutions on the separation efficiency are investigated. The linear dependence of absorbance of lead on its concentration in the initial solution is in the range of 0.5–40.0 ng/mL with the detection limit of 0.13 ng/mL (3s_b/m, n = 10). The proposed method is applicable to the analysis of red lipstick, pine leaves, and water samples for their lead contents.     

Evaluation of carbon nanotubes as a solid-phase extraction adsorbent for the extraction of cephalosporins antibiotics, sulfonamides and phenolic compounds from aqueous solution

The adsorptive potential of carbon nanotubes (single-walled carbon nanotubes and multi-walled carbon nanotubes) for solid-phase extraction of three groups of highly polar compounds (namely cephalosporins antibiotics, sulfonamides and phenolic compounds) was tested in this article. The analytes were strongly retained by the carbon nanotubes. And acceptable recoveries were obtained with the addition of ammonium acetate into eluents. The effects of solution pH on the recoveries of the antibiotics and phenolic compounds were examined. To check the retention abilities of three groups of compounds on carbon nanotubes, fixed amount of each analyte was added to different volumes (up to 500 mL) of aqueous solution, and then extracted by the sorbents. Comparative studies showed that the carbon nanotubes were much superior to C₁₈ for the extraction of the highly polar analytes. For the cephalosporins antibiotics and sulfonamides, the carbon nanotubes showed stronger retention capability than graphitized carbon blacks, but for some of the phenolic compounds graphitized carbon blacks seemed to be more suitable, indicating different retention mechanisms of these analytes. To further assess the enrichment ability of carbon nanotubes for highly polar compounds, the solid-phase extraction method of multi-walled carbon nanotubes packed cartridge was well developed, and the sulfonamides were used as model compounds. Under the optimal procedures, the detection limits of sulfonamides were in the range of 27-38 ng L⁻¹. The spiked recoveries from several real water samples obtained for sulfathiazole and sulfadiazine ranged from 55% to 79% and 72% to 92%, respectively, while the recoveries of sulfapyridine and sulfamethazine were in the range of 85-102%.     

A solid-phase extraction procedure for the clean-up of thiram from aqueous solutions containing high concentrations of humic substances

A simple solid-phase extraction (SPE) procedure with an octadecyl bonded phase silica (C₁₈) was developed for clean-up of the fungicide thiram from aqueous solutions containing high concentrations of humic substances, for future studies of thiram adsorption onto solid humic substances or soils. Suspensions of humic acids and soil, in aqueous 0.01 M CaCl₂ solution, were prepared and used as samples. These extracts were spiked with thiram and immediately applied to a C₁₈-SPE cartridge. Thiram was eluted with chloroform and its concentration measured by spectrophotometry at 283 nm. Non-spiked aqueous extracts (blanks) and a control sample of thiram in 0.01 M CaCl₂ aqueous solution were also prepared and submitted to the same SPE procedure. The results show that humic substances are extensively retained by the C₁₈ cartridge but are not eluted with CHCl₃. Recoveries of 100-104% were obtained for thiram in the presence of humic substances. The SPE procedure described in this work is an efficient clean-up step to remove the interference of humic substances absorbance and to be coupled to any spectrophotometric or HPLC-UV method, usually used for thiram analysis in food extracts.