Chloroform Ethanol Research Articles

Human carbonic anhydrase: Purification and characterization study in thalassemia major patients compared to healthy subjects

Background: Carbonic anhydrase (CA) catalyzes the reversible reaction of converting carbon dioxide to bicarbonate. Objective: This study was aimed to isolate and purify human erythrocytes CA and study its physicochemical properties of the enzyme reaction for s-thalassemia major patients. Materials and Methods: The blood samples included 61 samples of blood (31 males and 30 females) from s-thalassemia patients visited Azadi Hospital/Kirkuk city. Healthy individuals as control group included 40 participants. The separated fractions were obtained using four steps: extraction by ethanol and chloroform, ammonium sulfate precipitation, dialysis, and gel filtration chromatography; finally, the CA was analyzed by polyacrylamide gel electrophoresis. Results: The CA activity showed significant (P ≤ 0.05) decrease, total protein showed nonsignificant (P ≥ 0.05) increase, and specific activity significantly (P ≤ 0.05) increased in patients group compared to healthy individuals. CA was partially purified with a factor of 22.5 and 18 by extraction with ethanol and chloroform and 1.5,1.4 for Fraction I and 1,2 for Fraction II using gel filtration chromatography. The optimum conditions for the CA reaction in patients group were enzyme concentration (6 μl), substrate concentration (6 Mm), pH = 7.4, and temperature 37°C. The electrophoresis study indicated that the bands of CA in patients group showed bands with less intensity than the bands in healthy individuals. Conclusion: The best method to purify CA from human erythrocytes with high recovery and fold of purification was ethanol–chloroform extraction.

Optical and morphological studies of 3-methyl-2,6-diphenylpiperidinium picrate

Single crystals of 3-methyl-2,6-diphenylpiperidinium picrate, a proton-transfer complex, are grown from ethanol–chloroform (2:1, v/v) at room temperature by slow evaporation. The vibrational patterns, as measured by Fourier transform infrared spectroscopy, show the formation of complexes. The crystal exhibits good transmittance in the visible region, and the lower optical cutoff wavelength is ∼495 nm. The structure and the crystallinity of the material were further confirmed by powder X-ray diffraction analysis. The scanning electron microscopy study indicated defect-centered surface morphology. Second-harmonic generation efficiency was estimated by the Kurtz and Perry powder method. Thermal analysis by thermogravimetry/differential thermal analysis shows the stability of the material.

Development of Singlet Oxygen Absorption Capacity (SOAC) Assay Method Using a Microplate Reader.

Recently, a new assay method that can quantify the singlet oxygen absorption capacity (SOAC) of natural antioxidants and food extracts was developed. The SOAC values were measured in ethanol-chloroform-D2O (50 + 50 + 1, v/v/v) solution at 35°C using a UV-Vis spectrophotometer equipped with a six-channel cell positioner and an electron-temperature control unit. In the present study, measurement of the SOAC values was performed for eight representative carotenoids and three vegetable extracts (tomato, carrot, and red paprika) using a versatile instrument, the microplate reader. A 24-well glass microplate was used for measurements because a plastic microplate, commonly used in the laboratory, dissolves in the ethanol-chloroform-D2O solution. The SOAC values of eight carotenoids and three vegetable extracts measured using a microplate reader were in good agreement with the corresponding values measured using a UV-Vis spectrophotometer, suggesting that the microplate reader is an applicable instrument for the measurement of reliable SOAC values for general antioxidants and food extracts in solution.

Extraction of superoxide dismutase, catalase, and carbonic anhydrase from stroma-free red blood cell hemolysate for the preparation of the nanobiotechnological complex of polyhemoglobin–superoxide dismutase–catalase–carbonic anhydrase

We report a novel method to simultaneously extract superoxide dismutase (SOD), catalase (CAT), and carbonic anhydrase (CA) from the same sample of red blood cells (RBCs). This avoids the need to use expensive commercial enzymes, thus enabling a cost-effective process for large-scale production of a nanobiotechnological polyHb–SOD–CAT–CA complex, with enhancement of all three red blood cell functions. An optimal concentration of phosphate buffer for ethanol–chloroform treatment results in good recovery of CAT, SOD, and CA after extraction. Different concentrations of the enzymes can be used to enhance the activity of polyHb–SOD–CAT–CA to 2, 4, or 6 times that of RBC.

SCREENING AND DETERMINATION OF ANTIOXIDANT SCAVENGING ACTIVITY OF PIPER LONGUM AND EUCALYPTUS CAMALDULENSIS

The herbal medicine is used from time immemorial to treat various common ailments like cold, cough and gastric problems. India has various system of treatment like Ayurveda, Siddha, Unani and Homeopathy which are devoid of side effects and treat the person with higher benefits. Piper longum fruitsand Eucalyptus camaldulensis Dehnh., leaves has been used as spices and for medicinal oil preparation for various treatment. We have made an attempt to elevate the importance of these herbal sources by understanding the antioxidant scavenging activity. We have extracted and processed the plants fruit andleaves using different solvents in 1:10 dilution such as petroleum ether, chloroform ethanol, hexane, methanol and distilled water. It was found that ethanol and methanol extract of Piper longum and Eucalyptus camaldulensis Dehnh., has higher antioxidant activity when compared with the standard Vitamin C and IC50value was observed to be nearly 70μg/ml for all the tests

WITHANIA SOMNIFERA: DETERMINATION OF ANTIOXIDANT SCAVENGING POTENTIAL ACTIVITY

Due to an increase in modernization, people suffer from various stresses related and sexual problems. In India, with various herbal reservoirs, Withania somnifera (Ashwagandha) plays an important role in treating the vital and vigour problem as well as in treating sexual problems. We made an attempt toelevate the importance of this herbal source by understanding its antioxidant scavenging activity. We have utilized the powder of this plant fruit and processed using different solvents in 1:10 dilution by dissolving with 3g of powder with 30ml of each solvent such as petroleum ether, chloroform ethanol, hexane, methanol and distilled water. It was found that methanol and chloroform extracts of W. somnifera, has higher antioxidant activity when compared with the standard Vitamin C was observed using the graph plotted as the percentage of inhibition of scavenging activity vs concentration and IC50 value was observed to be nearly 50 μg/mL for all the tests.

Growth and characterization of 2-amino-5-nitrobenzophenonium picrate crystals

Single crystals of 2-amino-5-nitrobenzophenonium picrate (ANBP) were grown by slow evaporation solution growth technique from a mixed solvent system ethanol–chloroform–acetic acid (1:1:1, v/v). The 1H and 13C signals of the grown crystal are identified by the nuclear magnetic resonance analyses. Fourier transformed infrared spectroscopy confirms the presence of characteristic functional groups present in the grown crystal. Powder X-ray diffraction was carried out to determine the structure and crystallinity. The crystal belongs to monoclinic system. The optical properties of the grown crystals were analyzed by UV–Vis spectroscopy. Thermogravimetric and differential thermal analysis studies reveal no decomposition up to the melting point. The surface morphology of the as-grown crystals was studied by scanning electron microscopy.

Crystal structure of 8-hy-droxy-quinoline: a new monoclinic polymorph.

In an attempt to grow 8-hy­droxy­quinoline–acetamino­phen co-crystals from equimolar amounts of conformers in a chloro­form–ethanol solvent mixture at room temperature, the title compound, C9H7NO, was obtained. The mol­ecule is planar, with the hy­droxy H atom forming an intra­molecular O—H⋯N hydrogen bond. In the crystal, mol­ecules form centrosymmetric dimers via two O—H⋯N hydrogen bonds. Thus, the hy­droxy H atoms are involved in bifurcated O—H⋯N hydrogen bonds, leading to the formation of a central planar four-membered N2H2 ring. The dimers are bound by inter­molecular π–π stacking [the shortest C⋯C distance is 3.2997 (17) Å] and C—H⋯π inter­actions into a three-dimensional framework. The crystal grown represents a new monoclinic polymorph in the space group P21/n. The mol­ecular structure of the present monoclinic polymorph is very similar to that of the ortho­rhom­bic polymorph (space group Fdd2) studied previously [Roychowdhury et al. (1978 ▶). Acta Cryst. B34, 1047–1048; Banerjee & Saha (1986 ▶). Acta Cryst. C42, 1408–1411]. The structures of the two polymorphs are distinguished by the different geometries of the hydrogen-bonded dimers, which in the crystal of the ortho­rhom­bic polymorph possess twofold axis symmetry, with the central N2H2 ring adopting a butterfly conformation.

Extracting DNA from within intact foraminiferal shells

Abstract Here we present an evaluation of several methods for extracting DNA from within intact foraminiferal shells. We tested 12 lysis buffers under carefully optimised incubation conditions, evaluating their success in terms of efficiency in evacuating cellular material, crude DNA yield, PCR success, and final shell integrity. A number of the buffers tested produced excellent results. The most successful method utilized a lysis buffer containing Sodium N-lauroyl sarcosine, Guanidinium isothiocyanate, Isopropanol, TRIS and NaCl, incubated at 75 °C for 24 h, followed by chloroform extraction and ethanol precipitation. High yields of DNA were produced, with no signs of PCR inhibition, and the foraminiferal shells were left completely intact. Retaining the shell of individual specimens presents a significant advance, allowing for direct comparisons between shell morphology and genotype data, which could greatly enhance the utility of foraminifera as palaeoproxies of past climate change.

Single-cell analysis of K562 cells: An imatinib-resistant subpopulation is adherent and has upregulated expression of BCR-ABL mRNA and protein

In chronic myeloid leukemia (CML) cells from different stages of maturation may have differential expression of BCR-ABL at both messenger RNA (mRNA) and protein level. However, the significance of such differential expression to clinical disease behavior is unknown. Using the CML-derived, BCR-ABL expressing cell line, K562, distinct plastic-adherent (K562/Adh) and nonadherent (K562/NonAdh) subpopulations were established and then analyzed both as single cells and as bulk cell populations. BCR-ABL mRNA was upregulated in K562/Adh compared with K562/NonAdh cells in both single cell and bulk population analyses (p < 0.0001). Similarly, phosphorylation of BCR protein was upregulated in K562/Adh, compared with K562/NonAdh cells (63.42% vs. 23.1%; p = 0.007), and these two K562 subpopulations were found to express significantly different microRNA species. Furthermore, treatment with the BCR-ABL tyrosine kinase inhibitor, imatinib, reduced cell viability more rapidly in K562/NonAdh compared with K562/Adh cells (p < 0.005) both at single and bulk cell levels. This discovery of an adherent subpopulation of K562 cells with increased BCR-ABL mRNA, increased phosphorylated BCR protein expression, differential microRNA expression, and increased imatinib resistance suggests that a similar subpopulation of cells can also mediate clinical resistance to imatinib during treatment of patients with CML.

Abstract 5043: Comparison of the efficacy and analytical performance of isolation procedures for circulating plasma microRNAs

Abstract Introduction: The efficiency and reproducibility of plasma miRNAs extraction protocol consists a critical step concerning the quantification of circulating miRNAs in plasma. Plasma is a specimen with high concentrations of lipids and proteins, so specific handling is required in order to achieve an enriched fraction of small RNAs at the end of the procedure. In the present study, we conducted a comparative investigation regarding RNA yielding among different commercially available experimental approaches. Our evaluation approach relied on the comparative extraction yielding of endogenous human miR-21 and exogenous spiked-in C.elegans miR-39 from pooled plasma samples from healthy individuals. Materials and Methods: In the present study we evaluated the extraction potential of small RNAs using three different commercially available kits for miRNAs extraction from plasma samples according to the manufacturer's protocols: a) miRNeasy mini Kit (Qiagen), b) mirVana PARIS kit (Ambion), c) microRNA purification Kit (Norgen Biotek) and d) Trizol LS Reagent (Invitrogen). We firstly pooled plasma samples from healthy individuals and used them for spiking experiments. In all cases, the initial plasma volume was 200μL and in each sample we spiked 25fmol of the exogenous standard C.elegans cel-miR-39. The isolation of miRNAs from plasma was conducted as per kit protocols and all extraction methods were performed three independent times from the same initial pooled plasma sample. A fixed volume of the eluted RNA sample was used as an input for the reverse transcription reaction. After isolation, quantification of cel-miR-39 was performed using real-time PCR. Recovery of cel-miR-39 in each case was estimated in respect to the levels of an equivalent amount of C.elegans copies added to total RNA after each extraction method (representing 100% recovery). In parallel, in all these samples the endogenous hsa-miR-21 was also quantified by real-time qPCR. The results were compared between the different isolation protocols concerning this with the greatest miRNAs yield for expression levels of endogenous miRNAs. Results: We found that the phenol:chloroform extraction and ethanol precipitation of RNA protocol (Trizol LS) recovers small RNAs with some loss whereas silica-column based RNA extraction methods provide a more effective and reliable mechanism for the isolation of small RNAs. Although all column protocols have indications to be very effective, in our hands the mirVana PARIS (Ambion) protocol appeared giving higher yields of circulating miRNAs from plasma samples. This was the case after quantifying for both the exogenous (cel-miR-39) and endogenous (miR-21) less abundant levels of miRNAs. Conclusion: According to our results, the mirVana PARIS kit (Ambion) was the most efficient among different commercially available kits for the isolation of circulating plasma miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5043. doi:1538-7445.AM2012-5043

Synthesis and Spectroscopic Evaluation of Novel N-, S-, and O-Substituted 1,4-Naphthoquinone Derivatives

Novel O- S,O- S,S- N,N- and N,S-substituted naphthoquinone compounds were prepared by reaction of 2,3-dichloro-1,4-naphthoquinone and the corresponding nucleophiles in the presence of chloroform and triethylamine or ethanol solution of Na2CO3. The structures of the novel naphthoquinone compounds were characterized by micro analysis, FT-IR, UV/Vis, 1H NMR, 13C NMR, MS, and fluorescence spectroscopy.GRAPHICAL ABSTRACT

Purification and characterization of Cu, Zn-superoxide dismutase from black soybean

A superoxide dismutase (SOD) was purified from seeds of the Chinese black soybean(Glycine max cv). The purification procedure comprised ammonium sulfate precipitation, ion-exchange chromatography on CM-Sephadex C-50, affinity chromatography on Affi-gel blue gel and HP LC on DEAE 650-C. The final enzyme was purified 15.40 fold with a specific activity of 4619.3U/mg protein and a recovery of enzyme was 29.41% from (NH4)2SO4 precipitation. The SDS-PAGE of the purified protein exhibited a molecular mass of 31.0kDa and could be depolymerized spontaneously to a band at about 14.5kDa in non-reducing conditions, and a diffusion band at about 15.5kDa in reducing conditions, indicating a double-strand protein with intra-molecular disulfide bridges. The isoelectric point was determined to be 5.6, indicating it's an acidic protein. The enzyme is a type of copper plus zinc superoxide dismutase (Cu, Zn-SOD) because it was greatly inhibited by hydrogen peroxide, insensitive to chloroform–ethanol, and the maximum ultraviolet absorbance was found to be 266nm. Thermostability experiments showed that the activity of the purified enzyme was stable after exposure to temperatures below 50°C, and the activity could be well maintained between pH 6.0 and 8.0. Moreover, the SOD exerted an obvious protective effect against oxidative stress of UVC radiation in normal human liver cell strain L-02 cells.

In vitro study of the PLA2 inhibition and antioxidant activities of Aloe vera leaf skin extracts

BackgroundIn the present work we determined the total phenolic content of Aloe vera leaf skin (AVLS) extracts by using various solvents (hexane, chloroform-ethanol (1/1), ethyl acetate, butanol and water). We have also evaluated the antioxidant and the anti-PLA2 properties of these extracts by measuring their inhibition potency on the human pro-inflammatory phospholipase A2 (group IIA).ResultsThe water extract exhibits the highest inhibitory effect with an IC50 = 0.22 mg/ml and interestingly no effect was observed on the digestive phospholipase A2 (group IB) even at a concentration of 5 mg/ml. Antioxidant activities were also analyzed and the most active extracts were observed when using chloroform ethanol (1/1) and ethyl acetate (IC50 = 0.274 and 0.326 mg/ml, respectively). Analysis of the total phenolic content reveals that the water extract, with the best anti-PLA2 effect, was poor in phenolic molecules (2 mg GAE/g). This latter value has to be compared with the chloroform-ethanol and the ethyl acetate extracts (40 and 23.8 mg GAE/g, respectively), mostly responsible for the antioxidant activity.ConclusionA significant correlation was established between the total phenolic content and the antioxidant capacity but not with the anti PLA2 activity. Results from phytochemical screening suggest that the anti PLA2 molecules were probably catechin tannins compounds.

Statistical mixture design — Principal component determination of synergic solvent interactions for natural product extractions

Ethanol, ethyl acetate, dichloromethane, acetone and chloroform mixtures of a simplex centroid design have been used to extract total crude material as well as the neutral, organic, basic, fiber and residue fractions of Mikania laevigata Sch. Bip. leaves. The quantity of crude extracted material is found to depend on the solvent proportions according to a special cubic model. Synergic binary solvent effects between the ethanol–ethyl acetate, ethanol–dichloromethane, ethanol–chloroform, ethyl acetate–chloroform and ethyl acetate–dichloromethane pairs are found to explain the superior extraction results of the mixtures compared to results for the pure solvents. These significant synergic binary effects are seen to occur between solvents having disparate acidity, basicity and/or dipolarity parameters located at extremities of the domain defined by the mixtures within the Snyder-Rohrschneider solvent selectivity triangle. So molecular interactions known to be important for chromatographic separations are also significant for separating crude material from its Mikania laevigata Sch. Bip. leave matrix. Principal component analysis shows these interaction effects to be important for obtaining higher yields of the fractionated materials.

Comparison of solid dispersions produced by supercritical antisolvent and spray-freezing technologies

Oxeglitazar is a new orally administered poorly water soluble active substance used in the treatment of type II diabetes. The objective of this work was to improve its dissolution kinetics using supercritical antisolvent (SAS) and spray-freezing (SF) techniques. Oxeglitazar was formulated with various excipients, including: Poloxamer 188 and 407, polyethylene glycol (PEG) 8000 and polyvinilpyrrolidone (PVP) K17 in a 1:1 weight ratio. In the SAS technology, pharmaceutical ingredients were dissolved in an appropriate solvent, and the feed solution was dispersed through a capillary nozzle in supercritical CO 2 (SC CO 2). Dichloromethane (DCM), chloroform (CHCl 3), and a binary co-solvent system of chloroform–ethanol (EtOH/CHCl 3 50:50, v/v%) were tested. In the SF process, tert-butanol ( tBuOH) was used as solvent. The feed solution was injected into liquid nitrogen through a capillary nozzle located above the surface of the boiling nitrogen. Frozen particles were collected and freeze-dried for 30 h. Formulations were compared in terms of particle morphology, particle size, flow properties, crystallinity, polymorphic purity, residual solvent content, precipitation yield, drug content, specific surface area and dissolution kinetics. SAS and SF processed formulations exhibited enhanced dissolution rates. Within 5 min, the amount of dissolved drug varied from 31.6 to 64.3% for SAS and from 77.9 to 96.9% for freeze-dried formulations while only 30.5% was dissolved from raw drug. Apart from oxeglitazar/PVP K17, SAS prepared solid dispersions were characterized by high crystallinity and acicular shape. Freeze-dried formulations consisted of porous spherical particles with high amorphous content (94.2–100%).

SPE-HPTLC of procyanidins from the barks of different species and clones of Salix

A SPE-HPTLC method was developed for the qualitative and quantitative analysis of procyanidin B 1 in willow barks. The chromatography was performed on HPTLC silica gel layer with the mobile phase chloroform–ethanol–formic acid (50:40:6 v/v/v), in the Automatic Developing Chamber—ADC 2. The methanol extracts from willow barks were purified by SPE method on RP-18 silica gel columns with methanol–water (7:93 v/v) as the eluent. The presence of procyanidin B 1 was revealed in the majority of investigated willow barks. The content of procyanidin B 1 varied from 0.26 mg/g in the extract of Salix purpurea clone 1067–2.24 mg/g in the extract of Salix alba clone 1100. The method was validated for linearity, precision, LOD, LOQ and repeatability.

Isolation, purification and characterization of superoxide dismutase from garlic

A simple and cost-effective way to purify superoxide dismutase (SOD) from garlic was proposed. The stepwise isolation and purification procedure consists of phosphate buffer and chloroform–ethanol extraction, acetone precipitation followed by ion exchange chromatography on DEAE column. The specific enzyme activity was 4124 U/mg protein with the recovery rate of 53.3% and concentration factor of 192, respectively. SDS-PAGE analysis showed high consistency of garlic SOD with cattle blood SOD. The enzyme activity of garlic SOD was greatly inhibited by cyanide and hydrogen peroxide while it could be well maintained at pH values ranging from 4 to 11. Only slight decrease of enzyme activity was identified at temperatures lower than 50 °C or by treatment with ultrasonic wave for 30 min. The ultraviolet absorbance of garlic SOD was found to be 269 nm.

Separation and purification of verticine and verticinone from Bulbus Fritillariae Thunbergii by high-speed counter-current chromatography coupled with evaporative light scattering detection

High-speed counter-current chromatography (HSCCC) coupled with evaporative light scattering detection (ELSD) was successfully applied to preparative separation and purification of verticine and verticinone from crude extracts of Bulbus Fritillariae Thunbergii by a one-step separation, using chloroform–ethanol–0.2 mol L −1 hydrochloric acid (3:2:2, v/v/v) as a solvent system. HPLC analysis of the fractions collected on the preparative HSCCC of 200 mg of crude extracts showed that the purity of verticine (25.6 mg) was 96.8% and that of verticinone (10.3 mg) was 95.4%. The chemical identities of these components were confirmed by 1H NMR and EI–MS.

Accurate Calculation for Liquid–Liquid Equilibria of Typical Solvent Systems Used in CCC

The properties of solvent systems in CCC can be estimated using calculations. In this work, liquid–liquid equilibrium (LLE) data of several typical solvent systems used in CCC are correlated and predicted using a molecular thermodynamic model called the Non‐Random Theory of Liquids, NRTL. The parameters in this model are regressed only with the special system considered. The selected solvent systems are widely used in CCC: chloroform–methanol–water; chloroform–ethanol–water; hexane–methanol–water; ethyl acetate–methanol–water; hexane–ethyl acetate–methanol–water; hexane–ethanol–water; ethyl acetate–ethanol–water; butanol–acetic acid–water. Detailed comparison with experimental data shows that LLE of these systems can be predicted with greatly improved accuracy. Together with these results, volume ratio, density, viscosity, dielectric constant, and interfacial tension can be predicted with improved accuracy.