The use of non-aqueous chloroform/methanol extraction for the delipidation of brain with minimal loss of enzyme activities.
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ABSTRACT:
SUBMITTER: Kurihara T
PROVIDER: S-EPMC1164878 | biostudies-other | 1977 Jul
REPOSITORIES: biostudies-other
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Project description:Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.
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Project description:Accurate and reliable analysis of gene expression depends on the extraction of pure and high-quality RNA. However, while the conventional phenol-chloroform RNA extraction is preferable over silica-based columns, particularly when cost is a concern or higher RNA yield is desired, it can result in significant RNA contamination. Contaminants including excess phenol, chloroform, or salts, can have significant impacts on downstream applications, including RNA quantification and reverse transcription, that can skew data collection and interpretation. To overcome the issue of RNA contamination in the conventional phenol-chloroform based RNA extraction method, we have optimized the protocol by adding one chloroform extraction step, and several RNA washing steps. Importantly, RNA quality and purity and accuracy in the quantification of RNA concentration were significantly improved with the modified protocol, resulting in reliable data collection and interpretation in downstream gene expression analysis. •Our protocol is customized by the addition of a second chloroform extraction step. Chloroform is carefully pipetted so as to not disturb the interphase layer. Any contaminants accidentally removed from interphase will be present in subsequent steps and can result in RNA contaminated with protein or phenol. The additional chloroform step increases RNA purity.•Additionally, the addition of 2 additional ethanol washes, initially intended to remove any residual salts from the isopropanol RNA precipitation step, also removed residual phenol contamination, enhancing RNA purity.•In summary, these modifications serve to enhance not only the purity of the RNA but, also increase the accuracy and reliability of RNA quantification.
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Project description:BackgroundA study carried out by World Health Organization revealed that around 80% of individuals globally depends on herbal forms of medication with 40% of pharmaceutical products being sourced from medicinal plants. The study objective was to evaluate the phytochemicals composition, in vitro antimicrobial and antioxidant properties of the leaves of Terminalia catappa L. aqueous and methanolic extracts.MethodsAntimicrobial activity was analyzed by disk diffusion, the minimum inhibitory concentration in-vitro assays with ciprofloxacin as the standard for antibacterial assay while nystatin for antifungal assay. Ferric reducing antioxidant power and 2,2-diphenyl-1-picryl-hydrazyl-hydrate assays were used for the evaluation of antioxidant properties of the crude extracts while the groups responsible for this activity identified using Fourier transform infrared spectrophotometer.ResultsThe study found that the leaves of Terminalia catappa contained alkaloids, tannins, steroids, cardiac glycosides, flavonoids, phenols, saponins, and coumarins, but terpenoids were absent. Presence of functional groups associated with this class of compounds such as OH vibrational frequencies were observed in IR spectrum of the crude extracts. Methanolic extract from Terminalia catappa exhibited greater antibacterial properties against Pseudomonas aeruginosa, Escherichia coli and Staphylococcus aureus, whereas aqueous extract displayed greater antibacterial activity against Bacillus subtilis for all concentrations tested. The amount of the sample that scavenged 50 percent of DPPH (IC50) was found to be 8.723, 13.42 and 13.04 µg/mL for L-ascorbic acid, Terminalia catappa L. methanolic and aqueous extracts respectively. The antimicrobial and antioxidant activities varied with the extract concentration and solvent used in extractions.ConclusionTerminalia catappa L. leaves are prospective for use as a source of therapeutic agents that could lead to the advancement of new antimicrobial and antioxidant products.
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Project description:ObjectiveThe aim of this study was to investigate the antiplasmodial effects of the crude aqueous, methanol and chloroform extracts of the leaves of Vernonia adoensis in Plasmodium berghei infected Swiss albino mice using Peters' 4-day suppressive test.ResultsThe number of mice used for the toxicity test was 20 (5/group) and for each extract and control groups 5 mice per group was used. The aqueous, methanol and chloroform extracts of V. adoensis leaves indicated statistically significant (P?<?0.05) suppression of parasitaemia in the treated mice. The highest inhibition was that of the methanol extract treated mice (83.36%) followed by aqueous (72.26%) and chloroform (54.34%) at an oral dose of 600 mg/kg b.wt. Each extract prevented body weight loss and packed cell volume (PCV) reduction as compared to the negative control groups. The survival time of the mice treated with chloroform based on Kaplan-Meir analysis was 12.53?±?0.37 at 600 mg/kg b.wt, while the negative control was 7.93?±?0.37 days. The LD50 of the extracts was greater than 3000 mg/kg body weight. In conclusion, the crude leaves extract of V. adoensis have demonstrated antiplasmodial effect in vivo. P. berghei infection is suppressed in a dose-dependent manner showing relevance of the traditional use of the plant.
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Project description:The almond cake is a protein-rich residue generated by the mechanical expression of the almond oil. The effects of the aqueous (AEP) and enzyme-assisted aqueous extraction processes (EAEP) on the biological properties of the almond cake protein were evaluated. Total phenolic content (TPC), antioxidant capacity, inhibitory effects against crucial enzymes related to metabolic syndrome, antimicrobial potential, and in vitro protein digestibility profile were assessed. EAEP provided the best results for antioxidant capacity by both ORAC (397.2 µmol TE per g) and ABTS (650.5 µmol TE per g) methods and also showed a high (~ 98%) potential for α-glucosidase inhibition. The AEP resulted in protein extracts with the highest lipase inhibition (~ 70%) in a dose-dependent way. Enzymatic kinetic analyses revealed that EAEP generated uncompetitive inhibitors against α-glucosidase, while EAEP, AEP, and HEX-AEP (used as control) generated the same kind of inhibitors against lipase. No protein extract was effective against any of the bacteria strains tested at antimicrobial assays. An in silico theoretical hydrolysis of amandin subunits corroborated with the results found for antioxidant capacity, enzyme inhibitory experiments, and antimicrobial activity. Digestibility results indicated that the digestive proteases used were efficient in hydrolyzing almond proteins, regardless of the extraction applied and that HEX-AEP presented the highest digestibility (85%). In summary, EAEP and AEP skim proteins have the potential to be used as a nutraceutical ingredient. The biological properties observed in these extracts could help mitigate the development of metabolic syndrome where EAEP and AEP skim proteins could be potentially used as a prophylactic therapy for diabetes and obesity, respectively.
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Project description:BACKGROUND:Though blood is an excellent biofluid for metabolomics, proteins and lipids present in blood can interfere with 1d-¹H NMR spectra and disrupt quantification of metabolites. Here, we present effective macromolecule removal strategies for serum and whole blood (WB) samples. METHODS:A variety of macromolecule removal strategies were compared in both WB and serum, along with tests of ultrafiltration alone and in combination with precipitation methods. RESULTS:In healthy human serum, methanol:chloroform:water extraction with ultrafiltration was compared to methanol precipitation with and without ultrafiltration. Methods were tested in healthy pooled human serum, and in serum from patients with sepsis. Effects of long-term storage at -80 °C were tested to explore the impact of macromolecule removal strategy on serum from different conditions. In WB a variety of extraction strategies were tested in two types of WB (from pigs and baboons) to examine the impact of macromolecule removal strategies on different samples. CONCLUSIONS:In healthy human serum methanol precipitation of serum with ultrafiltration was superior, but was similar in recovery and variance to methanol:chloroform:water extraction with ultrafiltration in pooled serum from patients with sepsis. In WB, high quality, quantifiable spectra were obtained with the use of a methanol: chloroform precipitation.
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Project description:Dacryodes edulis (G. Don) H.J. Lam) is the most popular species under the genus Dacryodes. It is well known for its nutritional and ethno-medicinal uses in South-eastern and South-western Nigeria. This study was aimed to evaluate the toxicity of the aqueous-methanol fraction of crude methanol extract of Dacryodes edulis leaves (AMDE). The test rats were randomized to groups of single oral treatment of AMDE (10-5000 mg/kgbw) for the acute toxicity study. They were monitored for obvious signs of behavioural change and mortality. For the subacute toxicity study, the rats were randomized to three daily treatment groups (of 200, 400 and 600 mg/kgbw of AMDE) for 28 days. The fourth group (control) received 2.5 %v/v DMSO. At the end of the experiment, blood samples were collected for hematology and clinical chemistry evaluation. The histopathology of the livers and kidneys were assessed using the excised organs. The cytotoxicity and genotoxicity of AMDE were also evaluated using Allium cepa model. The result showed that acute administration of AMDE, up to a dose of 5000 mg/kgbw did not result in mortality of the test rats. The observed median lethal dose (LD50) was greater than 5000 mg/kgbw. The subacute oral administration of AMDE for 28 days showed no significant (p > 0.05) effect on liver function, kidney function indices, organ - body weight ratio, but significantly (p < 0.05) decreased erythrocytic indices: red blood cells, haematocrit, and haemoglobin at 600 mg/kgbw. The Allium cepa assay revealed a non-significant reduction in mitotic index and low chromosomal aberrations of the treated groups. In conclusion, the aqueous-methanol solvent fraction of methanol extract of Dacryodes edulis leaves, AMDE is relatively safe. However, there are strong indications that it may contain compounds that are cytotoxic and reduces erythrocytic indices including red blood cells at high doses. Thus, adequate care should be taken in dosing and administering the extract to avert anaemic condition.
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