Isopropanol Extraction Research Articles

One-step intracellular triglycerides extraction and quantitative measurement in vitro

Introduction Measurement of intracellular triglyceride content is traditionally performed using Oil Red O stain and isopropanol extraction. However, the assay is semi-quantitative and its simplicity needs improving. Therefore, a novel assay is described to address these problems. Methods Intracellular triglycerides (TG) were extracted directly by organic solvent Dimethyl Sulfoxide (DMSO) without stain. TG concentration dissolved in DMSO was subsequently measured with a commercial assay kit. Results A series of experiments were conducted to verify the validity of the new protocol such as the extraction efficiency of DMSO, linear range of the kit and sensitivity of the assay. Our results suggest that this novel assay may be applied in the measurement of intracellular triglyceride accumulation induced by pre-adipocyte differentiation or compounds such as peroxisome proliferator-activated receptor γ (PPARγ) agonists. Conclusion The simplicity and efficiency of the method were validated in this study. Our findings show that this novel assay has potential application in the discovery of anti-diabetic compounds or in the research of cell TG metabolism.

P4-280: Marine polyunsaturated fatty acids as BACE inhibitors

The marine environment represents an enormous resource for the discovery of potential chemotherapeutic agents. It is the source of a large group of structurally unique natural products that are mainly accumulated in invertebrates such as sponges, tunicates, bryozoans, and molluscs. Several of these compounds show pronounced pharmacological activities and are interesting candidates for new drugs in several areas of treatment. Within a systematic screening program aimed at isolating compounds from marine organisms showing biological activities relevant for preventing and/or treating neurodegenerative diseases, such as Alzheimer's disease (AD), we have found that isopropanolic extracts from a number of marine organisms, including sea stars and blue crabs, presented interesting biological activities, particularly BACE (β site AβPP cleaving enzyme) inhibition. This enzyme is involved in formation of amyloid β peptide and has become an important target for the development of therapeutic compounds against AD. Isolation and structure elucidation led to find out that some of the compounds which were responsible for the inhibitory activity in the initial isopropanolic extracts were polyunsaturated fatty acids (PUFAs). Particularly, the monodimensional and bidimensional proton and carbon nuclear magnetic resonance studies and mass spectra performed to elucidate the structure of the more frequently isolated compounds correspond to those of known PUFAs, namely linolenic acid, linoleic acid, eicosapentaenoic acid and arachidonic acid. The positive results of the biological activity obtained from the natural extracts were confirmed by the additional testing of some commercially available PUFAs.

Determination of Doxorubicin Levels in Whole Tumor and Tumor Nuclei in Murine Breast Cancer Tumors

Pharmacokinetic studies on liposomal drugs have previously measured total drug levels in tumors, which include non-bioavailable drug. However, drugs must be released from liposomes to have activity. We have developed a method for measuring levels of bioavailable (released) doxorubicin in vivo in tumors that will allow therapeutic activity to be correlated with bioavailable drug levels. Mice orthotopically implanted with mammary carcinoma (4T1) were injected i.v. 10 days after implantation with free doxorubicin or formulations of liposomal doxorubicin with different drug release rates. Tumors were excised at various times after injection, and total tumor doxorubicin levels were determined by acidified isopropanol extraction of whole tumor homogenates. Bioavailable doxorubicin levels were determined by extraction of doxorubicin from isolated tumor nuclei. Free doxorubicin had high levels of bioavailability in tumor tissue; 95% of the total doxorubicin in tumors was bound to nuclear DNA by 24 hours after injection. Administration of Doxil, a slow release liposomal formulation of doxorubicin, gave an area under the time-versus-concentration curve (AUC) for total doxorubicin 7 days after injection that was 87-fold higher than that obtained for free doxorubicin, and 49% of the liposomal doxorubicin was bioavailable. For liposomes with a more rapid doxorubicin release rate, by 7 days after injection, the AUC(0-7 days) for total doxorubicin was only 14-fold higher than that for free doxorubicin and only 27% of liposomal doxorubicin was bioavailable. This technique allows correlations to be made between drug bioavailability and therapeutic activity and will help in the rational design of drug carriers.

The effects of remifemin® on subjective symptoms of menopause

The authors studied the changes in subjective symptoms of menopause in 2016 Hungarian women who had been treated with an isopropanol extract of Cimicifuga racemosa (black cohosh). The inclusion criteria were age (40-65 y). Kupperman index (20), and refusal or contraindication for estrogen therapy. The severity of the symptoms was evaluated at the start of the study and at the end of 4, 8, and 12 weeks of treatment. The average decrease in Kupperman index after 12 weeks of therapy was 17.64 points (P<.001). Based on the weighted symptom scores, the most favorable changes were found in hot flashes (-6.31 points), sweating (-2.86 points), insomnia (-2.27 points), and anxiety (-2.00 points) (P<.001 in each case). The isopropanol extract of C racemosa was found to be effective in the alleviation of menopausal symptoms.

Surface Lipid and Free Fatty Acids (FFA) Content of Head and Broken Rice Produced by Milling After Different Drying Treatments

ABSTRACTThe surface lipids and free fatty acids (FFA) content of head and broken rice samples generated through milling after various drying treatments were studied. Long grain cultivars Francis, Wells, and Cypress, and medium grain cultivar Bengal were dried under three air conditions (mild 25°C, 50% rh; moderate 45°C, 40% rh; and stressed 65°C, 20% rh) for two durations (10 and 30 min). Immediately after drying, the rough rice samples were placed in a conditioning chamber to continue drying slowly to ⋍12.5% moisture content (MC), which occurred within three to five days. After dehulling, a McGill No. 2 mill was used to mill the samples for 30 sec. The head rice yield (HRY) for all rice samples were within the range of 40–68%. Rice surface lipid was extracted with isopropanol (IPA) and the lipid and FFA content of the IPA extracts were determined. Broken rice kernels had significantly greater surface lipid and FFA content than head rice kernels. The surface FFA contents of broken kernels were within the range of 0.045–0.065% of broken rice mass, while that of head rice was 0.027–0.040%. Broken ricehad greater b values indicating greater yellow color than did head rice.

Cimicifuga racemosa Extract Inhibits Proliferation of Estrogen Receptor-positive and Negative Human Breast Carcinoma Cell Lines by Induction of Apoptosis

Hormone replacement therapy is contraindicated in women with breast cancer. Extracts from the rhizomes of Cimicifuga racemosa, have gained acceptance as a natural alternative for the treatment of menopausal symptoms. In the present study we investigated the antiproliferative activity of C. racemosa extracts (isopropanolic and ethanolic) on the estrogen receptor positive MCF-7 and estrogen receptor negative MDA-MB231 breast cancer cells by WST-1 assay. Down regulation of the proliferative activity and cell killing by isopropanolic and ethanolic extracts occurred in a clear dose-dependent response with a 50% growth inhibitory concentration of 54.1 +/- 11.4 and 80.6 +/- 17.7 micro g/ml in MCF-7 cells and of 29.5 +/- 3.0 and 58.6 +/- 12.6 microg/ml in MDA-MB231 cells, respectively. Further, the mode of cell death was identified as apoptosis by microscopic inspection and confirmed by light scatter characteristics and by detection of Annexin V adherence to phosphatidylserine by flow cytometry. In addition, the involvement of activated caspases was supported by the cleavage of cytokeratin 18 detected with M30 antibody. Increases in the level of M30-antigen of about 4-fold and 2-fold over untreated controls were observed in C. racemosa -treated MCF-7 and MDA-MB231 cells. These results indicate that C. racemosa extract exerts no proliferative activity, but kills the estrogen receptor positive MCF-7 as well as estrogen receptor negative MDA-MB231 cells by activation of caspases and induction of apoptosis.

Physicochemical and physiological properties of 5α-cyprinol sulfate, the toxic bile salt of cyprinid fish

5alpha-Cyprinol sulfate was isolated from bile of the Asiatic carp, Cyprinus carpio. 5alpha-Cyprinol sulfate was surface active and formed micelles; its critical micellization concentration (CMC) in 0.15 M Na+ using the maximum bubble pressure device was 1.5 mM; by dye solubilization, its CMC was approximately 4 mM. At concentrations >1 mM, 5alpha-cyprinol sulfate solubilized monooleylglycerol efficiently (2.1 molecules per mol micellar bile salt). When infused intravenously into the anesthetized rat, 5alpha-cyprinol sulfate was hemolytic, cholestatic, and toxic. In the isolated rat liver, it underwent little biotransformation and was poorly transported (Tmax congruent with 0.5 micromol/min/kg) as compared with taurocholate. 5alpha-Cyprinol, its bile alcohol moiety, was oxidized to its corresponding C27 bile acid and to allocholic acid (the latter was then conjugated with taurine); these metabolites were efficiently transported. 5alpha-Cyprinol sulfate inhibited taurocholate uptake in COS-7 cells transfected with rat asbt, the apical bile salt transporter of the ileal enterocyte. 5alpha-Cyprinol had limited aqueous solubility (0.3 mM) and was poorly absorbed from the perfused rat jejunum or ileum. Sampling of carp intestinal content indicated that 5alpha-cyprinol sulfate was present at micellar concentrations, and that it did not undergo hydrolysis during intestinal transit. These studies indicate that 5alpha-cyprinol sulfate is an excellent digestive detergent and suggest that a micellar phase is present during digestion in cyprinid fish.

Processing of spent nickel catalyst for fat recovery

Spent nickel catalyst (SNC) have the potential of insulting the quality of the environment in a number of ways. The disposal of SNC will have a pollution effect. Optimum recovery of fat from SNC , could save the environment and reduce the oil loss. Hexane has been the solvent of choice for oil extraction. Alternative solvents that are considered safer have been evaluated. Hexane, isopropanol, ethanol, and heptane were examined using soxhlet extraction. While hexane was more efficient in oil recovery from SNC, isopropanol proved to be very good, to clarifying separation of oil from waste material and also provide high solvent recovery compared to other solvents. Isopropanol extraction with chill provided separation of miscella into two phases: lower oil–rich and an upper solvent – rich. It saved much energy of vaporization for distilling. An aqueous extraction process with immiscible solvent assisted was tested. Solvent like hexane added to SNC, and water added later with continuous stirring. The mixture was stirred for about 30 minutes, prior to centrifugation. Aqueous process extracted less amount of oil compared to solvent extraction.

Antiestrogenic activities of Cimicifuga racemosa extracts

Despite the wide use of extracts from the rhizome of black cohosh ( Cimicifuga racemosa) for the treatment of menopausal complaints, surprisingly little is known on their potential estrogenic properties, e.g. on estrogen dependent gene transcription. In addition, available informations on the effects on cell proliferation are contradictory. We therefore, tested for estrogenic and antiestrogenic effects of Cimicifuga racemosa extracts on proliferation of MCF-7 cells and on gene expression using ethanolic and iso-propanolic extracts of this medical plant. Estrogenic properties of plant extracts could neither be detected in proliferation assays, nor on gene expression using an estradiol-inducible yeast assay or the estrogen-inducible MVLN cells. In contrast, in all three experimental systems Cimicifuga racemosa antagonized estradiol induced activities. Estradiol induced stimulation of proliferation was inhibited by a dosage >1 μg/ml of extract concentration, gene expression was suppressed by doses of 100–1000 μg/ml of Cimicifuga racemosa extracts. From these results we conclude, that extracts from the rhizome of Cimicifuga racemosa contain compounds with antiestrogenic properties.

Rapid Methods for Milled Rice Surface Total Lipid and Free Fatty Acid Determination

ABSTRACTTotal lipids and free fatty acid (FFA) determination is widely used in the food industry to assess the quality of milled rice. An improved rapid ambient temperature isopropanol (IPA) extraction method to determine milled rice surface lipid was more effective than Soxhlet solvent extraction. The improved method was probably due to better extraction of polar lipids and antioxidants by IPA. A colorimetric method to determine FFA requiring only 30 μL of sample is also described. The new technique provides results similar to those obtained using the slower, conventional acid‐base titration method. The colorimetric FFA method requires a smaller sample size, has greater precision, and is more objective. The new methods are particularly suitable for industrial use in providing rapid results for large numbers of samples.

Effect of adsorption and solvent extraction process on the percentage of carotene extracted from crude palm oil

AbstractPalm carotene was successfully concentrated from crude palm oil (CPO) by a batch adsorption process using a synthetic (polymer) adsorbent followed by solvent extraction. Carotene was concentrated to about 20,000 ppm, or about 33.3 times the original concentration in CPO. Carotene recovery varied from 16 to 74% depending on the process conditions. Adsorption times, isopropanol (IPA) extraction times, temperatures of adsorption and solvent extraction process, effect of agitation during IPA extraction process, and adsorbent lifespan were evaluated to determine their effects on the percentage of carotene extracted and carotene concentration. The minimum adsorption time required was 0.5 h. However, an adsorption time of 1.5 h gave a significantly higher carotene concentration than adsorption times of 0.5, 1.0, and 0.2 h. The IPA extraction time was determined based on the final carotene concentration desired. The suitable temperature for adsorption and solvent extraction process was 40°C. There was no significant difference in the percentage of carotene extracted and carotene concentration between the IPA extraction process with and without agitation.

Processing of spent nickelcatalyst for fat recovery

Spent nickel catalyst (SNC) has the potential of insulting the quality of the environment in a number of ways. Its disposal has a pollution effect. Optimum recovery of fat from SNC, could save the environment and reduce the oil loss. Hexane has been the solvent of choice for oil extraction. Alternative solvents considered to have been safer have been evaluated. Hexane, isopropanol, ethanol and heptane were examined using soxhlet extraction. While hexane is more efficient in oil recovery from SNC, isopropanol proved to be very good in clear separation of oil from waste material and also provides high solvent recovery compared to other solvents. Isopropanol extraction with chill separation of miscella into lower oil-rich phase, and an upper, solvent-rich recyclable phase save mush energy of vaporization for distilling. An aqueous extraction process with immiscible solvent assisted was tested. Solvent like hexane added to SNC, and water added later with continuous stirring. The mixture was stirred for about 30 minutes, prior to centrifugation. Aqueous process extracted less amount of oil compared to solvent extraction.

Chemical Composition And Biological Activity Of Extracts From Arrabidaea Bilabiata

Consumption of Arrabidaea bilabiata fresh leaves produces a paraplegic syndrome in cattle. For isolation of the active principle, isopropanol and methanol extracts were prepared from the aerial parts of the plant; these were administered to Sprague-Dawley rats at doses of 0.5, 1, and 2 mg/kg, orally, to determine the presence of substances affecting spontaneous motor activity. The isopropanol extract produced significant increases of long-pause and short-pause movements (112 and 54%, respectively). The methanol extract tended to increase motor activity, but the effect did not reach statistical significance. Chemical and spectroscopic analysis of the isopropanol extract showed allantoin was the major constituent. It was administered to rats, at doses of 1.2, 12, and 120 mg/kg. A complex dose–response curve was observed, but at the highest dose, there was a 52% increase in the number of long-pause movements. It was inferred that allantoin and the constituents of the alcholic extracts might not be responsible for the paraplegic synndrome in cattle. The alteration of cattle motor activity could be caused by other non-alcoholic constituents. Additionally, extracts were assayed for in vitro activity against Escherichia coli , Klebsiella pneumonae , Pseudomonas aeruginosa , Staphylococcus aureus , Bacillus sp. and Candida albicans . All microbial strains were found to be resistant to the extracts with the exception of C. albicans , suggesting a possible antifungal activity.

Chemical Composition And Biological Activity Of Extracts From Arrabidaea Bilabiata

Consumption of Arrabidaea bilabiata fresh leaves produces a paraplegic syndrome in cattle. For isolation of the active principle, isopropanol and methanol extracts were prepared from the aerial parts of the plant; these were administered to Sprague-Dawley rats at doses of 0.5, 1, and 2 mg/kg, orally, to determine the presence of substances affecting spontaneous motor activity. The isopropanol extract produced significant increases of long-pause and short-pause movements (112 and 54%, respectively). The methanol extract tended to increase motor activity, but the effect did not reach statistical significance. Chemical and spectroscopic analysis of the isopropanol extract showed allantoin was the major constituent. It was administered to rats, at doses of 1.2, 12, and 120 mg/kg. A complex dose–response curve was observed, but at the highest dose, there was a 52% increase in the number of long-pause movements. It was inferred that allantoin and the constituents of the alcholic extracts might not be responsible for the paraplegic synndrome in cattle. The alteration of cattle motor activity could be caused by other non-alcoholic constituents. Additionally, extracts were assayed for in vitro activity against Escherichia coli , Klebsiella pneumonae , Pseudomonas aeruginosa , Staphylococcus aureus , Bacillus sp. and Candida albicans . All microbial strains were found to be resistant to the extracts with the exception of C. albicans , suggesting a possible antifungal activity.

Determination of Ethosuximide in Plasma By Derivatization and High Performance Liquid Chromatography

A simple and sensitive liquid chromatographic method is described for the determination of ethosuximide in plasma as a highly sensitive derivative. Ethosuximide in plasma, after separation with isopropanol extraction, was derivatized with strong chromophore reagent, 4-bromomethyl-7-methoxy-coumarin. The resulting derivatives were separated on a Nova-Pak C18 column with water-acetonitrile-methanol(60:20:20, v/v) as the mobile phase. The linear range for the determination of ethosuximide in spiked plasma was over 2-40 nmol. The limit of detection for ethosuximide was about 7.0 ± 1.2 pmol per 20 μL injection (S/N= 5). The intraday relative standard deviation (n = 6) and the interday relative standard deviation (n = 12) were all less than 2.5% for ethosuximide. The recovery for ethosuximide in plasma was greater than 96%.

Comparison of isopropanol and hexane for extraction of vitamin E and oryzanols from stabilized rice bran

AbstractThe effects of solvent‐to‐bran ratio (2∶1 and 3∶1, w/w), extraction temperature (40 and 60°C), and time (5, 10, 15, 20, and 30 min) were studied for hexane and isopropanol extraction. Increasing the solvent‐to‐bran ratios and extraction temperature increased the amounts of crude oil, vitamin E and oryzanol recovered for both solvents. An extraction time of 15 min was sufficient for optimum crude oil, vitamin E, and oryzanol extraction. Preheated isopropanol (3∶1 solvent/bran ratio and 60°C) extracted less crude oil (P&lt;.05) but more vitamin E (P&lt;.05) and similar amounts of oryzanol (P&gt;.05) relative to preheated hexane. The data suggest that isopropanol is a promising alternative solvent to hexane for extraction of oil from stabilized rice bran.

Characterization of a chemical anoxia model in cerebellar granule neurons using sodium azide: protection by nifedipine and MK-801.

Induction of chemical anoxia, using sodium azide in cerebellar granule cells maintained in primary culture, was evaluated as an in vitro assay for screening of potential neuroprotective compounds. The purpose of this study was to evaluate sodium azide as an alternative to cyanide salts, compounds which, despite their unfavorable characteristics, are often used in assays for chemical anoxia. The viability of neuronal cultures after treatment with azide, with or without preincubation with calcium channel blockers, tetrodotoxin (TTX), or glutamate receptor antagonists, was monitored by subsequent incubation with the tetrazolium dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), followed by isopropanol extraction and spectrophotometric quantification of cellularly reduced MTT. The azide-induced degeneration of neurons was shown to be dependent on the concentration as well as on the duration of incubation with submaximal concentrations of azide. Incubation of the neurons with nifedipine, a blocker of L-type voltage-sensitive calcium channels (L-VSCC), or with the noncompetitive N-methyl-D-aspartate (NMDA) subtype glutamate receptor antagonist MK-801, prior to addition of submaximal concentrations of azide, significantly attenuated azide-induced neuronal death. Blockers of N-type and Q-type VSCC (omega-conotoxin MVIIA and MVIIC, respectively) and the P-type VSCC blocker omega-agatoxin IVA had no effect in this assay. The sodium channel blocker TTX was without effect when added to neurons under depolarizing conditions, but potently and effectively protected cells when experiments were performed in a nondepolarizing buffer. The results show that chemical anoxia induced by incubation of cultured neurons with azide leads to detrimental effects, which may be quantitatively monitored by the capability of the cells to reduce MTT. This procedure is a suitable method for screening of compounds for possible protective effects against neuronal death induced by energy depletion. In addition, the results suggest involvement of L-type VSCC as well as of glutamate receptors in the pathways leading to neuronal degradation induced by energy depletion in cerebellar granule neurons. This would further support the notion that these pathways might be important in neurodegeneration induced by cerebral ischemia or anoxia.

Measurement of the ratio between the reduced and oxidized forms of coenzyme Q10 in human plasma as a possible marker of oxidative stress.

It has been postulated that lipid peroxidation plays a crucial role in the pathogenesis of atherosclerosis. As CoQ10H2 (reduced form of coenzyme Q10) is easily oxidized to CoQ10 (oxidized form of coenzyme Q10), it has been proposed that the CoQ10H2/CoQ10 ratio may be used as a possible marker of in vivo oxidative stress. However, sample preparation has an important effect on the redox status of coenzyme Q10 due to the extreme sensitivity of CoQ10H2 towards oxidation. We now report a rapid, simple isocratic HPLC procedure for the determination of CoQ10H2 and CoQ10 in plasma isopropanol extracts, and we used this method to investigate conditions by which the CoQ10H2/CoQ10 ratio can be reliably measured. Our results indicate that CoQ10H2 is unstable in whole blood, plasma, and isopropanol extracts; subsequently the CoQ10H2/CoQ10 ratio changes considerably soon after a blood sample has been obtained. The time period since blood sampling and HPLC analysis, as well as the sample pretreatment procedure, are two factors that have a profound effect on the pre-analytical variation in the determination of the CoQ10H2/CoQ10 ratio. If these two factors are properly controlled, the CoQ10H2/CoQ10 ratio may be a sensitive and practical way to measure in vivo oxidative stress. Furthermore, this indicator is independent from plasma total cholesterol concentrations, implying that groups who differ with respect to cholesterol levels may be compared directly.

Foaming properties of modified faba bean protein isolates

The foaming properties of native and modified faba bean ( Vicia faba) protein isolates were investigated. Foam stability was studied using both a micro-conductimetric/sparging and a whipping method. Samples were defatted by either supercritical carbon dioxide or isopropanol extraction, both of which produced an increase in the foam stability. Increases in foam stability were also observed for acetylated and mechanolyzed (ball milled) samples. The latter produced the more marked effect. Samples with differing degrees of mechanolyzation were examined, the most highly mechanolyzed samples displaying the greatest foam stability. Studies using circular dichroism spectroscopy showed a correlation between the observed change in functional properties and the secondary structure of the faba bean protein. The robustness of foams formed from several of the treated samples was investigated by assessment of the stability of the foams against destabilization by competitive adsorption of the lipid analogue lysophosphatidylcholine. Again the mechanolyzed sample possessed the best foaming properties in the presence of this competitive surfactant.

A rapid colorimetric assay for determination of leukocyte-mediated inhibition of mycelial growth of Candida albicans.

A staining method with crystal violet (CV) was demonstrated to be useful for a simple, quick and objective assessment of in vitro growth inhibitory activity of leukocytes against Candida albicans cells. Candida cells incubated with murine neutrophils or macrophages for 14 hr in microwells were stained with CV and, after washing with 0.25% sodium dodecyl sulfate (SDS), treated with isopropanol containing HCl (0.04 N) to extract Candida cell-bound CV. Then the absorbance at 590 nm of the isopropanol extract was photometrically measured. The results showed that the photometrical absorbance was proportional to the amount of 3H-glucose taken up by C. albicans cells, which reflected the number of viable Candida cells.