Butylated hydroxyl-toluene, 2,4-Di-tert-butylphenol, and phytol of Chlorella sp. protect the PC12 cell line against H2O2-induced neurotoxicity.

Oxidative stress is considered the main cause of cellular damage in a number of neurodegenerative disorders. One suitable ways to prevent cell damage is the use of the exogenous antioxidant capacity of natural products, such as microalgae. In the present study, four microalgae extracts, isolated from the Persian Gulf, were screened to analyze their potential antioxidant activity and free radical scavenging using ABTS, DPPH, and FRAP methods. The methanolic extracts (D1M) of green microalgae derived from Chlorella sp. exhibited potent free radical scavenging activity. In order to characterize microalgae species, microscopic observations and analysis of the expression of 18S rRNA were performed. The antioxidant and neuroprotective effects of D1M on H2O2-induced toxicity in PC12 cells were investigated. The results demonstrated that D1M significantly decreased the release of nitric oxide (NO), formation of intracellular reactive oxygen species (ROS), and the level of malondialdehyde (MDA), whereas it enhanced the content of glutathione (GSH), and activity of heme oxygenase 1 (HO-1), NAD(P)H: quinone oxidoreductase 1 (NQO1), and catalase (CAT) in PC12 cells exposed to H2O2. The pretreatment of D1M improved cell viability as measured by the MTT assay and invert microscopy, reduced cell apoptosis as examined by flow cytometry analysis, increased mitochondrial membrane potential (MMP), and diminished caspase-3 activity. The GC/MS analysis revealed that D1M ingredients have powerful antioxidant and anti-inflammatory compounds, such as butylated hydroxytoluene (BHT), 2,4-di-tert-butyl-phenol (2,4-DTBP), and phytol. These results suggested that Chlorella sp. extracts have strong potential to be applied as neuroprotective agents, for the treatment of neurodegenerative disorders.

Antioxidant activity of rosemary essential oil fractions obtained by molecular distillation and their effect on oxidative stability of sunflower oil.

The objective of this study was to evaluate the antioxidant activity of rosemary essential oil fractions obtained by molecular distillation (MD) and investigate their effect on the oxidative stability of sunflower oil. MD fractions were prepared in a series of low-pressure stages where rosemary essential oil was the first feed. Subsequently, a distillate (D1) and residue (R1) were obtained and the residue fraction from the previous stage used as the feed for the next. The residue fractions had the largest capacity to capture free radicals, and the lowest peroxide values, conjugated dienes and conjugated trienes. The antioxidant activity of the fractions was due to oxygenated monoterpenes, specifically α-terpineol and cis-sabinene hydrate. Oxidative stability results showed the residues (R1 and R4) and butylated hydroxytoluene had greater antioxidant activity than either the distillate fractions or original rosemary essential oil. The residue fractions obtained by short path MD of rosemary essential oil could be used as a natural antioxidants by the food industry.

Investigations towards new antidiabetic drugs from fungal endophytes associated with Salvadora oleoides Decne.

The nature has provided abundant natural resources which can be explored for their medicinal uses. The present study was undertaken to investigate the antidiabetic and hypolipidemic activity of various extract's fractions obtained from mycelia of seventeen endophytic fungi in different solvents (methanol, acetone and aqueous) isolated from Salvadora oleoides Decne (Salvadoraceae) in glucose loaded fasting and alloxan induced diabetic Wistar albino rats. Only four extracts namely; unidentified fungus (aqueous), Aspergillus sp.JPY2 (methanol), Aspergillus sp.JPY1 (methanol) and Phoma sp. (acetone) significantly reduced blood glucose levels as revealed by glucose tolerance test. It has been observed that in alloxan induced diabetic rats, the maximum reduction in blood glucose level was after 5 hours in the acute treatment experiment and on14th day in sub acute treatment at a dose of 250mg/kg of body weight (P<0.05). The reduction in blood glucose in long term treatment experiment was ranged from 11.3% to 28.04%, whereas standard drug tolbutamide reduced the blood glucose level up to 40%. In long term treatment, unidentified fungus (aqueous) extract showed significant improvement in parameters like body weight and lipid profile of alloxan induced diabetic rats. The gas chromatography mass spectrometer (GCMS) analysis of bioactive fraction (aqueous) of unidentified fungus and methanolic extract fraction of Aspergillus sp.JPY1 revealed that the main constituents were 2, 6-di-tert-butyl-p-cresol and Phenol, 2, 6-bis (1, 1-dimethylethyl)-4-methyl respectively. The results have also suggested that the above four bioactive fractions have good margin of safety and did not show any lethal effects on the animals up to the doses of 1000mg/kg b.w. along with safe doses up to 500 µg/ ml to human erythrocytes.

Coenzyme Q10 production by Sphingomonas sp. ZUTE03 with novel precursors isolated from tobacco waste in a two-phase conversion system.

Coenzyme Q10 (CoQ10) is a widely used supplement in heart diseases treatment or antioxidative dietary. The microbial production of CoQ10 was enhanced by addition of solanesol and novel precursors recovered from waste tobacco. The novel precursors were separated by silica gel and identified as alpha-linolenic acid (LNA) and butylated hydroxytoluene (BHT) based on the effect on CoQ10 production and GC-MS. The effects of novel precursors on CoQ10 production by Sphingomonas sp. ZUTE03 were further evaluated in a two-phase conversion system. The precursor's combination of solanesol (70 mg/l) with BHT (30 mg/l) showed the best effect on the improvement of CoQ10 yield. A maximal CoQ10 productivity (9.5 mg l-1 h-1) was achieved after 8 h conversion, with a molar conversion rate of 92.6% and 92.4% on BHT and solanesol, respectively. The novel precursors, BHT and LNA in crude extracts from waste tobacco leaves, might become potential candidates for application in the industrial production of CoQ10 by microbes.

Cloud-point extraction and reversed-phase high-performance liquid chromatography for the determination of synthetic phenolic antioxidants in edible oils.

A cloud-point extraction (CPE) method using Triton X-114 (TX-114) nonionic surfactant was developed for the extraction and preconcentration of propyl gallate (PG), tertiary butyl hydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT) from edible oils. The optimum conditions of CPE were 2.5% (v/v) TX-114, 0.5% (w/v) NaCl and 40 min equilibration time at 50 °C. The surfactant-rich phase was then analyzed by reversed-phase high-performance liquid chromatography with ultraviolet detection at 280 nm, using a gradient mobile phase consisting of methanol and 1.5% (v/v) acetic acid. Under the studied conditions, 4 synthetic phenolic antioxidants (SPAs) were successfully separated within 24 min. The limits of detection (LOD) were 1.9 ng mL(-1) for PG, 11 ng mL(-1) for TBHQ, 2.3 ng mL(-1) for BHA, and 5.9 ng mL(-1) for BHT. Recoveries of the SPAs spiked into edible oil were in the range 81% to 88%. The CPE method was shown to be potentially useful for the preconcentration of the target analytes, with a preconcentration factor of 14. Moreover, the method is simple, has high sensitivity, consumes much less solvent than traditional methods, and is environment-friendly. Practical Application: The method established in this article uses less organic solvent to extract SPAs from edible oils; it is simple, highly sensitive and results in no pollution to the environment.

Dosage of 2,6-bis (1.1-dimethylethyl)-4-methylphenol (BHT) in the plant extract Mesembryanthemum crystallinum.

A naturally occurring BHT was identified in the leaves of the halophyte plant Mesembryanthemum crystallinum. This phenol was extracted in this study by two methods at the different plant growth stages. One of the methods was better for BHT extraction; the concentration of this phenol is plant growth stage dependent. In this study, the floraison stage has the highest BHT concentration. The antioxidant activity of the plant extract was not related to BHT concentration. The higher antioxidant activity is obtained at seedlings stage.

Determination of butylated hydroxytoluene in food samples by high-performance liquid chromatography with ultraviolet detection and gas chromatography/mass spectrometry.

A reversed-phase high-performance liquid chromatography (RP-HPLC) method was developed and compared with a gas chromatography/mass spectrometry (GC/MS) method for determining butylated hydroxytoluene (BHT) in foodstuffs as a result of migration from plastic packaging. Similar extraction procedures were used in both methods. BHT was quantitated using an external standard in the HPLC method and an internal standard in the GC/MS method. Both methods presented good linearity (r(2) > or = 0.9917) and low detection limits. Recoveries obtained with the HPLC method (chicken meat, 95.8%, and Gouda cheese, 83.9%) were better than with the GC/MS method (chicken meat, 85.6%, and Gouda cheese, 71.3%).

Screening of the antioxidative properties and total phenolic contents of three endemic Tanacetum subspecies from Turkish flora.

Methanolic extracts of three different Tanacetum subspecies [Tanacetum densum (Lab.) Schultz Bip. subsp. sivasicum Hub-Mor and Grierson, Tanacetum densum (Lab.) Schultz Bip. subsp. eginense Heywood and Tanacetum densum (Lab.) Schultz Bip. subsp. amani Heywood] which are endemic to Turkish flora were screened for their possible antioxidant activities by two complementary test systems namely DPPH free radical scavenging and beta-carotene/linoleic acid. In DPPH system, the most active plant was T. densum subsp. amani with an IC(50) value of 69.30+/-0.37 microg/ml. On the other hand, T. densum subsp. sivasicum exerted greater antioxidant activity than those of other subspecies in beta-carotene/linoleic acid system (79.10%+/-1.83). Antioxidant activities of BHT, curcumine and ascorbic acid were also determined as positive controls in parallel experiments. Total phenolic constituents of the extracts of Tanacetum subspecies were performed employing the literature methods involving Folin-Ciocalteu reagent and gallic acid as standard. The amount of total phenolics was highest in subsp. sivasicum (162.33+/-3.57 microg/mg), followed by subsp. amani (158.44+/-2.17 microg/mg). Especially, a positive correlation was observed between total phenolic content and antioxidant activity of the extracts.

Simultaneous analysis of antioxidants and preservatives in cosmetics by supercritical fluid extraction combined with liquid chromatography-mass spectrometry.

This study evaluated supercritical fluid extraction (SFE) combined with liquid chromatography-mass spectrometry (LC-MS) to determine trace preservatives and antioxidants including methylparaben (MP), ethylparaben (EP), propylparaben (PP), butylparaben (BP), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), alpha-tocopherol (alpha-t) and alpha-tocopherol acetate (alpha-ta) in cosmetic products. A supercritical fluid extraction procedure was used to isolate four paraben preservatives and four antioxidants from the cosmetic matrix before quantitative analysis. The optimum extraction condition was performed with static extraction for 5 min, then dynamic extraction for 20 min by using carbon dioxide supercritical fluid at 14,000 kPa and 65 degrees C. Methanol was used as collection solvent and the sea sand was chosen as a filling material. The analytes were separated on a C18 reversed-phase column using methanol-water as mobile phase and quantified by measuring its mass spectrometry. The linearity range is from 10 to 20,000 ng/g with RSD values below 18%. Detection limits are achieved at the level of 4.7-142 ng/g. It was successfully applied to the determination of paraben preservatives and antioxidants in cosmetics without tedious pretreatment.

Cyclodextrin-modified microemulsion electrokinetic chromatography for separation of alpha-, gamma-, delta-tocopherol and alpha-tocopherol acetate.

Different forms of tocopherols, together with tocotrienols, are collectively named as vitamin E, and each possesses different degree of medical, biological and physiochemical significance. The main difficulty of separating different forms of tocopherols lay in their highly structural similarities and hydrophobicities. Microemulsion electrokinetic chromatography (MEEKC), claimed to attain high peak efficiency with great solubilization power, has not previously been applied to the separation of tocopherols. The effects that various parameters, such as buffer system, type and concentration of cyclodextrins, temperature, and sample matrix, have on the separation of tocopherols by MEEKC have been investigated. By using a buffer mixture of 4% (w/w) sodium dodecyl sulfate (SDS), 6.6% (w/w) 1-butanol, 0.8% (w/w) n-octane, 20% (w/w) 2-propanol, 68.6% (w/w) phosphate (25mM, pH 2.5), and 25mM heptakis(2,6-di-O-methyl)-beta-cyclodextrin (DM-beta-CD), the separation of alpha-, gamma-, and delta-tocopherol, alpha-tocopherol acetate, as well as the antioxidant butylated hydroxytoluene (BHT) at -26kV, 25 degrees C was completed within 35min. The practical potential of the present approach has been further validated by the determination of tocopherols in a vitamin E preparation, with the result of 132.63 (RSD 1.25%), 176.51 (RSD 0.29%), and 64.32mg (RSD 3.34%) per 500mg capsule for alpha-, gamma- and delta-tocopherol, respectively.

Determination of antioxidants in cosmetics by micellar electrokinetic capillary chromatography with electrochemical detection.

A new and efficient method for the determination of antioxidants [Propyl gallate (PG), tert-butylhydroquinone (TBHQ), butylated hydroxyanisole (BHA), and butylated hydroxytoluene (BHT)] in cosmetics has been established by using micellar electrokinetic capillary chromatography with electrochemical detection (MECC-ED). Under the optimum conditions of the method, all analytes were successfully separated within 13 min at the separation voltage of 18 kV in a 20 mmol/L borate running buffer (pH 7.4) containing 25 mmol/L sodium dodecyl sulfate. The excellent linearity was obtained in the concentration range from 5.0 x 10(-4) to 2.0 x 10(-6) mol/L and the detection limits (S/N = 3) of PG, TBHQ, BHA, and BHT range from 3 x 10(-7) to 3 x 10(-6) mol/L.

Rapid assay of vitamin E in vegetable oils by reversed-phase capillary electrochromatography.

A rapid capillary electrochromatographic (CEC) method for the analysis of vitamin E in vegetable oils is reported. Vitamin E consists of a group of eight isomers, tocopherols (TOHs) and tocotrienols. The separation of four TOHs (alpha-, gamma-, delta-TOH), alpha-tocopherol acetate (alpha-TOH-Ac), and an antioxidant compound, butylated hydroxytoluene (BHT) used to prevent TOH autoxidation, was optimized. The CEC experiments were carried out in a 75 microm inner diameter (ID) fused-silica capillary, partially packed with 3 microm C(18 )stationary phase (33 cm total length, 8.4 cm and 7 cm effective and packed lengths, respectively). The optimum mobile phase was a polar organic phase composed of a mixture of methanol-acetonitrile in the ratio 50/50 v/v containing 0.01% ammonium acetate, applying a voltage and temperature set at -25 kV and 20 degrees C, respectively. The tocopherols and the BHT were successfully separated within 2.5 min using the short-end injection method. Under these experimental conditions, repeatability of retention time and peak area, analyte detection and quantitation limits, linearity, precision, and accuracy were studied. The CEC method was applied to determine the content of TOHs in different commercially available oils of virgin olive, hazelnut, sunflower, and soybean. The extraction of vitamin E isomers from oil samples was achieved using methanol and a methanol-isopropanol mixture.

Ability of surfactant micelles to alter the partitioning of phenolic antioxidants in oil-in-water emulsions.

In oil-in-water emulsions, the physical location of antioxidants can be an important determinant in their activity. Surfactants can potentially influence the physical location of antioxidants in oil-in-water emulsions by causing solubilization of lipid-soluble antioxidants into the aqueous phase. Excess Brij micelles in an oil-in-water emulsion were found to increase the partitioning of phenolics into the continuous phase with polar antioxidants (propyl gallate) partitioning more than nonpolar antioxidants (butylated hydroxyltoluene). Solubilization of propyl gallate was rapid coming to equilibrium in less than 5 min. Increasing surfactant micelle concentrations from 0.3 to 2.8% increased the solubilization of propyl gallate by 2.3-fold. Solubilization of phenolic antioxidants into the aqueous phase by Brij micelles did not alter the oxidative stability of salmon oil-in-water emulsions, suggesting that surfactant micelles influenced oxidation rates by mechanisms other than antioxidant solubilization.

Total phenolics concentration and antioxidant potential of extracts of medicinal plants of Pakistan.

Thirty-seven plant organs, traditionally used as drugs, collected in Pakistan, were extracted with 70% acetone and analyzed for their total phenolics concentration and antioxidant potential. Seven extracts showed more than 85% inhibition of lipid peroxidation in vitro as compared with blank. Butylated hydroxytoluene (BHT) (IC50 = 233.6 microg/l +/- 28.3) was the strongest antioxidant in our test system. The IC50 results indicate that the extracts of Nymphaea lotus L. flowers, Acacia nilotica (Linn.) Delile beans, Terminalia belerica Roxb. fruits, and Terminalia chebula Retz. (fruits, brown) were stronger antioxidants than alpha-tocopherol, while Terminalia chebula Retz. (fruit coat), Terminalia chebula Retz. (fruits, black) and Ricinus communis L. leaves were weaker antioxidant extracts than alpha-tocopherol and BHT. Total phenolics concentration, expressed as gallic acid equivalents, showed close correlation with the antioxidant activity. High performance liquid chromatographic analysis with diode array detection at 280 nm, of the seven extracts indicated the presence of hydroxybenzoic acid derivatives, hydroxycinnamic acid derivatives, flavonol aglycones and their glycosides as main phenolics compounds. This information, based on quick screening methods, enables us to proceed towards more detailed chemical and pharmacological understanding of these plant materials.

Purification of fatty acid methyl esters by high-performance liquid chromatography.

The determination by gas chromatography (GC) of fatty acid methyl esters (FAMEs) prepared from complex biological samples is subject to interference from cholesterol. During sample injection on the GC system of FAMEs prepared from tissues that contain cholesterol, we observed a major contaminant that co-eluted with docosahexaenoic acid (DHA, 22:6n-3). To address this problem, FAMEs were purified on an amino-phase high-performance liquid chromatography (HPLC) column using a hexane-isopropanol gradient. The HPLC retention times for both the FAME fraction and cholesterol were stable and reproducible when the amino column was used for sample purification. The purified extracts were analyzed by GC without artifacts or impurity peaks after 50 analytical runs. The method described here will be useful for measurement of 22:6n-3 and other fatty acids important for studies of nutrition or pathology.

GERI-BP002-A, novel inhibitor of acyl-CoA: cholesterol acyltransferase produced by Aspergillus fumigatus F93.

A new inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), designated GERI-BP002-A, was isolated from the culture broth of Aspergillus fumigatus F93 by acetone extraction, EtOAc extraction, SiO2 column chromatography and reverse phase HPLC. Spectroscopic analyses of the compound identified bis (2-hydroxy-3-tert-butyl-5-methylphenyl) methane as the structure and its molecular weight and formula to be 340 and C23H32O2, respectively. GERI-BP002-A inhibited ACAT activity by 50% at the concentration of 50 microM in an enzyme assay system using rat liver microsomes.

Characterization and identification of substances isolated from dialyzer extracts.

The organic phase of dialyzer dried extracts obtained from Cuprophan hollow fiber and polyacrylonitrile AN 69 parallel-plate dialyzers, all sterilized by ethylene oxide, were submitted to light and polarized light microscopy, infrared (IR) spectrophotometry, and gas chromatography coupled with mass spectrometry. Colorless polygonal (approximately 5 X 20 micron) and needle-like (approximately 3 X 50 micron) crystals were found in great quantity on microscopy examination. IR spectrophotometry of the crystals embedded in potassium bromide disc showed specific spectra in the 1400-800 cm-1 wave number range and typical peaks on wave number assigned for hydroxy or amine (3420 cm-1), aromatic hydrogen (3060 cm-1), methyl, methylenic, or methenyl (2960-2860 cm-1), and carbonyl (1715 cm-1) groups can be demonstrated. Seven peaks were detected on gas chromatography of the organic solution containing crystals. Fatty acids and undefined compounds could originate from the dialyzer. Phtalates may leach from blood tubing, used in the rinsing procedure, and BHT is a stabilizer of ethyl ether used as solvent. These compounds could also originate from the dialyzer since phtalates are widely used as a plasticizer and BHT an antioxidant for various polymers.

Methods of extraction and high-performance liquid chromatographic analysis of butylated hydroxytoluene from the tissues and serum of rats.

Butylated hydroxytoluene (BHT) is a phenolic antioxidant which is widely used in foods and has been shown to inhibit chemical carcinogenesis in the mammary gland induced by 7,12-dimethylbenz(a)anthracene. However, its mechanism of action as a tumor inhibitor is unclear. The purpose of this work was first to develop a method for extracting and quantitating BHT and then to determine the amounts that accumulate in the tissues and serum of rats as a starting point for looking at mechanistic possibilities in the inhibition of mammary carcinogenesis. Methodology of extracting BHT from rat tissues and serum was developed using a modified lipid extraction procedure. The sensitive nature of reverse-phase high-performance liquid chromatography proved useful in detecting and quantifying BHT after its extraction from biological tissues. All tissues were taken from animals consuming semipurified diets with and without 0.3% BHT for various periods of time (weeks). BHT was found in much higher levels in mammary tissue than in the liver and serum of rats. The lipid content in mammary tissue appears to be predictive of the amount of BHT found in this tissue, presumably because of the lipophilic character of the antioxidant.