Quenching mechanism and kinetics of ascorbyl palmitate for the reduction of the gamma irradiation-induced oxidation of oils

The effects of 0, 250, 500, and 1000 ppm (wt/vol) ascorbyl palmitate (AP) on the gamma irradiation-induced oxidation of soybean oil, cottonseed oil, corn oil, tallow, lard, or linoleic acid either in a solvent mixture (benzene/methanol, 4:1 vol/vol) or in methanol, was studied immediately after gamma irradiation with a dose of 1–5 kGy. Steady-state kinetic approximation was used to determine a quenching mechanism and quenching rate constant of AP on the gamma irradiation-induced oxidation of purified soybean oil in a solvent mixture (benzene/methanol, 4:1 vol/vol). Irradiation greatly increased oxidation of all oils, as was expected. AP was extremely effective at minimizing oxidation in all oils, and its effectiveness was concentration dependent. AP showed significantly greater antioxidative activity than α-tocopherol for the reduction of oxidation in all oils (P<0.05). The steady-state kinetic studies indicated that AP quenched oxygen only to minimize the oxidation of oils. The calculated total quenching rate of AP was 7.51×10⁷ M⁻¹s⁻¹. The present results clearly show the effective oxygen quenching ability of AP for the reduction of gamma irradiation-induced oxidation of oils.     

Quenching mechanism and kinetics of ascorbyl palmitate for the reduction of the photosensitized oxidation of oils

Effects of 0, 500, 1000, and 1500 ppm (wt/vol) ascorbyl palmitate (AP) on the methylene-blue- and the chlorophyll-sensitized photooxidations of linoleic acid or soybean oil, either in methanol or in a solvent mixture (benzene/methanol, 4:1, vol/vol), were studied during storage under 3300 lux fluorescent light for 5 h. Steady-state kinetic approximation was used to determine a quenching mechanism and quenching rate constant of AP in the chlorophyll-sensitized photooxidation of methyl linoleate in a solvent mixture (benzene/methanol, 4:1, vol/vol). Both methylene blue and chlorophyll greatly increased the photooxidation of linoleic acid and soybean oil, as was expected. AP was extremely effective at minimizing both methylene-blue-and chlorophyll-sensitized photooxidations of linoleic acid and soybean oil, and its effectiveness was concentration-dependent. The addition of 500, 1000, and 1500 ppm AP resulted in 69.3, 83.6, and 94.6% inhibition of methyleneblue-sensitized photooxidation of linoleic acid, respectively, after 5-h storage under fluorescent light. AP showed significantly greater antiphotooxidative activity than α-tocopherol for the reduction of methylene blue-sensitized photooxidation of linoleic acid (P < 0.05). The steady-state kinetic studies indicated that AP quenched singlet oxygen only to minimize the chlorophyll-sensitized photooxidation of oils. The calculated total quenching rate of AP was 1.0 × 10⁸ M⁻¹s⁻¹. The present results clearly showed, for the first time, the effective singlet oxygen quenching ability of AP for the reduction of photosensitized oxidation of oils.     

Simultaneous extraction of oil and antinutritional compounds from flaxseed

A two-phase solvent extraction process, developed in our laboratory for rapeseed, was used to simultaneously extract oil and toxic, antinutritional components from flaxseed to produce a meal suitable for animal feed. The most effective solvent systems consisted of hexane in combination with a solution of methanol that contained 10% (vol/vol) water and 2.5–5% (w/w) ammonia, or methanol that contained 10% water (vol/vol) and 0.08% (w/w) NaOH. The treatments were carried out both at laboratory and semipilot-plant scales. The success of the test with a pilot-scale Karr liquid-liquid extraction column suggested that this process could be readily carried out on an industrial scale. The resulting flax meal had a high protein content (40–47%) and low levels of cyanogenic glycosides (reduced by 90–100% from the starting material). The methanol-ammonia extraction reduced the total polyphenol content by ≈20%. The oil extraction efficiency of the Karr column was high, resulting in meal residual oil contents of ≈1%.     

Optimization and validation of high-performance liquid chromatographic method for the determination of dowtherm ATM in edible oils and oleochemicals

A method using high-performance liquid chromatography and fluorescence detection is optimized and validated for the determination of Dowtherm A^TM in spiked oleochemicals and edible oils. The samples are directly injected into a reversed-phase C18 column, and Dowtherm A is detected using a fluorescence detector set at 247 nm excitation and 310 nm emission wavelengths. The simple isocratic mobile phase used is a mixture of methanol and water (90∶10, vol/vol) at a flow rate of 1 mL/min. The limits of quantitation are from 0.1 to 0.2 μg/g. Mean recoveries ranged from 93.0 to 116% with reproducibilities of 1.29–3.84%. The procedure provides a simple, reliable and sensitive method for determining Dowtherm A residue in oleochemicals and edible oils without prior sample cleanup or extraction.     

Effect of extraction methods on lipid yield and fatty acid composition of lipid classes containing γ-linolenic acid extracted from fungi

The effect of extraction procedures on the lipid yield and fatty acid composition of total lipid and main lipid structures (phospholipids, diacylglycerols, triacylglycerols, free fatty acids, and sterol esters) of fungal biomass (Mucor mucedo CCF-1384) containing γ-linolenic acid (GLA) was investigated. Seventeen extraction methods, divided into three groups, were tested: six with chloroform/methanol, five with hexane/alcohols, and six with common solvents or mixtures. The chloroform/methanol procedure (2∶1) was selected as standard, where lipid yield (TL/DCW, total lipid per dry cell weight) was 17.8%, considered to be 100% of lipids present. All chloroform/methanol extractions yielded more than 83% recorvey of lipids. Use of hexane/isopropanol solvent systems led to a maximum of 75% recovery. The best lipid yield was achieved by a two-step extraction with ethanol and hexane (120%). Extraction efficiency of the other solvent systems reached a maximum of 73%. Triacylglycerols were the main structures of lipid isolated; only methanol-extracted lipid contained 58.5% phospholipids. The fatty acid content of total recovered lipid was variable and depended on both the lipid class composition and the solvent system. GLA concentrations in total lipids isolated by hexane/alcohol procedures (7.3–10.7%) are comparable with classical chloroform/methanol systems (6.5–10.0%). The maximal GLA yield was obtained with chloroform/methanol/n-butanol/water/0.1 M ethylenediaminetetraacetic acid (EDTA) (2∶1∶1∶1∶0.1, by vol) and after two-step extraction with ethanol and hexane (14.3 and 13.7 g GLA/kg DCW, respectively). The highest GLA content was analyzed in the phospholipid fraction (16.1%) after using chloroform/methanol/n-butanol/water/0.1 M EDTA (2∶1∶1∶1∶0.1, by vol). Remarkably low concentrations of polyunsaturated fatty acids were determined in the free fatty acid fraction.     

Analysis of soybean lecithins and beef phospholipids by HPLC with an evaporative light scattering detector

Linear (r > 0.99) calibration curves were obtained for 10–150 μg of phosphatidylethanolamine (PE), 10–75 μg of phosphaditylinositol (PI), phosphaditylserine (PS) and lysophosphatidylethanolamine, 10–100 μg of phosphatidic acid (PA) and 10–250 μg of phosphatidylcholine (PC) by high-performance liquid chromatography analyses with an evaporative light scattering detector, a Zorbax 7-μm silica column and gradient elution with two solvents. One solvent (A) contained 415 mL isooctane (IOCT), 5 mL tetrahydrofuran (THF), 446 mL isopropanol (IPA), 104 mL CHCl₃ and 30 mL H₂O; and the other solvent (B) contained 216 mL IOCT, 4 mL THF, 546 mL IPA, 154 mL CHCl₃ and 80 mL H₂O. The gradient in which 100% A linearly changed to 100% B in 20 min followed by 12 min of 100% B and then a linear change to 100% A during 5 min separated PE, PS and PC in soybean lecithins and beef lipids, but failed to resolve PI and PA. In these same samples, less polar lipids were separated from phospholipids (PL) by elution from Bond-Elut silica columns with diethyl ether/hexane (20:80, vol/vol), and PL were recovered by elution with methanol. This procedure is useful for concentration of minor lipid components. Levels of PE, PI-PA, PS and PC were higher in granular than in liquid lecithin, and PC was the most abundant PL in soybean lecithins and beef lipids.     

Determination of squalene in olive oil using fractional crystallization for sample preparation

A simple, rapid method for the determination of squalene in virgin olive oil was developed using RP-HPLC with detection at 208 nm. Fractional crystallization from methanol/acetone (7∶3, vol/vol) was applied to obtain squalene in the liquid fraction of the oil prior to HPLC. Elution of squalene was then carried out isocratically with acetone/acetonitrile (40∶60 vol/vol) within 11 min. The detection limit was 23 mg/kg, and the limit of quantification 79 mg/kg. The precision of the crystallization procedure (CV%=3.76, n=7) and the mean recovery (92.5 and 81.5% for the 7,000 and 700 mg/kg levels of addition, respectively) were satisfactory. The method is easily applicable to fulfill future needs for nutrition labeling.     

Fractionation of oligomeric triacylglycerides and the relation to rejection limits for used frying oils

Precipitates enriched in oligomeric triacylglycerides were separated from thermally oxidized olive residue oil, conventional and high-oleic sunflower oils, and soybean oil by solvent fractionation in methanol/acetone at 4–5°C for 16 h. Different fractionation conditions were evaluated in an effort to isolate the oligomeric triacylglycerides (OTG). OTG, formed in frying oils upon heating at low concentations, were not detectable with conventional methods to determine polymeric compounds. The best conditions found from the different assays were the following: (i) weight of oil sample-to-solvent volume ratio of 1∶20; and (ii) solvent system methanol/acetone 10∶90 (vol/vol) for monounsaturated oils and 15∶85 (vol/vol) for polyunsaturated oils. Precipitates, enriched in oligomers, were formed when heated oils and used frying oils contained more than 27% polar compounds, a value which is widely accepted as the upper limit for use of frying oils.     

Antioxidant activity of extracts of defatted seeds of niger (<Emphasis Type="Italic">Guizotia abyssinica</Emphasis>)

Niger (Guizotia abyssinica) seed was ground and then defatted with hexane. The meal remaining after oil extraction was tested as a source of antioxidants. Three solvent systems, A [80∶20 (vol/vol) ethanol/water], B [80∶20 (vol/vol) acetone/water], and C (water) were evaluated as extraction media. Crude extracts were examined for their antioxidant activity in a β-carotene-linoleate and a meat model system. Extract A exhibited superior antioxidant activity, compared to extracts B and C, and its composition was studied further by using column chromatography and HPLC. Four fractions (I–IV) were obtained, of which fractions III and IV showed activity in the β-carotene-linoleate model system. Fraction IV was also highly effective in scavenging the 2,2-diphenyl-1-picrylhydrazyl radical but was less active when used in a bulk oil model system. Preparative TLC showed fraction IV as consisting of two components. UV spectroscopy suggested that the major active component pressent was a chlorogenic acid-related compound. Furthermore, HPLC analysis established that chlorogenic acid was dominant in the free phenolics fraction (2.6 mg/g). Upon hydrolysis, however, a substantial amount of caffeic acid (42.8 mg/g) was released, presumably from esterified and glycosylated chlorogenic acid. Thus, niger extracts derive their antioxidant activity, at least in part, from the chlorogenic acid-related compounds.     

A simple and precise method for the routine determination of platelet-activating factor in blood and urine

A simple and precise method is described for the measurement of platelet-activating factor (PAF) in blood and urine. The method involves the isolation of PAF from blood samples by two successive steps. In the first step, blood proteins are precipitated with ethanol and the “free” PAF, i.e. the PAF which is extractable with ethanol, is recovered. In the second step, “bound” PAF, i.e., PAF not extractable with ethanol, is extracted from the protein precipitate with chloroform/methanol/water. The extraction of PAF from urine samples requires only the ethanol extraction step. “Free” and “bound” PAF are then each fractionated by silicic acid column chromatography, and the methanol/water eluent containing PAF is then further fractionated by high-performance liquid chromatography using an isocratic solvent system of acetonitrile/methanol/water. PAF is then quantitated by measuring its ability to induce platelet aggregation in an aggregometer. Application of the method to blood and urine samples from twenty-three healthy volunteers revealed PAF levels in blood of 140–480 pg/mL (630–254.4 pg “free” PAF/mL and 64–225.6 pg “bound” PAF/mL), and of 1.2–4.0 pg PAF/mL in urine. The method overcomes various technical problems and was shown to be very precise. It should prove useful for monitoring PAF levels in various disease conditions.     

Quantitation by radioimmunoassay of PAF in human saliva

Approximately 75% of the PAF present in saliva is recovered on extraction of whole saliva (0.8 vol) with chloroform/methanol/water (2∶2∶1 v/v/v). PAF levels, determined by our recently developed radioimmunoassay, in saliva extracts ranged from 0.5–21 ng/mL with 59% between 2–6 ng/mL. These figures, for apparently healthy subjects, are higher than previously reported levels obtained by platelet assays. The validity of our radioimmunoassay results was checked by isolating and quantitating the PAF fraction from whole saliva. In addition, when we examined our saliva samples by platelet aggregation, low levels of PAF, comparable with the values found in the literature, were detected. Investigations revealed the presence of a substance(s) which inhibited PAF-induced platelet aggregation but which did not affect the radioimmunoassay.     

杀螟丹高效液相色谱法检测条件

建立了高效液相色谱法检测杀螟丹的分析检测条件。采用甲醇-水（体积比60：40）的混合溶液为流动相,用盐酸（1mol、L）调pH至3。0,流速为0．8mL／min,检测波长为220nm。方法检测线性范围为1．25-80mg／L,决定系数为0．9989,检出限为0．1mg／L,相对标准偏差为0．73％。     

Rapid analysis of oxidized cholesterol derivatives by high-performance liquid chromatography combined with diode-array ultraviolet and evaporative laser light-scattering detection

Extensive evidence of the deleterious biological effects of oxidized 5-cholesten-3β-ol (cholesterol) derivatives has led to great interest in their detection. We observed that known oxidized cholesterol derivatives can be rapidly quantitated by combining reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorption and evaporative laser light-scattering (ELSD) detection. Using a 20 × 0.46 cm C18 HPLC column and methanol/acetonitrile (60:40, vol/vol) as the mobile phase at 1.0 mL/min, 10 species of oxidized cholesterol derivative were measured by UV (205, 234, and 280 nm) while 5-cholestan-5α,6α-epoxy-3β-ol (5α-epoxycholesterol), 5-cholestan-5β,6β-epoxy-3β-ol (5β-epoxycholesterol), and 5-cholestan-3β,5α,6β-triol (cholestanetriol) were detected by only ELSD. The minimal limits of detection ranged from 100 to 500 ng depending on sterol and detector. The response was linear in the range 0–1000 or 0–2000 ng depending on detector. These oxidized cholesterol derivatives were also identified by HPLC/mass spectrometry analysis combined with UV detector. Heated tallow contained cholestanetriol, 5-cholesten-3β,7α-diol (7α-hydroxycholesterol), 5-cholesten-3β,7β-diol (7β-hydroxycholesterol), 5-cholesten-3β-ol-7-one (7-ketocholesterol), 5α- and 5β-epoxycholesterols under the developed analysis condition. Photooxidized cholesterol had cholestanetriol, 7α- and 7β-hydroxycholesterols and 3,5-cholestadien-7-one. On the other hand, 7α- and 7β-hydroxycholesterols, 7-ketocholesterol, 5α- and 5β-epoxycholesterols and 3,5-cholestadien-7-one were observed in copper-oxidized low-density lipoprotein. Thus, this developed HPLC analysis method could be applied to identification of oxidized cholesterol derivatives in food and biological specimen.     

Rapid separation of the major phospholipid classes on a single aminopropyl cartridge

A rapid method for the separation of the individual phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) by a single solid-phase extraction was developed. PC, PE, PS and PI were sequentially eluted from aminopropyl bonded silica with acetonitrile/n-propanol (2∶1, vol/vol), methanol, isopropanol/methanolic HCl (4∶1, vol/vol) and methanol/methanolic HCl (9∶1, vol/vol). Standard recoveries were over 95% for PC and PE and over 85% for PS and PI with undistorted fatty acid composition. The separation of complex lipid mixtures on aminopropyl minicolumns can be refined to the level of individual phospholipid classes.     

Production of an eicosapentaenoic acid-containing oil by a Δ12 desaturase-defective mutant ofMortierella alpina 1S-4

A Δ12 desaturase-defective mutant of an arachidonic acid (AA)-producing fungus,Mortierella alpina 1S-4, converted α-linolenic acid (18:3ω3) to 5(Z),8(Z),11(Z),14(Z),17(Z)-eicosapentaenoic acid (EPA). On submerged cultivation at 20°C for 10 d in a 5-L fermentor containing medium comprising 1% glucose, 1% yeast extract and 3% (vol/vol) linseed oil, EPA production amounted toca. 1 g/L culture broth (64 mg/g dry mycelium), which accounted forca. 20% of the total mycelial fatty acids. AA content was 26 mg/g dry mycelium (0.4 g/L), accounting for 7.8% of the total mycelial fatty acids. The other major mycelial fatty acids were palmitic acid (4.5%), oleic acid (20.4%), linoleic acid (10.0%), 18:3ω3 (20.3%) and lignoceric acid (4.3%). Most of the EPA produced (ca. 90 mol%) was in triglyceride form.     

Possible fatty acyl pheromone precursors in Spodoptera littoralis. Search for 11- and 12-hydroxytetradecanoic acids in the pheromone gland

Lipidic extracts of Spodoptera littoralis pheromone glands submitted to acid methanolysis using: (i) sulfuric acid/methanol/benzene (0.1∶4∶2, by vol) at 90°C for 1 h; (ii) 12 N HCl/methanol (1∶2; vol/vol) at 90°C for 1 h, or (iii) 14% BF₃-MeOH at 90°C for 1 h did not reveal the presence of either 11- or 12-hydroxytetradecanoic acid in the extracts, as concluded from the gas chromatography-mass spectrometry analyses. Under the above methanolysis conditions, a synthetic sample of methyl (14, 14, 15-²H₃) 12-hydroxytetradecanoate remained unaltered. These results may indicate that formation of (E)-11-tetradecenoic acid from tetradecanoic acid does not occur in the pheromone gland by dehydration of an intermediate hydroxyacid. Acid methanolysis of a lipidic extract using BF₃-MeOH led to the formation of a mixture of methoxy fatty acid methyl esters, identified by gas chromatography-mass spectrometry. These methoxy derivatives should arise from BF₃-catalyzed addition of methanol to the double bond of the natural monounsaturated fatty acyl derivatives present in the gland. Thus, under the same conditions, a synthetic sample of methyl (Z)-11-tetradecenoate was partially transformed into methyl 11-methoxytetradecanoate and methyl 12-methoxytetradecanoate. This reaction might be a useful alternative procedure to obtain methoxy derivatives of olefins, which are very helpful for the structural characterization of the parent alkenes.     

An evaluation on PAH degradation and characteristics as media of PVA-derivative hydrogels prepared by using a CGA technique

We manufactured PVA-derived hydrogels wth some crosslinkers by using a foam generation technique. Amino acids gels showed remarkably higher swelling ratios, probably because of the highly crosslinked network along with hydrogen bonds. Boric acid and starch would catalyze dehydration while structuring to result in much lower water content and accordingly high gel content, leading to less elastic, hard gels. Bulky materials such as ascorbic acid or starch produced, in general, large pores, and also nicotinamide, highly hydrophobic, was likely to enlarge its pore size, thus leading to reduced swelling. Hydrophilic (or hydrophobic), functional groups which are involved in the reaction or physical linkage, and bulkiness of crosslinkers were found to be more critical to the crosslinking structure and its density than molecular weights that seemed to be closely related to pore sizes. The average sizes of pores were 20 μm for methionine, 10–15 μm for citric acid, 50–70 μm for L-ascorbic acid, 30–40 μm for nicotinamide, and 70–80 μm for starch. Also, amino acid and glucose gels were more elastic than the others. The elasticity of a gel was reasonably correlated with its water content or swelling ratio. On the other hand, L-ascorbic acid among glucose, methionine, citric acid and vitamins, imparted not only the most favorable physical properties and the greatest cell density but also the highest PAH degradation on its derivative gels. The higher biomass ensured the higher degradation rate. The maximum cell density was 0.267 mg/g-hydrogel and degradation rates and efficiencies ranged 0.013–0.007 mM/mg/day and 92-48%, respectively.     

Swelling behavior of poly(α-hydroxy acrylic acid) gel in water/organic solvent mixtures

Summary Swelling behavior of poly(α -hydroxy acrylic acid) (PHA) gel was investigated in water/organic solvent mixtures (acetone, dioxane, dimethyl sulfoxide: DMSO) in comparison with poly(acrylic acid) (PAA) gel. The swelling degrees of PHA and PAA gels significantly decreased with increasing acetone or dioxane content in the mixed solvents, while the deswelling in aqueous DMSO was less apparent. The deswelling region was dependent on the dissociation state of the carboxyl group; when the carboxyl group in the gels was dissociated (-COO), significant deswelling occurred in 30–60 vol.% of acetone or dioxane for PHA gel and in 40–80 vol.% for PAA gel. When the carboxyl group was in an acid form (-COOH) or only slightly dissociated, the deswelling region was 0–30 vol.% for PHA and 80-100 vol.% for PAA. The unique swelling behavior of PHA gel was ascribed to intermolecular hydrogen bonding. Effects of dielectric constant on the swelling are also discussed. Received: 6 December 1999/Revised version: 17 January 2000/Accepted: 28 January 2000     

Determination of citric acid in fats

The direct analysis of small amounts (2–50 ppm) of citric acid in refined glyceride oils and fats is described. Fat, containing citric acid, is titrated potentiometrically in a pyridine/ acetone mixture with tetrabutylammonium hydroxide (TBAH). The method is relatively simple and rapid with an accuracy better than ±5%.     

Separation and determination of glycolipids from edible plant sources by high-performance liquid chromatography and evaporative light-scattering detection

Glycolipids from edible plant sources were accurately quantified by silica-based, normal-phase high-performance liquid chromatography using an evaporative light-scattering detector. Five major glycolipid classes (acylated steryl glucoside, steryl glucoside, ceramide monohexoside, monogalactosyldiacylglycerol, and digalactosyldiacylglycerol) were separated and determined with a binary gradient system consisting of chloroform and methanol/water (95∶5, vol/vol) without any interference from other lipid classes and pigments. The described method was applied to 48 edible plants available in Japan including cereals, legumes, vegetables, and fruits. Examined plant species contained glycolipids in wide concentration ranges, such as 5–645 mg/100 g tissue.