Lipoxygenase inhibitory activity of anacardic acids

6[8'(Z)-pentadecenyl]salicylic acid, otherwise known as anacardic acid (C15:1), inhibited the linoleic acid peroxidation catalyzed by soybean lipoxygenase-1 (EC 1.13.11.12, type 1) with an IC50 of 6.8 microM. The inhibition of the enzyme by anacardic acid (C15:1) is a slow and reversible reaction without residual activity. The inhibition kinetics analyzed by Dixon plots indicates that anacardic acid (C15:1) is a competitive inhibitor and the inhibition constant, KI, was obtained as 2.8 microM. Although anacardic acid (C15:1) inhibited the linoleic acid peroxidation without being oxidized, 6[8'(Z),11'(Z)-pentadecadienyl]salicylic acid, otherwise known as anacardic acid (C15:2), was dioxygenated at low concentrations as a substrate. In addition, anacardic acid (C15:2) was also found to exhibit time-dependent inhibition of lipoxygenase-1. The alk(en)yl side chain of anacardic acids is essential to elicit the inhibitory activity. However, the hydrophobic interaction alone is not enough because cardanol (C15:1), which possesses the same side chain as anacardic acid (C15:1), acted neither as a substrate nor as an inhibitor.     

Oxidation of resveratrol catalyzed by soybean lipoxygenase

In this work the oxidative degradation of resveratrol catalyzed by lipoxygenase-1 (LOX-1) has been studied. The process has been characterized by spectroscopic and polarographic measurements. The oxidation of resveratrol was dependent on the concentration of resveratrol and the enzyme. When resveratrol was incubated in the presence of lipoxygenase at pH 9.0, the reaction displayed a k(M) value of 18.6 x 10(-)(6) M and a catalytic efficiency (k(cat)/k(M)) of 4.3 x 10(4) s(-)(1) M(-)(1). These values are close to those shown by the enzyme when linoleic acid is used as the substrate. The effect of lipoxygenase inhibitors on the lipoxygenase-catalyzed resveratrol oxidation was also evaluated. The rate of resveratrol oxidation was markedly decreased by the presence of NDGA in the incubation mixture. From HPLC measurements, it can be deduced that resveratrol is oxidatively decomposed to a complex mixture of products similar to those obtained when the molecule is oxidized by hydrogen peroxide.     

Enrichment of conjugated linoleic acid in oats (Avena sativa L.) by microbial isomerization

A method for microbial isomerization of oat linoleic acid to conjugated linoleic acid (CLA) was developed. The method includes hydrolysis of oat lipids in aqueous flour slurries by the endogenous oat lipase. Then, the flour slurry containing free linoleic acid is utilized as a substrate for the isomerization reaction carried out by resting cells of Propionibacterium freudenreichii ssp. shermanii. The isomerization reaction progressed most effectively when, after the lipid hydrolysis period, the pH of the slightly acidic oat slurry was elevated to 8.0-8.5 and maintained at this range. With slurries containing 5% (w/v) oat flour, the amounts of CLA formed per dry matter were up to 10.1 mg/g corresponding to 102 mg/g lipids or 0.44 mg/mL slurry. Increments in the flour content up to 15% increased the volumetric production of CLA to 0.85 mg/mL. The proportion of the cis-9,trans-11 isomer was 80% of the total CLA formed. CLA could be concentrated into the solid material of the oat slurry by acidification.     

Effect of calcium on the oxidation of linoleic acid by potato (Solanum tuberosum var. Desiree) tuber 5-lipoxygenase

When the effect of calcium on the oxidation of linoleic acid by potato tuber 5-lipoxygenase (LOX) was investigated, it was seen to promote the enzyme's activity at pH values higher than the optimum pH of 6.3, resulting in an enzyme activation at alkaline pH. Kinetic analysis of calcium activation at different pH values revealed that the cation abolished the inhibition by high substrate concentration, which occurs in the absence of Ca(2+), thus leading to activation at high substrate concentration. Studies were conducted to investigate the influence of Ca(2+) on the physicochemical nature of the substrate and its effect on the LOX activity expression. It was concluded that the aggregation mode rather than the aggregation state of linoleic acid is responsible for potato 5-LOX changes.     

Microbially safe utilization of non-inactivated oats (Avena sativa L.) for production of conjugated linoleic acid

A microbially safe process for the enrichment of conjugated linoleic acid (CLA) in oats was developed. The process consists of hydrolysis of oat lipids by non-inactivated oat flour, followed by propionibacterium-catalyzed isomerization of the resulting free linoleic acid to CLA. The first stage was performed at water activity (a(w)) 0.7, where hydrolysis of triacylglycerols progressed efficiently without growth of the indigenous microflora of flour. Thereafter, the flour was incubated as a 5% (w/v) aqueous, sterilized slurry with Propionibacterium freudenreichii ssp. shermanii. The amount of CLA produced in 20 h was 11.5 mg/g dry matter corresponding to 116 mg/g lipids or 0.57 mg/mL slurry. The oat flour had also the capability to hydrolyze exogenous oils at a(w) 0.7. Sunflower oil, added to increase linoleic acid content in triacylglycerols 2.7-fold, was hydrolyzed rapidly. Isomerization of this oil-supplemented flour as a 5% slurry gave final CLA content of 22.3 mg/g dry matter after 50 h of fermentation, corresponding to 118 mg/g lipids or 1.14 mg/mL slurry. Storage stability of CLA in fermented oat slurries at 4 degrees C was good.     

Antioxidative capacity of extracts and constituents in Cornus capitata adventitious roots

Radical scavenging activities of extracts and constituents in Cornus capitata adventitious root cultures were evaluated by using 1,1-diphenyl-2-pycrylhydrazyl (DPPH) and superoxide anion radicals. Inhibitory activity against peroxidation of linoleic acid was assayed by using the thiobarbituric acid (TBA) method. Ethyl acetate and aqueous fractions were prepared from adventitious roots cultured in Murashige-Skoog liquid medium with 0.1 microM Cu(2+) (0.1CuMS) or 10 microM Cu(2+) (10CuMS). The highest scavenging activities on DPPH and superoxide anion radicals were observed in the ethyl acetate fraction from 0.1CuMS. In the inhibitory activity against linoleic acid oxidation, the ethyl acetate fraction from 10CuMS was highest among the fractions tested. The ethyl acetate fraction of adventitious roots cultured in 0.1CuMS contained mainly galloylglucoses (1,2,3,6-tetragalloylglucose and 1,2,3,4,6-pentagalloylglucose). The ethyl acetate fraction of adventitious roots cultured in 10CuMS contained mainly ellagic acid derivatives [3,3'-di-O-methylellagic acid 4-(5"-acetyl)-alpha-L-arabinofuranoside and stenophyllin H1]. Aqueous fractions prepared from both media contained iridoid glycosides (dihydrocornin and cornin). Tetra- and pentagalloylglucoses showed strong inhibitory activities (61.9 and 85.2%, respectively) against linoleic acid oxidation relative to those of butylated hydroxytoluene (BHT) (91.1%) or alpha-tocopherol (49.5%) at 50 microM concentration. Although both ellagic acid derivatives had weak activities (<50%) on DPPH and superoxide anion radical scavenging, 3,3'-di-O-methylellagic acid 4-(5"-acetyl)-alpha-L-arabinofuranoside was stronger (74.7%) than alpha-tocopherol (49.5%) in inhibiting linoleic acid oxidation at 50 microM concentration. Iridoid glycosides exhibited little activity against DPPH and superoxide anion radicals or against oxidation of linoleic acid.     

Caseins and casein hydrolysates. 2. Antioxidative properties and relevance to lipoxygenase inhibition

The antioxidant activity of caseins and casein-derived peptides was evaluated by using three free radical producing reactions-the lipoxygenase- and AAPH-catalyzed oxidation of linoleic acid and the hemoglobin-catalyzed oxidation of linoleic acid hydroperoxide. Caseins and casein-derived peptides were able to inhibit enzymatic and nonenzymatic lipid peroxidation, suggesting they were preferred targets for the free radical intermediates. The antioxidative feature was not lost with the dephosphorylation or the proteolysis of the proteins. The fractionation of the tryptic beta-casein digest yielded peptides with antioxidant activity. A structure-function relationship between the amino acid sequence and the antioxidant capacity and effectiveness is proposed. In addition, indirect evidence suggested that the trapping of free radicals by the proteins/peptides was accompanied by the oxidation of proteins/peptides, according to a sequence-specific mechanism.     

Characterization of betacyanin oxidation catalyzed by a peroxidase from Beta vulgaris L. roots

A protein fraction with peroxidase (EC 1.11.1.7) activity against guaiacol from Beta vulgaris L. roots oxidized both betanidin and betanin (betanidin 5-O-beta-D-glucoside), the former being the more efficient substrate for the enzyme. The protein fraction contained three strongly basic perxidase isoenzymes. Betanidin quinone was formed as the only product in the course of enzymatic betanidin oxidation, whereas betalamic acid and several oxidized cyclo-DOPA 5-O-beta-D-glucoside polymers were generated during the oxidation of betanin. In accordance with the catalytic properties of peroxidase, a possible mechanism for betanidin oxidation is proposed. This mechanism includes the formation of a betanidin radical, which, by further dismutation, yields betanidin quinone and betanidin. The betanidin oxidation rate showed a Michaelis-type dependence on the substrate concentration. The apparent K(M) for the reaction was 0.46 mM. On the basis of the spectral properties of the enzyme responsible for both betanidin and betanin oxidations, its peroxidase nature is suggested.     

Slow-binding inhibition of soybean lipoxygenase-1 by dodecyl gallate

Dodecyl gallate inhibited the soybean lipoxygenase-1 (EC 1.13.11.12, type-1) catalyzed peroxidation of linoleic acid with an IC50 of 0.007 microM without being oxidized. The progress curves for enzyme reactions were recorded by both spectrophotometric and polarographic methods, and the inhibition kinetics revealed competitive and slow-binding inhibition. Both the initial velocity and steady-state rate in the progress curve decreased with increasing dodecyl gallate. The kinetic parameters that described the inhibition by dodecyl gallate were evaluated by nonlinear regression fits.     

Avenanthramides in oats (Avena sativa L.) and structure-antioxidant activity relationships

Eight avenanthramides, amides of anthranilic acid (1) and 5-hydroxyanthranilic acid (2), respectively, and the four cinnamic acids p-coumaric (p), caffeic (c), ferulic (f), and sinapic (s) acid, were synthesized for identification in oat extracts and for structure-antioxidant activity studies. Three compounds (2p, 2c, and 2f) were found in oat extracts. As assessed by the reactivity toward 1,1-diphenyl-2-picrylhydrazyl (DPPH), all avenanthramides except 1p showed activity. Initially, the antioxidant activity of the avenanthramides decreased in a similar order as for the corresponding cinnamic acids, that is: sinapic > caffeic > ferulic > p-coumaric acid. The avenanthramides derived from 2 were usually slightly more active than those derived from 1. All avenanthramides inhibited azo-initiated peroxidation of linoleic acid. 1c and 1s were initially the most effective compounds. The relative order of antioxidant activities was slightly different for the DPPH and the linoleic acid assays run in methanol and chlorobenzene, respectively.     

Dual positional and stereospecificity of lipoxygenase isoenzymes from germinating barley (green malt): biotransformation of free and esterified linoleic acid

The lipoxygenase isoenzymes LOX1 and LOX2 from green malt were separated by isoelectric focusing, and their catalytic properties regarding complex lipids as substrates were characterized. The regio- and stereoisomers of hydroperoxy octadecadienoates (HPODE) resulting from LOX1 and LOX2 enzymatic transformations of linoleic acid, methyl linoleate, linoleic acid glycerol esters monolinolein, dilinolein, and trilinolein, and 1-palmitoyl-2-linoleoyl-glycero-3-phosphocholine (PamLinGroPCho) were determined. In addition, biotransformations of polar and nonpolar lipids extracted from malt were performed with LOX1 and LOX2. The results show that LOX2 catalyzes the oxidation of esterified fatty acids at a higher rate and is more regioselective than LOX1. The dual position specificity of LOX2 (9-HPODE:13-HPODE) with trilinolein as the substrate (6:94) was higher than the resultant ratio (13:87) when free linoleic acid was transformed. A high (S)-enantiomeric excess of 13-HPODE was analyzed with all esterified substrates confirming the formation of 13-HPODE through the LOX2 enzyme; however, 9-HPODE detected after LOX2 biotransformations showed (R)-enantiomeric excesses. PamLinGroPCho was oxygenated by LOX1 with the highest regio- and stereoselectivities among the applied substrates.     

Quality of Fats in Cookies as Affected by Storage and Addition of Oat Flakes

Changes in fats extracted from cookies stored for three or five weeks and containing different levels of oat flakes (10, 20, 30%) were investigated. The fats used for baking differed in fatty acid (FA) composition and contained 17.6–57.6% saturated (SFA), 0.8–46.8% trans FA (TFA), and 0.6–6.6% polyunsaturated FA (PUFA). Oat flakes were steam stabilized and were in the middle of their shelf‐life. Oat flakes contained 9.5% lipids in which linoleic acid (C 18:2 9c 12c) predominated, amounting to 42.7%. Increasing the content of oat flakes and the storage time of cookies resulted in increased values of primary and secondary fat oxidation products. Highest increases in PV values were found in cookies stored for three or five weeks, especially with a high content of oat flakes (30%). Secondary lipid oxidation products measured by AnV values increased with the time of storage. Highest increases in hydrolytic and oxidation products were associated with higher content (6.6%) of PUFA in utilized fats. Regarding the degree of oxidation, it appears safe to add as much as 30% of oat flakes to cookies when the storage time does not exceed three weeks.     

Improvement of oxidative stability of conjugated linoleic acid (CLA) by microencapsulation in cyclodextrins

Oxidative stability of conjugated linoleic acid (CLA) encapsulated in alpha-, beta-, and gamma-cyclodextrins (designated CLA/CDs microencapsules) was studied by measuring the headspace-oxygen depletion in airtight serum bottles and by measuring the peroxide values (POV). The rate of oxygen depletion was reduced from 41.0 (control) to 21.5, 2.1, 1.2, and 1.1 micromol/L.h(-)(1) by CLA/alpha-CD microencapsules at 1:1, 1:2, 1:4, and 1:6 mole ratios, respectively, indicating that CLA oxidation was completely protected by a 1:4 mole ratio of CLA/alpha-CD. Such a protective effect by CLA/beta-CD or CLA/gamma-CD microencapsules was achieved at a 1:6 mole ratio, but the effect by CLA/beta-CD was slightly greater than that by CLA/gamma-CD. The protective effect of alpha-, beta-, and gamma-CDs for CLA oxidation was confirmed by their POV-reducing abilities in CLA/CDs. These results suggest that alpha-CD was the most effective for the protection of CLA oxidation by microencapsulation, followed by beta-CD and gamma-CD.     

缺刻缘绿藻ω3脂肪酸去饱和酶基因(ω3FAD)在酿酒酵母中的低温诱导表达

ω3 脂肪酸去饱和酶(fatty acid desaturase, FAD)能使藻类细胞产生一系列具有高附加值的ω3 脂肪酸。在已克隆到缺刻缘绿藻(Myrmecia incisa Reisigl)的ω3FAD 基因基础上,为进一步了解其功能,本研究首先利用反转录 PCR(RT-PCR)技术,克隆其开放阅读框(open reading frame, ORF)片段,然后亚克隆到穿梭表达载体 pYES2 中,以构建重组酵母表达载体 pY-ω3FAD;通过电穿孔法将该重组载体转入酿酒酵母(Saccharomyces cerevisiae)INVSc1 菌株中,经筛选与序列验证得到含有 pY-ω3FAD 重组质粒的酵母转化株。在 5℃,添加底物亚麻酸及半乳糖诱导表达,连续培养 72 h,经脂肪酸甲酯的气相色谱分析及气相色谱 - 质谱联用证明,转目的基因的酵母能将外源添加的亚油酸在 15 位脱氢生成α- 亚麻酸,表明ω3FAD具有Δ15 脂肪酸去饱和作用的功能。在不同温度条件下诱导培养时,气相色谱结果显示,在 30℃培养的转基因酵母中没有检测到α- 亚麻酸;但在不高于 25℃培养的转基因酵母中,发现ω3FAD 能将外源添加的底物去饱和为α- 亚麻酸,且随着温度的降低,其去饱和能力增强,5℃时的底物转化效率达到29.73%。将转目的基因酵母在 5℃温度下诱导培养不同时间,结果显示,随着培养时间的增加,LA(linoleic acid,亚油酸)转化为α- 亚麻酸的效率也提高,培养 4 d 时其转化效率达到 38.86%。该研究结果提示,缺刻缘绿藻ω3FAD 基因编码的酶蛋白为一个低温诱导酶。缺刻缘绿藻ω3FAD 基因之所以能在酵母细胞中被低温诱导表达,可能因为后者存在一个低温诱导的脂肪酸去饱和系统。     

Synthesis of conjugated linoleic acid by human-derived Bifidobacterium breve LMC 017: utilization as a functional starter culture for milk fermentation

This study was performed to discover bifidobacteria isolated from human intestines that optimally convert linoleic acid (LA) to conjugated linoleic acid (CLA) and to optimize the culture conditions of milk fermentation. One hundred and fifty neonatal bifidobacteria were screened for CLA-producing ability, and Bifidobacterium breve LMC 017 was selected as it showed about 90% conversion of free LA in MRS broth. The selected strain showed resistance at 0.5% LA in microaerophillic conditions. When monolinolein (LA 90%) was used as a substrate for CLA production, the conversion rate was lower compared to free LA, but the growth rate was unaffected during the milk fermentation. There was no significant difference in CLA production between aerobic and anaerobic conditions, and little decline in CLA was shown after the maximal CLA level had been reached. CLA production increased by 80% with 24 h of incubation in milk containing additional skim milk (5%), where the proteins may have facilitated the production of CLA by enhancing the interaction of substrate with the bacteria. CLA production did not decline after 9 h of fermentation and an additional 12 weeks of storage with other commercial starters. This demonstrates the possibility of using this strain as a costarter in the production of CLA-enriched yogurt.     

Honey as a protective agent against lipid oxidation in ground turkey.

Lipid oxidation is a major deteriorative factor in meats. Sources of natural antioxidants that are as effective as commercially available antioxidants are desired. The objective of this research was to investigate honey as an inhibitor of lipid oxidation in ground poultry. The antioxidant content of different varieties of honey was investigated spectrophotometrically and honey's effectiveness in reducing oxidation of ground poultry determined by monitoring thiobarbituric acid reactive substances (TBARS). Buckwheat honey had the highest antioxidant content and acacia honey the lowest. Honeys of different floral sources differed in their protection against lipid oxidation. Buckwheat honey (5%, w/w) reduced TBARS approximately 70%, whereas acacia honey reduced TBARS approximately 34% at 3 days of storage at 4 degrees C. In comparison to butylated hydroxytoluene and tocopherol (0.02% of total fat), honey (at 5% of the weight of the meat) was much more effective at preventing oxidation. Honey has great potential as an antioxidant source and may result in greater acceptability of meat products and prevent negative health implications of oxidized meats.     

Chemical composition, iron bioavailability, and antioxidant activity of Kappaphycus alvarezzi (Doty)

Kappaphycus alvarezzi, an edible seaweed from the west coast of India, was analyzed for its chemical composition. It was found that K. alvarezzi is rich in protein (16.24% w/w) and contains a high amount of fiber (29.40% w/w) and carbohydrates (27.4% w/w). K. alvarezzi showed vitamin A activity of 865 mug retinal equivalents/100 g of sample. It contained a higher quantity of unsaturated fatty acids (44.50% of the total), in which relative percentage of oleic acid was 11%, cis-heptadecanoic acid 13.50%, and linoleic acid 2.3% and 37.0% of saturated fatty acids (mainly heptadecanoic acid). K. alvarezziwas also found to be good source of minerals, viz 0.16% of calcium, 0.033% of iron, and 0.016% of zinc, which are essential for various vital biological activities. Bioavailability of iron by in vitro methods showed a higher efficiency in intestinal conditions than in stomach conditions. Ascorbic acid influenced higher bioavailability of iron. Successive extracts of n-hexane, acetone, ethyl acetate, ethanol, and direct extractables of chloroform/methanol (1:1 and 2:1) were screened for antioxidant activity using a beta-carotene linoleic acid model system (B-CLAMS), DPPH (alpha,alpha-diphenyl-beta-picrylhydrazyl) model system and hydroxyl radical scavenging activity. The chloroform/methanol (2:1) extract has shown 82.5% scavenging activity at 1000 ppm. Acetone fraction extracts at the 1000 ppm level showed 63.31% antioxidant activity in beta-carotene linoleic acid system. The acetone extract showed 46.04% scavenging activity at 1000 ppm concentration. In the case of hydroxyl radical scavenging activity, all the extracts showed better activity at the concentrations of 25 and 50 ppm, where at the 50 ppm level ethyl acetate extract showed 76.0%, acetone 75.12%, and hexane 71.15% activity, respectively. Results of this study suggest the utility of K. alvarezzi (Eucheuma) for various nutritional products, including antioxidant for use as health food or nutraceutical supplement.     

Capabilities of Oat Extracts in Inhibiting Cholesterol and Long Chain Fatty Acid Oxidation During Heating

The antioxidant activities of oat groat extracts using different solvents were investigated. For acetone, hexane, and methanol extracts, the total phenolic compound contents were 12.83, 8.56, and 26.17 μg of catechin equivalent/g, respectively. Their corresponding free radical scavenging activities measured using the DPPH (2,2'‐diphenyl‐1‐picrylhydrazyl) method were 0.68, 0.35, and 0.79 μmol of Trolox equivalent/g, respectively. All oat extracts presented significantly greater capability in preventing cholesterol oxidation than the control (no extract added) during heating at the three levels of addition (1, 5, and 10 mg). The capability of the hexane or acetone extract in preventing cholesterol oxidation was not as effective as the methanol extract. In the docosahexaenoic acid (DHA, C22:6) oxidation test, all oat extracts at the level of 5 and 10 mg had greater inhibition capability on DHA oxidation than the control (DHA only). No significant inhibition was found for the hexane and acetone extract at the level of 1 mg, except for the methanol extract. The capabilities of oat extracts to inhibit oxidation of cholesterol and DHA from high to low was methanol, acetone, and hexane extract, which agreed with their free radical scavenging activities and total phenolic compound contents.     

Lipoxygenase and hydroperoxide lyase activities in ripening strawberry fruits

The enzymes lipoxygenase and hydroperoxide lyase have been identified in strawberry (Fragariax ananassa Duch.) var. Camarosa. Their subcellular localization, substrate preference, and product specificity were determined in mature strawberry fruits. The activity of both enzymes was located mainly in the microsomal fraction. Linolenic acid was the preferred substrate for strawberry lipoxygenase, forming 13- and 9-hydroperoxides of this acid in the proportion 70:30. The strawberry hydroperoxide lyase cleaves 13-hydroperoxide of linoleic (13% relative activity) and linolenic (100% relative activity) acids to form hexanal and (3Z)-hexenal, respectively. Both enzyme activities and endogenous content of volatile aldehydes formed by sequential action of lipoxygenase-hydroperoxide lyase were evaluated during strawberry development and ripening. A sequential enzymatic pathway for the formation of green odor compounds in strawberry is proposed.     

Effects of paprika pigments on oxidation of linoleic acid stored in the dark or exposed to light

We examined the antioxidant effects of paprika pigments on oxidation of linoleic acid and on decoloration of the sample when stored at 37 degrees C in the dark or exposed to fluorescent light for 8 h per day. (1)H nuclear magnetic resonance with dioxane as an external proton reference was used to estimate the oxidative deterioration of linoleic acid. Oxidation was estimated by observing the ratio of the divinylmethylene proton signal area in linoleic acid vs the proton signal area in dioxane. The addition of paprika pigments suppressed the oxidation of linoleic acid during storage in the dark, and the effect was markedly increased with increasing concentrations (0.02, 0.2, and 2%). When the linoleic acid with added paprika pigments was exposed to light, only a slight suppression of oxidation was observed, and the color of the sample disappeared more rapidly than that in the dark. At the time of decoloration of the sample with added pigments, considerable oxidation of linoleic acid occurred. As the color change is due to degradation of the pigment, an increase in oxidation at the time of discoloration is consistent with the pigments functioning as antioxidants. The addition of alpha-tocopherol to paprika pigments stabilized degradation of the pigments by light. Although the addition of alpha-tocopherol to linoleic acid with added paprika pigments prolonged the decoloration of the sample under light, the prevention of oxidation under the light condition was not as effective as for the samples stored in the dark.