Antioxidant effects of chlorophyll and pheophytin on the autoxidation of oils in the dark. I. Comparison of the inhibitory effects

The effects of chlorophyll and pheophytin on the autoxidation of oils in the dark were investigated by oven tests. The results indicated that both chlorophyll and pheophytin show antioxidant activity when methyl linoleate is used as substrate. Furthermore, chlorophyll retarded the oxidative deterioration of triglycerides in rapeseed and soybean oils at 30 C. Among the four chlorophyll derivatives (chlorophylls a and b and pheophytins a and b), chlorophyll a showed the strongest antioxidant activity. The antioxidant effects of chlorophyll and pheophytin depended on the storage temperature and the kinds of oil used as substrate.     

Structure of monohydroperoxides formed by chlorophyll photo-sensitized oxidation of methyl linoleate

The structures of the hydroperoxides prepared by photochemical oxidation of methyl linoleate in the presence of chlorophyll were determined by gas liquid chromatography-mass spectrometry (GLC-MS) of the trimethylsilyl (TMS) derivatives of the hydroxyesters. The position of the double bonds were deduced via hydroxylation with OsO₄, followed by GLC-MS analysis of the TMS derivatives. Structures of the original hydroperoxides were elucidated from these data. One of 28 papers presented at the Symposium “Metal-Catalyzed Lipid Oxidation,” ISF-AOCS World Congress, Chicago, September 1970.     

Characterization of chlorophyll pigments present in canola seed,meal and oil

Chlorophyll pigments present in canola seed, meal and crude and degummed oils were analyzed by high-performance liquid chromatography (HPLC) with a fluorescence detector. Chlorophylls a and b, low levels of pheophytin a, and occasionally traces of pheophorbide and its methyl ester were present in canola seed. Meals and oils contained magnesium-deficient chlorophyll pigments such as pheophorbide a, methylpheophorbide a, pheophytins a and b, and pyropheophytins a and b but not chlorophyll a or b. The amounts of chlorophyll pigments were oil > seed >> meal. Both crude and degummed oils contained pheophytin a and pyropheophytin a as main components, but the ratio of pyropheophytin a to pheophytin a was markedly higher in degummed oils. No pheophorbides were detected in degummed oils. These results suggest that oil processing steps such as extraction and degumming affect the composition of chlorophyll pigments. Publication No. 678 Canadian Grain Commission.     

Effects of processing and storage on chlorophyll derivatives in commercially extracted canola oil

This study characterizes the chlorophyll pigments present in canola oil immediately after commercial extraction and following oil storage to determine the best storage conditions for analytical samples and to examine the changes that chlorophyll derivatives undergo during oil processing and storage. Samples of pressed, solvent-extracted, crude and degummed canola oils, obtained from a commercial crushing plant, were stored for one month under four different conditions—in the freezer, in a refrigerator and at room temperature both in the light and in the dark. Chlorophyll derivatives (chlorophylls, pheophytins, pyropheophytins) were measured by high-performance liquid chromatography immediately after sampling and then on a weekly basis. The main pigments present in commercially extracted canola oil were pheophytin a, pyropheophytin a, chlorophyll a and chlorophyll b. The “a” derivatives comprised 81 to 100% of total chlorophyll pigments in the fresh oil samples. During degumming, the remaining chlorophylls were converted to pheophytins and pyropheophytins. During oil storage, exposure to light at room temperature affected the composition of chlorophyll derivatives as chlorophyll b was converted to pheophytin b and chlorophyll a was converted first to pheophytin a, then to pyropheophytin a.     

Phenothiazine derivatives as new antioxidants for the autoxidation of methyl linoleate and their reaction mechanisms

The effect of new antioxidants, such as phenothiazine derivatives, on the autoxidation of methyl linoleate was evaluated by estimating the induction period using the weighing method. Peroxide values (iodometry and IR), refractive indices, mol wts, and UV and IR spectra were measured to investigate the extent of the autoxidation. The induction period evaluated from the weighing method gives almost the same value as that from the peroxide values. It was shown that some new phenothiazine derivatives are remarkably effective antioxidants, and, besides, the mechanism for the autoxidation of methyl linoleate containing phenothiazine derivatives as antioxidants is probably of the same type as that for the substrate alone. This study also investigated the reaction mechanism of phenothiazine derivative antioxidants by determining the electron spin resonance spectra for the antioxidants in the autoxidation of methyl linoleate. Then, the following mechanism was proposed. That is, within the induction period, these inhibitors hold stable nitroxide radicals (>NO*) in the reaction between the antioxidant amino radical (>N*), produced by the reaction of the antioxidant with ROO* or O₂, and the peroxy radical (ROO*). Besides, the more superior the phenothiazine derivative antioxidant, the more inactive the antioxidant makes oxygen and the peroxy radical for the methyl linoleate autoxidation and also for the antioxidant oxidation. Presented at the 12th World Congress of ISF, Milan, 1974.     

Prooxident activities of chlorophylls and their decomposition products on the photooxidation of methyl linoleate

Although chlorophylls in oils affect oxidative stabilities, little has been known about the prooxidant activity of the chlorophylls or their decomposition products. To evaluate the prooxidant activities of chlorophyll derivatives, chlorophyll (CHL) A and B, pheophytin (PHY) A and B and pheophorbide (PHO) A and B were added to methyl linoleate (ML). The sample oils were then subjected to photooxidation at O C and the peroxide value and absorbance at 234 nm were measured. Chlorophylls catalyzed oxidation of methyl linoleate at concentrations greater than 2.2×10⁻⁹mol/g ML, and CHL B showed a stronger prooxidant activity, ca. twice that of CHL A under the same light intensity. PHY and PHO exhibited stronger prooxidant activities than CHL, the prooxidant activity of PHO being the strongest among them. Moreover, photosensitive activity was found in PHY as well as in CHL. These results suggest that particular attention should be paid to the decomposition products of CHL that affect the quality of vegetable oils.     

Ion exchange resins and ethyleneimine polymer as antioxidants: I. Activity and mechanism

The antioxidant activity of ion exchange resins and ethyleneimine polymer was determined in methyl linoleate free from natural antioxidants and metals. Superior antioxidant activities were recognized in an ion exchange resin with NH groups and in the ethyleneimine polymer. The antioxidant activities in these heterogeneous reaction systems increased in linear proportion to the amount present. These antioxidant activities in the heterogeneous reaction system were mainly owing to their ability to donate hydrogen to the peroxy radicals produced in the autoxidation of methyl linoleate. The antioxidant radicals produced in these cases were very stable and generally couldn’t participate in the increase of the induction periods in the autoxidation of methyl linoleate by terminating the peroxy radicals. These results have never been obtained in the homogeneous reaction system but are characteristic in the heterogeneous one.     

Characterization of chlorophyll pigments in ripening canola seed (Brassica napus)

This study characterizes the chlorophyll pigments in ripeningBrassica napus seed. Seed samples, collected weekly as the crop ripened, were analyzed by high-performance liquid chromatography to characterize chlorophyll pigment composition. Chlorophyll A, chlorophyll B, pheophytin A and pheophytin B were the predominant pigments, while pheophorbide A, methylpheophorbide A and pyropheophytin A were minor components. No differences in pigment composition were observed between the three cultivars tested or between early and late seeding dates. There were differences in pigment composition between the two years of the study, which may result either from seed aging during storage or from environmental influences. Pigment composition was dependent on seed maturity, with physiologically mature green seeds containing both chlorophylls and pheophytins, but fully mature seeds containing only chlorophylls. Pheophytins and the minor components appeared transiently, presumably formed from the chlorophylls and subsequently degraded. The ratio of chlorophyll A/B increased during seed ripening, with fully mature canola seed having a chlorophyll A/B ratio twice that of physiologically mature green seed. The “B” derivatives degraded faster than the “A” derivatives, suggesting enzymatic reactions. The initial steps in the chlorophyll breakdown pathway in canola seed appear to be:      

Antioxidant activity of flavonol aglycones and their glycosides in methyl linoleate

The antioxidant activities of the flavonol aglycones, quercetin and myricetin, and their selected glycosides were compared in bulk methyl linoleate oxidized at 40°C. Methyl linoleate hydroperoxide formation, hydroperoxide isomer distribution, and ketodiene formation were followed by using high-performance liquid chromatography (HPLC) analysis. The aglycones, quercetin and myricetin, were consistently more active in bulk methyl linoleate than their glycosides and more active than α-tocopherol at 500 and 1000 μM. At 50 μM, the order of activity was myricetin > α-tocopherol > quercetin, and the order of activity of quercetin and its derivatives was quercetin > quercitrin > isoquercitrin > rutin. Myricitrin was slightly less active than myricetin. The sugar moiety was shown to have a marked effect on the antioxidant activity of flavonols. The rhamnoside derivatives, quercitrin and myricitrin, both possessed activity close to that of their corresponding aglycones. The different activities of glycosides could be partly explained by different solubilities and by differences in oxidizability of glycosides containing a monosaccharide or disaccharide at the C₃ position. The effect on hydroperoxide isomer distribution indicates that α-tocopherol was a more effective hydrogen donor than flavonoids, although flavonoids were more effective in inhibiting oxidation of methyl linoleate.     

Antioxidant effect of the constituents of susabinori (Porphyra yezoensis)

The antioxidant activities of several extracts from Susabinori (Porphyra yezoensis) were measured by the ferric thiocyanate method and the thiobarbituric acid method. The methanol, acetone, ethyl acetate, and hexane extracts, and the chloroform-soluble and water-soluble fractions from the chloroform-methanol extract exhibited higher activities than α-tocopherol. The hot water extract showed little activity. Thin-layer chromatography analysis of the active extracts suggested the existence of several antioxidants. The activity of the chloroform soluble fraction was due to chlorophyll analogs. A strong antioxidant was isolated from the methanol extract, accompanied by several amino acids such as leucine and phenylalanine. This compound was identified as usujilene, a kind of mycosporine-glycine like amino acid.     

Antioxidative components,xanthone derivatives,inSwertia japonica Makino

Substances with antioxidative properties were obtained from an ether extract ofSwertia japonica Makino. Six active components of the extract were isolated and identified as methylbellidifolin, methylswertianin, swertianin, bellidifolin, norswertianin and desmethylbellidifolin. These six xanthone derivatives were shown to possess different antioxidant activities by chemiluminescent assay. The antioxidative activities of bellidifolin, norswertianin and desmethylbellidifolin were higher than those of butylated hydroxytoluene (BHT) and α-tocopherol. On autoxidation of methyl linoleate, bellidifolin had activity similar to that of BHT. On the basis of the data present on antioxidative properties and data reported on the mutagenicities of the xanthones, both activities were shown to give a good correlation.     

Behavior of chlorophyll derivatives in canola oil processing

Chlorophyll derivatives in canola oil were analyzed quantitatively by reversed-phase high-performance liquid chromatography without any pretreatment. The main components were pheophytin (pheo) a and b and pyropheophytin (pyro) a and b. The factors affecting the types and concentration of chlorophyll derivatives in oil have been investigated during seed preparation, expelling, extraction, degumming and alkali-refining processes. Bleaching tests of alkali-refined canola oil with activated earth indicated the adsorption of each derivative to decrease in the following order: pheo a > pyro a >> pheo b > pyro b. In bleaching with activated carbon, however, the following order was observed: pyro b > pheo b > pheo a > pyro a.     

Use of chlorophyll and carotenoid pigment composition to determine authenticity of virgin olive oil

The chlorophyll and carotenoid pigment profile of 50 mono-variety virgin olive oils was used to develop an index of authenticity for the product. The presence of carotenoids other than those described, or chlorophyll derivatives at another level of degradation, were found to be determing elements of this index for “virgin” olive oil quality. In addition, the ratio of chlorophyll/carotenoid should be around 1, and the ratio of minor carotenoids/lutein should be about 0.5, with a limited variability. These characteristics may be expected of virgin olive oil in general and are independent of variety. Finally, the percentage of lutein, violaxanthin, and total pigment content may be used to distinguish between mono-variety virgin olive oils.     

The antioxidant activity of 3,5-di-tert-butyl-4-hydroxybenzyl derivatives

Various electron donating groups such as alkoxyl, alkylthio, and alkylamino groups were used to replace a hydrogen atom of the para-methyl group of 2,6-di-tert-butyl-4-methylphenol, BHT. The antioxidant activity of these 3,5-di-tert-butyl-4-hydroxybenzyl derivatives in stripped corn oil at 79.5°±1.0C was measured on the basis of the length of the induction period. A modified peroxide determination was devised and used in the present study. The overall average of the per cent accuracy, based on the standard error, was 2.5%. The results indicated that the tertiary methyl and ethylamine derivatives had the strongest potency among all compounds tested. Both amine derivatives were able to prolong the induction period of the corn oil twice as much as BHT. The alkoxyl derivatives were as effective as BHT. The alkylthio derivatives showed stronger activity than either BHT or the alkoxyl derivatives. The secondary alkylamine derivatives, except the n-hexadecylamine derivative, demonstrated nearly the same degree of potency as BHT. However, the n-hexadecylamine derivative exhibited far stronger activity than either the three lower alkylamine derivatives or BHT. In all three homologous series, the methyl derivatives showed significantly lower activity than the ethyl or n-propyl derivatives. Differences between the ethyl and n-propyl derivatives were not significant. 3,5-Di-tert-butyl-4-hydroxybenzylpyridinium bromide acted as a prooxidant. An attempt has been made to explain the variation in activity of these derivatives in terms of the inductive effect exerted by the nucleophilic substituents and/or in terms of the structural features of the derivatives. Supported by Research Grant HTS-5368 from the National Institutes of Health, United States Public Health Service, Department of Health, Education, and Welfare.     

Electrochemical processes in the photoelectrolysis of nicotinamide adenine dinucleotide on the chlorophyll electrodes

The photoelectrolysis, in which redox compounds are electrolysed on a pair of photo-excitable electrodes by supplying photo—energy in place of electric energy, has been performed. The photo—excitable electrodes were prepared by coating a platinum plate with a thin layer of a chlorophyll—quinone composite. These electrodes were called chlorophyll electrodes. The chlorophyll electrode of chlorophyll—naphthoquinone composite worked as a cathode and that of chlorophyll—anthrahydroquinone composite as an anode when they were illuminated. The chlorophyll electrode of chlorophyll—naphthoquinone composite was characterized by an electrochemical behavior of p-type semiconductor electrode. Reduction of nicotinamide adenine dinucleotide (NAD⁺) was carried out on the chlorophyll electrode under illumination at various controlled electrode potentials. NAD was reduced at extremely noble electrode potentials are compared with the reductive potential of NAD⁺ to NADH. Electron transfer accompanied with the photoelectrochemical reactions is discussed.     

Effect of Steric Structure on the Mechanism of Antioxidant Activity of Alkyl Gallates in Soybean Oil Triacylglycerols—A Kinetic Approach

From an interfacial phenomena standpoint, the effect of the alkyl chain length is evaluated on the mechanism of antioxidant activity of gallic acid ester derivatives (methyl, propyl, octyl, dodecyl, and stearyl gallates) in bulk phase oil. A combinational kinetic model is used to estimate the oxidation kinetic parameters, such as induction period, the maximum concentration of lipid hydroperoxides, and critical reverse micelle concentration. These kinetic parameters are estimated much better with the combinational model compared to the tangent method. The nonlinear behavior is observed for inhibitory activities of homologous series of antioxidants in soybean triacylglycerols. In terms of different kinetics parameters, including antioxidant effectiveness and activity, methyl gallate has the highest inhibitory effect during lipid peroxidation. Gallic acid and alkyl gallates are able to protect bulk oils against peroxidation (induction period > 336 h) in terms of the extent of their participation in the main reaction of chain termination and pro-oxidative side reactions of chain initiation, and anti-oxidative side reactions of chain propagation. Practical Applications: This work explains the effect of the esterification in the inhibitory activity of alkyl gallates, mechanism action of alkyl gallates in bulk oil systems, antiradical potency as a function of the interfacial phenomena, and free radical chain mechanism, the impact of antioxidant activity on critical reverse micelle concentration of hydroperoxides. Moreover, in this study, it is confirmed that the combinational kinetic model can be employed as a reliable method for determining the oxidation kinetic parameters.     

The influence of various emulsifiers on the partitioning and antioxidant activity of hydroxybenzoic acids and their derivatives in oil-in-water emulsions

The partitioning behavior of a series of hydroxybenzoic acids and their derivatives was determined in biphasic water-oil systems, emulsifier solutions, and oil-in-water (O/W) emulsions. The effect of gradually decreasing polarity on partitioning behavior and antioxidant efficiency in O/W emulsions was investigated by using gallic acid and its esters as antioxidants. Sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB), polyoxyethylene 20 cetyl ether (Brij 58), and partially hydrolyzed soybean lecithin (PHLC, Emultop^®) were used to investigate the influence of different classes of emulsifiers on the partitioning behavior. The antioxidant activity of gallic acid and its methyl, ethyl, propyl, butyl, and octyl esters showed markedly different trends in O/W emulsions depending on the emulsifier used. The results are discussed with respect to the properties of the emulsifiers, such as hydrogen bond basicity, hydrophobic interactions, and structural properties.     

Kinetics of lipid oxidation in the presence of cinnamic acid derivatives

The effects of seven (prenyl‐ and methoxy‐) derivatives of cinnamic acid (0.1 mM) on the kinetics of lipid (sunflower oil triacylglycerols, TGSO) bulk phase oxidation at 80 °C have been compared. Synthesis of prenyl cinnamic acid derivatives: 3‐prenyl‐4‐hydroxy‐cinnamic acid (PHC), 3,5‐diprenyl‐4‐hydroxy‐cinnamic acid (DPHC), 2,2‐di‐methyl‐6‐carboxy‐ethenyl‐2H‐benzopyran (DMCB), 2,2‐dimethyl‐6‐carboxy‐ethenyl‐8‐prenyl‐2H‐benzopyran (DCEPB) present in Brazilian propolis has been performed. The monoprenyl derivative (PHC) has been found to exert a higher antioxidant activity as compared to the diprenyl derivative (DPHC). However, cinnamic acid derivatives DMCB and DCEPB have caused no change in the kinetics of TGSO oxidation. The results obtained have been compared with those on related compounds containing a cinnamic acid moiety as a structural feature, such as 4‐hydroxy‐cinnamic (p‐coumaric), 3‐methoxy‐4‐hydroxy‐cinnamic (ferulic) and 3,5‐dimethoxy‐4‐hydroxy‐cinnamic (sinapic) acids, as well as with data on butylated hydroxytoluene (BHT) and α‐tocopherol (αToc). PHC has shown a stronger antioxidant efficiency than BHT, p‐coumaric and ferulic acid, but a weaker antioxidant efficiency than α‐Toc and sinapic acid. The observed antioxidant effect of DPHC was stronger than that of p‐coumaric and ferulic acids and weaker than that of α‐Toc, BHT and sinapic acid.     

2-Hydroxy-4-benzyloxylimine Resveratrol Derivatives as Potential Multifunctional Agents for the Treatment of Parkinson's Disease

A series of 2-hydroxy-4-benzyloxylimine resveratrol derivatives was designed, synthesized and evaluated as multifunctional agents for the treatment of Parkinson's disease. The results revealed that most derivatives possessed good multifunctional activities. Among them, representative compound (E)-5-[(4-fluorobenzyl)oxy]-2-{[(4-hydroxyphenyl)imino]methyl}phenol ( 7 h ) exhibited excellent MAO-B inhibition (IC₅₀=8.43×10⁻³ μM) and high antioxidant activity (ORAC=3.45 Trolox equivalent). Additionally, 7 h displayed good metal chelating ability, appropriate blood–brain barrier (BBB) permeability, significant neuroprotective effect, and great anti-neuroinflammatory activity. Furthermore, 7 h can also ameliorate 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease symptoms in mice. Therefore, compound 7 h was found to be a promising candidate for further development against PD.     

Surface activities,biodegradability and antimicrobial properties of glucosamine derivatives containing alkyl chains

Three series of D-glucosamine derivatives containing an alkyl chain with 8 to 14 atoms, methyl 2-acylamino-2-deoxy-D-glucopyranosides,n-alkyl 2-acetylamino-2-deoxy-D-glucopyranosides andn-alkyl 2-amino-2-deoxy-D-glucopyranoside hydrochlorides, were synthesized, and their surface properties (such as surface tension, critical micelle concentration (CMC), dynamic surface tension and foaming properties), biodegradability and antimicrobial activities were evaluated.n-Alkyl 2-amino-2-deoxy-D-glucopyranoside hydrochlorides containing C8 to C12 carbon chains showed surface activities, a CMC and excellent foaming properties. The α-anomers showed a slightly lower CMC than the β-anomers, indicating less hydrophilicity of the α-anomers. On the other hand, glucosamine derivatives containing amide groups showed poor surface activities in water due to their lower solubilities in water. All glucosamine derivatives containing alkyl chains were biodegraded as well as conventional ethoxylated nonionics by activated sludge from the municipal sewage treatment plant. Methyl 2-acylamino-2-deoxy-D-glucopyranosides andn-alkyl 2-amino-2-deoxy-D-glucopyranoside hydrochlorides showed a broader spectrum of antimicrobial activity than the correspondingn-alkyl glucopyranosides. Among them the C12 derivatives showed the best results.