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.     

Effects of ionizing radiations on fats. II. Accumulation of peroxides and other chemical changes

The accumulation of peroxides, carbonyl com-pounds and reducing substances during irradia-tion and post-irradiation storage of pure fatty acid methyl esters has been studied. Irradiation and storage of irradiated methyl myristate under vacuum results in formation of small quantities of these compounds. Irradiation under oxygen gives peroxides and carbonyl com-pounds in yields indicating that every ionization results in the formation of one molecule of each group, and antioxidants have no effect on the formation of these compounds during irradiation. Irradiation of methyl linoleate under vacuum results in destruction of pre-formed hydroperox-ides. During irradiation in oxygen, approximately one-eighth of the peroxides formed arises from the direct reaction of irradiation-induced free radi-cals with oxygen, while the rest is formed through a chain mechanism with an average chain length of 7. Peroxides continue to accumulate in irradiated methyl linoleate stored under oxygen at a rate increasing with initial irradiation dose. Antioxidants have some effect in retarding the formation of peroxides during irradiation of methyl linoleate and during post-irradiation stor-age, but the effect is small compared to their antioxygenic activity toward simple autoxidation. The effect varies with the nature of the antioxi-dant and with irradiation dose. Propyl gallate is much less effective than butylated hydroxy-anisole and appears to be easily destroyed during irradiation. For paper I of this series, see Ind. Eng. Chem.49. 1713 (1957). Presented at the AOCS Meeting in Minneapolis, 1963.     

Autoxidation of methyl esters of cyclopentenyl fatty acids

The early stages of the autoxidation of methyl hydnocarpate, chaulmoograte and gorlate in air have been examined at 40, 60 and 80 C, and the initial products have been compared by several methods with those derived from methyl oleate and linoleate autoxidized at 60 C. To supplement information about oxygen absorption and peroxide development in relation to time, other information about the early products, and some information about the reduced products, have been obtained by ultraviolet (UV) and infrared (IR) spectrophotometry, and by thin layer chromatography (TLC). The kinetic and other data presented in this study strongly support the conclusion that the methyl esters of cyclopentenyl fatty acids yield initial autoxidation products that, although they are primarily peroxides, differ in some ways (as expected) in the kinetics of their formation and their chemical nature, compared to those of oleate and linoleate. Nevertheless, all the data obtained strongly support the surmise that the peroxides are formed autocatalytically by a chain mechanism, and that secondary products not derived from peroxide decomposition, are formed pari passu in lesser, but increasing amounts with increasing temperature, probably from free radical intermediates. The autoxidation of esters of cyclopentenyl fatty acids has potential importance in several ways, 3 of which are mentioned briefly.     

Lipid oxidation: Mechanisms,products and biological significance

This paper reviews our studies of fatty acid hydroperoxides, their secondary products and mechanisms for their formation in the context of some of their possible biological consequences. The uneven distribution of isomeric hydroperoxides in oxidized linolenate and photosensitized oxidized linoleate is related to the formation of hydroperoxy cyclic peroxides. Interest in the hydroperoxy mono-and bi-cycloendoperoxides from oxidized linolenate stems from their structural relationship to the prostaglandins. However, the biological activity of hydroperoxy cyclic peroxides formed by autoxidation has not yet been reported. Thermal decomposition studies of secondary lipid oxidation products show they are important precursors of volatile compounds. An acid-acetalation decomposition procedure establishes that 5-membered hydroperoxy cyclic peroxides and 1,3-dihydroperoxides are important precursors of malonaldehyde. This approach provides a more specific test than the thiobarbituric acid (TBA) color reaction to evaluate lipid oxidation products as sources of malonaldehyde and its biological effects due to crosslinking. A better understanding is needed of the biological effects of a multitude of lipid oxidation decomposition products other than malonaldehyde.     

Effects of relative humidity on lipid autoxidation in a model system

A new model system was developed for the study of autoxidation of thin films of neat lipid and the effect of relative humidity on the oxidation reaction. In the model system, the surface-to-volume ratio of lipid was large and measureable, and the relative humidity (RH) and oxygen partial pressure were controlled. Methyl linoleate, oxidized at six different RH as a thin film in an atmosphere of pure oxygen, exhibited a maximum rate of oxidation at 32% RH and minimum rates at 0% and 100% RH. The rates of oxygen uptake, determined manometrically, were linear and reproducible at all six RH. The maximum rate at 32% RH was attributed to solvation and stabilization of the propagation transition state by water. Increasing the RH beyond 32% resulted in solvation of the peroxy radical, sterically hindering the radical from entering the propagation transition state.     

Autoxidation of ethyl eicosapentaenoate and docosahexaenoate

The extent of oxidation of ethyl esters of eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) was compared quantitatively with that of ethyl linoleate (Lo) and ethyl linolenate (Ln) by oxygen uptake and formation of conjugated diene, hydroperoxide and secondary oxidation products. EPA and DHA esters were oxidized rapidly even at 5 C in the dark after an induction period of 3–4 days, while the induction periods of Ln and Lo esters were 20 days and more than 60 days, respectively. Oxygen uptake of EPA and DHA esters after the induction period was 5.2 and 8.5 times faster than that of ethyl Ln, respectively. Hydroperoxides of EPA and DHA esters are much less stable than those of ethyl Lo. The peroxide value is not necessarily a good indication of oxidation in these polyenoic acids because a considerable amount of secondary products is formed at the early stage of oxidation. Polymers were found to be major secondary products in these polyenoic esters.     

Thermal oxidation of fractionated polypropylene in solution

Thermal oxidation of fractionated polypropylene was carried out in trichlorobenzene under atmospheric oxygen at 125°C with conventional oxygen uptake. The oxidizability of the polymers is discussed on the basis of the oxygen uptake curves and the properties of the polymers. Fractions of atactic polypropylene oxidized easily at the initial stage of the oxidation and showed neither autoxidation phenomena nor the induction period observed in the isotactic polymer. Hydrogenation of the ether-soluble fraction by a Wilkinson catalyst gave a polymer which was, according to infrared spectrometry, free from impurity groups such as hydroperoxide, carbonyl, and unsaturation groups. The hydrogenated fraction was more stable to thermal oxidation than the unhydrogenated fraction and showed an induction period. The results indicate that the initiation process of the oxidation of polypropylene is apparently dependent on the impurities such as hydroperoxide, carbonyl, and unsaturation and that stereoregularity of the polymer affects the kinetic dependence of the oxidation.     

Chemical lonization-mass spectrometry of secondary oxidation products from methyl linoleate and linolenate

Chemical ionization-mass spectrometry (CI-MS) with a direct exposure probe was used to analyze a series of hydroperoxy cyclic peroxides and dihydroperoxides obtained from methyl linoleate and linolenate by either autoxidation or photosensitized oxidation. The mass spectra obtained with isobutane and ammonia as reacting gases showed fragmentation patterns similar to those ditions of gas chromatography (GC)-electron impact (EI)-MS. Because the fragmentation patterns obtained under either CI-MS with a direct exposure probe or GC-(EI)-MS conditions are sufficiently predictable, these techniques are powerful analytical tools for the structural characterization of lipid oxidation products. These techniques are also useful to elucidate the cleavage pathways to volatile lipid oxidation products of flavor and biological significance.     

Reactions of fatty materials with oxygen. XVII. Some observations on the secondary products of autoxidation of methyl oleate

Summary Methyl oleate, autoxidized for short and long periods of time, has been fractionated with urea. Up to a peroxide content of about 15% the autoxidation mixture can be cleanly separated into a peroxide concentrate containing 90% peroxide and unoxidized methyl oleate. From about 15% peroxide to the maximum peroxide content (35–40%) concentration to only about 70% peroxide can be obtained, and the remaining material is largely a mixture of oxygenated compounds and residual methyl oleate. If the autoxidation is conducted beyond the peak value in peroxide content, little, if any, concentration of peroxide can be obtained. Also, beyond the peak in peroxide value and in the range of 30–20% peroxide, methyl oleate is substantially absent and the autoxidation mixture consists almost entirely of oxygenated compounds containing only one functional group in the chain. Evidence is presented which shows that in the autoxidation of methyl oleate substantially all of it undergoes single attack by oxygen (or peroxides) before any significant quantity of multiple attack occurs. α,β-Unsaturated carbonyl compounds are among the important secondary products of autoxidation. Paper XVI is reference 14. Presented at the Fall Meeting of the American Oil Chemists' Society, Minneapolis, Minn., Oct. 11–13, 1954. A labortory of the Eastern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

A possible role for singlet oxygen in the initiation of fatty acid autoxidation

A mechanism for the initiation of autoxidation in fatty acids is proposed which involves singlet state oxygen, formed through a photosensitization reaction, as the reactive intermediate. Both singlet oxygen generated in a radio-frequency gasdischarge, and photosensitization by natural pigments, were shown to catalyze the oxidation of methyl linoleate. The involvement of singlet oxygen was shown by the identification of nonconjugated hydroperoxides as products common to both photooxidation and singlet O₂ oxidation. Nonconjugated hydroperoxides could not be detected among the free radical autoxidation products. Further proof for the above mechanism was gained by showing that compounds known to react strongly with singlet oxygen, inhibited the photooxidation. With the exception of chlorophyll, all sensitizers could be completely inhibited. Although singlet oxygen formation can account for approximately 80% of the observed chlorophyll photooxidation, at least one other mechanism must be involved. It is postulated that proton abstraction by the photoactivated carbonyl group of chorophyll could account for the remaining 20% of the observed photooxidation. The conclusion is drawn that oxygen, excited to its singlet state by a photosensitization process, plays the important role of forming the original hydroperoxides whose presence is necessary before the normal free radical autoxidation process can begin.     

Photochemical oxidation of fatty acid esters with and without chlorophyll

1. It has been confirmed that the principal products formed in the oxidation of methyl oleate by oxygen under a variety of conditions are predominantlytrans hydroperoxides. However no inversion of the double bond occurs in unoxidized oleate. Hence the conversion ofcis totrans double bonds and peroxide formation occur together in the same molecules.
2. The autoxidation of methyl linoleate at low temperature yields predominantlycis,trans conjugated hydroperoxides. Autoxidation at 25°C., oxidation catalyzed by visible light, or ultraviolet light and copper soap catalyzed oxidation at temperatures appreciably above 0°C., lead to the formation primarily oftrans,trans conjugated hydroperoxides. The inversion of the second double bond in this case appears to be independent of the peroxide-forming reactions.
3. The photochlorophyll oxidation of methyl linoleate leads to the formation of some unconjugated hydroperoxides, some of which containtrans double bonds.
4. Under all of the conditions employed in the present investigation, the oxidation of methyl oleate and linoleate led primarily to the formation of monomeric peroxides which retained most of the unsaturation of the parent compound.

     

Kinetics of linoleate oxidation in model systems

Oxidation of methyl linoleate, trilinoleate and linoleic acid has been studied in model systems based on various solid supports. Oxidation was followed by measurement of oxygen absorption, peroxide values and products of oxidation as a function of moisture equilibrium relative humidity. Effects of various metals, histidine and the antioxidants propyl gallate and butylated hydroxytoluene were studied. The results indicate: (a) at 50 C oxidation of protein increases with increasing moisture content and the protein interacts with peroxides changing the overall oxidation rate; (b) increasing moisture content shows the same inhibitory effect on oxidation of trilinoleate as it does on methyl linoleate; (c) the effectiveness of antioxidants is increased with increasing humidity but some chelating agents complexed with metals become catalytic at the higher moisture content; and (d) at moisture contents in the region of capillary condensation, mobility of reactants is enhanced since the rate of oxidation increases significantly. Presented at the AOCS Meeting, New York, October, 1968. Contribution No. 1396, Dept. of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass.     

On the kinetics of the autoxidation of fats. II. Monounsaturated substrates

The autoxidation of methyl oleate and oleic acid shows some differences as compared to the autoxidation of linoleate,e.g., the formation of water at an early stage. Linearization of experimental data on the autoxidation to high oxidation degrees of methyl oleate and other monounsaturated substrates shows that the rate equations previously derived for methyl linoleate in the range of 1–25% oxidation are valid, provided the correct expression for the remaining unreacted substrate is used. With monounsaturated substrates, part of the oxygen is consumed by a secondary oxidation reaction almost from the beginning, and only a certain constant fraction α of the total O₂ consumption is consumed in hydroperoxide formation. The fraction α is different for methyl oleate, oleyl alcohol, oleic acid andcis 9-octadecene, but the rate constant for the hydroperoxide formation is the same for all of them when experimental conditions are the same. The main difference between oleate and linoleate autoxidation is the much faster decomposition of the oleate hydroperoxides relative to their slow formation.     

Structure of hydroperoxides obtained from autoxidized methyl linoleate

Summary A sample of debromination methyl linoleate has been autoxidized to a peroxide value of 671 m.e./kg. at approximately 0°C. in the dark. An essentially pure concentrate of methyl octadecadienoate monohy-droperoxide was quantitatively separated; infrared and ultraviolet spectral studies were made on the peroxide concentrate and on the corresponding hydroxyl derivative obtained by reducing the peroxides with stannous chloride. The infrared data showed no conjugated peroxides having geometric configurations other than cis, trans; the same data also showed that the peroxide concentrate contained at least 90% conjugated cis,trans forms. Calculations based on ultraviolet spectrophotometric methods also indicated that the peroxides were at least 90% conjugated. The remaining 10% of the sample is most likely nonconjugated diene hydroperoxide. Since analogous cis, cis conjugated dienes have not been isolated and their infrared and ultraviolet properties are unknown, their presence here in small amounts is possible. Ultraviolet and infrared spectra of the reduced compounds conform closely to those of the peroxides except for reduction in the intensity of the OH bond at 2.88 μ. The infrared absorption spectra of the C−H structure and carbonyl groups of an essentially pure conjugated cis, trans methyl octadecadienoate monohydroperoxide were recorded, using a LiF prism. The infrared absorption spectra of the C−H strucfraction isolated from methyl linoleate autoxidized in the dark at 24°C. indicated that appreciable amounts of conjugated trans, trans hydroperoxides were present, in addition to those of the cis, trans type. It is possible that the conjugated cis, trans isomers were formed originally but were labile at the higher temperature and in the presence of catalysts (e.g., peroxides) were transformed to the thermodynamically more stable conjugated trans, trans isomer. This work was supported in part by a contract between the Office of Naval Research, Department of the Navy, and the University of Minnesota. Hormel Institute publication No. 81, and paper No. 133, Journal Series, General Mills Research Laboratories.     

Detection and measurement of hydroperoxides by near infrared spectrophotometry

Summary Near-infrared spectra have been measured on a group of hydroperoxides of fatty acid esters and related substances. Only those substances having an −OOH group were found to absorb at 1.46 and 2.07 μ. Dialkyl peroxides and ozonized unsaturated substances had no such maxima in their near infrared spectra although they had high iodometric peroxide values. In a study of the thermal decomposition of methyl oleate hydroperoxide and a study of the autoxidation of methyl linoleate, the intensity of absorption at 1.46 and 2.07 μ paralleled the iodometric peroxide value. This work was aided by grants-in-aid from the Hormel Foundation, the Atomic Energy Commission (Contract AT-11-1-108), the Office of Naval Research (Contract N8 onr66218), the National Live Stock and Meat Board, and the National Diary Council. Hormel Institute Publication No. 170.     

Concurrent oxidation of accumulated hydroperoxides in the autoxidation of methyl linoleate

Summary 1. Kinetic studies showed that concurrent oxidation of preformed hydroperoxides may be expected to take place at all stages of the autoxidation of methyl linoleate. The rate of oxidation relative to the rate of autoxidation of unoxidized ester is determined chiefly by the extent of the accumulation of hydroperoxides. 2. Infrared spectral analysis of hydroperoxides oxidized to various degrees indicated thattrans, trans diene conjugation and isolatedtrans double bonds produced in the autoxidation of methyl linoleate are related to the concurrent oxidation of the accumulated hydroperoxides. 3. The low absorptivity observed for diene conjugation, compared to that which may be expected for the exclusive production ofcis, trans diene conjugated hydroperoxide isomers during the autoxidation of methyl linoleate is attributed to the concurrent oxidation of accumulated hydroperoxides. 4. The effect of antioxidants in giving a well-defined induction period in the oxidation of hydroperoxides isolated from autoxidized methyl linoleate indicated that the oxidation proceeds by a chain reaction. 5. The primary reaction products of the oxidation of hydroperoxides isolated from autoxidized methyl linoleate were found to be polymers formed in a sequence of reaction involving the diene conjugation. 6. Studies on the autoxidation of methylcis-9,trans-11-linoleate showed thatcis, trans isomerization of the conjugated diene took place with the concurrent production of isolatedtrans double bonds and loss of diene conjugation. Hormel Institute publication no. 138. Presented before the American Oil Chemists’ Society, Philadelphia, Pa., Oct. 10–12, 1955. This work was supported by a grant from the Hormel Foundation.     

Oxidation at intermediate moisture contents

In the study of the oxidation rate of methyl linoleate in protein and cellulose systems, a prooxidant effect was found at intermediate moisture contents. At low water content, water hydrates metals and hydrogen bonds with peroxides, and an overall decrease in the rate of lipid oxidation results. With an increase in the water content to the region with a water activity of 0.6 to 0.7, the water predominantly acts as a solvent to dissolve and mobilize previously unavailable trace metals with the result of increased oxidation rates. Use of chelating agents such as ethylenediaminetetraacetic acid and citric acid reduced oxidation significantly although some antioxidant activity was also observed for butylated hydroxyanisole. These results have important implications in the preparation of intermediate moisture foods. Presented at the AOCS Meeting, New Orleans, April 1970. Contribution No. 1674, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Mass.     

Reactions of fatty materials with oxygen. XVIII. Catalytic hydrogenation of autoxidized methyl oleate and oleic acid. Preparation of monohydroxystearic acids

Summary Autoxidation of methyl oleate and oleic acid beyond the peak peroxide values followed by catalytic hydrogenation gave mixed monohydroxystearic acids in high yield. The complicated autoxidation mixture which contains peroxides, hydroxy, carbonyl, and oxirane compounds was simplified considerably in composition by this procedure. For complete reduction of the double bond, and the carbonyl and oxirane groups, hydrogenation was conducted at about 150° and 150 lbs.. Peroxides were reduced at room temperature. Catalysts used were palladium on carbon and Raney nickel. The selective reduction of peroxides in autoxidation mixtures has been studied by chemical and catalytic means. Peroxides were converted largely to carbonyl compounds rather than to the anticipated hydroxy compounds. Palladium-lead on calcium carbonate is an excellent catalyst for reducing peroxides with hydrogen. tert-Butyl hydroperoxide, 12- ketostearic acid, stearone,cis-9,10-epoxystearic acid and methyl oleate peroxide concentrate were employed as model substances in determining hydrogenation conditions. Paper XVII. is reference 5. Presented at the fall meeting of the American Oil Chemists' Society, Minneapolis, Minn., Oct. 11–13, 1954. A laboratory of the Eastern Utilization Research Branch, Agricultural Research Service, U. S. Department of Agriculture.     

Degradation of monocarbonyls from autoxidizing lipids

In an attempt to account for carbonyls found in oxidized lipid systems, but not theoretically predicted from the decomposition of lipid hydroperoxides, a member from each of the monocarbonyl classes commonly observed in oxidizing lipids was oxidized at 45C in a Warburg apparatus and the carbonyl products studied. The carbonyl compounds used weren-nonanal,n-non-2-enal,n-hepta-2,4-dienal andn-oct-1-en-3-one. Nonanal was relatively stable to oxidation and was oxidized to nonanoic acid. Oct-1-en-3-one did not absorb oxygen during a 52-hr period; however, the unsaturated aldehydes oxidized at faster rates than methyl linoleate or linolenate. Non-2-enal upon absorption of 0.5 mole of oxygen was oxidized almost quantitatively to non-2-enoic acid. Hepta-2,4-dienal was polymerized at 0.5 mole of oxygen uptake. In addition both of the unsaturated aldehydes produced shorter chain mono- and dicarbonyls as oxidative degradation products. The identification of these compounds helps to explain the presence of carbonyls in oxidizing lipids and model systems that are not accountable through the decomposition of theoretically predictable isomeric hydroperoxide esters. The relatively large yield of malonaldehyde from the oxidized dienal suggests that these carbonyls may serve as a major source of malonaldehyde in oxidizing diene esters. Significant quantities of malonaldehyde are not observed in methyl linoleate until late stages of oxidation, and the dienals formed through degradation of primary hydroperoxides may in turn oxidize to give malonaldehyde. Technical Paper No. 1804, Oregon Agricultural Experiment Station. Submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy.     

Influence of cholesterol on the kinetics of lipid autoxidation and on the antioxidative properties of α‐tocopherol and quercetin

The autoxidation kinetics of triacylglycerols of sunflower oil (TGSO) in the presence of 10% cholesterol (Chol) at 80, 90 and 100 °C has been studied. The process was followed by monitoring the peroxide value and the formation of conjugated dienes. Cholesterol has been found to exhibit a prooxidative effect. During the oxidation of the mixture (TGSO/Chol), cholesterol peroxides were not registered. It is supposed that the initial amount of cholesterol peroxides formed decomposes to free radicals and that these radicals accelerate TGSO oxidation. A kinetic analysis of the antioxidative behavior of α‐tocopherol and quercetin (2.9 x 10^?4‐17.8 × 10^?4 M) in both TGSO and TGSO/Chol at 100 °C was performed. It was found that the effectiveness, strength, and activity of α‐tocopherol are greater in TGSO/Chol than in TGSO, while these parameters for quercetin are practically the same in both lipid systems. The differences in the mechanism of action of α‐tocopherol and quercetin are discussed.