Characterization of natural antioxidants of meals of borage and evening primrose

Wettasinghe, Mahinda (1999) Characterization of natural antioxidants of meals of borage and evening primrose. Doctoral (PhD) thesis, Memorial University of Newfoundland.

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Abstract

Antioxidant efficacy of borage and evening primrose meals in a cooked comminuted pork model system was investigated. Borage meal, at 2% (w/w), reduced the formation of thiobarbituric acid-reactive substances (TBARS), hexanal and total volatiles in treated samples by 27. 30 and 19%, respectively. Formation of TBARS. hexanal and total volatiles in samples containing 2% (w/w) evening primrose meal was reduced on day-7 of the storage by 44, 73 and 63%, respectively. Since meals demonstrated antioxidant properties, their crude extracts were prepared under optimum extraction conditions which were determined by employing response surface methodology (RSM). Effects of three variables, namely the solvent content in the aqueous extraction medium (x₁, %, v/v), extraction temperature (x₂, °C) and time (x₃, min) on antioxidant efficacy (Y) of the extracts were investigated. RSM predicted that the maximum antioxidant activity of borage extract was reached when x₁, x₂, and x₃, were 52% ethanol (v/v). 72°C and 62 min. respectively. The corresponding optimum extraction conditions for evening primrose were predicted to be 56% acetone (v/v), 71°C and 47 min. respectively. Fitted polynomial models for borage and evening primrose were significant (p ≤ 0.05) and reproducible (CV < 5%). Verification experiments carried out to determine the adequacy of the models showed that the predicted response values were well in agreement with the observed values (r < 0.95). Crude extracts prepared under optimum extraction conditions, were subjected to Sephadex LH-20 column chromatography. For both types of extracts, six fractions (I-VI) were obtained and their contents of total, hydrophobic and hydrophillic phenolics determined. Borage fractions I-VI consisted of 283, 129, 140, 366, 280 and 347 mg of phenolics as sinapic acid equivalents, respectively, while evening primrose fractions I-VI contained 158, 313, 369, 402, 279 and 445 mg of phenolics as catechin equivalents, respectively. Borage fractions contained more of hydrophillic than hydrophobic phenolics whereas evening primrose fractions contained high amounts of both types of phenolics. A qualitative vanillin test was employed to determine the presence or absence of condensed tannins in borage and evening primrose fractions. Borage fractions did not contain condensed tannins as evidenced by a negative vanillin test, but fractions III-VI of evening primrose did. Ultraviolet (UV) spectra of borage fractions indicated possible presence of phenolic acids while those for evening primrose fractions suggested possible presence of procyanidins. -- Antioxidant efficacies of borage and evening primrose crude extracts and their fractions (additives) were investigated in β-carotene-linoleate, cooked comminuted pork, bulk stripped corn oil and stripped corn oil-in-water emulsion systems. In general, all additives exhibited varying antioxidant activities in all four types of model systems investigated. After a 2 h assay period, borage and evening primrose additives were able to retain 27-79% and 37-84% of initial content of β-carotene, respectively, as compared to 10% retention in the control. In cooked comminuted pork model systems, borage and in evening primrose additives inhibited the formation of TBARS, hexanal and total volatiles (on day-3 of storage) to varying degrees (19-97%) as compared to the control. On day-3 of storage. 200 ppm (as sinspic acid equivalents) of borage additives inhibited the formation of conjugated dienes, hexanal and total volatiles in bulk stripped-corn oil and its oil-in-water emulsions by 21-95% as compared to the control. Inhibition of formation of oxidation products in samples treated with 200 ppm (as catechin equivalents) of evening primrose additives, on day-3, ranged from 17 to 94% as compared to the control. -- In general, borage additives were better antioxidants in bulk stripped corn oil systems than their emulsion counterparts. Evening primrose additives performed well in almost all systems examined. These effects were attributed to the different affinities of active compounds to various phases and interfaces of the model systems. In an attempt to investigate the antioxidant mechanisms of additives, iron (II) chelating, reactive-oxygen species (ROS) scavenging and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activities of borage and evening primrose additives were also determined. Iron (II) binding capacities of borage and evening primrose additives ranged from 33 to 100%, depending upon the type and concentration of additives investigated. In general, borage and evening primrose additives exerted strong ROS scavenging properties of 100% in most cases. Borage and evening primrose additives also exhibited strong DPPH radical scavenging activities. An attempt was made to correlate iron (II) chelating, ROS and DPPH scavenging activities of the additives to the antioxidant activities brought about by respective additives in model systems; correlations ranged from weak (r < 0.6) to very strong (r < 0.9), depending mainly upon the nature of the model system involved, as determined by linear regression analysis. -- For the first time, major phenolic antioxidants present in the borage and evening primrose extracts were identified. Presence of rosmarinic, syringic and sinapic acids in borage extract was confirmed by chromatographic as well as UV, mass and nuclear magnetic resonance (NMR) spectroscopic data. Instrumental analysis of isolated evening primrose phenolics allowed identification of (+)catechin, (-)epicatechin and gallic acid as the major active compounds responsible for antioxidant activity of its extracts.

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