Development of a Rapid and Robust Method to Measure Antioxidant Activity in a Lipid System

Md Ahsan Ghani

Research output: ThesisDoctoral Thesis

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Lipid oxidation is problematic in both the food industry (oxidative rancidity) and
human health (diseases such as atherosclerosis). Some lipids such as polyunsaturated fatty acids (PUFAs, e.g. (linoleic acid (LA)), are very susceptible to oxidation in the presence of heat, light or trace amount of metal ions. This oxidation produces harmful primary oxidation products such as peroxides, which breakdown to numerous toxic secondary oxidation products such as aldehydes, conjugated dienes, and furans. Of the products, aldehydic malondialdehyde (MDA) is most studied as it is associated with off-flavours and aromas in meat products, as well as being a marker of oxidative damage in physiological systems. MDA, and some other lipid oxidation products react with thiobarbituric acid (TBA) to give pink/red coloured adducts, which are measured in the thiobarbituric acid reactive substances (TBARS) assay. On the other hand, peroxides may be measured by the ferric thiocyanate (FTC) assay. These two assays are of the several methods such as conjugated diene (CD), ferrous oxidation xylenol orange (FOX) and hexanal to measure lipid oxidation products.

Antioxidants are compounds that can inhibit the formation of oxidation products
when present at a much lower concentration compared to an oxidisable substrate. Both FTC and TBARS assays can be used to measure the effectiveness of an antioxidant (i.e., antioxidant activity) in protecting a lipid system from oxidative damage. The above assays consist of two steps: lipid oxidation and colour development, and the former was found to be the source of variability in the widely used TBARS assay. The variability in the TBARS assay includes imprecision of results in intra and inter-laboratory trials. Both FTC and TBARS assays have the same oxidation step, which needs a long time for the lipid substrate to be oxidised. This makes the two assays slow. As TBARS assay has variability in results, and both TBARS and FTC assays are slow in screening antioxidant activity, there is a need to develop a method so the assays can be rapid and more robust.

In this thesis, a rapid and robust method incorporating both FTC and TBARS assays was developed. It was carried out in several stages. In the first stage, the variability in the TBARS assay was investigated in a multi-phase oxidation system using a literature method. Steps were taken to minimise or overcome the variability. Having no or little possibility of overcoming variability, an alternative LA-emulsion system was chosen and the system was tested for variability in results, as above. The LA emulsion showed good intra-and inter-batch precision for the FTC and TBARS assays in measuring oxidation and antioxidant activity. Therefore in this oxidation system, the conditions of reaction were altered so that the assay became rapid. After optimising the method to be more rapid and robust, it was applied to screen antioxidant activity of Australian native plants used in traditional Indigenous medicine.

In the first stage of this thesis, linoleic acid (LA) was oxidised in the multi-phase
system by atmospheric oxygen with vigorous shaking to promote interaction with a separate aqueous layer of Cu2+ catalyst and Trolox as antioxidant. Initial monitoring of the results showed variability in the TBARS assay. Therefore a systematic study was conducted to identify possible sources of variability including: order of addition of reactants; pre-formed peroxides in LA, and the effect of different concentrations of added antioxidant, Trolox. All contributed to variability for the measurement of antioxidant activity of Trolox. In this multi-phase system, breakdown of peroxides did not form TBARS consistently from one experiment to the next. Through the use of LA with pre-formed peroxides removed, the variability was not minimised. It was also found that antioxidant activities reported in other studies were most likely not due to the protective effect of antioxidant on the LA substrate, rather than the antioxidant inhibits the breakdown of pre-formed peroxides. In contrast to the multiphase system, oxidation of LA in an emulsion showed good intra-and inter-batch precision for the FTC and TBARS assays in measuring oxidation and antioxidant activity.

In the second stage of this thesis, the oxidation of the LA emulsion system was
modified by altering the reaction parameters such as temperature, catalyst, and
oxygen initiator in order to make the oxidation faster. To validate the method, the order of antioxidant activity for oxidation of LA at 37 °C for 20 h, with 250 µM antioxidants (Trolox, quercetin, ascorbic acid, and gallic acid) was used. Of the altered reaction parameters, only 50 °C provided an oxidation system consistent with the validation conditions reducing the assay time from 20 hours to 5 hours. Further validation of the method was achieved with antioxidants (+)- catechin, (-)- epicatechin, caffeic acid, and α-tocopherol. Similar order of antioxidant activity was observed under accelerated conditions. It showed that the method could distinguish between different levels of protection of LA and thus could be used for screening purposes.

The optimised method was used to screen the antioxidant activity of crude extracts and fractions (n-hexane, DCM, EtOAc and EtOH) of three species of Australian native plants, vis. Acacia implexa bark and leaves, Eucalyptus rossii and Exocarpos cupressiformis leaves. All crude extracts of all species showed appreciable antioxidant activity against formation of both peroxides and TBARS. The non-polar n-hexane and DCM fractions showed high antioxidant activity in both FTC and TBARS assays. For comparison, the Folin-Ciocalteu and 2,2’-azinobis (3-ethylbenzothiazolline-6-sulfonic acid) (ABTS) assays were also performed and the more hydrophilic fractions had high antioxidant activity in these assays, while the lipophilic fractions showed low activity. Thus screening with a lipid-based assay revealed antioxidants that were more potent in a lipid system, but which might have been missed if only aqueous-based tests had been used.

In order to identify potential antioxidants in the above extracts and fractions,
chromatographic analysis was undertaken by liquid chromatography diode array
detection quadrupole time of flight mass spectrometry (LC-DAD-qTOF-MS). Major peaks were tentatively assigned for each species. In this thesis, for the first time, several compounds such as rhamnetin, hederagenin, and quercetin diglucoside in Acacia implexa bark, (-)- epicatechin, rutin, and kaempferol in Acacia implexa leaves, ellagic acid, sideroxylin, and eucalyptin in Eucalyptus rossii, and (+)- catechin, and quercetrin in Exocarpos cupressiformis leaves have been reported. Some of these compounds are flavonoids, which are well studied antioxidants and are likely to contribute to the antioxidant activity as measured using the developed assay.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
  • Charles Sturt University
  • Prenzler, Paul, Co-Supervisor
  • Barril, Celia, Co-Supervisor
  • Bedgood, Danny, Co-Supervisor
Award date01 Mar 2016
Place of PublicationAustralia
Publication statusPublished - 2016

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