Anu Hopia

Antimicrobial effects of Finnish plant extracts containing flavonoids and other phenolic compounds

Plant phenolics, especially dietary flavonoids, are currently of growing interest owing to their supposed functional properties in promoting human health. Antimicrobial screening of 13 phenolic substances and 29 extracts prepared from Finnish plant materials against selected microbes was conducted in this study. The tests were carried out using diffusion methods with four to nine microbial species (Aspergillus niger, Bacillus subtilis, Candida albicans, Escherichia coli, Micrococcus luteus, Pseudomonas aeruginosa, Saccharomyces cerevisiae, Staphylococcus aureus and Staphylococcus epidermidis). Flavone, quercetin and naringenin were effective in inhibiting the growth of the organisms. The most active plant extracts were purple loosestrife (Lythrum salicaria L.) against Candida albicans, meadowsweet (Filipendula ulmaria (L.) Maxim.), willow herb (Epilobium angustifolium L.), cloudberry (Rubus chamaemorus L.) and raspberry (Rubus idaeus L.) against bacteria, and white birch (Betula pubescens Ehrh.), pine (Pinus sylvestris L.) and potato (Solanum tuberosum. L.) against gram-positive Staphylococcus aureus.

Effects of α- and γ-tocopherols on the autooxidation of purified sunflower triacylglycerols

The antioxidant effects of α- and γ-tocopherols were evaluated in a model system based on the autooxidation of purified sunflower oil (p-SFO) triacylglycerols at 55°C for 7 d. Both tocopherols were found to cause more than 90% reduction in peroxide value when present at concentrations >20 ppm. α-Tocopherol was a better antioxidant than γ-tocopherol at concentrations ≤40 ppm but a worse antioxidant at concentrations >200 ppm. Neither α- nor γ-tocopherol showed a prooxidant effect at concentrations as high as 2000 ppm. The amount of tocopherols consumed during the course of oxidation was positively correlated to the initial concentration of tocopherols, and the correlation was stronger for α- than for γ-tocopherol. This correlation suggested that, besides reactions with peroxyl radicals, destruction of tocopherols may be attributed to unknown side reactions. Addition of FeSO4, as a prooxidant, caused a 12% increase in the peroxide value of p-SFO in the absence of tocopherols. When tocopherols were added together with FeSO4, some increase in peroxide value was observed for samples containing 200, 600 or 1000 ppm of α- but not γ-tocopherol. The addition of FeSO4, however, caused an increase in the amount of α- and γ-tocopherols destroyed and led to stronger positive correlations between the amount of tocopherols destroyed during oxidation and initial concentration of tocopherols. No synergistic or antagonistic interaction was observed when α- and γ-tocopherols were added together to autooxidizing p-SFO.

Flavonoids quercetin, myricetin, kaemferol and (+)‐catechin as antioxidants in methyl linoleate

The antioxidant effect of the flavonoids quercetin, myricetin, kaemferol, (+)-catechin and rutin on methyl linoleate oxidation was investigated. In addition, the synergistic effects of flavonoids and alpha-tocopherol were studied. Oxidation was monitored by conjugated diene measurement and by determining the formation of hydroperoxide isomers by HPLC. The antioxidant activity of flavonoids in non-polar methyl linoleate differ from that previously reported in water-containing systems, such as LDL and liposome systems. The activity of antioxidants (10-1000 micromolar) measured by hydroperoxide formation decreased in the order: myricetin > quercetin > alpha-tocopherol > (+)-catechin > kaemferol = rutin. The antioxidant activity of flavonoids increased as the number of phenolic hydroxyl groups increased. In addition to the number of hydroxyl groups, other structural features such as the 2,3 double bond in the C-ring and a glycoside moiety in the molecule had an effect on the antioxidant activity. Myricetin and rutin, especially had a synergistic effect with alpha-tocopherol. Myricetin, quercetin and rutin protected alpha-tocopherol from decomposition, myricetin being the most protective. The relative hydrogen-donating activity measured by the ration of cis,trans- to trans,trans-hydroperoxide isomers formed during oxidation decreased in the order: alpha-tocopherol > myricetin > quercetin. Hydroperoxide isomeric distribution of the samples containing kaemferol or rutin did not differ from the control. Thus, although alpha-tocopherol was the most effective hydrogen-donor, myricetin and quercetin were more effective antioxidants in inhibiting the hydroperoxide formation in methyl linoleate.

Analysis of lipid classes by solid-phase extraction and high-performance size-exclusion chromatography

An improved method to analyze lipid classes of edible oils and fats by solid-phase extraction (SPE) and high-performance size-exclusion chromatography (HPSEC) is presented. A mixture of lipid standards was fractionated by the solid-phase extraction procedure (NH2 phase) into polar and nonpolar fractions; these were then submitted to analysis by HPSEC. The size-exclusion chromatographic columns were three styrene/divinylbenzene columns with pore sizes of 100 Å and 50 Å. Light-scattering was used for the detection system, and the parameters of the detector were optimized to minimize the difference between the responses of the compounds studied. With this procedure it was possible to separate the following lipid classes: triacylglycerols, diacylglycerols, monoacylglycerols and free fatty acids, sterols, sterol esters, tocopherols and carotenoids. Quantitative analysis was studied for a light-scattering detector with several lipid standards of different molecular weights and unsaturation levels.

Hydroperoxide formation during autoxidation of conjugated linoleic acid methyl ester

The aim of this study was to investigate whether hydroperoxides are formed in the autoxidation of conjugated linoleic acid (CLA) methyl ester both in the presence and absence of α-tocopherol. The existence of hydroperoxide protons was confirmed by D2O exchange and by chemoselective reduction of the hydroperoxide groups into hydroxyl groups using NaBH4. These experiments were followed by nuclear magnetic resonance (NMR) spectroscopy. The 13C and 1HNMR spectra of a mixture of 9-hydroper-oxy-10-trans,12-cis-octadecadienoic acid methyl ester (9-OOH) and 13-hydroperoxy-9-cis, 11-trans-octadecadienoic acid methyl ester (13-OOH), which are formed during the autoxidation of methyl linoleate, were studied in detail to allow the comparison between the two linoleate hydroperoxides and the CLA methyl ester hydroperoxides. The 13CNMR spectra of samples enriched with one of the two linoleate hydroperoxide isomers were assigned using 2D NMR techniques, namely Correlated Spectroscopy (COSY), gradient Heteronuclear Multiple Bond Correlation (gHMBC), and gradient Heteronuclear Single Quantum Correlation (gHSQC). The 13C and 1H NMR experiments performed in this study show that hydroperoxides are formed during the autoxidation of CLA methyl ester both in the presence and absence of α-tocopherol and that the major isomers of CLA methyl ester hydroperoxides have a conjugated monohydroperoxydiene structure similar to that in linoleate hydroperoxides.

Determination of hydroperoxides and structures by high-performance liquid chromatography with post-column detection with diphenyl-1- pyrenylphosphine

A high-performance liquid chromatographic method, using post-column detection with diphenyl-1-pyrenyl-phosphine (DPPP), was developed for the quantitative and qualitative determination of isomeric lipid hydroperoxides (OOH). The OOH eluted from a normal-phase column were passed through a photodiode array detector and then mixed with DPPP solution in a reaction coil heated at 80°C. DPPP oxide formed by the reaction with OOH was determined by monitoring the fluorescence intensity at 380 nm and excitation at 352 nm. The conjugated diene OOH (13-cis, trans- and 9-cis, trans-OOH) and nonconjugated OOH (12-cis-trans- and 10-cis, trans-OOH) from photosensitized oxidation of methyl linoleate were determined in a molar ratio of 31∶29∶19∶21, respectively. However, only the two conjugated hydroperoxides were detected by ultraviolet absorption at 234 nm. Further applications were carried out for the determination of OOH of methyl oleate and methyl linolenate. This method proved to be useful for the determination of the OOH containing both conjugated and nonconjugated diene structures.

Margarines, butter and vegetable oils as sources of polycyclic aromatic hydrocarbons

Concentrations of polycyclic aromatic hydrocarbons (PAH) were determined in Finnish butter, margarines and vegetable oils and their raw materials. In all the samples some degree of PAH contamination was found. The average per capita intake was estimated at 0.6 µg per day. The plant raw materials are supposed to be contaminated by combustion-derived atmospheric particles during the growing period. Inappropriate drying processes can substantially increase the contamination level. Deodorization processes used by food industry seem to decrease the total PAH levels significantly.

Application of High-Performance Size-Exclusion Chromatography to Study the Autoxidation of Unsaturated Triacylglycerols

A combination of solid-phase extraction (SPE) and high-performance size-exclusion chromatography (HPSEC) was used to study the autoxidation of triacylglycerol (TAG) mixtures separated from low-erucic acid rapeseed oil and butter oil. The samples were autoxidized in the dark at 40°C for four weeks. The polar compounds of the autoxidated samples were separated by SPE (NH2 stationary phase), and the polar fraction was further characterized by HPSEC with a series of three size-exclusion columns and an evaporative light-scattering detector. The polar fraction contained TAG polymers, polar TAG monomers (PTAG) and diacylglycerols. Peroxide values and anisidine values of the samples were also measured. By using three different types of TAG mixtures, it could be demonstrated that the PTAG content of the TAGs increases during autoxidation. A slight increase was also detected in polymer content. The correlation between PTAG content and the comparative measurements was considered significant. The results indicate that the measurement of PTAG and polymeric material content by HPSEC analysis can be used when studying the autoxidation level of edible oils and in characterizing the autoxidation products of different molecular sizes.

Postcolumn chemiluminescence, ultraviolet and evaporative light-scattering detectors in high-performance liquid chromatographic determination of triacylglycerol oxidation products

Postcolumn chemiluminescence (CL), ultraviolet (UV) and evaporative light-scattering (ELS) detectors were compared in the HPLC of triacylglycerol (TAG) oxidation products. Polar lipid fractions from autoxidized TAG samples were first separated from non-polar material by solid-phase extraction and then analysed by RP-HPLC in combination with UV-CL and UV-ELS detectors. TAG hydroperoxides were detected with a CL detector, oxidation products containing conjugated diene structures with a UV detector and all non-volatile oxidation products with an ELS detector. More accurate characterization of the oxidation products was possible by combining the information from different detectors. For samples at early stages of oxidation, the CL and UV detectors were more suitable because of their better sensitivity and selectivity compared with the ELS detector.

α-, γ- and δ- Tocopherols as inhibitors of isomerization and decomposition of cis,trans methyl linoleate hydroperoxides

The effects of α-, γ- and δ-tocopherols on the stability and decomposition reactions of lipid hydroperoxides were studied. Isomerization and decomposition of cis,trans methyl linoleate hydroperoxides (cis,trans ML-OOH) in hexadecane at 40 °C were followed by high-performance liquid chromatography. Due to its higher hydrogen donating ability, α-tocopherol was more efficient than γ- and δ-tocopherols in inhibiting the isomerization of cis,trans ML-OOH to trans,trans ML-OOH. α-Tocopherol stabilized hydroperoxides into the cis,trans configuration, whereas γ- and δ-tocopherols allowed hydroperoxides to convert into trans,trans isomers. Thus, the biological importance of α-tocopherol as compared to other tocopherols may be partly due to its better efficacy in protecting the cis,trans configuration of hydroperoxides formed, for example, in the enzymatic oxidation of polyunsaturated fatty acids. The isomeric configuration of hydroperoxides has an impact on biological activities of further oxidation products of polyunsaturated fatty acids. Paradoxically, the order of activity of tocopherols with regard to hydroperoxide decomposition was different from that obtained for hydroperoxide isomerization. γ- and δ-tocopherols were more efficient inhibitors of ML-OOH decomposition when compared to α-tocopherol. A loss of antioxidant efficiency, observed as the tocopherol concentration increased from 2 to 20 mM, was highest for α-tocopherol but was also evident for γ- and δ-tocopherols. Thus, the differences in the relative effects of tocopherols at differing concentrations seem to result from a compromise between their radical scavenging efficiency and participation in side reactions of peroxidizing nature.