Effect of phenolic compounds extracted from sunflower seeds on native lipoxygenase activity

Se obtuvieron extractos de lipoxigenasa de semillas de girasol usando 12 mezclas de extracción diferentes, y se determinó la actividad lipoxigenasa y el contenido en compuestos fenólicos. Este último se llevó a cabo mediante medida de la relación de absorbancias a 320/280 nm. El análisis estadístico de los resultados para los extractos mostró una correlación entre la actividad lipoxigenasa y la relación OD32onm/OD28onm con T = 0,619. Sin embargo, después de la diálisis de los extractos, no se observó esta correlación.


INTRODUCTION
Lipoxygenase (E.G. 1.13.11.12) ¡s an enzyme, which catalizes the oxidation of fatty acid containing CIS, cis 1,4 -pentadiene moieties into fatty acids hydroperoxides containing a conjugated double bond system.The hydroperoxides can futher be degraded either enzymaticaily or non-enzymatically (Eskin etaL, 1977).Lipoxygenase-generated products contribute to either desirable, fresh flavours arising from metabolic processes or undiserable off-flavours (Sessa, 1979).Lipoxygenase is generally distributed in oilseeds, which are characterized by a high content of phenolic compounds.Some of these compounds are inhibitors of enzymes as lipoxygenases activity (Amarowicz and Fornal, 1995;Zadernowski et al., 1998).
Sunflower seeds are a rich source of phenolic compounds (Leung et al., 1981;Sosulski, 1979) but the interaction of these compounds extracted together with lipoxygenase on the activity of this enzyme has not yet been characterized.Therefore, the aim of this study was to investigate the relationship between phenolics compounds and lipoxygenase activity in crude enzyme extracts from sunflower seeds.

MATERIALS AND METHODS
Sunflower seeds were obtained from Seeds Central Company in Olsztyn (Poland).Seeds were dehulled and defatted with acetone on MPW-120 homogenizer.
Enzymatic extracts with different lipoxygenase activity and content of phenolics compounds were prepared from the deffated seeds using the following extraction mixtures at a 1:10 (w/v) ratio (Gambor and Zaiik, 1958; Leoni et al., 1985;Shiba et. al., 1991;Price, 1993)  Extraction was performed at temperatures between 0° C and 4° C for 60 min.The mixture was then centrifuged at 24000 x g for 20 min.Pellets were discarded and supernatants were used for the enzyme assay.
Low molecular compounds, including phenolic compounds, were removed by dialysis against 6 litres of 0.1 M phosphate buffer, pH 7.0 carried out in dialysis bags with 16 kDa cuts off overnight.After dialysis, the extracts were centrifuged at 8000 x g for 20 min.
The relative content of phenolic compounds in the extracts was determined by optical density measurement at 280 and 320 nm (Beckman DU 7500 diode array spektrophotometer ) using cuvettes with a 1 cm path length.Results were expressed as the ratio of the optical density at 320 nm/optical density at 280 nm (i.e.OD32onm/OD28onm).Dlroct determination of phenolic compounds using colorimetric method was difficult because a Folin-Denis reagent gives also a positive reaction with aromatic amino acids for extracts originated from lipoxygenase or other proteins molecules.Many phenolic compounds, above all phenolic acids, show strong absorption band at 320 nm (Shahidi & Naczk, 1995).In contrast a wavelength 280 nm is used for protein determination.
Lipoxygenase activity in supernatants before and after dialysis was measured spectrophotometrically at 25° C (Zimmerman and Vick, 1970) with linoleic acid as the substrate.An emulsion of linoleic acid was prepared according to Surrey's (1964) method and as modified by Shiiba etal., (1991).Results are presented in activity units per mg protein (A.U./mg protein).One unit of lipoxygenase activity corresponds to the amount of the enzyme that produces one unit change of absorbance at 234 nm per minute.The protein content in extracts was determined according to Bradford's method (1976) and bovine serum albumin (Sigma) was used as a standard.
Regression analysis was performend on «Excel» statistical system.

RESULTS AND DISCUSSION
Lipoxygenase extracts with specific activities ranging from 2.38 (extraction mixture No 10) to 5.61 (No 11) activity units/mg protein could be selected from the various extracts so obtained (Table I A relationship between phenolic compounds present in the extracts and lipoxygenase activity was confirmed by regression analysis.The correlation coefficient for the relationship between the OD320nm/OD280nm Indox and lipoxygenase activity was statistically significant, but only for crude extracts (a = 0.05) (Fig. 1).For dialysed samples, the statistic dependence disappeared (Fig. 2).The highest OD32onm/OD28onm ratio after dialysis of lipoxygenase extracts were observed when an acetate buffer pH 5,0 or an acetate buffer pH 5,0 containing sodium bisulfite were employed.This suggests that the phenolic compounds were probably irreversibly bound to proteins to a great extent at pH 5.0 than at pH 7.0.Phenolic compounds can interact with proteins reversibly by means of hydrogen binding or irreversibly, when covalent binding of their oxidazed form to the protein takes place (Sabir et al., 1974;Sosulski, 1979).
The influence of phenolic compounds from the extract on lipoxygenase activity, observed in this study, is in agreement with literature data of other oilseeds.Inhibition of soybean lipoxygenase by chain-breaking antioxidants was noted by Maccarone atal., (1995).Arachidonate lipoxygenase was strongly inhibited by extract from olives.One of the compounds responsible for this inhibition was purified and identified as 2-(3,4-dihydrophenyl)ethanol (Kohyama et al., 1997).Caffeic acid phenylethyl ester was found as an inhibitor of lipoxygenase activity (Sud'ina etal., 1993).Soybean lipoxygenase was inhibited in a model system by lipo-and hydrophobic phenolic compounds extracted from rapeseed (Zadernowski etal., 1998).
In conclusion, evidence is presented that phenolic compounds extracted together with lipoxygenase inhibited enzyme activity.Partial identification of these compounds is in progress.

Figure 2
Figure 1 Correlation between lipoxygenase specific activity and theOD320nm/OD280nm ratio for crude extracts