ABSTRACT: A simple and relatively inexpensive procedure to obtain 90% eicosapentaenoic acid + docosahexaenoic acid concentrates from sardine oil involved a two-step winterization of the oil (10 and 4ºC), followed by saponification and selective precipitation of saturated and less unsaturated free fatty acids by an ethanolic solution of urea. Antioxidant effects of butylated hydroxytoluene, dl-α-tocopherol, and two natural antioxidants, quercetin and boldine, added to the concentrate (0.5% wt/vol), were compared in the Rancimat at 60°C. dl-α-Tocopherol was unable to inhibit concentrate oxidation. Butylated hydrox- yanisole and butylated hydroxytoluene had induction periods of 1.7–1.8 h compared to the control (1.0 h). A mixture of quercetin + boldine (2:1 w/w) significantly increased the induc- tion period to 4.5 h. JAOCS 75, 733–736 (1998). KEY WORDS: Boldine, natural antioxidant, polyunsaturated fatty acid concentrates, quercetin, Rancimat test, synthetic an- tioxidant. Nutritional and pharmacological effects of n-3 polyunsatu- rated fatty acids from marine origin have raised interest in de- veloping processes to concentrate eicosapentaenoic acid (20:5, EPA), and docosahexaenoic acid (22:6, DHA) (1). Samples enriched in these fatty acids are needed to further in- vestigate their nutritional, health, and biochemical effects. Procedures to obtain EPA and DHA include low-temperature crystallization (2), silver-resin chromatography (3), acidoly- sis catalyzed by immobilized lipases (4), and supercritical ex- traction (5). Although these procedures produce 75–80% EPA and DHA concentrates, they are suitable for laboratory-scale only and are expensive for industrial application. In the pres- ent work, we adapted a simple, rapid, and relatively inexpen- sive procedure to concentrate EPA + DHA as free fatty acids from fresh sardine oil. Polyunsaturated fatty acids are highly susceptible to ox- idative rancidity, which produces undesirable changes in the chemical and sensory (flavor) properties of the product and generates potentially toxic end-products (6). Oxidation may be avoided or delayed by addition of antioxidants, of either synthetic or natural origin. During the last two decades, syn- thetic antioxidants, such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT), have raised concern be- cause some deleterious and potentially toxic effects have been observed (7–9). Antioxidants of natural origin may offer an alternative to synthetic antioxidants (10). A number of prod- ucts, extracted mainly from vegetables, may have antioxidant properties (11). We demonstrated that flavonoids, such as morin, quercetin and rutin, and boldine, an aporphine ex- tracted from the bark of the Boldo tree (Peumus boldus Mol.), showed potent and synergistic antioxidative effects by in- hibiting metal- or temperature-induced oxidation of fish oil (12,13). In this work, we assayed the effectiveness of quercetin and boldine, compared to BHT, BHA, and dl-α-to- copherol, for stabilizing temperature-induced oxidation of EPA + DHA concentrate from sardine oil. EXPERIMENTAL PROCEDURES Materials. Recently refined sardine oil was obtained from a local fish meal and fish oil factory (CORPESCA S.A., Mejil- lones, Segunda Región, Chile). BHA, BHT, and quercetin were obtained from Sigma (St. Louis, MO). dl-α-Tocopherol, in free form, was obtained from Productos Roche (Santiago, Chile). Boldine was crystallized to chromatographic purity from the crude alkaloidal mixture extracted from Boldo bark (14). Boron trifluoride (14% ethanol) was purchased from Merck (Merck Quimica Chilena, Santiago, Chile). The sol- vents (analytical grade) were obtained from Romil Chemicals (Leicestershires, England), and urea (99%) was obtained from J.T. Baker (Phillipsburg, NJ). Procedure. Sardine oil was deodorized under high-vacuum distillation to reduce peroxides and cholesterol content (15). Recently distilled oil (20 L), with a peroxide value below 0.5 meq/kg (16) and a cholesterol content below 20 mg% (17), was winterized in a two-step temperature-controlled process, first at 10ºC for 48 h, and then at 4ºC for 72 h. After each win- terization step, the supernatant was recovered by centrifuga- tion (2500 × g, 10 min). Final volume recovered after winter- ization was 18.2 L of oil. Two liters of winterized oil was saponified by adding 2 vol of an ethanolic (70%) KOH (7 N) EDTA (5.0 mM) solution, heating at 90ºC under nitrogen in a stirred, steam-jacketed, 15-L kettle, and keeping under reflux for 2 h. The mixture was cooled to ambient temperature by Copyright © 1998 by AOCS Press 733 *To whom correspondence should be addressed. E-mail: avalenzu©uec.inta.uchile.cl Concentration and Stabilization of n-3 Polyunsaturated Fatty Acids from Sardine Oil Angélica Ganga a , Susana Nieto b , Julio Sanhuez b , Claudio Romo a , Hernán Speisky b , and Alfonso Valenzuela b, * a CECTA, Universidad de Santiago, and b Unidad de Bioquímica Farmacológica y Lípidos, Instituto de Nutrición y Tecnología de los Alimentos, Universidad de Chile, Casilla 138-11, Santiago, Chile