High performance liquid chromatographic determination of nine phenolic antioxidants in oils, lards, and shortenings

A simple, rapid technique is described for the determination of 2- and 3-tert-butyl-4-hydroxyanisole (BHA), tert-butylhydroquinone (TBHQ), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), 2,6-di-tert-butyl-4-hydroxymethylphenol (Ionox-100), 2,4,5-trihydroxybutyrophenone (THBP), propyl gallate (PG), octyl gallate (OG), dodecyl gallate (DG), and nordihydroguaiaretic acid (NDGA) in vegetable oils, lards, and shortenings. The antioxidants are partitioned from hexane-oil into acetonitrile, concentrated under vacuum, and determined by reverse phase, gradient elution, high performance liquid chromatography with detection at 280 nm. The mobile phase is water-acetonitrile with 5% acetic acid. With this system, only Ionox-100 and OG are not resolved. Recoveries from soya-sunflower seed oil spiked at 16 and 100 ppm and from lard at 32 ppm for 8 of the 9 antioxidants ranged from 96 to 103%, 100 to 102%, and 98 to 102%, respectively. The recoveries of BHT, due to incomplete extraction, were 84, 85, and 87%, respectively.     

Effect of antioxidants on soy oil conjugated linoleic acid production and its oxidative stability

Conjugated linoleic acid (CLA)-rich soy oil can be produced by photoisomerization of soy oil linoleic acid to produce a soy oil with up to 20% CLA. Recent studies indicate that mixed soy tocopherols added to refined bleached deodorized (RBD) oil produced significant increase in soy CLA yield during soy oil linoleic acid photoisomerization. However, the effect of common synthetic free radical scavenging antioxidants and specific tocopherols on CLA yield and its oxidative stability is not known. Therefore, this investigation evaluated the effects of various antioxidant systems on soy oil CLA yield and oxidative stability. Soy oil with added antioxidants consisting of combinations of mixed tocopherols (MT), ascorbyl palmitate (AP), butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ) was photoisomerized to produce CLA- rich soy oil. The CLA content was determined by GC-FID analysis and oxidative stability by peroxide value (PV). The soy oil in the presence of TBHQ, MT alone and MT with 500 ppm of AP produced significantly greater CLA yields and improved oxidative stability compared to a control without added antioxidants (p < 0.05). However, added mixed tocopherols produced the greatest CLA yield and also reduced PV relative to the control. Tocopherols in the form of α-, γ- and δ-tocopherols were then each examined as to their relative effect on CLA yields and PV. The largest increase in CLA yield was obtained with 1800 ppm of γ-tocopherols with reduced PV. Mixed tocopherols, TBHQ and γ-tocopherols can be used to increase CLA yield and reduce PV of soy oil during linoleic acid photoisomerization.     

Antioxidant evaluation in dessert spices compared with common food additives. Influence of irradiation procedure

The antioxidant properties of seven dessert spices (anise, cinnamon, ginger, licorice, mint, nutmeg, and vanilla) were compared with those of the common food antioxidants butylated hydroxyanisole (BHA) (E-320), butylated hydroxytoluene (BHT) (E-321), and propyl gallate (E-310). The influence of irradiation process on antioxidant activity was also evaluated. Mint and cinnamon exhibited a higher percentage of inhibition of oxidation than the other spices analyzed and the food antioxidants, as tested by the lipid peroxidation assay (LOO*). Nutmeg, anise, and licorice showed the strongest protection in the deoxyribose assay (OH*). Vanilla exhibited the highest antioxidant activity in the peroxidase-based assay (H2O2). Nutmeg, propyl gallate, ginger, and licorice improved the stability of oils (sunflower, corn, and olive) and fats (butter and margarine) against oxidation (110 degrees C Rancimat). Cinnamon was a better superoxide radical scavenger than the other analyzed spices and additives. When the Trolox equivalent antioxidant capacity (TEAC) assay was used to provide a ranking order of antioxidant activity, the result in decreasing order of antioxidant capacity was cinnamon approximately equal to propyl gallate > mint > anise > BHA > licorice approximately equal to vanilla > ginger > nutmeg > BHT. Irradiated samples did not show significant differences (p < 0.05) in the antioxidant activity with respect to the non-irradiated samples (1, 3, 5, and 10 kGy) in the assays used.     

Effect of antioxidants on oxidative stability of edible fats and oils: thermogravimetric analysis

Thermogravimetric analysis was used to determine the oxidative stability of various edible oils (olive oil, milkfat) and triacylglycerides (triolein, trilinolein), while the effect of natural (alpha-tocopherol, ascorbic acid) and synthetic antioxidants (butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tertiary butyl hydroquinone were evaluated by addition to trilinolein. Oil resistance to oxidation was obtained by measuring the increase in sample weight due to the uptake of molecular oxygen, the temperature at maximum sample weight, and the temperature at the onset of oxidation. When comparing sample weight increase, trilinolein proved to be oxidatively less stable than triolein, olive oil, and milk fat, while triolein was less stable than olive oil and milk fat. Olive oil showed significantly higher stability than milkfat when comparing the temperature at the onset of oxidation. When comparing effectiveness of antioxidants, a combination of 0.01% BHA and 0.01% BHT increased trilinolein stability the most.     

The relationship between the physicochemical properties of antioxidants and their ability to inhibit lipid oxidation in bulk oil and oil-in-water emulsions

Chain-breaking antioxidants differ in their effectiveness at inhibiting lipid oxidation because of their chemical properties and physical location within a food. Our objective was how the physicochemical properties of four structurally related lipid-soluble antioxidants were related to their antioxidant activity. Antioxidants differed in the number of methyl (alpha-tocopherol and delta-tocopherol) or hydroxyl (butylated hydroxytoluene (BHT) and 4-hydroxymethyl-2,6-ditertiarybutylphenol) groups. Surface activity of the antioxidants was in the order of delta-tocopherol > alpha-tocopherol approximately 4-hydroxymethyl-2,6-ditertiarybutylphenol > BHT. Free-radical scavenging activity was similar between alpha-tocopherol and delta-tocopherol as well as BHT and 4-hydroxymethyl-2,6-ditertiarybutylphenol. In bulk menhaden oil, BHT was a more effective antioxidant than 4-hydroxymethyl-2,6-ditertiarybutylphenol while alpha-tocopherol was more effective than delta-tocopherol. In menhaden oil-in-water emulsions, BHT was a more effective antioxidant than 4-hydroxymethyl-2,6-ditertiarybutylphenol while delta-tocopherol was more effective than alpha-tocopherol. These results indicate that the surface activity and polarity of lipid-soluble antioxidants were not the only determinants of their antioxidant effectiveness in food lipids.     

Liquid chromatographic determination of seven antioxidants in dry food

The liquid chromatographic determinative step of the official method for propyl gallate, trihydroxybutyrophenone, tert-butylhydroquinone, nordihydroguaiaretic acid, butylated hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxymethylphenol, and butylated hydroxytoluene (BHT) in fats and oils has been applied to their determination in a number of dry foods. A representative sample (10 g) is homogenized first with hexane (25 mL), then with 5 mL added water, and finally with 75 mL added acetonitrile. The hexane and acetonitrile are decanted, filtered, and separated; the hexane and rehydrated food are reextracted with 2 additional portions of acetonitrile, and the combined acetonitrile extracts are concentrated and diluted to 10 mL. An aliquot is analyzed as described in the official method, using a 150 x 4.6 mm 5 microns C-18 column. The need for rehydration to maximize the recovery of BHA and other antioxidants from marketplace dry food samples such as potato flakes, dry coffee whiteners, and dessert topping mixes was demonstrated. Rehydration was not required for cheese snacks, breakfast cereals, cake mixes, and some other foods. The need for rehydration should be determined by analyzing other foods with and without the addition of water. Potato and corn chips, popcorn and cheese snacks, breakfast cereals, dry beverage mixes, rice, potato flakes, french fried potatoes, and cake mixes were spiked with the above antioxidants at 10-50 ppm. Overall recoveries ranged from 64.3 to 105.6% and repeatabilities ranged from 0.7 to 10.8%. A total of 109 samples of the above foods were analyzed, and 64% contained detectable (greater than 1-2 ppm) antioxidants, mainly BHA and BHT.     

Evaluation of extracts from Gevuina avellana hulls as antioxidants

The antioxidant activity of the extracts from Gevuina avellana hulls was evaluated and compared with that of BHT (butylated hydroxytoluene) and BHA (butylated hydroxyanisole), using the beta-carotene bleaching assay, the accelerated oxidation of crude soybean oil, and the 2,2-diphenyl-beta-picrylhydrazyl (DPPH) radical scavenging method. Solvents of different polarity were used to obtain the extracts. Both the extraction yield and the antioxidant activity were strongly dependent on the solvent. The ethanol and diethyl ether soluble fractions were the most active with the beta-carotene assay. Ethanol and methanol extracts were the most active in hydrogen radical scavenging activity. Water and methanol inhibited more efficiently the oxidation of soybean oil at 70 and 80 degrees C, respectively. As a general trend, increased antioxidant activity was observed for increased extract concentration. Except the acetone extracts, all were stable after 6 months storage at 4 degrees C. The ethanol solubles from G. avellana hulls present antioxidant activity similar to that of synthetic antioxidants and to other reported residual agroindustrial materials.     

Ability of surfactant micelles to alter the partitioning of phenolic antioxidants in oil-in-water emulsions

In oil-in-water emulsions, the physical location of antioxidants can be an important determinant in their activity. Surfactants can potentially influence the physical location of antioxidants in oil-in-water emulsions by causing solubilization of lipid-soluble antioxidants into the aqueous phase. Excess Brij micelles in an oil-in-water emulsion were found to increase the partitioning of phenolics into the continuous phase with polar antioxidants (propyl gallate) partitioning more than nonpolar antioxidants (butylated hydroxyltoluene). Solubilization of propyl gallate was rapid coming to equilibrium in less than 5 min. Increasing surfactant micelle concentrations from 0.3 to 2.8% increased the solubilization of propyl gallate by 2.3-fold. Solubilization of phenolic antioxidants into the aqueous phase by Brij micelles did not alter the oxidative stability of salmon oil-in-water emulsions, suggesting that surfactant micelles influenced oxidation rates by mechanisms other than antioxidant solubilization.     

Antioxidant activity of capsaicinoid in canola oil

Interest in replacing synthetic antioxidants, namely, butylated hydroxytoluene (BHT) and butylated hydroxyanisole (BHA), with natural antioxidants is increasing. The present study examined the antioxidant activity of capsaicinoid from chili pepper in heated canola oil. The oxidation was conducted at 60, 90, 120, and 180 °C by monitoring oxygen consumption and the decrease in linoleic acid and α-linolenic acid in canola oil. At 60 °C, capsaicinoid was more effective against oxidation of canola oil compared with BHT. At higher temperatures of 90, 120, and 180 °C, capsaicinoid possessed an antioxidant activity similar to or slightly weaker that that of BHT. It was found that capsaicinoid prevented canola oil from oxidation in a dose-dependent manner. To study the structure-antioxidant relationship, it was found that the trimethylsiloxy (TMS) derivatives of capsaicinoid did not exhibit any antioxidant activity, suggesting the hydroxyl moiety was the functional group responsible for the antioxidant activity of capsaicinoid. It was concluded that capsaicinoid had the potential to be further explored as a natural antioxidant in foods, particularly spicy foods.     

Distribution of exogenous delta-tocopherol between the membrane lipids and triacylglycerols of a cod muscle-triacylglycerol model system

Membranes of muscle foods are more susceptible to oxidation than triacylglycerols. Hence, directing a lipid-soluble antioxidant into the membranes may reduce the oxidative deterioration of muscle tissue. The objective of this research was to use a model system of cod muscle and triacylglycerol to study the distribution of exogenous delta-tocopherol between the membranes and triacylglycerol fractions of muscle. When ethanol was the carrier solvent, more tocopherol was incorporated into the membranes than when oil was the carrier. Addition of tocopherol to the muscle before the triacylglycerol was added allowed more antioxidant to be incorporated into the membranes than for the case when the oil was added before the antioxidant. When the triacylglycerol was solid, the amount of tocopherol incorporated into the membranes was higher than if the triacylglycerol was liquid and the amount of tocopherol incorporated into the membranes was less dependent on the order of tocopherol and triacylglycerol addition. There was a competition between the membrane lipids and triacylglycerol for uptake of the delta-tocopherol. In some circumstances, some of the tocopherol did not enter either the membrane lipid or triacylglycerol phase.     

Chemical composition and antioxidant properties of clove leaf essential oil

The antioxidant activity of a commercial rectified clove leaf essential oil (Eugenia caryophyllus) and its main constituent eugenol was tested. This essential oil comprises in total 23 identified constituents, among them eugenol (76.8%), followed by beta-caryophyllene (17.4%), alpha-humulene (2.1%), and eugenyl acetate (1.2%) as the main components. The essential oil from clove demonstrated scavenging activity against the 2,2-diphenyl-1-picryl hydracyl (DPPH) radical at concentrations lower than the concentrations of eugenol, butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA). This essential oil also showed a significant inhibitory effect against hydroxyl radicals and acted as an iron chelator. With respect to the lipid peroxidation, the inhibitory activity of clove oil determined using a linoleic acid emulsion system indicated a higher antioxidant activity than the standard BHT.     

Antioxidizing potency of phenol compounds in olive oil mill wastewater

The antioxidizing potency of phenol compounds contained in olive oil mill wastewater (OOMWW) has been elucidated. Commercially available phenol standards at varying concentrations and the Rancimat oxidation test have been used. Refined purified olive oil was utilized as an oxidation lipid substrate. Synthetic antioxidants, such as 2,3-tert-butyl-4-hydroxyanisole (BHA), 3,5-di-tert-butyl-4-hydroxytoluene (BHT), l-ascorbic acid, and gallates (commonly used as food preservatives), and other known chemicals endowed with antioxidizing properties have been employed as reference compounds. The OOMWW phenol compounds have been classified into different groups depending on their antioxidizing potency. This was significantly affected by the tested concentrations of the standards. Mixtures of phenol standards and other antioxidants (l-proline, chlorophyll a, chlorophyll b, and alpha-, gamma-, and delta-tocopherol) have also been tested. Many phenol compounds present in OOMWW showed antioxidizing potency higher compared to that of the less safe synthetic antioxidants and could therefore replace these in the industrial preservation of food items. They could also be used in combination with other natural antioxidants (e.g., tocopherols). In fact, some mixtures of antioxidants, owing also to the synergistic phenomena, showed strong antioxidizing potency.     

Simultaneous stopped-flow determination of butylated hydroxyanisole and propyl gallate using a T-format luminescence spectrometer

A simple and fast luminescent method is used for the first time to resolve a mixture of two synthetic antioxidants, propyl gallate (PG) and butylated hydroxyanisole (BHA), by the joint use of the stopped-flow mixing technique and a T-format luminescence spectrometer. The determination of these compounds involves two different and independent reactions. On the one hand, PG determination is based on an energy transfer process that involves the formation of a lanthanide chelate with terbium in the presence of Triton X-100 and tri-n-octylphosphine oxide. On the other hand, BHA is determined using a reaction between the oxidized form of Nile Blue and the antioxidant. Both systems are excited at the same excitation wavelength (310 nm), and the emission wavelengths are 545 and 665 nm for PG and BHA, respectively. The absence of overlap in the emission spectra makes it possible to measure separately the analytes in each channel of the instrument. Initial rate and equilibrium signal are used as analytical parameters and measured in 0.1 and 1 s for PG and BHA, respectively. Calibration graphs are linear over the range 0.09-3.5 microg mL(-)(1) for PG and 0.3-15 microg mL(-)(1) for BHA. The relative standard deviations of both systems are close to 2%. The proposed method is applied to the determination of these two antioxidants in several commercial food samples with recoveries ranging between 94.8 and 102.9% for PG and between 94.1 and 102.1% for BHA.     

Sequestering ability of butylated hydroxytoluene,propyl gallate,resveratrol, and vitamins C and E against ABTS,DPPH, and hydroxyl free radicals in chemical and biological systems

The antioxidant capacity of butylated hydroxytoluene (BHT; 2,6-di-tert-butyl-p-cresol), propyl gallate (3,4,5-trihydroxybenzoic acid n-propyl ester), resveratrol (trans-3,4',5-trihydroxystilbene), and vitamins C (l-ascorbic acid) and E [(+)-alpha-tocopherol] was studied in chemical and biological systems. The chemical assays evaluated the capacity of these antioxidants to sequester 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.) and 1,1 diphenyl-2-picrylhydrazyl (DPPH.). A new colorimetric method to determine hydroxyl radical scavenging is also described. The biological tests use the eucaryotic cells of Saccharomyces cerevisiae treated with the antioxidants in the presence of the stressing agents apomorphine, hydrogen peroxide, and paraquat dichloride (methylviologen; 1,1'-dimethyl-4,4'-bipyridinium dichloride). The results in chemical systems showed that all of the antioxidants were able to significantly inhibit the oxidation of beta-carotene by hydroxyl free radicals. The assays in yeast showed that the antioxidant activity of the tested compounds depended on the stressing agent used and the mechanism of action of the antioxidant.     

Evaluation of occasional nonresponse of a washed cod mince model to hemoglobin (Hb)-mediated oxidation

An emerging model to test antioxidants for application in seafoods is washed cod mince fortified with hemoglobin (Hb) as a catalyst. This system has been used to test the antioxidative activity of certain muscle extracts and some pure compounds such as BHA, BHT, TBHQ, and propyl gallate during ice storage. However, the washed cod mince model has occasionally been resistant to Hb-mediated oxidation. This has been in cases when the moisture of the model has been minimized by washes at the protein isoelectric point (pH approximately 5.5) to allow for large additions of potentially antioxidative solutions. In this paper, noncontrollable and controllable factors for this intriguing occasional oxidation resistance were studied. Compositional analyses (lipid content, alpha-tocopherol, and lipid hydroperoxides) and structural analysis of a "normal" oxidizing model and a stable model were done to identify any differences among them. Some controllable factors related to the model preparation that were studied included different washing pH values (5.5-6.6), Hb concentrations (7.2 and 13.5 microM), final model moisture contents (75, 81, and 90%), and light exposure during ice storage (0 h, 3-4 h, or 24 h of light/day). Results revealed a 2-fold higher alpha-tocopherol content in the stable model than in the oxidizing model. Electron microscopy images showed a more and less disrupted myofibrillar structure in the stable and the oxidizing cod model, respectively. This indicated that "cold setting" (i.e., pre-gelation) of the stable model may have occurred and prevented Hb from diffusing freely in the model. Controllable factors that reduced lipid oxidation in the models were less Hb and lower moisture.     

Spray-drying of the microalga Dunaliella salina: effects on beta-carotene content and isomer composition.

The effects of spray-drying of the unicellular microalga Dunaliella salina on its beta-carotene content and geometric isomer composition have been studied. The efficacy of a range of synthetic and natural antioxidants in preventing degradation of beta-carotene has been determined. Losses of beta-carotene and isomerization were minimal during processing for both the control (no exogenous antioxidants) and the samples containing butylated hydroxytoluene (BHT) and tert-butylhydroquinone (TBHQ). However, the use of tocopherol-based antioxidants resulted in degradation of 52-72% of beta-carotene during the drying process. All dried powders of Dunaliella proved to be unstable during storage in the presence of light and air, with beta-carotene degraded according to a first-order kinetic model. Of the antioxidants studied, only TBHQ was successful in significantly minimizing degradation (degradation constants of 0.03 and 0.04 days(-)(1), compared to 0.53 days(-)(1) for the respective control). For control powders and those with BHT added to the feed, the degradation constants were reduced to values between 0.27 and 0.37 days(-)(1) by restricting light and flushing with nitrogen; however, storage in the dark alone had no effect. For more slowly degrading powders having TBHQ added to the feed, it was clear that degradation of beta-carotene was influenced by both light and oxygen. During storage the 9-cis isomer of beta-carotene was significantly more unstable than the all-trans form. TBHQ was, however, successful in reducing relative losses of this isomer for samples stored in the dark. The results suggest a dominant photodegradative mechanism for the loss of the 9-cis isomer of beta-carotene.     

Antioxidants modulate molecular mobility, oxygen permeability, and microstructure in zein films

The effect of octyl gallate and propyl gallate on the molecular mobility, oxygen permeability, and microstructure of zein/glycerol films was studied. Films were cast from 70% ethanol/water containing 20% (w/w) glycerol and different amounts of the antioxidants propyl gallate or octyl gallate. The oxygen permeability and local mobility of these films were measured using phosphorescence from the dispersed triplet probe erythrosin B. Although both antioxidants increased the local mobility of the zein matrix to about the same extent, octyl gallate and to a lesser extent propyl gallate dramatically increased the permeability of the film to oxygen. Atomic force microscopy imaging indicated that propyl gallate induced aggregation of zein complexes, which could lead to a more condensed film. These results indicate that the addition of specific functional ingredients, such as antioxidants, to edible films may significantly affect the physical properties and structure and, thus, functional properties in ways that influence their eventual use.     

Hydroxytyrosol prevents oxidative deterioration in foodstuffs rich in fish lipids

Hydroxytyrosol, a natural phenolic compound obtained from olive oil byproduct, was characterized as an antioxidant in three different foodstuffs rich in fish lipids: (a) bulk cod liver oil (40% of omega-3 PUFAs), (b) cod liver oil-in-water emulsions (4% of omega-3 PUFAs), and (c) frozen minced horse mackerel ( Trachurus trachurus) muscle. Hydroxytyrosol was evaluated at different concentration levels (10, 50, and 100 ppm), and its antioxidant capacity was compared against that of a synthetic phenolic, propyl gallate. Results proved the efficiency of hydroxytyrosol to inhibit the formation of lipid oxidation products in all tested food systems, although two different optimal antioxidant concentrations were observed. In bulk oil and oil-in-water emulsions, a higher oxidative stability was achieved by increasing the concentration of hydroxytyrosol, whereas an intermediate concentration (50 ppm) showed more efficiency, delaying lipid oxidation in frozen minced fish muscle. The endogenous depletion of alpha-tocopherol and omega-3 polyunsaturated fatty acids (omega-3 PUFAs) was also inhibited by supplementing hydroxytyrosol in minced muscle; however, the consumption of the endogenous total glutathione was not efficiently reduced by either hydroxytyrosol or propyl gallate. A concentration of 50 ppm of hydroxytyrosol was best to maintain a longer initial level of alpha-tocopherol (approximately 300 microg/g of fat), whereas both 50 and 100 ppm of hydroxytyrosol were able to preserve completely omega-3 PUFAs. Hydroxytyrosol and propyl gallate showed comparable antioxidant activities in emulsions and frozen fish muscle, and propyl gallate exhibited better antioxidant efficiency in bulk fish oil.     

Degradation products of cyanidin glycosides from tart cherries and their bioactivities

The bioactive anthocyanins present in tart cherries, Prunus cerasus L. (Rosaceae) cv. Balaton, are cyanidin 3-glucosylrutinoside (1), cyanidin 3-rutinoside (2), and cyanidin 3-glucoside (3). Cyanidin (4) is the major anthocyanidin in tart cherries. In our continued evaluation of the in vivo and in vitro efficacy of these anthocyanins to prevent inflammation and colon cancer, we have added these compounds to McCoy's 5A medium in an effort to identify their degradation products during in vitro cell culture studies. This resulted in the isolation and characterization of protocatechuic acid (5), the predominant degradation product. In addition, 2,4-dihydroxybenzoic acid (6) and 2,4,6-trihydroxybenzoic acid (7) were identified as degradation products. However, these degradation products were not quantified. Compounds 5-7 were also identified as degradation products when anthocyanins were subjected to varying pH and thermal conditions. In cyclooxygenase (COX)-I and -II enzyme inhibitory assays, compounds 5-7 did not show significant activities when compared to the NSAIDs Naproxen, Celebrex, and Vioxx, or Ibuprofen, at 50 microM concentrations. However, at a test concentration of 50 microM, the antioxidant activity of protocatechuic acid (5) was comparable to those of the commercial antioxidants tert-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT), and butylated hydroxyanisole (BHA), and superior to that of vitamin E at 10 microM concentrations.     

Cyclooxygenase inhibitory and antioxidant compounds from crabapple fruits

Crabapple trees belong to the Malus genus (Rosaceae) and bear fruits that are sparingly consumed and used in the preparation of fruit beverages. Cyclooxygenase (COX) enzyme inhibitory and antioxidant bioassay-guided fractionation of the aqueous and methanol extracts of Malus x kornicensis and Malus x Indian Summer yielded (+)-catechin (1), (-)-epicatechin (2), cyanidin-3-O-beta-galactopyranoside (3), and amygdalin (4). Pure compounds 1-4 were obtained by HPLC, identified by LC-ES/MS, CD, and NMR spectroscopic methods and evaluated for their COX enzyme inhibitory and antioxidant activities. In COX-1 and -2 enzyme inhibitory assays, compounds 1-3 (all at 80 microM) showed activities of 20.4, 46.3%; 57.6, 47.9%; and 8.2, 13.7%, respectively, compared to naproxen (54.3, 41.3%; 10 microM), ibuprofen (47.5, 39.8%; 10 microM), Celebrex (46.2, 66.3%; 1.67 ppm), and Vioxx (23.8, 88.1%, 1.67 ppm). In the antioxidant assay, the catechins (1-2) and anthocyanin (3) (all at 40 microM) showed activities of 61.3, 62.5, and 60.1%, respectively. The synthetic antioxidants, tert-butylhydroquinone (TBHQ), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and vitamin E (all tested at 10 microM), gave 75.2, 80.1, 70.0, and 10.2% activities, respectively. The cyanogenic glycoside, amygdalin (4), and its hydrolysis products, mandelonitrile (5) and benzaldehyde (6), were not active in the antioxidant or COX enzyme inhibitory assays at 80 microM concentrations.