Sulfur-Containing Heterocyclic Compounds with Antioxidative Activity

Chapter 20

Sulfur-Containing Heterocyclic Compounds with Antioxidative Activity Formed in Maillard Reaction Model Systems Jason P. Eiserich and Takayuki Shibamoto

Downloaded by FUDAN UNIV on February 17, 2017 | http://pubs.acs.org Publication Date: July 29, 1994 | doi: 10.1021/bk-1994-0564.ch020

Department of Environmental Toxicology, University of California, Davis, CA 95616-8588

Extracts of microwave heated glucose/cysteine Maillard reaction systems and headspace samples of heated peanut oil/cysteine and peanut oil/methionine mixtures were shown to possess strong antioxidative activity. Several common classes of sulfur-containing volatile heterocyclic compounds known to be formed in Maillard-type reactions were evaluated for antioxidative activity. Antioxidative activity was measured by a recently developed method involving the oxidation of heptanal to heptanoic acid in a dichloromethane solution. Alkyl thiophenes, thiazoles, thiazolidine, and 1,3-dithiolane inhibited heptanal oxidation for various periods of time. The degree of unsaturation in the heterocyclicring,as well as the substituent type had variable effects on the antioxidative capacity of these compounds. Thiophenes substituted with electron donating alkyl groups showed stronger activity than the unsubstituted thiophene. However, thiophene substituted with an electron withdrawing substituent such as the acetyl group showed little or no antioxidative activity. Saturated cyclic sulfides showed higher activity than either the thiazoles or thiophene derivatives. Cyclic sulfides reacted with t-butylhydroperoxide (t-BuOOH) and m-chloroperoxybenzoic acid (m-CPBA) to form S-oxides. Mechanisms of antioxidative activity are proposed. These results present the potential role of sulfur-containing heterocyclic compounds for inhibiting the oxidative degradation of lipid-rich foods.

Lipid peroxidation is the primary mechanism by which food deteriorates upon storage in the presence of oxygen. This process of oxidation can be initiated enzymatically, by metal ion catalysis, or by photochemical processes, to name a few. Free radicals including peroxyl, alkoxyl, and hydroxyl have been implicated in the mechanism of lipid peroxidation. The changes in the quality of processed foods are manifested by

0097-6156/94/0564-0247$08.00/0 © 1994 American Chemical Society

Mussinan and Keelan; Sulfur Compounds in Foods ACS Symposium Series; American Chemical Society: Washington, DC, 1994.


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deterioration of flavor, aroma, color, texture, nutritive value, and the formation of toxic components (7,2) including aldehydes and epoxides. These changes in food quality are therefore of significance to both the food industry and the consumer. Traditionally, the effects of lipid peroxidation are eliminated or suppressed with the use of synthetic antioxidants such as BHA and BHT. Recently, however, the safety of these compounds with respect to human health has been questioned, hence the search for "natural" alternatives. Maillard reaction model systems, usually involving the heat treatment of a sugar or lipid, and an amino acid, has given significant insight into the thermal interactions of food constituents, and the subsequent formation of flavor and aroma. Aside from the complex flavor and aroma that is generated in model and real food systems, Maillard reaction products (MRPs) have been shown to possess some very interesting chemical and biological properties, including both antimutagenic and antioxidative activity (3). The antioxidative activity of the MRPs was first observed by Franzke and Iwainsky (4), when they reported on the oxidative stability of margarine following the addition of products from the reaction of glycine and glucose. It has further been shown that antioxidative materials are formed as the result of heating foods. Yamaguchi et al. (5) found that the addition of glucose and amino acids to cookie dough prior to baking significantly increases oxidative stability. Shin-Lee (6) found that diffusate from beef browned extensively when heated at 180 °C for 2 hours, and inhibited lipid oxidation (TBA values) by 95% in cooked beef during storage. Identification of Maillard reaction antioxidants has focused primarily on the higher molecular weight melanoidins. The Maillard reaction, however, also produces hundreds of volatile compounds that are responsible for the aroma of cooked food. Recently, volatile MRPs prepared by heating a glucose-glycine solution were found to slow the oxidative degradation of soybean oil by increasing the oxidation induction period and by decreasing the oxidation rate constant (7,8). Macku and Shibamoto (9) identified 1-methylpyrrole as an antioxidant generated in the headspace of a heated corn oil/glycine model reaction system. Similarly, Eiserich et al. (10) showed that thiazoles, oxazoles, and furanones formed in an L-cysteine/D-glucose Maillard model system possessed antioxidative activity. These previous studies clearly show the potential of volatile MRPs to inhibit the oxidative degradation of lipid-rich foods. Natural sulfur-containing compounds such as cysteine, glutathione, and lipoic acid, as well as synthetic compounds including N-acetylcysteine and a-mercaptopropionylglycine have historically been known to protect against oxidative stress in biological systems through the scavenging and reduction of various oxidants (77). The particular chemical and physcial characteristics including hypervalency, multiplicity of oxidation state, and the potential role of 3d orbitals in the stabilization of free radicals (72) implicates sulfur-containing compounds in foods as potential antioxidants. With these facts in mind, the objective of this research was to identify volatile heterocyclic sulfur-containing MRPs from various model systems that possessed antioxidative activity; compounds which have previously been overlooked as potential antioxidants.


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Sulfur-Containing Heterocyclic Compounds 249

Experimental Procedures


Maillard Reaction Sample Preparation. Peanut Oil/Cysteine and Peanut Oil/Methionine Systems. Peanut oil (100 g) and 10.0 g of cysteine or methionine were mixed and placed in a 500-mL two-neck round-bottom flask, which was interfaced to a simultaneous purging and solvent extraction (SPE) apparatus developed by Umano and Shibamoto (13). The mixture was heated at 200°C for 5 hr while stirring. The headspace volatiles were purged into 250 mL of deionized water by a purified nitrogen stream at a flow rate of 10 mL/min. The volatiles trapped by the water were continuously extracted with dichloromethane (50 mL) for 6 hr. The water temperature was kept at 10°C by a Brinkman RM6 constant-temperature water circulator. The dichloromethane extract was dried over anhydrous sodium sulfate, and the extract was then concentrated to 2.0 mL by fractional distillation with a Vigreux column at atmospheric pressure. The concentrated extract was placed in a vial and stored under argon at -4°C until tested for antioxidative activity. Glucose/Cysteine Systems. The method of sample preparation was adapted from Yeo and Shibamoto (14). L-Cysteine (0.05 mol) and D-glucose (0.05 mol) were dissolved in 30 mL of deionized water, and the pH of each solution was adjusted to 2, 5,7, and 9 with either 6N HC1 or 6N NaOH. The solutions were then brought to a final volume of 50 mL with deionized water and covered with Saran Brand plastic wrap. The four solutions were irradiated at the high setting of a 700-W microwave oven for 15 min. At 4-min intervals, the irradiation was interrupted and the samples rotated 90° to ensure uniform irradiation. The irradiation time coincided with the onset of browning, whereas further irradiation led to charring and sample combustion. After microwave irradiation, each brown mass was dissolved in approximately 100 mL of deionized water and allowed to cool to room temperature. The resulting solutions were adjusted to pH 8 with 6N NaOH to enhance the extraction efficiency of nitrogen-containing heterocyclic compounds. The aqueous solution was extracted with 50 mL of dichloromethane using a liquid-liquid continuous extractor for 6 h and then dried over anhydrous sodium sulfate for 12 h. After removal of sodium sulfate, the dichloromethane extract was concentrated to 1 mL by fractional distillation with a Vigreux column at atmospheric pressure. Measurement of Antioxidative Activity. The antioxidative activity of the Maillard reaction products was evaluated by a method similar to that developed by Macku and Shibamoto (9) and later modified by Eiserich et al. (10). The antioxidative activities of 25 uL-aliquots of peanut oil/cysteine and peanut oil/methionine dichloromethane extracts and 5 uL-aliquots of the glucose/cysteine extracts were measured. 2-Alkylthiophenes, 2-thiophenethiol, 2-methyl-3-furanthiol, furfuryl mercaptan, thiazolidine, and 1,3-dithiolane were tested for antioxidative activity at a concentration of 1 mM. The above extracts and standards were added to dichloromethane solutions containing 25 mg of heptanal. Nonadecane (400 mg) was added as a gas chromatographic internal standard, and the resulting solutions were brought to a 5-mL final volume with


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dichloromethane. The solutions were transferred to small vials and stored at room temperature. The headspace of each vial was purged with air every 2 days. Controls containing only heptanal, the internal standard, and dichloromethane were prepared for each experiment. The experimental vials and controls were periodically analyzed by GLC/FID. A Hewlett-Packard (HP) Model 5890 gas chromatograph equipped with a flame ionization detector (FID) and a 30 m χ 0.25 mm i.d. DB-Wax bonded-phase fused silica capillary column (J&W Scientific, Folsom, CA) was used to quantitate heptanoic acid formed from heptanal oxidation. The GC peak areas of heptanoic acid obtained at various time intervals were divided by the GC peak area of the internal standard (nonadecane, 400 mg) to calculate a relative peak area (RPA). Reactions of Sulfur-Containing Compounds with m-CPBA and 1-BuOOH. m-CPBA (3 mM) and i-BuOOH (3 mM) were reacted separately with equal molar concentrations of 2-butylthiophene and 1,3-dithiolane. The reactions were conducted in sealed glass vials at 25 and 50°C for m-CPBA and f-BuOOH, respectively. The reactions were allowed to proceed for 12 hr (m-CPBA) and 24 hr (ί-BuOOH). The reaction products were subsequently analyzed by GC. Identification of m-CPBA- and 1-BuOOH-Induced Oxidation Products. The oxidation reaction products of the above listed sulfur-containing compounds were analyzed using a HP Model 5890 gas chromatograph equipped with a flame photometric detector (FPD) set in the sulfur mode. The injector and detector temperatures were both set at 250°C. A 30 m χ 0.25 mm i.d. DB-1 bonded-phase fused silica capillary column (J&W Scientific, Folsom, CA) was used to separate the reaction products. The GC oven temperature was programmed from 80 to 250°C at a rate of 5°C/min. The linear velocity of the helium carrier gas flow was 26.5 cm/s, with a split ratio of 1:25. Mass spectral (MS) identification of the oxidation products was carried out on a HP Model 5971 series mass selective detector (MSD) interfaced to a HP Model 5890 gas chromatograph. Mass spectra were obtained by electron impact ionization at 70 eV and a source temperature of 250°C. The capillary column and GC conditions were the same as described above. Results and Discussion Antioxidative Activity of Maillard Reaction Extracts. In the antioxidative assay system utilized in this study heptanal was readily oxidized to heptanoic acid in the dichloromethane solutions. However, the presence of a-tocopherol (Figure 1) inhibited this transformation in a concentration dependent manner. This system was then used to evaluate the antioxidative activity of dichloromethane extracts of several Maillard reaction model systems. Figure 2 shows the antioxidative activity of 5-uL aliquots of each pH extract from a microwave heated glucose/cysteine model system. The order of antioxidative effect of the extracts from the samples was as follows: pH 9 > pH 5 > pH 2 > pH 7. The Maillard reaction is catalyzed under both slightly basic and acidic conditions and may explain this trend. Volatiles from sugar/cysteine Maillard reaction


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EISERICH & SHIBAMOTO

Sulfur-Containing Heterocyclic Compounds

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< "δ Downloaded by FUDAN UNIV on February 17, 2017 | http://pubs.acs.org Publication Date: July 29, 1994 | doi: 10.1021/bk-1994-0564.ch020

G

50 ng/μΐ, 10

15

20

Time (days) Figure 1.

Antioxidative activity of various concentrations of α-tocopherol. The relative peak area (RPA) is equal to the G C peak area of heptanoic acid divided by the G C peak area of the internal standard, nonadecane.

Sulfur-Containing Heterocyclic Compounds with Antioxidative Activity

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