oO @ books.google.com.pk/book Chapter 1 Natural Antioxidants: An Overview F. Shahidi Department of Biochemistry, Memorial University of Newfoundland, St. John's, Newfoundland, Canada. Introduction Antioxidants are substances that when present in foods or in the body at low con- centrations compared with that of an oxidizable substrate markedly delay or pre- ‘ent the oxidation of that substrate (1). Food manufacturers have used food-grade antioxidants to prevent quality deterioration of products and to maintain their nutritional value, Antioxidants have also been of interest to biochemists and health professionals because they may help the body protect itself against damage caused by reactive oxygen species and degenerative diseases. Antioxidants are known to act at different levels in the oxidative sequence involving lipid molecules. They may act by decreasing oxygen concentration, intercepting singlet oxygen, preventing first-chain initiation by scavenging initial radicals such as hydroxy! radicals, binding metal ion catalysts, decomposing pri- mary products to non-radical compounds, and chain-breaking to prevent continued hydrogen abstraction from substrates. The extent to which oxidation of lipids occurs also depends on the chemical structure of the fatty acids involved as well as, other factors related to the storage of foods and reaction conditions Natural antioxidants from dietary sources include phenolic and polyphenolic compounds, chelators, antioxidant vitamins and enzymes, as well as carotenoids and camosine. The mechanism by which these antioxidants are involved in the control of food autoxidation and rancidity prevention may be different, However, their presence in the live plants may be for the sake of protecting tissues from inju- rious damage, Furthermore, the beneficial effects of consuming plant foods have been ascribed, in part, to the presence of antioxidants in the plant, which is associ- ated with counteracting risk of most cardiovascular diseases, cancer, and cataracts, among other degenerative diseases of aging. ‘The process of autoxidation and development of rancidity in foods involves a free radical chain mechanism proceeding via initiation, propagation, and termina- tion steps (2). While radicals are produced in the “initiation” step, they react with unsaturated fatty acids by abstracting a hydrogen atom from their molecules in the “propagation” step. The reactions in the propagation make up a chain reaction until “termination” reaction occurs.oO @ books.google.com.pk/book 2 F. Shahid Initiator Initiation; LH Loe Pp Propagation: L'+0; > LOO" LOO" + LH > LOOH + L* ‘Termination: LOO" + LOO" + LOO’ +L* > non-radical products +L Antioxidants (AH) interfere with the above process by different mechanisms ‘These involve inactivation of prooxidants in the medium, such as carotenoids that scavenge singlet oxygen or chelators that inactivate metal catalysts. These reac- tions lead to a delay in the onset of oxidation and the extension of the induction period. Antioxidants may also donate a hydrogen atom or an electron to radicals formed from unsaturated lipids, thus imparting stability to food lipi LOO* + AH + LOOH + A* A‘ + LOO + non-radical products A+ ATS In the body, free radicals may be involved in a number of diseases and tissue injuries such as those of the lungs, heart and cardiovascular system, kidneys, liver, gastrointestinal tract, blood, eye, skin, muscle, brain, and the process of aging (3-5), Oxidants and radicals which mediate various disorders are mainly reactive species which are formed from triplet oxygen, water, and unsaturated lipid mole- cules (Fig. 1.1) Therefore, lipid peroxidation is a problem not only in the edible oil and the food industry, but also in the human body. Excess production of oxygen radical species, particularly hydroxyl radicals, can affect lipid cell membranes to produce lipid peroxides and reactive oxygen species (ROS) which are linked to a variety of diseases as well as acceleration of the aging process (Fig. 1.2) While hydroperoxides, referred to as primary oxidation products, are colorless and odorless when present in foods, they are labile and readily produce a number ‘of secondary products such as alkanes, alcohols, aldehydes, and acids, some of which are highly odor-active and have low threshold values. In addition, polymers and other undesirable compounds that could affect the sensory characteristics asoO @ books.google.com.pk/book [Natural Antioxidants: An Overview 3 toe Or Me 0, 00H LL ont “ edt hw Sear “e 10" ue 0 tow Fig. 1.1. Formation of oxidant and radical species in food and biological systems and their inactivation, well as the safety of food products may be formed (see Fig. 1.3). Many of these secondary oxidation products are highly reactive themselves, ¢.g., malonaldchyde (MDA) and 4-hydroxynonenal (4-HN) (6,7). Both MDA and 4-HN are known to interact with biological and food components such as proteins, amino acids, amines, and DNA; the latter reactions are implicated in aging, mutagenesis, and carcinogenesis in the body (8-11). In foods, the above reactions are involved in changes in flavor, texture, color, and other sensory attributes as well as nutritional value of products and may lead to the destruction of essential fatty acids and lipid- soluble vitamins, in addition to being toxic by themselves. Thus, use of antioxi- dants in foods is recommended for controlling rancidity with its deleterious conse- quences. In addition, consumption of antioxidants in foods or as dietary supple- ‘ments may be advised in order to influence the primary causes of atherosclerosis, cancer and degenerative diseases of the aging (12) (see Fig. 1.4) Atherosclerosis Inflammation Arthritis ‘Shock Cancer Frost bite Infection; malaria, entamoeba, AIDS ‘Aging Parkinsonism Radiation damage Fig. 1.2. Diseases and damages caused by reactive oxygen species; courtesy of Dr. M.-T. Huang.ESV Acl 4s) oO @ books.google.com.pk/book 3 4 Shahid ui ant O oy S40, Son Abra oH tem Fe re Tnitiators: snonn | smart oy mt eat te 0 ip re eal Termination | Q\, rm dhe sation ogee, presi, stn DNA, stamine, ut Palymeneatn products oH (dark alread poably tx compounds) pid alsded Fig. 1.3, The mechanism and consequences of lipid oxidation in food and in the body and their possible neutralization by enzyme antioxidants Antioxidants from Natural Sources Plant-Based Antioxidants Plants provide a rich source of natural antioxidants (13). These include tocopherols, vitamin C, carotenoids, and phenolic compounds. Plant phenolics are thought to pro- tect the plants against tissue injuries as they oxidize and combine with proteins and ‘other components. In addition, phenolic compounds in plants may serve as defense systems against herbivory (14). By-produets of photosynthesis may also produce high levels of oxygen, free radicals, and reactive oxygen species (ROS) in profusion. Thus, plants use a myriad of antioxidant compounds to deal with these in order to survive. Many of these compounds have basic molecular similarities in that all have at least ‘one aromatic ring and a hydroxyl group. These include phenolic acids, flavonoids and isoflavones, gallate esters (hydrolyzable tannins), lignans, coumarines, stilbenes, flavonones, and oligomeric proanthrocyanidins. Together, these compounds produce an array of antioxidants, which may act by different mechanisms, to confer an effec-@ books.google.com.pk/book Natural Antioxidants: An Overview 7 TABLE 1.2 Antioxidant Activity of Selected Amino Acids! Concentration. M. 0.001 0.01 Glutamic acid 092 074 Methionine 0.88 072 Alanine 093 Asparagine oss 0.66 Valine 08: 057 Aspartic acid 086 056 Serine oat 0.47 Lysine 069 037 Tryptophan 045 031 Histidine ot 0.17 "Values represent oxygen uptake in herring oil emul to samples devoid of any amino acid. From Ref. 11 hydrophobic amino acids, namely, valine and leucine at the N-terminal position and proline, histidine, or tyrosine in their sequence (see Table 1.3). These sequences were also confirmed to be present in the B-conglycinin as deduced from the nucleotide sequence. These authors did not observe any antioxidative activity when the constituent amino acids were mixed (in proportions present in the pep- tides) and tested in their assay media, and thus concluded that the characteristic amino acid sequences of peptides were required for them to manifest antioxidant effects. The antioxidant activity of P, was similar to that of BHA when tested at 10° and 104 M. TABLE 1.3 Antioxidative Peptides of Soy 75 Protein and Egg White Albumin Hydrolyzates! Peptide ‘Amino acid sequence SOY 7S PROTEIN P, Val-Asn-Pro-His-Asp-His-Glu-Aso P, Leu-Val-Asn-Pro-His-Asp-His-Glu-Asn P Lew-Leu-Pro-His-His, Py Leu-Leu-Pro-His-His-Ala-Asp-Ala-Asp-Tyr Ps Val-lle-Pro-Ala-Gily-Tyr-Pro % Lew-Gln-Ser-Gily-Asp-Ala-Lew-Arg-Val-Pro- Ser-Gly-Thr-The-TyrTyr EGG WHITE ALBUMIN 1 Ala-His 2 Val-His-His 3 Val-His-His-His-Ala-Asn-Glu-Asn ‘From Refs. 32 and 33,@ > books.google.com.pk/book 8 F. Shahidi ‘The antioxidant activity of histidine-containing peptides has also been report- ed by Uchida and Kawakishi (34) and Murase et al. (35). The activity of peptides examined was thought to be related to their metal-chelating ability as well as the lipid radical-trapping potential of the imidazole ring. Furthermore, the antioxidant activity of histidine-containing peptides was higher than that of histidine itself, per- hhaps due to the increased hydrophobicity of peptides which leads to their increased interaction with lipid fatty acids. Tyrosine moieties present in some other active peptides are thought to offer strong hydrogen donating ability The antioxidant peptides isolated from egg white albumin hydrolyzates were recently analyzed and confirmed the presence of histidine moieties in their struc- tures (See Table 1.3). Recently, we have isolated a number of peptides arising from the hydrolysis of capelin, seal meat, and shrimp proteins and have found that com- pounds with moderate hydrophobicity were active antioxidants (e.g., 36) However, the presence of prooxidative peptides was also noted in these prepara- tions. When added to a meat emulsion system, all hydrolyzates were effective in protecting meat against oxidation, although to different degrees Other effective histidine-containing antioxidative dipeptides are carnosine, homocamosine, and anserine. Carnosine and anserine are found in relatively high amounts in muscles, and their dominance is in the white tissues where high levels of anaerobic metabolism are common (37). The antioxidant role of carnosine and anserine seems t olve singlet oxygen and free radical scavenging as well as metal chelation (38-40). Antioxidants Activity of Phytates Phytates are strongly negatively charged compounds found in many cereal-based foods. They are associated with proteins or metal ions and as such are capable of imparting antioxidant activity to many food systems, presumably by chelation of prooxidant metal ions. Antioxidant activity of phytic acid has been demonstrated in several model and food systems (41-43). Phospholipids as Antioxidants Phospholipids are obtained as by-products of oil-refining, especially from soybean. Phospholipids are known to affect lipid oxidation, Soybean lecithin and the ethanol- soluble fraction of a phospholipid are particularly effective in stabilizing lard and sunflower oil when used at a 0.1~1% level. Swift (44) found that purified phos- phatidylethanolamine from cottonseed oil was a good antioxidant and increased the antioxygenic action of a-tocopherol. Later, Dalton ef al. (45) suggested that the antioxygenic action of phospholipids was due in part to the metal-scavenging effect of the fat-soluble phosphoric acid. Therefore, use of lecithin and other phospho- lipids as antioxidants has been practiced (46). Liliger and Saucy (47) have patented 4 process in which a mixture of tocopherols, ascorbates, and lecithin was used for stabilization of bulk oils. However, use of such mixtures in emulsion-type productsa @ books.google.com.pk/book Natural Antioxidants: An Overview 9 was ineffective (48). Many other specialized applications of phospholipids remain proprietary within the industry, Vitamins and Enzyme Antioxidants Vitamins may act as antioxidants in foods and in the body, both individually and in combination. Fat-soluble vitamins, such as vitamin E and vitamin A, and water- soluble vitamins such as vitamin C as well as B-carotene (vitamin A precursor) are known to exert antioxidant activity. In addition, regeneration of a-tocopherol by vitamin C is a well-known phenomenon, Due to the importance of this topic, chap- ter 14 has been devoted to vitamin antioxidants. Enzyme antioxidants of interest to food and biological systems include super- oxide dismutase (SOD) in different sources, catalase, glutathione and glutathione peroxidase, glutathione reductase, glutathione-5-transferase and phenol oxidase. Involvement of selenium in exerting antioxidant activity in conjunction with some enzyme antioxidants is well documented. Details of enzyme antioxidants are sum- marized in chapter 15. Methodologies for Assessing Lipid Oxidation and Antioxidant Activity Methods to assess lipid oxidation are manifold, and these may also depend on the system under investigation. Chemical, sensory, and instrumental methods are fre quently employed. Therefore, monitoring of superoxide-, hydrogen peroxide hydroxyl radical- and peroxyl radical-scavenging effects of antioxidants is common- place (e.g., 49-51). Assay media used may vary, depending on the type of study. In foods, both chemical and instrumental procedures are used; these include determina- tion of peroxide value, individual peroxides, conjugated dienes, para-anisidine value, and 2-thiobarbituric acid (TBA) value (51). Sensory analysis of foods as well as analysis of total and selected volatile decomposition products of lipids may be per- formed (52). To monitor lipid oxidation, the loss of polyunsaturated fatty acids, espe- cially in marine or some vegetable oils with highly unsaturated lipids, may also be followed. Meanwhile, use of novel techniques involving electron spin resonance (ESR), nuclear magnetic resonance (NMR), and infrared (IR) is of interest. In addi- tion, employing oxygen uptake and weight gain, and use of the Rancimat, Oxidative Stability Instrument (OSE, and Oxidograph have been traditionally practiced Several chapters in this monograph are devoted to the application of different methodologies for assessing lipid oxidation and antioxidant activity References 1, Halliwell, B.. Aeschbach, R., Loliger, J., and Aruoma, 0.1. (1995) Food Chem. Toxicol. 33, 601 2. Shahid, F., and Nacek, M, (1995) in Food Phenolics: Sources, Chemistry, Effects and Applications, Technomic Publ. Co., Lancaster and Basel, pp. 235-277