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AgroFOOD industry hi-tech November/December 2006 Anno 17 - No. 6

Antioxidant activities of carnosol and carnosic acid in food and biological model systems - A review

Part 1

SUBHASHINEE S. K. WIJERATNE*, SUSAN L. CUPPETT *Corresponding author Department of Food Science and Technology University of Nebraska-Lincoln, Lincoln, Nebraska, 68583-0919, USA

OXIDATIVE STRESS

Lincoln, Nebraska, 68583-0919, USA OXIDATIVE STRESS Oxidative stress in a biological system describes the

Oxidative stress in a biological system describes the disturbance in the cellular prooxidant-antioxidant balance in the favour of the former (1). It is imposed on a cellular system when the generation of ROS exceeds the system's ability to neutralize and/or eliminate them (2). An imbalance of production and removal of ROS can result from a lack of antioxidant protection, overabundance of ROS from the environment and/or a failure to repair oxidative damage. If not regulated properly, excess ROS can damage cellular components and inhibit normal function, which can lead to the pathogenesis of several human diseases (3). Living cells are continuously involved in redox pathways that lead to the production of ROS, such as free radicals and other non-radical reactive oxygen derivatives such as peroxides. A free radical is a chemical species capable of independent existence that possesses one or more unpaired electrons (2). Free radicals are produced from endogenous sources as by- products of electron transport, oxygen-utilizing systems, and other processes involved in normal aerobic metabolism, lipid peroxidation, viral, fungal and bacterial infections, and detoxification reactions involving the liver cytochrome P-450 enzyme system (4, 5). Exogenous sources include cigarette smoke, environmental pollutants, such as emission from automobiles and industries (ozone and nitrous oxide), asbestos, consumption of alcohol in excess, and exposure to ionizing radiation (from industry, sun exposure, cosmic rays, and medical X-rays) (6, 7). Four types of damage are initiated by ROS: (i) damage to fat compounds: free radicals mainly target polyunsaturated fatty acids and cholesterol located in the membranes surrounding the cells, thus a damaged membrane loses its ability to transport oxygen, nutrients, and water to the cells; (ii) damage to protein molecules: results in defective cell membranes and loss of enzyme activities; (iii) damage to nucleic acids:

alterations in nucleic acids, which comprise the genetic code

ABSTRACT

Oxidative stress occurs in biological systems due to an increase in oxidant generation, a decrease

Oxidative stress occurs in biological systems due to an increase in oxidant generation, a decrease in antioxidant activity, and/or failure to repair oxidative damage. Reactive oxygen species and other free radicals present in the environment and that are derived from dietary sources are oxidative stress inducers. Dietary antioxidants play an important role in restoring the oxidant-antioxidant balance in biological systems when the existing cellular antioxidant defence mechanisms fail to combat increased levels of oxidizing agents. Antioxidants, carnosol and carnosic acid, derived from rosemary (Rosemarinus officinalis L.) leaf extracts, have shown to exert strong antioxidant activities against reactive oxygen species and free radical attacks in food as well as biological model systems.

within each cell, can initiate the growth of abnormal cells, which is the first step in cancer development, and (iv) damages to lysosomes: lysosomes contain cellular degenerative enzymes, which leak out when the cell membrane is damaged. The enzymes start digesting the cell

itself, causing a chain reaction of destruction that eventually, will lower the immune system resistance (8, 9). Such damages to various molecules and cells have been implicated in several diseases, including cancer, arthritis, atherosclerosis, ischemic damage following stroke, premature aging, and a variety of neurodegenerative disorders (2, 6, 10-12). Some of the major ROS found in biological systems are listed in Table 1. Superoxide

radical (O2 . ) is produced by the activity of oxidases during oxidative metabolism in living cells (13). When an electron is transferred to oxygen during reduction of oxygen (O2) to water (H2O),

O2 . is generated as shown in Figure 1. This mechanism protects animals by killing bacteria in activated phagocytes (6). Superoxide radical formation can also occur by the transfer of an electron from a transition metal ion, such as ferrous (Fe 2+ ), to O2 (13). Superoxide is less reactive compared to many of the free radicals listed in Table 1. It is not particularly reactive with lipids, carbohydrates and nucleic acids but exhibits some activity against proteins (14), especially proteins that contain heme-moieties (15) or iron-sulfur clusters (16). The main destruction by superoxide radical is due to the formation of hydroxyl radicals (HO . ) (9).

Table 1. Free radicals and non-radical products found in biological systems

Table 1. Free radicals and non-radical products found in biological systems

Figure 1. Role of oxygen in producing reactive oxygen species

Figure 1. Role of oxygen in producing reactive oxygen species

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Hydrogen peroxide (H2O2), present in low concentrations in

aerobic cells as a metabolite, is less reactive because it is not

a free radical. It is generated during superoxide dismutase-

catalyzed dismutation reactions (17), oxidation of D-amino acids by D-amino acid oxidases, and action of xanthine oxidase upon xanthine and hypoxanthine to form uric acid (18). Hydrogen peroxide is also converted to hypochlorous acid by myeloperoxidase in neutrophils. Hypochlorous acid is

a strong oxidant that acts as a bactericidal agent in phagocytic

cells. However, the biologically significant reaction of H2O2 is its spontaneous conversion, catalyzed by Fe 2+ (Fenton reaction) (Reaction I), to the highly reactive HO . (6).

substrate (6). Porter (20) considered an antioxidant as any acidic compound (including phenols) usable in foods which can readily donate an electron or a hydrogen atom to a peroxyl or alcoxyl radical to terminate lipid peroxidation chain reaction or to regenerate a phenolic compound, or which can effectively chelate a prooxidant transition metal. Biological systems are protected against oxidative damage which can effectively chelate a prooxidant transition metal. Biological systems are protected against oxidative damage by their built-in antioxidant defence system and by dietary antioxidants. The endogenous antioxidant defence system is composed of two components: antioxidant enzymes that will be discussed in the following sections, and low molecular weight antioxidants that include vitamins A and E, ascorbate, glutathione, ubiquinone and thioredoxin, and proteins such as transferrin, ferritin, ceruloplasmin, hemopexin, hepatoglobin and albumin that are capable of chelating metal ions (9). These substances are the body's natural defence against ROS generated by endogenous and external environmental factors (21, 22). Naturally occurring antioxidants are primarily plant phenolic compounds that may be present in any plant part. There are also synthetically derived antioxidants permitted for food use such as butylated hydroxyanisole (BHA),

butylated hydroxytoluene (BHT), propyl gallate (PG), dodecyl gallate and tertiary butyl hydroquinone (TBHQ) (23). The growing interest in replacing synthetic antioxidants by natural ones has brought about research on plant parts, both edible and non-edible, for identifying new antioxidants. Examples of compounds isolated from plant parts that possess antioxidant activity include tocopherols, carotenoids, flavonoid compounds (flavones, flavonols, isoflavones, catechins, flavonones, chalcones), diterpenes, cinnamic acid derivatives (caffeic acid, ferulic, chlorogenic acid), and coumarins (24, 25). To be used in a food system an antioxidant must (i) be safe; (ii) not impart any colour, odour or flavour; (iii) be effective at low

H2O2 + Fe 2+ −−−−−> HO .

+ OH - + Fe 3+

[I]

Hydroxyl radical can also be generated by exposing water to high-energy ionizing radiation (6). This radical reacts instantaneously with any biological molecule from which it can abstract a hydrogen atom, including DNA, proteins, lipids, and carbohydrates (9). Figure 2 depicts the effects of HO . in a biological system.

Figure 2. Effects of hydroxyl radical on biological molecules [Adapted from Acworth et al. (9)]

Figure 2. Effects of hydroxyl radical on biological molecules [Adapted from Acworth et al. (9)]

Hydroperoxyl is another highly reactive radical derived from O2. Production of this radical is favoured under acidic conditions, and its lipophilicity enables it to move across cell membranes easily to initiate peroxidation of polyunsaturated fatty acids in cell membranes (9). Nitric oxide (NO . ) is a weak oxidizing agent, whereas, nitrogen dioxide (NO2 . ) is a powerful oxidizing agent found in polluted air, cigarette smoke, and smoke of burning organic material (6). Oxides of nitrogen are important agents in defending living cells against attack by foreign organisms. However, excess production of NO . during infections can lead to formation of NO2 . , which abstracts hydrogen atoms from cell membrane lipids initiating lipid peroxidation (18). Lipid peroxyl radicals and hydroperoxides, produced during food lipid oxidation, are implicated in many intestinal pathological conditions, such as inflammation and cancer. Studies have shown that existing cellular antioxidant defence mechanisms are incapable of neutralizing the damaging effects of dietary lipid hydroperoxides (19), which highlights the importance of dietary antioxidants in combating excessive oxidants and free radicals.

ANTIOXIDANTS

An antioxidant is defined as a substance that when present at low concentrations compared to those of an oxidizable substrate, significantly delays or inhibits oxidation of that

concentrations; (iv) survive processing conditions; (v) be stable in finished products; (vi) be fat soluble; and (vii) be readily available at a low cost (24).

ROSEMARY ANTIOXIDANTS

Many herbs and spices have been found to exhibit antioxidant activity in food systems (26). Commercially available rosemary extracts that are widely used in the food industry exhibit potent antioxidant activity (27). Among the many flavones, diterpenes, triterpenes, and steroids isolated from rosemary extracts, the main two compounds that account for 90 percent of its antioxidant activity are carnosic acid and carnosol (26). Several other diterpenes such as rosmanol, epirosmanol, isorosmanol (28), rosmaridiphenol (29), and rosmariquinone (30, 31) have also been reported to possess antioxidative activity. Carnosic acid is first converted to carnosol, and then to rosmanol, epirosmanol or 7-methyl-epirosmanol when subjected to heat or oxygen in the presence of light (32, 33). Therefore, the presence of high amounts of carnosol relative to carnosic acid is expected in commercial rosemary extracts, as heating is involved in the extraction process (24). Antioxidant activity of rosemary extracts and compounds isolated from these extracts has been extensively tested in food model systems. Rosemary extracts have effectively inhibited lipid oxidation as measured by conjugated diene

hydroperoxide formation in bulk vegetable and fish oil modelin food model systems. Rosemary extracts have effectively inhibited lipid oxidation as measured by conjugated diene

AgroFOOD industry hi-tech November/December 2006 Anno 17 - No. 6

systems but were inactive in oil-in-water emulsion systems (34). The reason for this response has been described by the 'polar paradox' phenomenon which indicates that polar antioxidants are more effective in non-polar lipid systems, whereas non-polar antioxidants are more active in lipid emulsions (35). Hydrophilic antioxidants offer more protection in bulk oil systems by orienting in the air-oil interface, whereas in oil-in-water emulsions partitioning into the water phase dilutes the effectiveness of the polar antioxidants in inhibiting lipid oxidation (34). Lipophilic antioxidants are distributed more in the oil phase and orient at the oil-water interfaces, and therefore are more effective in emulsions compared to hydrophilic antioxidants (36). Similar effects were observed when carnosic acid and carnosol isolated from rosemary extracts were tested in oil model systems. Carnosic acid is more hydrophilic than carnosol due to its free carboxylic acid group (37); however, carnosic acid has a greater affinity towards the oil phase than to the water phase in water-oil mixtures that did not contain an emulsifier (36). Carnosic acid was relatively more active in inhibiting lipid oxidation in bulk oil, while less-polar carnosol performed better in oil-water emulsions (38). Also when antioxidative capacities were evaluated in lard at 100 °C, carnosic acid was more capable of preventing lipid oxidation than carnosol (39). The antioxidant activity of rosemary compounds is due to their ability to donate hydrogen and neutralize free radicals (40, 41). The antioxidant mechanism proposed by Masuda et al. (40) shows that carnosic acid undergoes oxidative coupling reactions with peroxyl radical at 12- and 14- carbon atoms followed by degradation reactions to form o-quinone and p- hydroxyl quinone derivatives. The antioxidant activity is determined not only by their hydrogen donating ability but also by their relative oxidative stability in different phases as observed in several food model systems. In bulk oil and oil emulsions, carnosic acid was less stable compared to carnosol, but the oxidative products of carnosic acid still possessed similar or increased antioxidant activities in these systems (36). In a given system, carnosic acid and carnosol have also shown significant differences in their antioxidative activities with changing pH. In corn oil emulsions, both carnosol and carnosic acid were highly active at pH 4 and 5, whereas no activity against lipid hydroperoxide and hexanal formation, which are by-products of lipid oxidation, was exhibited at pH 7 (34). These results suggest that carnosol and carnosic acid are more stable in low pH conditions. Apart from antioxidant activities rosemary compounds also possess many other biological activities. They have exhibited capabilities in inhibiting experimental carcinogenesis in animal models (42-44). Carnosic acid and carnosol inhibit lipid absorption in the gastrointestinal tract

inhibit lipid absorption in the gastrointestinal tract by inhibiting lipase, an enzyme that breaks down dietary
inhibit lipid absorption in the gastrointestinal tract by inhibiting lipase, an enzyme that breaks down dietary
inhibit lipid absorption in the gastrointestinal tract by inhibiting lipase, an enzyme that breaks down dietary

by inhibiting lipase, an enzyme that breaks down dietary fats (45), indicating the potential use of these compounds in weight loss management. Studies have shown that carnosic acid stimulates the synthesis of nerve growth factor (NGF), which is an agent that can help reverse the nerve cell damage and death caused by Alzheimer's disease (46). We recently investigated the effects of carnosol and carnosic acid against lipid hydroperoxide induced oxidative stress in Caco-2 cells. Our studies showed that incubating Caco-2 cells for 24 h with carnosic acid and carnosol significantly reduced lipid hydroperoxide mediated cell toxicity. We also observed that carnosic acid was more effective compared to carnosol in inhibiting lipid hydroperoxide induced lipid peroxidation and DNA damage in the Caco-2 cells. The effects of carnosol and carnosic acid on antioxidant enzymes, catalase, superoxide dismutase and glutathione peroxidase were dependent upon the compound and their concentrations at supplementation. Our results also showed that carnosic acid compared to carnosic acid was able to increase glutathione peroxidase activity in Caco-2 cells.

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