Boonton, New Jersey
It’s ironic that the oxygen we need to survive is also the catalyst for the production of free radicals. The job of antioxidants is to “quench” free radicals, meaning that they neutralize their electrical charge and prevent the free radical from taking electrons from other molecules. Antioxidants can slow down some of the physical signs of aging by minimizing wrinkles and preserving skin’s natural “glow.” The mechanisms by which skin damage occurs generally involve (1) direct oxidative alterations of physiologically critical molecules, including proteins, lipids, carbohydrates, and nucleic acids, along with modulation of gene expression and the inflammatory response; and (2) indirect oxidative response first by stimulating production of oxidase enzymes and then generating radicals with the alteration of physiologically critical molecules. This chapter is focused on addressing three key areas: (A) major causes and consequences of UV-induced photo-aging to skin, (B) role of endogenous antioxidant defense system, and (C) use of conventional and nonconventional antioxidants for skin protection and reversal of signs of aging.
Antioxidants are used widely, but rarely defined. They are used in food to stop rancidity; in polymer synthesis to control polymerization during manufacturing and to protect polymer (plastics) against UV damage; and as a nutritional supplement for a wide variety of health benefits. In personal care, antioxidants are used to protect formulation ingredients and to protect skin from sun-induced damage as well as reversing aging signs. An antioxidant is defined by Haliwell and Gutteridge as “any substance that, when present at low concentrations compared with those of an oxidizable substrate, significantly delays or prevents oxidation of that substrate”.
Aging is a natural phenomenon; it happens to all of us, and more signs of aging show up the longer we live. Antioxidants can slow down some of the physical signs of aging by reducing wrinkles and preserving skin’s natural “glow.” Use of “antioxidant for anti-aging” alone cannot provide the results we are looking for. A positive outlook on life and an optimistic perspective do contribute to beauty, both inside and out. We also need to take a holistic approach regarding the health and beauty of skin.
Free radicals promote beneficial oxidation that produces energy and kills bacterial invaders, but in excess—through accumulation—they can disturb cell structure, resulting in cellular damage in protein, fat, and DNA molecules. This is believed to contribute to aging, as well as various other health problems, because the human body’s functions depend on these biomolecules.
The skin is at risk of photo-oxidative damage due to the generation of singlet oxygen, hydroxyl radicals, hydrogen peroxides, and other reactive oxygen species (ROS). The most severe consequence of photo-oxidative damage is skin cancers. Less severe photo-aging changes result in wrinkling, scaling, dryness, and uneven pigmentation consisting of hyper- and hypopigmentation [2–4]. The mechanisms by which skin damage occurs generally involve (1) direct oxidative alterations of physiologically critical molecules, including proteins, lipids, carbohydrates, and nucleic acids, along with modulation of gene expression and the inflammatory response, and (2) indirect oxidative response first by stimulating production of oxidase enzymes and then generating radicals with the alteration of physiologically critical molecules.
This chapter is focused on three key areas: (A) major causes and consequences of UV-induced photo-aging to skin, (B) the role of endogenous antioxidant defense system and (C) the use of conventional and nonconventional antioxidants for skin protection and reversal of signs of aging.
Photosensitizer and Reactive Oxygen Species (ROS)
Singlet oxygen is generally accepted to be the first ROS formed from triplet excited states of endogenous photosensitizers. There are many endogenous chromophores in human skin, which in the presence of UVA radiation can generate ROS. Porphyrins (protoporphyrin, coproporphyrin, and uroporphyrin), flavins (riboflavin), quinone (ubiquinone), and the pyrimidine nicotinamide cofactors (nicotinamide adenine dinucleotide and nicotinamide adenine dinucleotide phosphate, NADH and NADPH) are examples of common photosensitizers in mammalian cells . The identification of the epidermal UVA-absorbing chromophore trans-urocanic acid that quantitatively accounts for the action spectrum of photo-aging has been reported . The excited photosensitizer subsequently reacts with oxygen, resulting in the generation of ROS including superoxide anion radical and singlet oxygen. Superoxide anion radical and singlet oxygen are also produced by neutrophiles that are present in increased quantities in photo-damaged skin, and contribute to the overall pro-oxidant state. Superoxide dismutase (SOD) converts superoxide anion radical to hydrogen peroxide. Hydrogen peroxide is able to cross cell membranes easily and, in conjunction with Fe2+, generates highly toxic hydroxyl radicals. Both singlet oxygen and hydroxyl radical can initiate lipid peroxidation. Many organic sunscreens also act as triplet sensitizers that convert harmless triplet oxygen into the highly reactive singlet oxygen . As a consequence of their high reactivity, ROS react nonspecifically with nearly every cellular target and may damage DNA, proteins, lipids, and carbohydrates .
While ROS are short-lived and extremely reactive, RNS are longer-lived and more specific in the reactions they undergo . Nitrogen oxide (NO) reacts with superoxide to form the peroxynitrite anion, resulting in oxidative stress. This results in the release of cytochrome c from the mitochondria to the cytoplasm and the subsequent conjugation of the cytochrome c with caspase-9 and Apaf-1 to form apoptosomes that activate caspase-3 and caspase-7. Activated caspase-3 then activates caspase-2, -6, -8, and -10, resulting in apoptosis .
The balance between cell cycle arrest and damage repair on one hand and the initiation of cell death, on the other hand, could determine if cellular or DNA damage is compatible with cell survival or requires cell elimination by apoptosis. Defects in these processes may lead to hypersensitivity to cellular stress, and susceptibility to DNA damage, genomic defects, and resistance to apoptosis, which characterize cancer cells .
Several oxidoreductases have been identified as potential sources of superoxide in mammalian cells . These include cyclooxygenase, lipoxygenase, cytochrome P450 enzymes, nitric-oxide synthase, xanthine oxidase, mitochondrial NADH:ubiquinone oxidoreductase (complex I) , and NADPH oxidase (Nox). Unlike other oxidoreductases, NADPH oxidase is a distinct enzymatic source of cellular ROS generation, because this enzyme is a “professional” ROS producer  whereas the other enzymes produce ROS only as by-products along with their specific catalytic pathways.
NADPH oxidase is a major source of UVA-induced ROS in keratinocytes  and fibroblasts . NADPH oxidase catalyzes the one-electron reduction of O2 to superoxide anion (O2–) using NADPH as an electron donor. Valencia and Kochevar have demonstrated that the mechanism for activation of Nox1 is mediated by an increase in intracellular calcium. Ceramide, which has been proposed to mediate responses to UVA in human keratinocytes, also activated NADPH oxidase. These results indicate that UVA activates Nox1-based NADPH oxidase to produce ROS that stimulate PGE2 synthesis, and that Nox1 may be an appropriate target for agents designed to block UVA-induced skin injury. Glucose can also stimulate production of NADPH oxidase .
Free Iron, Copper, and Calcium
In mammalian cells, the level of iron-storage protein is tightly controlled by the iron-regulatory protein-1 at the post-transcriptional level. This regulation prevents iron acting as a catalyst in reactions between ROS and biomolecules. It has been shown that both UVB and UVA can cause biological damage in exposed tissues via iron-catalyzed oxidative stress [17, 18]. The iron content is substantially elevated in the sun-exposed skin of healthy individuals . The underlying mechanism appears to be the UVB-induced formation of superoxide radical anion and its attack on ferritin, resulting in the release of free iron . Furthermore, a superoxide anion radical can react with hydrogen, which again enters the Fenton reaction. Interestingly, Reelfs et al. identified the immediate iron release as a key modulator of the activation of NFkB in fibroblasts after UVA-irradiation .
The key question is the availability of catalytic amounts of iron and copper in the skin. UV light and sweat are the two dominant sources for iron and copper . Water is also a source for iron in the skin. It is also easy to see from these data how athletes following an intensive training might become anemic due to loss of iron. Iron-chelating agents have been shown as protectants against UV radiation–induced free radical production .
UVA exposure of skin has been shown to increase intracellular calcium [Ca2+] in fibroblasts , which activates Nox1 thereby increasing the ROS that stimulate a further increase in calcium. The ROS formed initiate production of additional ROS from oxidation of mitochondrial and possibly other membranes and induce synthesis of PGE2, an inflammatory mediator after UVA irradiation, by a Ca2+-dependent mechanism .
Antioxidants Act as Pro-Oxidant
When a general use of antioxidants is advocated, it is often disregarded that these compounds not only function as antioxidants, but (intrinsically) have pro-oxidant action, especially in the presence of transition metals. There is pro-oxidant action even in well-known antioxidants, such as vitamin C (ascorbate), vitamin E (tocopherols), glutathione, and proanthocyanidins (from pine and grape). The pro-oxidant activity of vitamin C results from the reduction of Fe3+ to Fe2+ and its reaction with H2O2 to generate OH radical . Pro-oxidant effects are not unique to vitamin C; they can be demonstrated with many reducing agents in the presence of transition metal ions, including vitamin E, glutathione, and several plant phenolics. Thus if vitamin C’s pro-oxidant effects are relevant, the pro-oxidation effects of these other reductants may also be expected to occur .
High concentrations of vitamin E accelerate lipid auto-oxidation in vitro [25, 26]. Other authors also reported pro-oxidant effects in vitro for a-tocopherol [27, 28]. It is quite possible that a-tocopherol can generate tocopheroxyl radicals on skin under UV radiation and may thereby act as a pro-oxidant. Indeed, adverse biological effects of a-tocopherol are documented . A pro-oxidant activity of carotenoids has also been reported 
Pro-oxidant properties of antioxidants are not all that bad. For example, oxidation of proline to hydroxyproline, the key building block of collagens, is catalyzed by ascorbic acid due to its pro-oxidant activity. It is the excessive production of ROS that causes irreversible skin damage.
In order to counteract the harmful effects of reactive oxygen species (ROS), the skin is equipped with antioxidant defense systems consisting of antioxidant defense enzymes and non-enzymatic antioxidants, forming an “antioxidant network.” The antioxidant network is responsible for maintaining the equilibrium between pro-oxidants and antioxidants. However, the antioxidant defense can be overwhelmed by increased exposure to exogenous sources of ROS. Such a disturbance of the pro-oxidant/antioxidant balance may result in oxidative damage to lipids, proteins, and DNA. Thus, a comprehensive and integrated antioxidant skin defense mechanism is considered to be crucial for protecting this organ from ROS, and consequently for preventing the aging process of skin .
Antioxidant defenses are comprised of a number of species :
(1) Low-molecular-weight antioxidants that scavenge ROS and RNS. These antioxidants can be subdivided into two groups: endogenous (synthesized in the body) and exogenous (derived from the diet). Human skin contains both lipophilic [vitamin E (tocopherols and tocotrienols), ubiquinones (coenzyme Q), and carotenoids], and hydrophilic [(vitamin C (ascorbate), glutathione (GSH), and uric acid (urate)] antioxidants. Vitamin E, vitamin C, and carotenoids are derived from the diet, whereas the other three are synthesized in vivo.
(2) Enzymes that catalytically remove free radicals and other ROS. Examples are the enzymes superoxide dismutase (SOD, converts superoxide to hydrogen peroxide and oxygen), catalase (converts hydrogen peroxide to water and oxygen), glutathione peroxidase (GPx, converts hydrogen peroxide to water and oxygen).
(3) Proteins that minimize the availability of pro-oxidants such as iron ions, copper ions. Examples are transferins, metallothionein.
(4) Proteins that protect biomolecules against oxidative and other damages, e.g., heat shock proteins.
The composition of antioxidant defense differs from tissue to tissue and cell type to cell type and even from cell to cells of the same type. On a molar basis, vitamin C is the predominant antioxidant in skin; its concentration is 15-fold higher than glutathione, 200-fold higher than vitamin E, and 1,000-fold higher than ubiquinones . Concentrations of antioxidants are higher in epidermis than dermis; sixfold for vitamin C and glutathione, and twofold for vitamin E and ubiquinones. Some antioxidants are also present in the stratum corneum, such as vitamin E, which is the predominant antioxidant in the human stratum corneum .
In aged and photo-aged human skin, the components of the antioxidant defense system are regulated differently. In the epidermis, SOD and GPx remain completely unchanged, whereas catalase and glutathione reductase tend to increase. In the dermis, of the four antioxidant enzymes examined by Rhie et al., only catalase activity was significantly decreased . Non-enzyme antioxidants such as α-tocopherol, ascorbic acid, and glutathione were decreased in the epidermis and/or dermis. The decreased antioxidant capacity of aged skin may cause an increased accumulation of ROS, which will affect cell-signaling pathways leading to skin aging .
Ideally, an antioxidant or an antioxidant blend that can quench radicals and nonradicals, chelate free iron, copper, and calcium, inhibit activities of NADPH oxidase and other oxidase enzymes, and stimulate antioxidant defense system, is able to provide true broad-spectrum skin protection.
Skin aging entails drastic changes in the extracellular dermal matrix (ECM) and dermal-epidermal junction (DEJ) areas. In order for an antioxidant to provide anti-aging benefits, it has to work on two fronts: Protect ECM and DEJ from further degradation due to the sun-induced generation of matrix-degrading metalloprotease (MMPs) and stimulate synthesis of ECM and DEJ proteins to provide the bulk of the dermis lost due to the photo- as well as chronological-aging.
Matrix Metalloprotease (MMPs) are involved in the remodeling and degradation of extracellular matrix (ECM) proteins, such as collagens, elastins, fibronectin, and proteoglycans, both as part of normal physiological processes and in pathological conditions. At this time 24 different MMPs have been identified and classified .
Several studies carried out by researchers using dermal fibroblast cells show that both UVA and UVB cause a four- to fivefold increase in the production of MMP-1 and MMP-3 [35–37]. Brennan et al. have shown by punch biopsies of human skin after UV irradiation that MMP-1 rather than MMP-13 as the major collagen-degrading enzyme responsible for collagen damage in photo-aging . In contrast, the synthesis of tissue-inhibitory metalloprotease-1 (TIMP-1), the natural inhibitor of matrix metalloprotease, increases only marginally. This imbalance is one of the causes of severe connective tissue damage resulting in photo-aging of the skin. Many of the molecular alterations observed following UV irradiation occur in sun-protected chronologically aged human skin in vivo.
Extracellular Matrix (ECM)
The skin consists of two main layers, epidermis and dermis, separated by the basement membrane. The ECM is the material that forms the bulk of the dermis, excluding water and cells. Proteins and complex sugars form most of the dermal ECM and they are arranged in an orderly network fibers and ground substances. ECM is a compilation of different macromolecules organized by physical entanglements, opposing ionic charges, chemical covalent bonding, and cross-linking into a biomechanically active polymer. These matrices provide a gel-like form and scaffolding structure with regional tensile strength provided by collagens, elasticity by elastins, adhesiveness by structural glycoproteins, compressibility by proteoglycans – hyaluronans, and communicability by a family of integrins, which exchanges information between cells and between cells and the dermal extracellular matrix.
From a biological point of view in aged skin, significant histological changes occur within the dermis accompanied by an increased degradation of ECM through up-regulation of MMP production as well as a decrease in collagen content and synthesis [39, 40]. This reduction of collagen levels results in skin thinning and increased fragility.
Although collagen content decreases, collagen synthesis in sun-damaged skin appears to remain similar to that of sun-protected sites . Thus, evidence suggests that the decrease in collagen content in photo-damaged skin results from increased collagen degradation, by matrix metalloprotease, without significant changes in collagen production. The skin repercussion on the degradation of the ECM proteins may then be revealed in many ways depending on age, genetic predisposition, lifestyle, and of course, on the general health status of the individual.
Dermal and Epidermal Junction (DEJ)
Structural modifications of the superficial dermis during the aging process draw a parallel with considerable alterations of dermal epidermal junction (DEJ). The epidermis forms an undulating appearance, with intermittent regular protrusions of the epidermis layer (rete pegs) into the upper layers of the underlying dermis. One of the major morphological features of aged skin is a flattening of DEJ with the loss of rete pegs and reduplication of the lamina densa . Flattening of the rete pegs makes the skin more fragile and easier for the skin to shear. This process also decreases the amount of nutrients available to the epidermis by decreasing the surface area in contact with the dermis, also interfering with the skin’s normal repair process. Thus, DEJ changes are considered as crucial markers of skin aging .
At the structural level, DEJ consists of four distinct zones between epidermis and dermis: 1) plasma membrane and hemidesmosomes belonging to basal keratinocytes, 2) lamina lucida containing laminin 5, 3) lamina densa mostly consisting of type IV collagen, perlecan, and nidogen, and 4) a sublamina fibrillar zone containing anchoring fibrils of type VII collagen .
Several of these components have been shown to be altered and reduced in aged skin. Collagen VII, which is responsible for anchoring the basement membrane onto the dermal matrix, decreases with aging . This reduction has been shown to represent an important biochemical marker of wrinkles. During aging, the papillary dermis tends to be reduced, which is related to decreased expression of pro-collagen 1 and 3  and fibrilin 1 . Collagen IV content decreased with age over 35 years. The epithelial basement membrane thickness increased significantly with age and there is an inverse correlation between these two parameters .
Direct application of antioxidants on to skin has the advantage over oral administration because targeting antioxidants to the area of skin needing the protection is easier to achieve. It seems desirable to add low-molecular-weight antioxidants to the skin reservoir by applying antioxidants topically as they protect skin against oxidative stress. A review of the protective effects of topical antioxidants in humans has been published [2, 49].
Tocopherols and Its Derivatives
Tocopherols are a mixture of four lipid-soluble tocopherols (a, b, g, and d) and four lipid-soluble tocotrienols (a, b, g, and d