REVIEWS

The role of antioxidants in photoprotection: A critical review

Lucy Chen, BA,a Judy Y. Hu, MD,b and Steven Q. Wang, MDa New York, New York, and Hermitage, Tennessee
Free radicals have long been studied as a contributor to aging and disease processes. Endogenous production of radicals from cellular metabolism and exogenous sources from ultraviolet radiation and pollution can damage the skin on the cellular and tissue levels. Although the body possesses an elegant defense system to prevent radical damage, this innate system can be overwhelmed and lead to a state of oxidative stress or immunosuppression, and can even trigger carcinogenesis. Topical supplementation of antioxidants can provide additional protection to neutralize reactive oxygen species from both endogenous and exogenous sources. This review will discuss our current understanding of the mechanisms of free radical damage and evaluate the potential benet of topical antioxidants in sunscreens and skin care products. ( J Am Acad Dermatol 2012;67:1013-24.) Key words: antioxidants; free radicals; photoaging; photoprotection; reactive oxygen species; sunscreen.

verexposure to ultraviolet (UV) radiation (UVR) from the sun plays an important role in the development of skin cancers and skin aging. Over the past decade, there has been an increasing understanding on the mechanism by which UVA damages the skin. This awareness is reected in the development of newer sunscreen formulations with protection extending to the long range of UVA wavelengths. This insight, combined with the knowledge that UVA induces free radicals, has led to a renewed research focus on the detrimental role of free radicals on skin health. Although the body has an innate antioxidant (AOx) defense system to neutralize these radicals generated from both the exogenous and endogenous sources, this AOx reservoir can be quickly depleted. Hence, topical supplementation of AOxs, at least in theory, holds the promise of providing extra benet to the skin, especially under oxidative stress from excessive amount of UVA exposure. In this review, we will discuss the sources of free radicals, explain the mechanisms of damage from these radicals, and highlight the cellular and clinical consequences. In addition, we will review common AOxs with demonstrated benets. Lastly, we will

Abbreviations used: AOx: AP-1: ATP: GSH: H2O2: LC: MMP: NF-kB: O2d: OHd: ROS: SOD: UV: UVR: 1 O2: antioxidant activation protein-1 adenosine triphosphate glutathione hydrogen peroxide Langerhans cell matrix metalloproteinase nuclear factor-kB superoxide anion hydroxyl radical reactive oxygen species superoxide dismutase ultraviolet ultraviolet radiation singlet oxygen

examine the limitations in formulating sunscreen and skin care formulations with active AOxs.

PART I: FREE RADICALS

A free radical is dened as a species that can exist independently with one or more unpaired electrons.1 In living systems, free radicals are predominantly represented as reactive oxygen species (ROS), taking form as oxygen-centered oxidizing agents.

From the Dermatology Service, Memorial Sloan-Kettering Cancer Center, New York,a and Cumberland Skin Surgery and Dermatology, Hermitage.b Funding sources: None. Disclosure: Dr Wang is a consultant for LOreal. Ms Chen and Dr Hu have no conflicts of interest to declare.

Reprint requests: Steven Q. Wang, MD, Dermatology Service, Memorial Sloan-Kettering Cancer Center, 160 E 53 St, New York, NY 10022. E-mail: [email protected]. Published online March 12, 2012. 0190-9622/$36.00 2012 by the American Academy of Dermatology, Inc. doi:10.1016/j.jaad.2012.02.009

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The most common oxygen-based ROS are: superoxcause nonspecific cellular damage to DNA, protein, ide anion (O2d), peroxide, hydroxyl radical (OHd), and lipid structures. Environmental pollutants such hydroxyl ion, and singlet oxygen (1O2), an excited as polycyclic aromatic hydrocarbons from fossil fuel state of molecular oxygen. ROS are volatile and combustion can be activated and converted into unstable. In biological systems, ROS add electrons endogenous ROS via quinone intermediates.12 In vitro and in vivo studies demonstrate that a (oxidize) to other nearby molecules to release the common polycyclic aromatic hydrocarbons, benzoextra energy and return to stable states. When not apyrene and its intermediquenched by AOxs, the oxiates, act as photosensitizers, dation reactions can conCAPSULE SUMMARY which upon UVA expotinue, or unravel into sure, synergistically increase cascades with damaging Free radicals from endogenous and production of superoxide consequences. exogenous sources can damage DNA, and 1O2.12-16 A signicant source of enlipid membrane, and protein structures, dogenous ROS comes from and can also induce photocarcinogenesis the byproduct of oxidative CELLULAR DAMAGE and photoaging. metabolism in the mitochonFROM FREE RADICALS Topical antioxidants have the potential dria where adenosine tripExposure to excess UV to supplement the bodys innate defense hosphate (ATP) is generated irradiation and pollutants to neutralize free radicals. from glucose.2 In a coordileads to a pro-oxidant state. nated reaction, electrons The resulting oxidative stress Challenges remain in effectively pass through 4 complexes of can impact the genetic integincorporating antioxidants into the electron transport chain rity of a living organism. sunscreens and skin care products. to generate ATP and water Whereas UVB directly dam(Fig 1). As a side reaction, ages DNA, UVA acts by ROS molecular oxygen is also converted to O2d, a volatile intermediates. ROS-induced DNA damages can lead and potent ROS.3 It is estimated that 1% to 2% of the to the formation of a modied guanine nucleotide oxygen present in the cell divert to these side (8-hydroxyguanine), single-stranded breaks, and reactions.4 Aside from the ATP-generation process, oxidized pyrimidine bases.17,18 These damages, d O2 can also be generated by xanthine oxidase for although predominantly UVA related, have been the degradation of purine nucleotides and by nitric observed in UVB-irradiated cells.19 Incorporation of oxide synthase for the production of nitric oxide, a 8-hydroxyguanine into DNA strands has been implisecondary messenger. O2d is converted into hydrocated in tumor promotion, suggesting that permagen peroxide (H2O2) by spontaneous conversion or nent DNA damage leads to mutagenesis and superoxide dismutase (SOD) (Fig 2). H2O2 is the key carcinogenesis.20,21 In addition to nuclear DNA, the agent in the Fenton reaction, which readily occurs in 4977-base pair mitochondrial DNA deletion, known the presence of metal catalysts (iron or copper) and as the common deletion, is prevalent in human produces OHd, one of the most unstable ROS that skin irradiated with UVA.22 The mechanism has been 5 exists in a biological system. The half-life of OHd is attributed to the generation of 1O2.23 9 so short (10 seconds) that it can exert its damaging Cellular phospholipid membranes and proteins effects at nearly exclusively the site of its generation.6 are also targets of oxidative reactions incurred by UV Exogenous ROS production comes from environrays and ROS. Lipid peroxidation is initiated by an mental sources such as UVR, pollutants, and xenounstable OHd that abstracts a hydrogen atom from biotics (Fig 3). Measurable levels of H2O2 and OHd nearby unsaturated fatty acid. This forms lipid moloccur within 15 minutes after UV exposure and ecules with extra electrons, which form peroxyl continue for up to 60 minutes.7,8 The action specradicals in the presence of molecular oxygen. If not trum for ROS generation is predominately in the UVA quickly terminated, a chain reaction can occur, range (320-400 nm), although there is some overlap wreaking havoc on neighboring lipids and disintewith UVB.9 UVA reacts with photosensitizers or grating the cell membrane. Oxidative damage at the chromophores in the skin, such as cytochromes, protein level is reected in modication of the riboflavin, heme, and porphyrin. These chromopolypeptide chain to form carbonyl derivatives. phores absorb the energy from the UVA wavelength Protein oxidation products appear to accumulate and transition into an excited, unstable state. The and persist preferentially in the dermis.24 As DNA, energy expelled upon return to the stable state is lipid, and protein damages accrue in a cell undergotransferred to nearby oxygen molecules to generate ing oxidative stress, events can potentially spiral 1 O2 and other ROS.10,11 Collectively, these ROS can toward apoptosis. The role of 1O2 and O2d in
d d d

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Fig 1. Formation of superoxide in mitochondrial respiratory chain. Complexes in mitochondrial respiratory chain leak electrons to oxygen-producing superoxide anion (O2d). Increased concentrations of O2d may reduce transition metals, which in turn react with hydrogen peroxide (H2O2)-producing hydroxyl radicals (OHd) or may react with nitric oxide to form peroxynitrite. Both OHd and peroxynitrite are strong oxidants that indiscriminately react with DNA, lipids, and proteins. O2d can be converted into H2O2 and oxygen in both intermembrane space and matrix of mitochondria. Reprinted with permission from Turrens.114 Cyt c, Cytochrome c; SOD, superoxide dismutase.

apoptosis has been demonstrated in cell culture studies.25

CUTANEOUS DAMAGE FROM FREE RADICALS

Photoaging Harman26 first proposed the free radical theory of aging in 1956 stating that free radical accumulation was contributing to the cumulative changes seen in aging. Indeed, free radical damage on the skin by chronic ROS and UV stress plays a major role in photoaging (Fig 4). After UV exposure, ROS trigger the release of proinflammatory cytokines and growth factors.8,27 Specifically, factors activation protein1 (AP-1) and nuclear factor-B (NF-kB) up-regulate key matrix metalloproteinases (MMP) such as MMP1, MMP-3, MMP-8, and MMP-9. Collectively, these proteases degrade the collagen and elastin fibers of the extracellular matrix.28 Interestingly, MMP-1 expression is associated with the presence of mitochondrial DNA common deletion, reinforcing the possibility that ROS affects many points along this pathway.29,30 Furthermore, UVR-induced ROS have been shown to decrease transforming growth factoreb expression, which decreases collagen production and enhances elastin production.31-33 Hence, ROS degrade the structural integrity of skin by way of altering the collagen and elastin components of the extracellular matrix.

Immunosuppression It is known that both UVA and UVB can initiate immunosuppression of the skin.34 The mechanism of UVA immunosuppression is not completely known but a ROS-dependent mechanism has been implicated. UVA-induced ROS can lead to lipid peroxidation, disturb redox potential, initiate AP1 and NF-kB transcription, and eventually activate downstream cytokines (interleukin-4 and -10), which are responsible for systemic immunosuppression.35,36 Mechanistic studies using sunscreens and AOxs specifically implicate ROS in UV-induced immunosuppression, measured by depletion of epidermal Langerhans cells (LC) and suppression of contact hypersensitivity in skin studies.37 With the application of sunscreen, depletion of epidermal LC is prevented and delayed hypersensitivity is improved. The degree of protection is directly related to the level of UVA protection.38-41 In mice studies, Halliday et al42 used AOxs to evaluate UVA-induced immunosuppression. In the presence of topical L-NMMA (nitric oxide inhibitor), iron chelator 2,2dipyridl, and the SOD-mimicking agent 4-hydroxytempol, antigen induction on irradiated skin was reduced to undetectable levels. Similar results using biologically active AOxs, such as green tea polyphenols, have shown a reduction in markers of immunosuppression.43-45 In human studies, application of a formulation of topical AOxs, even in the absence of

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Fig 2. Generation of reactive oxygen species (ROS). Oxygen molecule can be converted into singlet oxygen (1O2) or superoxide anion (O2d). O2d is extremely unstable and can be further converted to hydrogen peroxide (H2O2) either spontaneously or enzymatically by superoxide dismutase (SOD). H2O2 is more stable than O2d and can permeate through lipid membrane of cells. ROS can be neutralized to form water and oxygen or hypochlorous acid. H2O2 can also be converted to hydroxyl radical (OHd) in presence of iron (Fe21) via Fenton reaction (ie, Fe21 1 H2O2 / Fe31 1 OHd 1 hydroxyl ion). OHd can react with nucleotides, unsaturated lipids, and amino acids or be neutralized to water. GSH, Glutathione; O2, molecular oxygen.

sunscreen, can also prevent LC depletion.46 Considerably more mechanistic work needs to be done in this field to determine the role of ROS in immunosuppression. Photocarcinogenesis Although the relationship between UVR and photoaging is well described, the mechanistic connection between ROS and skin cancer is still unclear. At the molecular level, it has been demonstrated that ROS interfere with normal cell signaling by affecting expression of signal transduction genes.47 Aberrant AP-1 and NF-kB pathways have been implicated in cell proliferation and apoptosis leading to carcinogenesis. Halliday34 examined DNA from human actinic keratoses and squamous cell carcinomas for signature ROS mutations. A large number of mutations in both groups were found to be ROS induced on the p53 gene, suggesting that ROS can be a mutagen, driving precursor lesions to malignancy. In addition, the presence of topical AOxs and ROS

inhibitors reduced UV-induced skin carcinogenesis in mice, suggesting a method to attenuate carcinogenesis by reducing ROS.42,48,49

INNATE DEFENSE SYSTEM AGAINST FREE RADICALS

Human skin has an elaborate enzymatic and nonenzymatic AOx defense network against ROS (Table I). The key AOx enzymes include SOD, catalase, and glutathione (GSH) peroxidase. SOD catalyzes the conversion of two volatile superoxide radicals into less volatile H2O2 and oxygen. H2O2 is further reduced to water and oxygen with the aid of catalase and GSH peroxidase (Fig 2). The nonenzymatic AOxs can occupy lipid- and water-soluble compartments of the cell, and the concentration and activity levels of these AOxs are higher in the epidermis than dermis. Both the enzymatic and nonenzymatic AOxs work in a coordinated fashion to neutralize ROS. For example, GSH reductase can regenerate GSH from GSH disulfide, the oxidized

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Fig 3. Cellular and clinical effects of reactive oxygen species (ROS ). ROS are generated from exogenous and endogenous sources. On cellular level, ROS has potential to cause DNA mutation, lipid peroxidation, and protein oxidation. On clinical level, ROS plays a role in photoaging, immunosuppression, and photocarcinogenesis. Antioxidants maintain redox state by quelling these harmful ROS. UV, Ultraviolet.

form of GSH. In turn, GSH can restore vitamins C and E from the oxidized to the reduced state, thereby activating these two AOx to neutralize additional ROS. At the molecular level, another key mechanism against oxidative damage is the transcription factor, NF-E2-related factor 2(Nrf2), and its transcriptional activation of AOx enzymes. Most recent studies have demonstrated that Nrf2 is protective of both skin keratinocytes and fibroblasts against UVA-oxidative damage.50,51 This may be a promising field for therapeutic applications targeting the innate defenses of AOx. Despite these innate defenses, increased oxidative stress can overwhelm the skins AOx reserves and enzymatic machinery. Shindo et al52,53 demonstrated decreased levels of both enzymatic (SOD, GSH peroxidase, catalase activity) and nonenzymatic (a-tocopherol, GSH, and L-ascorbic acid) AOxs on mice skin when the animals were exposed to acute UV irradiation. In human beings, even at suberythmogenic UVR doses, the AOxs in the stratum corneum are susceptible to depletion.54 Aging also diminishes AOx levels: compared with young human subjects, elderly subjects had 70% less concentration of a-tocopherol, L-ascorbic acid, and total GSH in their skin.55

PART II: TOPICAL ANTIOXIDANTS

There is a growing trend in incorporating AOxs in sunscreens and skin care products to replenish the natural reservoirs in the skin. Topical AOxs have the potential to diminish the ROS generated from the UVA radiation. In the following section, common topical AOxs and their effectiveness as a component of photoprotection are reviewed, and additional compounds with AOx properties are featured in Table II.

Vitamin C Vitamin C is a water-soluble AOx and it is the predominant AOx in the skin based on molar concentrations.54 Vitamin C neutralizes free radicals in aqueous compartments of the skin, and also plays a role in regenerating vitamin E. Aside from serving as an AOx, it is also a cofactor for critical enzymes in collagen synthesis and can inhibit elastin biosynthesis to reduce elastin accumulation.56 It also reduces pigment darkening by inhibiting tyrosinase and maintains hydration by protecting the epidermal barrier of the skin.57 At the molecular level, addition of topical 1% vitamin C increases collagen synthesis and reduces MMP (collagenase) expression.58

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Fig 4. Role of reactive oxygen species (ROS ) in photoaging. ROS from exogenous (eg, ultraviolet [UV] radiation) and endogenous sources initiates signal transduction cascade resulting in up-regulation of AP-1, NF-kB, and down-regulation of transforming growth factor (TGF )-b. Downstream, NF-kb signals increase in interleukin-1 and tumor necrosis factor-alfa levels, and AP-1 activates matrix metalloproteinases (MMP). Decrease in TGF-b expression leads to decrease in collagen synthesis. Cumulatively, these changes lead to increase in collagen breakdown, and increase in elastin production in extracellular matrix.

Table I. Endogenous antioxidants

Nonenzymatic antioxidants a-Tocopherol (vitamin E) Ascorbic acid (vitamin C) Glutathione Carotenoids Ubiquinone Flavonoids Uric acid Enzymatic antioxidants Superoxide dismutase Glutathione peroxidase Glutathione reductase Catalase

unstable. As a result, many formulators use more stable esteried substitutes, such as magnesium ascorbyl phosphate and ascorbyl-6-palmitate. Compared to L-ascorbic acid, the AOx activities of these substitutes are inferior and do not achieve the same activity levels in vivo.57,61,62 Vitamin E Vitamin E is a lipid-soluble AOx, and it exists as 8 major compounds (4 tocopherols and 4 tocotrienols) with the most abundant form being a-tocopherol. Its main function is to protect the cell membranes from oxidative stress. The highest concentration of vitamin E is delivered to the deepest layers of the stratum corneum by sebaceous gland secretion. The level of vitamin E can be depleted even after a single suberythemogenic dose of UVR exposure.54 A multitude of animal and human studies have demonstrated a reduction in lipid peroxidation,63 photoaging,64,65 immunosuppression,48,66,67 and photocarcinogenesis48,49 after topical vitamin E application. On the molecular level, topical a-tocopherol decreases MMP-1 transcription levels and

Application of topical L-ascorbic acid has been shown to have photoprotective effects including the reduction of erythema,59 sunburn cell formation,59 and immunosuppression.60 Delivery of topical application into the skin is a challenge. To penetrate the stratum corneum, L-ascorbic acid must lose its ionic charge and be in a formulation with a pH less than 3.5. At these pH settings, the hydroxyl group of L-ascorbic acid is

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Table II. Benefits of antioxidants in topical formulation

Antioxidant compound Sources Clinical end points studied

Vitamin C (ascorbyl palmitate, magnesium, ascorbyl phosphate)

Fruits, vegetables

Vitamin E (a-tocopherol acetate, a-tocopherol succinate)

Vegetable oil, seeds, nuts, meats

Vitamin A (retinols, carotenoids) Selenium Silymarin Green tea polyphenols (epicatechin, epicathechin-3-gallate, epigallocatechin, epigallocatechin-3-gallate) Soy isoflavones (genistein, daidzein, equol)

Colored fruits and vegetables (eg, tomatoes, sweet potatoes) Corn, wheat, soybean Milk thistle Fractions isolated from tea

Erythema59 Immunosuppression60 Photoaging92 Photocarcinogenesis64 Erythema93,94 Photoaging64,65 Immunosuppression48,66 Photocarcinogenesis48,49 Photoaging95 Erythema77,96 Photocarcinogenesis78,96 Photocarcinogenesis80,97 Immunosuppression98 Erythema44 Immunosuppression 43-45 Photoaging99 Photocarcinogenesis83 Erythema84,100,110 Photoaging84,102 Immunosuppression101 Photocarcinogenesis84,103 Erythema104 Immunosuppression105 Photoaging106 Photocarcinogenesis107 Erythema108 Photoaging109,110 Photocarcinogenesis109 Inflammation111 Immunosuppression111 Photocarcinogenesis111 Erythema112 Photocarcinogenesis113

Soy, red clover, ginkgo biloba

Caffeic acid (ferulic acid, caffeic acid phenethyl ester) Apigenin Polypodium leucotomos extract

Coffee beans, propolis, plant seeds Fruits and leafy vegetables, tea, wine Tropical fern plant Polypodium leucotomos Extract from bark of maritime pine tree

Pycnogenol

Resveratrol

Skin and seeds of grapes, nuts, fruits, red wine

inhibits thymine dimer formation, thereby slowing down the process of collagen breakdown and mutagenesis, respectively.68,69 The protection against dimer formation has been postulated to be a result of the AOx interplay with ROS rather than a UVBabsorbing sunscreen effect.70 Vitamins C and E work in conjunction in an elaborate network of redox reactions to stave off oxidative stress. Vitamin C regenerates oxidized vitamin E at sites of lipid peroxidation. Oxidized vitamin C requires GSH for its own regeneration. This interaction maintains the AOx reservoir in the skin tissues. Compared with vitamin C alone, the combination of 15% L-ascorbic acid and 1% a-tocopherol doubles the protection against UV-induced erythema, sunburn cell formation, and thymine dimer formation.71 Moreover, stabilizing agents such as 1.5% ferulic acid and phloretin, two powerful plant AOxs, provide even greater benefit in vitamin C

and E combination formulas, possibly by enhancing vitamin uptake into skin.67,72 This combination inhibits tanning and immunosuppression in mice and tanning in human beings. Vitamin A The two main forms of vitamin A used in topical form are retinoids and carotenoids. The carotenoids on the skin scavenge 1O2 and quench lipid peroxidation.73 Upon UV irradiation, the concentrations of human skin carotenoids, b-carotene and lycopene, are markedly reduced.74 In the topical form, retinoids are commonly found in sunscreens and skin care cosmetics. The safety of retinyl palmitate, the storage form of vitamin A (retinol) has come under scrutiny because of animal studies suggesting it has photocarcinogenic effects upon UV irradiation. However, evidence from long-standing use of topical retinoids in clinical medicine demonstrates that they

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are safe.75 Retinol and its forms (tretinoin, isotretinoin, and tazarotene) are marketed as having antiaging properties. The mechanism of action of these molecules is to bind to the nuclear receptors, retinoic acid receptors, and retinoid X, which will inhibit AP-1 and MMP-1 expression.56 The benefits are increased collagen production and increasing epidermal thickness. Selenium Selenium is an essential element to optimize the activity of GSH peroxidase and thioredoxin reductase, and it also serves as a cofactor for vitamin E regeneration. In general, selenium sulde and L-selenomethionine are the common forms used for topical delivery. The latter form has shown to have superior transepidermal delivery.76 Topical L-selenomethionine increases the minimal erythemal dose in human subjects.77 When combined with vitamin E, selenium has shown to diminish UVinduced blistering, pigmentation, and skin tumors in mice studies.78 Silymarin Silymarin from the milk thistle plant contains a combination of 3 avonoids, silybin, silydianin, and silychristin. Of these, silybin has the highest biologic potency to scavenge ROS and prevent lipoprotein oxidation. Topical application of silymarin inhibits sunburn cells, decreases pyrimidine dimers, and decreases skin tumors in hairless mice.79,80 Tea polyphenols Tea contains a rich level of polyphenols in the forms of epicatechin, epicatechin-3-gallate, epigallocatechin, and epigallocatechin-3-gallate. Unfermented tea extract has a very high antioxidative activity, which diminishes in the making of commercial green, black, and oolong tea. Like other AOxs, tea polyphenols are inherently unstable and a large portion of their biological activity is lost over a short duration. The topical formulation of polyphenol has been stabilized by butylated hydroxytoluene to reduce its susceptibility to oxidation.81 Hence, it is important to note that not all products containing tea extracts exhibit the same level of AOx properties. As AOxs, tea polyphenols are more potent than vitamins C and E in scavenging ROS.82 In addition, tea polyphenols, specifically epigallocatechin-3-gallate, has anti-inflammatory and anticarcinogenic effects 43,83 and can inhibit collagenase activity. In human studies, erythema and LC depletion have been examined.44

Soy isoavones Soybeans contain isoavones in the forms of genistein and daidzein. Diets high in soybeans are protective against various cancers and cardiovascular disease.84 Isoflavones have been found to be anticarcinogenic through scavengers of peroxyl and lipid radicals. Topical application of genistein has shown to decrease UV-induced oxidative damages, such as immunosuppression and inflammation.80,85,86

PART III: ANTIOXIDANTS IN PHOTOPROTECTION

Sunscreen remains one of the most widely adopted strategies by the public to protect themselves from UVR. However, because of inadequate application and compensatory exposure where users of sunscreens tend to stay out in the sun longer, the degree of UV protection is much lower in practice than stated in the product labels. Furthermore, current sunscreens on the market tend to offer more UVB than UVA protection. Sunscreens may not offer adequate protection against UVA-induced ROS. In fact, Haywood et al87 has shown that sunscreens with broad-spectrum UV protection only reduce free radical formation by 55%. Therefore, topical delivery of AOx can provide additional benefit to complement the protection from UV filters. The protective benet derived from combining AOxs with sunscreen has been demonstrated in human studies. In a study by Matsui et al,88 participants received two topical products: one sunscreen with an SPF 25 (SS) and the same sunscreen with an AOx mixture of caffeine, vitamin E and vitamin C, Echinacea pallida extract, gorgonian extract, and chamomile essential oil (SS1AOx). After UVR to the skin, the SS1AOx group had a 17% greater reduction in MMP-1 levels compared with the SS group. Both the SS and SS1AOx groups also protected against the depletion of LC. Wu et al46 used a similar study design with an AOx preparation containing vitamin C, vitamin E, chamomile extract, Echinacea pallida extract, and caffeine. The investigators found the SS1AOx group had significant protection against MMP-9 induction, pigment formation, and markers associated with epidermal hyperproliferation, when compared with SS or AOx alone. These data add to the growing knowledge that AOxs can add value to sunscreens but more in vivo research is needed to determine the best AOxs to use in sunscreen formulations. Despite the potential benet, formulating products that combine AOxs with sunscreen is a challenge. To ensure the efcacy of AOxs in the nal products, a number of technical requirements must

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be fullled. First, AOxs need to have a high antioxidative capacity and be present in high concentration. Second, AOxs need to be stable in the nal formulation. In general, AOxs are inherently unstable. In the case of vitamin E and C, tocopheryl acetate (a stabilized form of tocopherol) and ascorbyl palmitate (a stabilized form of ascorbic acid) are used as substitutes. However, these substitutes have very low biological activity. Other AOxs, such as ubiquinone, idebenone, and kinetin are degraded upon UV exposure.89,90 Third, AOxs need to penetrate the stratum corneum and maintain adequate concentrations in the epidermis and dermis. On the other hand, it is desirable to keep UV filters on the skin surface and not penetrate the skin. The conflicting goals for delivering AOxs and UV filters create additional challenges in the final formulation. Wang et al91 showed that sunscreens contained AOxs protected against free radicals, but nearly all of these tested sunscreen products had no or very minimal AOx capacity to neutralize the free radicals. In fact, the study showed that the radical protection is entirely from the UVA filters in the sunscreens. Many sunscreen products on the market claim to offer AOx protection, but they have inadequate or no AOx capacity to achieve any meaningful protection against free radicals.

CONCLUSION
ROS from endogenous and exogenous sources, such as UVR and pollution, can damage the DNA, lipid membrane, and protein structures, and also play a role in the acceleration of photoaging and the development of skin cancer. Although the bodys innate AOx defense can neutralize ROS, these protective agents may be overwhelmed and depleted when faced with an excessive amount of oxidative stress. Delivery of topical AOxs has the potential to provide additional benets, but there remain many challenges in effectively incorporating AOxs in skin care and sunscreen formulations.
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