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THE IMPACT OF ANTIOXIDANTS AND GLYCATION ON OXIDATIVE STRESS AND AGING by Michele M. Sonier A thesis submitted to the Graduate Faculty of THE UNIVERSITY OF BRIDGEPORT In partial fulfillment of the requirements For the degree of MASTER OF SCIENCE In Human Nutrition January 15, 2010 Bridgeport, CT Approved by Dr. Barry Kendler Advisor

Table of Contents Table of Contents ...................................................................................................................... ii Abstract .................................................................................................................................... iii Introduction ............................................................................................................................... 1 Literature Review...................................................................................................................... 3 Discussion ............................................................................................................................... 31 Conclusion .............................................................................................................................. 34 References ............................................................................................................................... 35  


Abstract This literature review discusses the impact of oxidative stress on the aging of human skin cells and demonstrates that antioxidants and a diet low in Advanced Glycation End Products (AGEs) can prevent and reverse the visible signs of skin aging. The review explains the process of cellular aging and discusses the role UV-induced oxidative stress and reactive oxygen species (ROS) play in skin aging. Recent research shows that oral and topical antioxidants, particularly vitamins A, C, E, B3, green tea, pycnogenol and CoQ10, can prevent and reverse the signs of aging and UV exposure. Also discussed is the process of glycation and AGE formation and how this natural process accelerates aging by damaging collagen repair mechanisms. Glycation, or the natural covalent bonding process of linking a sugar to a protein molecule, is further induced by UV exposure and a high sugar diet. Lastly, it reviews the role that AGEs have in damaging skin and discusses nutritional compounds that may reduce or prevent the damage. Based upon the findings, oral antioxidant supplementation has limited benefits to diminish the clinical signs of aging. However, topical application of antioxidants, alone or in combination, can prevent UV-induced aging and can reverse its effects. Further, a diet that is low in simple sugars and low in AGEs may also be a useful tool in preventing the clinical signs of aging.


Introduction This review discusses the impact of oxidative stress on the aging of human skin cells and demonstrates that antioxidants and a diet that is low in Advanced Glycation End Products (AGEs) can prevent and reverse the visible signs of skin aging. Also discussed is the process of cellular aging and how oxidative stress combines with exposure to sunlight to cause damage to human skin. Recent research is reviewed showing how antioxidant vitamins, both ingested and applied topically, can prevent and reverse the negative effects of aging and sun damage on the skin. The process of glycation is explained and how it impacts collagen repair mechanisms in skin, thus contributing to the aging process. It concludes that Advanced Glycation End Products damage skin and are a contributing factor to visible signs of aging. Human skin can age prematurely due to oxidative stress caused by human biochemical composition and oxidative stress caused by environmental factors. With scientific innovation feeding the cultural quest for continual youth, the anti-aging industry has become a booming business. Cosmeceuticals are defined as personal care products that in addition to their value as cosmetics, provide an added health benefit such as UV skin protection, moisturizing, or wrinkle or acne reduction. Demand in the United States for cosmeceutical products is expected to increase 7.2% per year to $9.4 billion in 2012. 1 Within the anti-aging market, the cosmeceutical industry is growing nearly twice as fast as the overall cosmetics and toiletries market. The largest and the fastest growing segment of the cosmeceutical area is skin care. Within skin care, anti-aging is the main driver both in revenue growth and innovation.1 While cosmeceutical products may claim added benefits, they do not claim a therapeutic effect and this has been somewhat criticized as it relates to marketing of these products. The bulk of the anti-aging industry is fueled by affluent women of the baby boomer generation in the U.S. and Europe with disposable incomes. As such, the marketing of these products is largely targeted to this segment of the population. As the world economy has faltered in recent years, research has shown that there is some elasticity of demand for anti-aging products. However,


the research showed that while consumers do tend to spend money on these products, they do tend to gravitate toward spending on products that are effective, yet reasonably priced and widely available. As a result, there is increased pressure on the industry not only to be innovative, but also to market only those products that are backed by scientific evidence and proven results. Many of the anti-aging products marketed today to this population are antioxidants or contain antioxidants. Many of these products take advantage of the fact that the general public has a positive connection with the effects of vitamins with respect to their antioxidant capabilities. An impressive amount of clinical research has been conducted in an attempt to mitigate the effects of sun damage as well as substantiate the addition of vitamins to skin care products.1 As nutritionists, it is increasingly important to be able to provide informed counseling regarding any marketing claims. Cultivating an understanding of the research is important for not only the industry, but also for the general population as they look to make informed purchasing decisions.


Literature Review There are two basic ways that skin aging occurs. Aging can occur through the inevitable chronological process of aging as well through the addition of exogenous factors such as sunlight. Chronological skin aging is not completely understood; however, it is understood that changes in appearance occur over time. As with every cell in the body, skin chromosomes are capped with identical repeating sequences called telomeres, which exist because cells do not have a mechanism to replicate the terminal portion of their DNA. Over time, telomeres are continuously shortening to protect the functional chromosome from degradation. Once the telomere is significantly shortened, cells either enter the biological aging process or undergo apoptosis, both of which lead to a reduction in functional ability. Since skin cells are some of the most actively dividing cells in the body, one hypothesis is that their telomeres may shorten more rapidly and age before other types of cells, thus leading to many of the manifestations of chronological aging. In sun exposed skin, UV irradiation also damages DNA and accelerates telomere shortening. Aging and photodamage appear to share a common final pathway following disruption of the telomere. These telomere-initiated responses, in combination with UV-induced damage to critical regulatory genes may lead to the familiar picture of photoaging.2 Another hypothesis is that repetitive exposure to reactive oxygen species formed during a lifetime of oxidative metabolism leads to an accumulation of cellular damage. Research has shown that older people tend to have accumulated more DNA damage than younger people, as evidenced by increases in levels of both matrix metalloproteinase (MMP) messenger RNA and protein as well as decreased levels of collagen synthesis. When there are increased levels of MMPs, there are imbalances between the creation and destruction of dermal collagen, which leads to increased signs of aging.3 The mechanisms by which premature exogenous skin aging occurs are much better understood than that of chronological aging. One of the most studied mechanisms by which premature skin aging develops is through ultraviolet induced photodamage. The contribution of


ultraviolet light to the skin’s natural aging process is known as photoaging. Skin that has been overly exposed to the sun develops such characteristics as fine and coarse wrinkles, telangiectasias (small dilated blood vessels near the surface of the skin or mucous membranes), irregular pigmentation and rough textures. Histologically speaking, healthy skin is replaced with disorganized collagen fibrils and abnormal amorphous elastin-containing material. Thinning of the outer layers of skin also becomes apparent. Additionally, after excessive long-term exposure to the sun, actinic keratosis may develop. There is also a clinically demonstrated relationship between UV exposure and nonmelanoma skin cancers.4 UV irradiation leads to the changes through a variety of means, including but not limited to reactive oxygen species formation, protein oxidation and mitochondrial damage. Both UVA and UVB have been linked with the photoaging process. Even though UVB is primarily responsible for UV-induced erythema, commonly referred to as sunburn, UVA is emerging as the greater contributor to photoaging due to its ability to penetrate significantly deeper into the dermis than UVB and its 10fold greater abundance in sunlight.5 When human skin is exposed to UV irradiation, reactive oxygen species (ROS) are formed. The type of ROS generated depends on the UV wavelength. UVB tends to stimulate the production of superoxide anion radicals by way of activation of NADPH oxidase and respiratory chain reactions. UVA generates singlet oxygen through a photosensitizing reaction with internal chromophores such as riboflavin and porphyrin. UVA also produces superoxide anion radicals through NADPH oxidase activation and photosensitization of advanced glycation products.6 The major type of ROS produced on the skin’s surface is singlet oxygen, which is generated by a photosensitizing reaction with UVA and porphyrins from bacterial flora living in the skin. Singlet oxygen is oxidized to squalene, cholesterol and to unsaturated acyl residues in the sebum to yield lipid hydroperoxides.6 Oxidized lipids and proteins cause alterations in skin conditions. The topical application of oxidized squalene in the form of squalene monohydroperoxide on the skin disrupts the skin barrier


function acutely and causes the skin to become rough in response. Alkyl aldehydes further oxidize lipid hydroperoxides and proteins to produce carbonylated proteins in the stratum corneum (SCCP). The SCCP levels go up after UV exposure and in the winter season. They are also increased in the skin of patients suffering from atopic dermatitis. Thus SCCP levels appear to reflect the amount of oxidative stress in the skin that is caused by the environment. Oxidative stress caused by ROS does in fact alter skin conditions.6 UVB radiation induces erythema or redness on the skin. A nitric oxide synthase (NOS) inhibitor and a cyclooxygenase (COX) inhibitor both mitigate sunburn. ROS, including nitric oxide, induce skin erythema through prostaglandin E2 synthesis. Expression of COX-2, an important enzyme in prostaglandin E2 synthesis, is up-regulated by ROS to stimulate the inflammation process.6 Hydrogen peroxide, an ROS itself as well as a propagator of more ROS, increases in the body within 15 minutes of UV exposure and continues to form for up to 60 minutes after the sun exposure has ended. These ROS lead to increases in pro-inflammatory cytokines and up-regulation of MMPs, which in turn decrease collagen production and increase collagen breakdown. This is observed in skin without evidence of sunburn.4 ROS can both degenerate pigmentation and increase pigmentation. An example where ROS can degenerate pigmentation is in the skin disorder vitiligo. ROS can increase skin pigmentation in the form of melasma or sun spots. In the presence of ROS, keratinocytes next to melanocytes contribute to melanogenesis by increasing the amount of tyrosinase and tyrosinase-related protein 1, both of which are key components of melanocytes. The hydrogen peroxide generated by UVB radiation activates epidermal phenylalanine hydroxylase (PAH), which is an enzyme that makes the amino acid L-tyrosine from L-phenylalanine. It is a contributing factor in melanogenesis because it increases the pool of available L-tyrosine. Exposure to one minimal dose of UVB increases PAH activity for up to a full day.6 The contribution of ROS to melanogenesis has been demonstrated using antioxidants. Îąmelanocyte stimulating hormone is increased by UVB but is stopped by the addition of N-acetyl


cysteine, a precursor to glutathione. Additionally, an endogenous antioxidant metallothionein also suppresses melanogenesis.6 ROS have an established role in UV-induced skin aging, characterized by wrinkles. Generally speaking, wrinkles are caused by alterations of the dermal matrix in which collagen levels are decreased by way of accelerated collagen breakdown and reduced collagen synthesis.6 The singlet oxygen generated by UVA irradiation stimulates the expression of matrix metalloproteinase (MMP)-1 in dermal fibroblasts through the secretion of interleukin (IL)-1Îą and interleukin (IL)-6. Oxidized lipids, such as linoleic acid hydroperoxide, also facilitate the expression of MMP-1 and MMP-3.6 UV exposure also lowers the synthesis of new collagen. Collagen synthesis is also decreased when human dermal fibroblasts are exposed to ROS, so it can be stated that ROS also regulate collagen synthesis.6 The skin has enzymes in it that function as antioxidants. These enzymes include catalase, copper-zinc superoxide dismutase and manganese superoxide dismutase, all of which are present in higher concentrations in the epidermis than in the dermis. Skin from subjects with evidence of sun damage shows significantly lower expression of antioxidants than skin from younger subjects or from subjects without evidence of sun damage. It has been found that both acute and chronic UV exposures reduce levels of antioxidants in the skin and allow oxidative damage of resident proteins to occur. In theory, the replacement of these antioxidants may reverse or slow the signs of clinical and histopathologic signs of photoaging.4 Studies have determined that many vitamins possess strong antioxidant capabilities and may play a role in processes involved in skin growth and repair mechanisms. As such, the benefits of vitamins consumed orally as well as applied topically have been widely explored.4 For the majority of the population, overall oral vitamin intake is dependent upon dietary consumption. As such, clinical trials are difficult to perform since they are heavily reliant upon recollection of diet, which can be unreliable. Additional factors such as nicotine use, exercise, topical


product use and UV exposure can also vary the results.4 Of the few studies that have been conducted on dietary vitamins and photoaging, the largest used the National Health and Nutrition Examination I Survey database, from a nationwide survey of approximately 32,000 people conducted between 1971 and 1974.7 Of the original sample, 4,025 women between the ages of 40 and 74 years received comprehensive dermatologic examinations and adequately completed 24-hour dietary recalls as part of the survey. Skin-aging appearance was defined as having a wrinkled appearance, senile dryness and skin atrophy.7 There was also an attempt to estimate lifetime sun exposure based upon factors such as history, occupation and recreational habits. Other mitigating circumstances were also measured including smoking habits, body mass index, menopausal status, supplement intake and race. Higher vitamin C intakes were associated with a lower likelihood of a wrinkled appearance and senile dryness. Higher linoleic acid intakes were associated with a lower likelihood of senile dryness and skin atrophy. A 17-gram increase in fat and a 50-gram increase in carbohydrate intakes increased the likelihood of a wrinkled appearance and skin atrophy. These associations were independent of age, race, education, sunlight exposure, income, menopausal status, body mass index, supplement use, physical activity, and energy intake. Thus researchers concluded that higher intakes of vitamin C and linoleic acid and lower intakes of fats and carbohydrates are associated with better skin-aging appearance. Researchers encouraged promoting healthy dietary behaviors to enhance and benefit skin appearance in addition to other health outcomes in the population.7 However, no definitive conclusions can be drawn considering that the results relied solely on one 24-hour dietary recall. The one dietary recall may or may not be representative of one’s regular diet and was not specifically designed to measure all signs of photoaging.4 In more recent years, oral vitamin supplementation has become more common. As such there have been a few research studies attempting to examine different combinations of oral vitamin supplementation in the treatment and prevention of photoaging.4 Although two clinical trials have reported positive results, they were conducted with very small sample sizes and they used formulations with a number of different ingredients. Sixty-two women aged 45-73 years participated


in a double-blind, placebo-controlled trial testing the efficacy of a proprietary oral supplement for skin nutrition, for improvement of skin elasticity and roughness. The active ingredients were vitamins C and E, carotenoids, selenium, zinc, amino acids and glycosaminoglycans, blueberry extract and pycnogenol.8 Skin elasticity, measured using an optical cutometer, was found to be increased by 9% after 6 weeks of treatment compared with placebo. Skin roughness, as evaluated by three-dimensional microtopography imaging, was found to be lowered by 6% compared with the control group after 12 weeks of treatment.8 A 6-month study of 40 women, with 20 taking a placebo and 20 taking a supplement found that 18 subjects taking the supplement reported positive results. The supplement contained vitamin C, vitamin E, vitamin B3, vitamin B5, zinc, copper, soy extract, pine bark extract, lycopene, red clover extract, marine protein complex, and ι-lipoic acid.9 Objective measurements of skin thickness and elasticity, together with subjective clinical assessments of various parameters (fine wrinkles, coarse wrinkles, tactile roughness and telangiectasia) were used to evaluate changes after 2, 4 and 6 months’ treatment. Self-evaluations were also made by the study participants. There was a significant improvement in skin quality in both objective and subjective parameters after treatment with the preparation compared with placebo.9 However, there is more data in existence that examines the use of individual vitamins, mainly in topical formulations, for the treatment of photoaging. Vitamin C, also known as ascorbic acid, is a water-soluble vitamin found predominantly in citrus fruits and dark green leafy vegetables. It is widely known for its involvement in many different reactions in the body, mainly as a cofactor providing electrons necessary for reducing molecular oxygen. Ascorbic acid is a necessary component in reactions ranging from folate, dihydrofolate, and tetrahydrofolate reduction, norepinephrine synthesis, maintenance of the functional activity of vitamin E, metabolism of prostaglandin and prostacyclin and long-chain fatty acid transport across membranes through its role in carnitine synthesis.4


Vitamin C is a natural compound produced in most plants and animals. However, humans lack the enzyme to produce it. Vitamin C is also found in the skin. However, even small amounts of sun exposure (i.e. 1.6 minimum erythema dose) decreases skin levels of vitamin C by a third.10 Ascorbic acid eliminates most ROS due to the oxidation of ascorbate to monodehydroascorbate and then to dehydroascorbate.6 In the skin, ascorbic acid plays an important role in collagen and elastin synthesis as a cofactor required for the enzymatic activity of prolyl and lysyl hydroxylases, which hydroxylates prolyl residues in procollagen and elastin and catalyzes the formation of hydroxyproline and hydroxylysine.6 Both proline and lysine are major components of collagen and their hydroxylation facilitates the excretion of procollagen from the fibroblast.4 Ascorbic acid also modulates the effects of UV-induced ROS damage. In fact ascorbic acid is one of the most efficient antioxidants in aqueous compartments, exerting its effects both intracellularly and extracellularly. It is due to its role in collagen production and ability to eliminate ROS that ascorbic acid has been studied as a therapy against the effects of photoaging.4 UV irradiation depletes ascorbic acid levels by depleting and reducing transcription of sodium-dependent vitamin C transporters found in the epidermis.4 However the transporters found in the dermis where collagen production takes place are unaffected and may still be fully used by increasing concentrations of ascorbic acid. Theoretically speaking, adding sufficient levels of ascorbic acid back into the skin may help to reverse or negate the effects of UV irradiation.4 Of the few clinically controlled trials in humans, some have shown favorable results, but all have been carried out with very small sample sizes. One double-blind, clinically controlled trial compared the effects of 3 months of daily L-ascorbic acid treatment on half the face with vehicle treatment on the other half. There were 19 volunteers between the ages of 36 and 72 and the study used clinical assessment, patient self-appraisal questionnaires and optical profilometry analysis to assess the effects of treatment.11 Fine wrinkling, tactile roughness, visual dryness, telangiectasia, laxity/tone, pigmentation and keratosis were all considered in the evaluation. Clinical assessment demonstrated significant improvement with active treatment versus control for fine wrinkling, tactile


roughness, coarse rhytids, skin laxity/tone, sallowness/yellowing, and overall features. Patient questionnaire results demonstrated statistically significant improvement, with the active treatment 84.2% greater than control. Photographic assessment also demonstrated significant improvement, with active treatment 57.9% greater than control.11 A topically applied cream containing 5% vitamin C and its excipient were tested on healthy female volunteers presenting with photoaged skin on their lower neck and arms to evaluate efficacy and safety of such treatment. A double-blind, randomized trial was performed over a 6-month period, comparing the action of the vitamin C cream versus the excipient on photoaged skin.12 Clinical assessments included evaluations at the beginning and then after 3 and 6 months of daily treatment. After 3 months, both patients and clinicians noticed a significant improvement in global score. The global score was a calculation based on scores of hydration, roughness, laxity, suppleness, fine wrinkles and coarse wrinkles. A highly significant increase in the density of skin microrelief and a decrease of the deep furrows were demonstrated. Structural evidence of the elastic tissue repair was also obtained and corroborated the favorable results of the clinical and skin surface examinations. Topical application of 5% vitamin C cream was effective, suggesting a positive influence of topical vitamin C on parameters characteristic of sun-induced skin aging.12 A separate trial looked at the effects of 6 months of daily topical 5% L-ascorbic acid treatment at the molecular level, substantiating in vitro findings regarding the role of ascorbic acid. Ten post-menopausal women were treated with the active formulation on one arm and a placebo on the other.13 At 6 months, messenger RNA levels of collagen type I and type III were significantly increased in the treated biopsy specimens compared with placebo. However, the stimulating activity of topical vitamin C was most conspicuous in the women with the lowest dietary intake of the vitamin and unrelated to the level of actinic damage. The results indicate that the functional activity of the dermal cells is not maximal in postmenopausal women and can be increased.13 Another placebo-controlled trial of 10 female patients applied in a double-blind manner a newly formulated vitamin C complex having 10% ascorbic acid (water soluble) and 7%


tetrahexyldecyl ascorbate (lipid soluble ascorbic acid derivative) in an anhydrous polysilicone gel base to one-half of the face and the inactive polysilicone gel base to the opposite side.14 Clinical evaluation of wrinkling, pigmentation, inflammation and hydration was performed prior to the study and at weeks 4, 8, and 12. Biopsies of the lateral cheeks were performed at 12 weeks in to test the levels of mRNA for type I collagen. A questionnaire was also completed by each patient.14 A statistically significant improvement of the vitamin C-treated side was seen in the decreased photoaging scores of the cheeks. The peri-orbital area improved bilaterally, probably indicating improved hydration. The overall facial improvement of the vitamin C side was statistically significant. Biopsies showed increased Grenz zone collagen, as well as increased staining for mRNA for type I collagen.14 No patients were found to have any evidence of inflammation. Hydration was improved bilaterally. Four patients felt that the vitamin C-treated side improved unilaterally. No patient felt the placebo side showed unilateral improvement.14 Ascorbic acid is also used widely as a depigmentation agent due to its inhibitory effect on tyrosinase. Studies have also shown that ascorbic acid can contribute to the formation of the skin barrier by enhancing epidermal differentiation and stimulating blood flow through production of nitric oxide.6 Although ascorbic acid is widely used in topical preparations for the skin in order to achieve such clinical improvements, poor skin penetration and instability in formulations reduces its clinical efficacy. It is unclear how much, if any, in tact molecules remain on the skin when applied.6 It is completely absorbed by the distal small intestine when ingested in doses of up to 100 milligrams per day. If dosages are increased, for instance with vitamin supplementation, the body only absorbs a fraction of the total amount of the vitamin available, as levels of ascorbic acid in the blood are regulated by renal filtration.4 To overcome these disadvantages, several alternatives have been synthesized and evaluated for their potential as pro-ascorbic acid derivatives. These derivatives involve esterification of the hydroxyl group in order to form a more stable product.10 Some of these alternatives include magnesium L-ascorbyl-2-phosphate, ascorbic acid 2-O-Îą-glucoside, 6-acylated


ascorbic acid 2-O-α-glucoside, and tetra-isopalmitoyl ascorbic acid.6 In order for the body to use ascorbic acid, it must convert it to L-ascorbic acid and many of the stabilized commercial forms have not been tested to determine if this conversion is possible or if they penetrate tissues to the extent of L-ascorbic acid.4 One study looked at topical vitamins C and E, as well as topical selenium to protect skin against sunburn, suntan and skin cancer and also reverse the mottled pigmentation and wrinkles of photoaging.15 It found for effective topical application, vitamin C must be non-esterified, acidic and optimally at 20% concentration and with a pH of 3.5. It showed that vitamin E must be the nonesterified isomer d-α-tocopherol at 2–5% concentration.15 Selenium is only percutaneously absorbed and active when applied topically as l-selenomethionine, optimally at 0.02–0.05%.15 It confirmed there are two great advantages in applying an active formulation of topical antioxidants to the skin. First, the skin attains far higher levels of each antioxidant than can be achieved by only taking these vitamins orally. The level of vitamin C attained in the skin by topical application is 20–40 times that achievable with oral vitamin C.15 With topical application, the concentration of vitamin E in the skin increases by a factor of 10.6 and selenium increases by a factor of 1.7. Second, topical application arms the skin with a reservoir of antioxidants that cannot be washed or rubbed off, a protection which stays in the skin for several days after application. This study also showed that ascorbic acid derivatives did not increase levels of L-ascorbic acid in the skin, placing their efficacy in question.15 At this point, it seems reasonable to encourage the use of L-ascorbic acid for the treatment of photoaging due to the lack of side effects as well as for its promising clinical results. That said, there does not exist sufficient evidence to support the use of the more stable derivatives which are the ingredients most commonly found in today’s cosmeceutical products.4 However, a topical application seems to be a more effective method for delivering vitamin C to the skin compared with oral administration.10 Vitamins C and E are often used together in topical formulations. Vitamin C acts to regenerate vitamin E from the tocopheroxyl radical, thus restoring the antioxidant effects of vitamin E. Thus, there is some rationale for the combination of vitamins C and E in many topical


formulations. Because of this important role that ascorbic acid plays in maintaining active vitamin E stores, some trials have looked at the combined effects of these two vitamins.10 One randomized, placebo-controlled study looked at the combined effect on 20 patients of 8 days of daily oral ascorbic acid in doses of 2 grams per day and d-α tocopherol in doses of 1000 IU per day.12 Ten subjects were randomized to receive the two vitamins, while 10 subjects received the placebo. Minimal erythema dose, or dose of UV exposure required to illicit redness, was measured at days 0 and 8. At day 8, a clinically significant increase in minimal erythema dose was seen among the vitamin group. A similar placebo-controlled study looked at the effect of 50 days of either 3 grams daily of oral ascorbic acid or 2 grams daily of α−tocopherol or the combination of the two on the minimal erythema dose. No statistically significant difference was seen with either vitamin used alone. However, after 50 days, those receiving the combination therapy showed a 78% increase in minimal erythema dose.12 In another study that compared L-ascorbic acid 15% and α-tocopherol 1% administered either alone or in combination compared with vehicle control, the individual ingredients were associated with a 2-fold increase after 4 days of treatment in the antioxidant protection factor compared with vehicle control. However, when combined, the two ingredients produced a 4-fold increase in antioxidant protection factor when compared to vehicle control. The combination also provided protection against DNA alterations as evidenced by almost complete inhibition of thymine dimer formation.16 However, there have not been any long-term studies examining what effect on photoaging the combination therapy might be able to offer.10 In 1969 the FDA recognized vitamin E, or tocopherol, as an essential nutrient. Since then, 8 naturally occurring vitamin E compounds have been recognized. Vitamin E is found in a variety of natural sources such as nuts, vegetable oils and green leafy vegetables. Nonesterified RRR-αtocopherol has the greatest biologic activity and is the form that is most commonly found in human tissues.4


The synthetic form of vitamin E, all-rac-α-tocopherol, is the form found in many commercial preparations and usually consists of the esterified form of all 8 stereoisomers of α-tocopherol in equal amounts in order to create a more stabilized form of the vitamin. However, these stereoisomers are not active until they have been hydrolyzed into a biologically active form. Ester hydrolysis occurs quite readily in the stomach after oral ingestion. However, it occurs much more slowly in the skin after topical application. Thus esterified forms of vitamin E may have little antioxidant potential in topical formulations.10 Orally ingested RRR-α-tocopherol takes approximately 7 days to affect concentrations in the skin, whereas all-rac-α-tocopherol ingestion never affects concentrations in the skin.4 One of the most important roles of vitamin E is to protect the viability of cell membranes. Polyunstaurated fatty acids are major components of cell membranes and often undergo lipid peroxidation from free radicals. Vitamin E is incorporated into biological membranes and works as an antioxidant, stopping peroxidation by donating hydrogen molecules to lipid and lipid peroxyl radicals. Vitamin E also works as a direct antioxidant by donating electrons to singlet oxygen and superoxide anions.4 Oxidative stress from either the sun or the environment can deplete the stores of vitamin E in the skin. This is important because the concentration of tocopherols in biologic membranes is low, necessitating its regeneration. Thus the antioxidant properties of vitamin E are dependent upon its continued regeneration by other biological agents in order to retain antioxidant efficacy. Both ascorbic acid and glutathione are essential for the sustained action of vitamin E as they donate necessary hydrogen ions when the tocopherol radical is formed.10 Because of its antioxidant properties and its contribution to the regulation of collagen breakdown, vitamin E is thought to play an important role in skin aging. As previously discussed, the levels of MMP-1 transcription are normally elevated in older persons compared to younger people. When fibroblasts from older people are infused with α-tocopherol, the levels of MMP-1 transcription


are significantly reduced. As MMP-1 increases lead to increases in collagen breakdown and destruction of supportive matrix, these findings suggest that α-tocopherol may have the ability to help protect the integrity of the dermis.4 Topical application of a vitamin E preparation has been shown to provide effective amounts of α-tocopherol to the stratum corneum. In a randomized, double-blind study of 13 volunteers, a preparation of 0.15% rinse-off α-tocopherol solution or a placebo was applied to forearm skin. An analysis of skin lipid extracts revealed that α-tocopherol levels were significantly higher in the actively treated group and the elevated levels were maintained for at least 24 hours. Further, the active group showed significant inhibition of UVA-induced photooxidation of squalene, indicating that the preparation decreased the amount of photooxidative stress.17 Vitamin E has also been studied as a protectant to be used prior to sun exposure. The protective effect of α-tocopherol in humans was first studied in psoriasis patients receiving psoralen plus UVA light therapy. The application of α-tocopherol prior to the psoralen and UVA light protected subjects against erythema in a dose-dependent fashion.4 In one study, human skin treated with 5% vitamin E and N-acetyl cystein (20%) before UV exposure inhibited the normal upregulation of human macrophage metalloelastase by 47%. Human macrophage metalloelastase is involved in the degradation of elastin and is found primarily in areas of the skin exposed to the sun.18 There seems to be a growing body of evidence suggesting that oral supplementation with high doses of vitamin E may be harmful. Oral supplements of vitamin E in excess of 1000 IU daily may negatively impact platelet aggregation. Because vitamin E is metabolized by the cytochrome P450 system, it could also possibly interact with the metabolism of other drugs. Additionally, a high intake of vitamin E may be associated with an increased risk of developing basal cell carcinoma and an increased rate of mortality.4 Unlike oral supplementation, topical application is generally considered to be safe and the side effects are limited to slight skin irritation. While it has shown promise as an antioxidant and


regulator of collagen breakdown, there is no published data demonstrating that vitamin E improves skin wrinkling, texture and discoloration despite the fact that there are a number of products on the market containing varying forms of vitamin E. There is also a lack of data showing which, if any, commercial form has the best skin penetration and bioavailability. If there is any benefit that vitamin E may have on the visible signs of photoaging, it may be when it is used alone as a protectant applied prior to sun exposure or when vitamin E is combined with vitamin C in a topical preparation.4 Vitamin A is known for its scientifically proven benefits in treating a wide variety of skin conditions. Both oral and topical vitamin A derivatives are widely used in the treatment of mild to severe acne in addition to conditions such as rosacea, solar keratosis, melasma, psoriasis, Kaposi sarcoma, cutaneous T-cell lymphoma and nonmelanoma skin cancer.4 Retinols and carotenoids are the two main forms of vitamin A found in nature. Vitamin A is a fat-soluble vitamin and it is stored in the liver within lipid globules in hepatic stellate cells. Retinol, also known as preformed vitamin A, is the most biologically active form of the vitamin. It is found in animal sources such as liver, milk and eggs. Retinol and its natural and synthetic derivatives form a class of substances known as retinoids.10 Carotenoids are organic pigments that are naturally produced by plants, algae, some types of fungus and some bacteria. Carotenoids, also called provitamin A, have strong antioxidant capabilities due to their ability to quench singlet oxygen.6 They are found in carrots, tomatoes and other colorful fruits and vegetables. The most common carotenoids are β-carotene, α-carotene, and β-cryptoxanthin. Various carotenoids may be converted into retinol through oxidation reaction. However, β-carotene is the form most efficiently and readily converted into retinol. In the United States, about 26-34% of vitamin A is consumed in the form of carotenoids.4 The carotenoids do not have a well-established role in the treatment of photoaging, but they may have a role in its prevention. Carotenoids are thought to help protect plants from


photosensitization by their own chlorophyll. This role in plants led to the investigation of carotenoids as possible protective agents against the harmful effects of UV irradiation in humans.4 Multiple human studies have attempted to determine whether oral supplementation with βcarotene raises the minimal erythema dose. The findings have varied. One double blind placebocontrolled study examined the effect of β-carotene supplementation on sunburn, specifically on the induction of sunburn cells at the time of peak reaction intensity (24 h) after a single simulated light exposure of 3 times the individually determined minimal erythema dose.19 The β-carotene was administered orally for 23 days either as a single 120 milligram dose to dietarily restricted subjects or as a daily 90 milligram supplement to subjects on standard diets. The study showed that β-carotene increased plasma and skin levels of β-carotene compared to both pretreatment levels and placebotreated controls, but provided no clinically or histologically detectable protection against a 3 MED sunburn reaction. Thus, these data suggest that oral β-carotene supplementation is unlikely to modify the severity of cutaneous photodamage in normal individuals to a clinically meaningful degree.19 Other trials that provided β-carotene supplementation over a longer period did show a significant increase in minimal erythema dose over time, which was further increased by the addition of vitamin E supplementation. A carotenoid supplement of 25 milligrams of total carotenoids daily and a combination of the carotenoid supplement and vitamin E in a dose of 500 IU of RRR-αtocopherol daily were given for 12 weeks to healthy volunteers. Erythema was induced by illumination with a blue-light solar simulator. Serum β-carotene, α-tocopherol concentrations and skin carotenoid levels were tested before and after the trial. The study showed that serum β-carotene and α-tocopherol concentrations increased with supplementation. Erythema on dorsal skin was significantly diminished after week 8 and erythema suppression was greater with the combination of carotenoids and vitamin E than with carotenoids alone.20 At this point, the evidence is not strong enough to offer any definitive support for the use of dietary carotenoids for photoprotection. Since the effectiveness of carotenoids may only be seen after


long periods of continuous supplementation with naturally occurring forms, there seems to be evidence supporting further trials to explore the possible photoprotective benefits of carotenoids since they have demonstrated ability to raise the minimal erythema dose in humans.4 In contrast to carotenoids, there is a substantial amount of research that supports the use of retinol and its derivatives for treatment of the clinical manifestations of photoaging. Retinol is the naturally occurring form of vitamin A and it can readily penetrate the epidermis.10 In the body, retinol is naturally converted to numerous derivatives including retinoic acid, the most biologically active form of the vitamin. Other derivatives include retinaldehyde, retinyl esters and the group of oxoretinoids. Synthetic derivatives of retinol have also been designed and marketed including tazarotene, acitretin, etretinate and adapalene.4 The biologic properties of retinoids include antioxidant activity via free radical scavenging, increased fibroblast proliferation, modulation of cellular differentiation and proliferation, increased collagen and hyaluronate and decreased matrix metalloproteinase-mediated extracellular matrix degradation. In sun-exposed skin, retinoic acid stimulates the growth of keratinocytes and fibroblasts resulting in an increase in the production of extracellular matrix. This translates into a number of effects on the skin including an improvement in fine and coarse facial wrinkling, a decrease in tactile roughness and actinic keratoses and a lightening of solar lentigines, or sun spots.10 Retinoids bind to two groups of receptors belonging to the nuclear receptor superfamily which are the retinoic acid receptors (RARs) and the retinoid X receptors. Each receptor group has 3 receptor subtypes: ι, β and γ. Different retinoids exert their effects through different combinations of receptors.4 In human studies, activation of RARs and retinoid X receptors results in molecular changes favoring collagen deposition and increasing dermal thickness. One study proposed that elevated metalloproteinases, resulting from activation of AP-1 and NF-KB by low-dose solar irradiation, degrade collagen and elastin in skin. Researchers found that when human skin is pretreated with


topical tretinoin (all-trans-retinoic acid) at least 16 hours before UV irradiation, the normal UVinduced up-regulation of AP-1 and MMPs was substantially reduced.21 Vitamin A has also shown positive effects on collagen production on skin that has not been exposed to UV radiation. In one study, biopsies of photodamaged skin from the forearm and skin from the buttocks, which had been protected from the sun, were performed on 26 healthy subjects. Patients were treated for 10 to 12 months with a daily application of 0.1% tretinoin cream or vehicle cream. Skin biopsy specimens obtained at baseline and after treatment were assessed immunohistologically for evidence of dermal collagen I formation. The study showed that treatment of photodamaged skin with tretinoin produced an 80% increase in collagen I formation, as compared with a 14% decrease in collagen formation with the use of vehicle alone.22 The most abundant clinical evidence in support of retinoids in the treatment of photoaging is in prescription drug formulations. Tretinoin cream of 0.02% and 0.05% and tazarotene cream of 0.1% are already approved by the FDA for the treatment of fine line wrinkles, skin roughness and mottled hyperpigmentation caused by aging and sun exposure. The first report that topical retinoids may be beneficial for aged skin was in 1983. It was not until 1986 when a clinical trial showed significant improvement in photoaged skin with the use of tretinoin that other researchers as well as the pharmaceutical and cosmeceutical industries began to conduct additional studies of their own.4 One other early study confirmed the benefits of topical tretinoin in the treatment of photoaging in a 16-week randomized, double-blind, vehicle-controlled study. The study showed a statistically significant improvement in photoaging of the treated forearms in all of the 30 patients and in 14 of the 15 patients receiving facial treatment.4 The positive results of these early trials with tretinoin were duplicated many times and sparked an interest that led to the testing of other retinoids.4 In a double-blind clinical trial to test the efficacy and safety of 0.1% isotretinoin versus vehicle cream, 800 patients applied either 0.1% isotretinoin or matching vehicle cream to the face, forearms, and hands once nightly for 36 weeks. Local irritation and adverse events were assessed, and plasma retinoid levels were measured before


and during treatment. Treatment response increased throughout the 36-week treatment period and the overall appearance of photodamaged skin was significantly better than with the vehicle alone.23 There have been large-scale clinical trials supporting the positive effects of topical 0.01% tazarotene cream on fine wrinkles and mottled hyperpigmentation.4 One of these trials compared the efficacy of tazarotene cream in concentrations of 0.1%, 0.05%, 0.025% and 0.01% with 0.05% tretinoin cream. The study demonstrated that the highest concentration of tazarotene (0.1%) had significantly greater effects on photoaging than the tretinoin at weeks 12 and 20. The 0.05% tazarotene had similar efficacy as tretinoin. Both the 0.025% and 0.01% tazarotene were less effective than 0.05% tretinoin. The study did not discuss whether or not there was a significant difference in side effects between the tazarotene-treated groups and the 0.05% tretinoin group, although it did state that higher instances of burning, irritation, peeling and dryness were noted as the concentration of tazarotene was increased.24 Unlike prescription drug formulations, there is less clinical evidence supporting the effectiveness of over-the-counter formulations to combat photoaging. The retinoids that can be found in over-the-counter cosmeceutical formulations include retinol, retinaldehyde, retinyl esters and the oxoretinoids. Of the options, retinol is the preferred choice.4 There was a study on the use of topical retinol in a randomized, placebo-controlled trial of 36 elderly patients. 0.4% retinol was applied to the sun-protected inner portion of one upper arm up to 3 times a week for 24 weeks. The treated areas were found to have significantly improved fine wrinkling scores at 24 weeks and the biopsy specimens showed significantly increased glycosaminoglycan expression and procollagen I immunostaining versus the placebo. Most of the subjects reported at least one side effect including swelling, peeling, dryness, burning or stinging.25 A synthetic derivative of retinol was found to be effective at improving skin roughness and fine wrinkles in a placebo-controlled study.26 A total of 24 patients completed a 24-week trial of retinyl N-formyl aspartamate twice daily on the left half of the face and a placebo on the right half of the face. A clinical evaluation, photographs, and silicone replicas of both crow's-feet areas were taken


at baseline and at weeks 12, 20, and 24. Skin replicas were then analyzed using an optical profilometry technique. The standard wrinkle and roughness features were then calculated and statistically analyzed. The tolerance profile of the product was also clinically evaluated during the study. The 24 women who completed this study showed more improvement on the left side of the crow's-feet area in terms of the signs of photodamage than on the right side according to their own observations as well as the investigator's evaluations. The study showed minimal side effects for this formulation.26 There is evidence showing that not all retinols are created equal in terms of efficacy. Clinical trials examining the use of retinyl esters as well as retinyl palmitate have been disappointing. While there was initially promising in vitro data for retinaldehyde, clinical trials have not shown it to be effective either. There have been no clinical trials conducted as of September 2010 using oxoretinoids.4 While topical retinoids are scientifically proven to be effective, one of the drawbacks leading to treatment noncompliance is the prevalence of side effects such as skin irritation, redness, peeling and burning.4 One trial tested the efficacy of 0.1% tretinoin versus 0.025% tretinoin. Both 0.1% and 0.025% tretinoin produced statistically significant overall improvement in photoaging of the face compared with vehicle. There were no clinically or statistically significant differences in efficacy between the two concentrations of tretinoin. However, irritant side effects (redness and scaling) were statistically significantly greater with 0.1% tretinoin than with 0.025% tretinoin.27 Additionally, the clinical benefits may not become apparent for 3 to 4 months and will likely plateau after 8-12 months. Improvements are likely to diminish after treatment is discontinued, but one trial showed that after 24 weeks of daily topical retinoid treatment, 3 times per week treatment, but not once a week, was sufficient to maintain and at times further improve the clinical results.4 The longest study of retinoic acid use in humans had a mean duration of 2.3 years with a maximum follow up of 4.3 years.4 This study supported the safety of topical retinoic acid use over this time period. It also showed that retinoic acid may have additional benefits aside from treatment


of photoaging. The study demonstrated that a long-term retinoic acid treatment of photoaged skin is not associated with an increase in epidermal cellular atypia, which would connote greater risk of skin cancer formation. Rather, it reduces the atypia, which is consistent with its appreciated chemopreventive effect.28 An added benefit of long-term retinoic acid use in photoaged skin is that of partially replenishing collagen deficiency. This ability to deposit new collagen matrix appears to be responsible for the wrinkle effacement that accompanies retinoic acid treatment of photoaging.28 Another study tested longer term treatment of tazarotene cream on photodamaged skin. In this multicenter, double-blind, randomized, vehicle-controlled study, 50 patients with photodamaged facial skin, defined by mild to moderate fine wrinkling and mottled hyperpigmentation, were randomized to apply tazarotene 0.1% cream or vehicle cream once daily for 24 weeks. Blinded assessments showed that tazarotene was less likely than vehicle to be associated with an increase in keratinocytic and melanocytic atypia and more likely than vehicle to be associated with a reduction in atypia. Compared with the vehicle, tazarotene was associated with an increase in epidermal polarity and epidermal thickness as well as a tendency for stratum corneum compaction.29 These studies highlight the need for further study to determine if topical retinoids may have a place in the prevention of skin cancers. There is relatively little data examining oral retinoids when compared with the amount of data on the topical treatments. However, there have been 3 small clinical trials examining the effect of isotretinoin on skin aging.4 The first study looked at oral isotretinoin combined with different procedures of facial rejuvenation and the effect on skin aging. Sixty patients ranging in age from 35 to 65 years, in whom additional modalities of rejuvenation were also used, were randomly assigned to receive treatment with oral isotretinoin in doses of 10–20 milligrams 3 times a week for 2 months. Their results were compared with 60 patients who had undergone the same surgical procedures but with no oral isotretinoin. All patients treated with oral isotretinoin noted improvement in wrinkles, thickness and color of the skin, size of pores, skin elasticity, tone, and reduction in pigmented lesions and mottled


hyperpigmentation. The side effects were minimal. However, there was no use of a placebo in this study.29 In the second study, 50 women aged 40 to 60 years were selected to receive 20 milligrams of isotretinoin orally 3 times a week for a 3-month period. Patients underwent clinical and laboratory evaluation whereby 2 biopsies and photographic control were performed. Patients were requested to use only topical photoprotecting agents during the treatment. Improvements in photoaging clinical parameters were evaluated through photos as well as a patient assessment. The results showed an improvement of photoaging in 20 patients out of the 30. As such, the researchers felt that isotretinoin improves photoaging with few adverse events because of the low dose used and the short treatment duration.30 The third trial studied 30 female patients, aged 40 to 55 years, with moderate to severe photoaging. The group was randomly assigned to 2 groups of 15 each. Group I patients were treated with 10 milligrams of isotretinoin and group II with 20 milligrams of oral isotretinoin three times a week for 3 months. The increase in the amount of collagen fibers was statistically significant with both dosage regimens. A pattern pointing toward a decrease in the number of elastic fibers was found. In addition, there was improvement in the general aspect of the skin, regarding texture, wrinkle depth and skin coloration. However, the study may also be less accurate due to a lack of a control group using placebo.31 Efficacy of oral supplementation of isotretinoin to treat photoaging remains to be seen, but there are significant barriers in place for prescribing it due to the proven negative side effects of longterm and short-term treatment. Topical retinoids are a better choice than oral supplementation of isotretinion given their proven efficacy and long-term safety.4 The B vitamins consist of 8 different water-soluble vitamins that often co-exist in the same foods. They are all grouped under the vitamin B name because of the historical belief that they were the same vitamin. All 8 of the B vitamins play distinct roles on cell metabolism. They are readily obtained in the diet and can be found in a variety of foods including potatoes, bananas, lentils, chili


peppers, liver, turkey, tuna, yeast, and molasses. This thesis will focus specifically on vitamin B3 as it is the one with the most relevance to the treatment of photoaging.4 The term vitamin B3 is used to refer to niacinamide and nicotinic acid, which is also known as niacin. The terms can be used interchangeably because they are readily converted into each other in vivo. It is an important component of coenzymes such as nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), both of which supply hydrogen to the respiratory chain for oxidation and energy production in mitochondria.10 At this point, the most well-established role of vitamin B3 is in reducing blood cholesterol and in treating atherosclerosis. New information is leading scientists to study the possibility that vitamin B3 may be a viable treatment for certain skin conditions including acne vulgaris, rosacea, alopecia and photoaging. Niacinamide increases collagen production in human fibroblast cell cultures.4 In a clinical trial, the application of topical 5% nicotinamide before UV irradiation prevented UV-induced immunosuppression, but found the effect was significantly greater in men than in women.32 Healthy volunteers were UV-irradiated on their backs, with 5% nicotinamide or vehicle applied to different sites in a randomized, double-blinded manner. Testing at irradiated and nearby sites that were not irradiated enabled measurement of UV-induced immunosuppression with and without nicotinamide. Men were immunosuppressed by doses three times lower than those required to immunosuppress women. This may be an important cause of the higher skin cancer incidence and mortality observed in men. Researchers concluded that topical nicotinamide prevented immunosuppression and that nicotinamide is a safe and inexpensive compound that could be added to sunscreens or after-sun lotions to improve protection from immunosuppression.32 Niacin is a precursor to NAD, which has a large influence on DNA surveillance and repair proteins. UV irradiation lowers levels of NAD in the skin and leads to decreased DNA surveillance and repair mechanisms. Both oral and topical niacin can increase levels of NAD in cutaneous tissues even in the presence of UV irradiation.4


A placebo-controlled clinical trial looked at the effects of a daily application of a topical lipophilic derivative of nicotinic acid called myristyl nicotinate. In 96 subjects, after 12 weeks of treatment the levels of NAD in the treated skin were increased by 25%, minimal erythema dose was increased by 10%, epidermal thickness increased by 20% and stratum corneum thickness increased by an average of 70% when compared with a placebo. Additionally, there were improvements in barrier function such that transepidermal water loss was reduced up to 20% compared with placebo. These results confirm that nicotinic acid was delivered to the skin and suggest that topical nicotinic acid preparations may be effective in the treatment of photodamaged skin and useful in other conditions with skin barrier malfunctions.32 The clinical trials examining the use of topical niacin and niacinamide to treat photoaging have begun only recently but the data appears promising. However, the sample sizes are small and the study designs are not optimal so they cannot be deemed conclusive.4 A trial in Japan involving 18 women aged 25 to 60 with a variety of hyperpigmentation disorders found that 12 weeks of treatment with topical niacinamide resulted in lightening of pigmented areas.33 The clinical trials included 18 subjects with hyperpigmentation who used 5% niacinamide moisturizer and vehicle moisturizer in a paired design, and 120 subjects with facial tanning who were assigned to two of three treatments: vehicle, sunscreen and 2% niacinamide and sunscreen. Changes in facial hyperpigmentation and skin color were objectively quantified by computer analysis and visual grading of high-resolution digital images of the face. In the clinical studies, niacinamide significantly decreased hyperpigmentation and increased skin lightness compared with vehicle alone after 4 weeks of use. The study concluded that the data suggest niacinamide is an effective skin lightening compound that works by inhibiting melanosome transfer from melanocytes to keratinocytes.33 In another trial, 39 women used either a 2% or a 5% topical niacinamide preparation to half of their face and a placebo to the other half twice a day for 8 weeks. Researchers found that topical application of niacinamide resulted in a dose-dependent and reversible reduction in hyperpigmented lesions.34 This trial additionally found that there was a significant


decrease in hyperpigmentation on the areas treated with 5% niacinamide, but not the 2% preparation compared with placebo.34 There has been one clinical trial conducted that offered the most convincing results on the efficacy of topical niacinamide to date.4 In this study, 50 women applied 5% niacinamide to one side of their face and a placebo to the other half two times a day for 12 weeks. Facial images and instrumental measures were obtained at baseline and at 4-week intervals. The results showed significant reductions in the appearance of hyperpigmented spots, redness, wrinkles, yellowing and elasticity.35 The noted improvements were not as great as with retinoid treatment but the side effects of skin irritation, dryness, peeling, and burning were not seen with the niacinamide treatment.4 Both oral and topical formulations of vitamin B3 have been shown to increase skin levels of NAD. However, there have only been trials on the efficacy of vitamin B3 to treat photoaging with formulations used topically.4 Oral niacin supplementation can have such undesirable side effects as peripheral vasodilation and cutaneous flushing, thus making topical use a much more desirable option. Considering the data that is available today supporting a favorable safety profile as well as promising initial clinical results, topical niacinamide may be recommended to prevent UV-induced molecular alterations in skin physiology. However, larger scale clinical trials should be conducted in order to establish its role as a definitive treatment for the visible signs of photoaging.4 In addition to vitamins, there are a few other antioxidant substances that have been studied relative to ROS and UV irradiation. Exploring the entire list is out of the scope of this literature review. Rather, it will focus on a few of the natural substances that a nutritionist may encounter that are also being used in topical cosmeceutical anti-aging preparations. Polyphenols are a group of chemical molecules found in plants and they are characterized by the presence of phenol units in their molecular structure.6 The tea plant, Camellia sinensis, is a potent source of polyphenols. Green tea contains a variety of polyphenols that have been shown to offer significant photoprotection in addition to serving as antioxidants.36


Green tea contains primarily monomeric catechins (flavonols), including epicatechin (EC), epicatechin-3-gallate (ECG), epigallocatechin (EGC) and epigallocatechin-3-gallate (EGCG). Of these catechins, EGCG is the most abundant and the most biologically active.37 ECGC inhibits the generation of intracellular hydrogen peroxide, one of the most active DNA-damaging reactive oxygen species. Additionally it inhibits the formation of cyclobutane pyrimidine dimers, a known source of UV-induced DNA damage. These findings suggest that green tea may have anti-aging properties through its ability to scavenge free radicals and limit inflammation.36 In a study completed in 2000, researchers found that EGCG reduced UVB-induced DNA damage by increasing DNA repair ability, suggesting a protective effect of EGCG on photoaging.38 Six male and female Caucasian volunteers topically applied a formulation of green tea extracts to buttock skin 20 minutes prior to sun exposure. The green tea extracts inhibited the formation of UV light induced sun damage, thus reducing risk of skin cancers.38 In another study, oral administration of ECGC was given for 8 weeks and it significantly increased the minimal erythema dose to UV and prevented further disruption of the epidermal barrier function. These findings suggest that ECGC strengthens the tolerance of the skin to UV initiating stress.39 Furthermore, it reduces UVB-induced MMP-1, MMP-8 and MMP-13 in a dose-dependent manner, further suggesting that ECGC lowers the UVB-induced production of MMP.40 Pycnogenol is a standardized extract of the bark of the French maritime pine, Pinus pinaster, a well-known, potent antioxidant.41 Studies in vitro show that pycnogenol is several times more powerful than vitamin E and vitamin C.41 In addition, it recycles vitamin C, regenerates vitamin E, increases the endogenous antioxidant enzyme system41 and protects against ultraviolet radiation.37 Therefore its efficacy in the treatment of melasma, or dark pigmentation in sun-exposed areas of the face, was investigated. Thirty women with melasma completed a 30-day clinical trial in which they took one 25 milligram tablet of pycnogenol with meals three times daily, or 75 milligrams pycnogenol per day. These patients were evaluated clinically by parameters such as the melasma area index, pigmentary intensity index and by routine blood and urine tests. After a 30-day treatment, the


average melasma area and the average pigmentary intensity decreased by 80% without any side effects.41 In another study, 21 volunteers were given an oral supplementation of pycnogenol in the amount of 1.10 milligrams per kilogram of body weight per day for the first 4 weeks and 1.66 milligrams per kilogram of body weight per day for the next 4 weeks. The minimal erythema dose was measured twice before supplementation, once after the first 4 weeks of supplementation and a last time at the end of the study. The UV dose necessary to achieve 1 MED was significantly increased during supplementation, thus indicating that oral supplementation of pycnogenol reduces erythema in the skin.42 Coenzyme Q10 (CoQ10) is also recognized for its antioxidant capacities and ability to mitigate ROS. It has been shown to reduce DNA damage triggered by UVA irradiation of human keratinocytes in vitro.43 In one study, researchers speculated that UVB radiation-induced cytokine production in keratinocytes may be inhibited by CoQ10. If this was the case, they felt CoQ10 could result in the reduction of MMPs in fibroblasts leading to wrinkle reduction. The in vitro studies showed that UVB-induced IL-6 production of normal human keratinocyte decreased in the presence of CoQ10. Furthermore, MMP-1 production of fibroblasts cultured with the medium containing CoQ10 collected from UVB-irradiated normal human keratinocyte significantly decreased during the 24-hour culture.43 In the clinical trial portion of the same study, the researchers found that the use of 1% CoQ10 cream for five months reduced the wrinkle score grade observed by a dermatologist. Those results indicate that CoQ10 may have potent anti-aging and anti-photoaging effect on skin by reducing wrinkle formation in vitro and on sun-exposed facial skin.43 In addition to the negative effects of the sun on skin, there has been increased interest and concern about the effects of sugar on the skin’s aging process. Researchers noted the elevated amount of sugar in the North American diet as early as 1942. Today, with the American diet being high in sugars and processed foods, coupled with the prevalence of sugar in the form of high fructose corn syrup, this is a growing concern. In 1945, researchers Urbach and Lentz showed a correlation


between a high sugar diet and elevated sugars in the skin. They also showed that a low-sugar diet resulted in low levels of sugar in the skin.44 An important concept in the relationship between sugars and aging lies in their cross-linking. When two collagen fibers are cross-linked, that act renders both of them incapable of being repaired through the usual process of remodeling. The more cross-linking that occurs, the more compromised the ability to repair and maintain becomes. Thus, cross-linking should be avoided since the appearance of youthful skin depends on the maintenance of youthful, flexible, repairable collagen fibers.44 The damage begins with a process called glycation. There are two types of glycation. Endogenous glycation is the binding of sugars to proteins, which results in cross-linking and rigidity of cellular structures. Glycation is responsible for wrinkles on the skin, hardening of arteries and tissue degeneration. Exogenous glycation is the result of cooking sugars with proteins or fats.44 Glycation is a covalent bonding process that links, using glucose and fructose, the amino acids present in the collagen and elastin that support the dermis. Collagen and elastin are normally linked in a pattern and a manner that allows them to be repaired. However, glycation adds cross-links that interfere with the repair mechanism. The glycation process in the skin is accelerated by excess sugar in the diet. UV exposure also increases cross linking in the skin and it decreases our natural defenses against free radicals. Because it is nearly impossible to repair glycated collagen, prevention is the best defense.44 Sugar is not the only problematic dietary cause. The protein-bound sugar that is involved in glycation, once heated, results in a complex called an advanced glycation end product or AGE. In simple terms, certain foods contain carbohydrates in the form of sugars, while others contain amino acids in the form of proteins. These sugars and amino acids can exist next to each other or be blended together in foods. Once the food is heated, a chemical reaction occurs to combine the carbon molecules in the sugars with the amino acids in the proteins. When these combine the result is that the food turns brown, for example the crust on a piece of bread, the black marks found on grilled


vegetables or the crisp skin on roasted turkey. Grilling, frying, deep frying or roasting foods produce high levels of AGEs. Water-based cooking methods such as boiling, steaming, poaching or stewing food produces far less AGEs. Cooking methods involving water tend to produce lower levels of AGEs than do methods involving high levels of heat. These AGEs are able to be absorbed and are able to enter the circulation where they can react with cellular and extracellular components thus increasing the burden of aging. AGEs derived from food can induce the protein cross-linking, inflammation and intracellular oxidative stress as much as excess dietary sugar.44 Continuous intake of AGE contributes to the excessive accumulation of these products into body tissues. Collagen and myelin are among the main targets of AGEs. In these tissues, AGEs lead to the synthesis of insoluble compounds that severely alter cellular functionality.45 It has been suggested that long-term results obtained with cosmetic procedures are likely to be much better if healing is not compromised by a diet that is high in sugars and preformed AGEs. In addition, AGEs are thought to also interfere with the normal human intestinal microflora. Glycated food can lead to lowered levels of good bacteria in the gut. Without sufficient good bacteria, other nutrients and phytochemicals that have AGE-inhibiting properties may not be absorbed. Further, lowering AGEs in the diet can help reduce systemic oxidative stress and inflammation.44 At this point, nothing dietary or nutritive has been found to break up or eliminate AGEinduced adducts. But some compounds have shown they can actually inhibit the production of AGE. Compounds such as cinnamon, cloves, oregano and allspice can help protect against the fructoseinduced AGE formation. Ginger, garlic, Îą-lipoic acid, carnitine, taurine, carnosine, some flavonoids, and benfotiamine have also shown protective benefits.44 The effects of glycation and damage done to the dermis have been heavily studied in patients with diabetes. This literature review did not reveal any studies looking at the effects of glycation in the healthy population, particularly among patients primarily concerned with maintaining a youthful appearance. Research that is being done today on glycation and AGEs in patients with diabetes may at some point have more widespread applicability to aging in the healthy population.


Discussion Although there is a large amount of research available on antioxidant therapy and skin aging, there are a number of opportunities for additional studies that might provide definitive answers or more substantive recommendations as to the efficacy of the many antioxidants and antioxidant formulations found on the market today. It was striking that the largest study on oral vitamin supplementation and aging was conducted nearly 40 years ago in the 1970s. Now that both processed foods and vitamin supplementation are more prevalent in today’s U.S. society it would be interesting to discover what a more recent large-scale study might find. It would also be very interesting to have a large-scale study that looked at antioxidant intake along with lifestyle choices such as nicotine impact, alcohol consumption, exercise habits, body mass index and race. Although there are challenges with data collection, the study should attempt to include a daily food and beverage diary over a longer period of time rather than simply one day’s dietary recall. Most of the studies across the board had weaknesses in the form of very small sample sizes. The need for larger sample sizes is important given the large populations of people who are purchasing cosmeceutical anti-aging formulations today. Additionally, the studies were often done with small populations of very specific ethnic groups. It is difficult to determine whether or not the positive results would be achieved in other ethnic groups, as different natural skin pigmentations might have special needs or respond differently to treatments. Another suggestion for further study is to look more specifically at the synthetic ascorbic acid derivatives. The natural form of L-ascorbic acid shows high levels of efficacy but can be unstable in formulations. Thus it is necessary to further study synthetic formulations for efficacy and ability to penetrate the skin. For instance, in order for the body to use ascorbic acid it must be converted to Lascorbic acid. A study looking at whether or not this conversion is possible with the stabilized commercial forms, or if the synthetic forms penetrate the tissues to the extent of L-ascorbic acid


would be beneficial to the marketplace looking to incorporate an over-the-counter vitamin C or combination vitamin C and vitamin E topical preparation. Given the market demand for inexpensive anti-aging products that are effective, it might be easy for savvy marketers to manipulate the good results gained by many natural antioxidant therapies so it is important for there to be further studies on the synthetic retinol derivatives in particular. There have been few, if any, studies comparing a prescription retinol to an over-the-counter version. Additionally, there have been no trials on the use of oxoretinoids to date. Clinical trials on the use of topical niacin and niacinamide are only just beginning and appear promising. However, additional large scale trials with better study design are needed to establish its efficacy. There have not been any studies to date on oral supplementation of vitamin B3 and skin aging, so studies looking at varying oral formulations might be useful as well. While most of the studies discussed in this literature review were double-blind, a few of the studies comparing one form of treatment to another did not use a placebo. While the results of those studies looked promising, additional studies using a placebo control would also be informative. Given the quest for eternal youth, particularly by the aging baby boomer population, there seems to be a cultural mission for the next magic elixir to erase the signs of aging. Since the general public has a positive identification with vitamins and “natural� substances, many anti-aging preparations containing ingredients such as soy, pomegranate, acai, coffee berry and others are often marketed by touting the antioxidant capacity of these ingredients. Reviewing the literature on all of these antioxidants was out of the scope of this review. However, this literature review demonstrated that not all powerful antioxidants are equally effective at reversing signs of aging and all do not contribute to collagen genesis, a very important anti-aging factor. Thus the general public would benefit from sound research studies on the anti-aging impact of these ingredients in either topical or oral formulations. Another observation was that virtually all of the research on glycation and aging has been done on patients with diabetes. While diabetes patients certainly are an important proxy for studying


the impact of glycation and sugar in the diet on aging cells, with the prevalence of processed foods and high fructose corn syrup in the North American diet, it would be beneficial to perform research on the impact of glycation and dietary sugar on healthy individuals as well. It would also be interesting to look at the effects of topical antioxidants versus more radical anti-aging treatments such as laser. Further, it is a plausible assumption based upon the literature review that the lasers used to erase photodamage (or even those that remove hair or perform other cosmetic benefits) can further create reactive oxygen species. It would be beneficial to study the amount of ROS generated with cosmetic laser procedures and determine if these lasers promote further skin damage. If so, it would be interesting to study if topical antioxidants applied prior to or after treatment would mitigate, prevent or reverse these ill effects. Lastly, most of the studies at least minimally discussed that the various antioxidant preparations do not replace overall healthy habits such as proper diet and exercise, smoking cessation and use of sunscreens. All of the research studies on ways to erase the clinical signs of aging do not take the place of common sense lifestyle choices to prevent and slow down the aging process, or at least minimize the clinical manifestations of it.


Conclusion In conclusion, the literature review demonstrated that antioxidants, particularly when applied topically, can prevent and reverse the clinical signs of photoaging. Further, it showed that glycation caused by dietary sugars and AGEs interfere with collagen repair mechanisms causing irreversible cellular damage and aging. Topical antioxidant formulations such as prescription retinol, vitamins C, E and B3 as well as green tea polyphenols, pynogenol and CoQ10 can be useful tools to maintain youthful skin quality, but they do not replace sunscreens and sound lifestyle choices.


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Michele's Masters Thesis  
Michele's Masters Thesis  

Michele Sonier's Masters in Human Nutrition Thesis