INTRODUCTION

Vitamin C is a naturally occurring antioxidant incorporated into cosmeceuticals for the purpose of preventing and treating sun-damaged skin. Most plants and animals have the capacity to synthesize vitamin C. In humans, however, vitamin C cannot be synthesized because of loss of the ability to produce L-glucono-gamma-lactone oxidase, the enzyme necessary for its production. Vitamin C must instead be obtained from dietary sources such as citrus fruits and leafy green vegetables (Fig. 8.1). Interestingly, oral supplementation with vitamin C produces only a limited increase in skin concentration. This is because even with ingestion of massive doses, the absorption of vitamin C is limited by active transport mechanisms in the gut. Therefore, vitamin C has become a popular topically applied cosmeceutical.

Fig. 8.1 Citrus fruits, such as oranges, are a rich dietary source of vitamin C

Three forms of vitamin C are found in cosmeceuticals marketed as creams, serums, and patches. The first form is the active form of vitamin C, L-ascorbic acid. Early formulations of L-ascorbic acid often turned yellow due to the oxidation byproduct dehydroascorbic acid produced upon exposure to air. For this reason, many cosmetic chemists turned to more stable esterified derivatives, such as ascorbyl-6-palmitate and magnesium ascorbyl phosphate. Stability studies comparing all three compounds have demonstrated that magnesium ascorbyl phosphate is the most stable in solution and emulsion followed by ascorbyl-6-palmitate, while L-ascorbic acid is least stable. In spite of these findings, cosmeceuticals containing all forms of vitamin C can be purchased in the marketplace today.

OXIDATIVE STRESS, AGING SKIN, AND VITAMIN C

Antiaging research has elucidated the role of reactive oxygen species in the pathogenesis of photoaging. Reactive oxygen species (ROS) including superoxide anion, peroxide, and singlet oxygen are generated when human skin is exposed to ultraviolet (UV) light. These ROS mediate their deleterious effects by causing direct chemical alterations of DNA, cell membranes, and proteins including collagen.

Oxidative stress can also activate certain cellular events mediated by transcription factors. ROS upregulate transcription factor activator protein-1 (AP-1). AP-1 increases matrix metalloproteinase (MMP) production resulting in collagen breakdown. Nuclear transcription factor kappa B (NF-κB) is also induced by oxidative stress and produces a number of inflammatory mediators that contribute to skin aging. Additionally, ROS increase elastin mRNA levels in dermal fibroblasts which may provide an explanation for the elastotic changes found in the photoaged dermis.

The skin relies on a complex system of enzymatic and nonenzymatic antioxidants to protect itself from harmful ROS. L-ascorbic acid is the most plentiful antioxidant in human skin. This water-soluble vitamin functions in the aqueous compartment of the cell. Vitamin C sequentially donates electrons, neutralizes free radicals, and protects intracellular structures from oxidative stress. Following the donation of the first electron, a more stable ascorbate free radical is formed and after the second electron is donated, dehydroascorbic acid remains. Dehydroascorbic acid can be converted back to L-ascorbic acid by dehydroascorbic acid reductase or may be broken down as the lactone ring opens. Vitamin C also helps regenerate the oxidative form of vitamin E, a potent lipid-soluble antioxidant. In this regard, these two vitamin antioxidants appear to function synergistically within the cell.

In a compounding manner, while UV light increases production of intracellular ROS, it is at the same time impairing the skin’s ability to neutralize them. UVB exposure depletes skin of many key antioxidants, including vitamin C. It is known that exposure to UV light depletes the skin reservoir of vitamin C in a dose-dependent manner. Even minimal exposure to 1.6 MED (minimal erythema dose) can decrease vitamin C levels to 70% of normal, while exposing murine skin to 10 times MED further increases depletion to 54% of normal. In addition, ozone depletes stores of vitamins C and E in epidermal cells. Thus environmental exposure impairs the skin’s natural defense mechanisms against oxidative stress.

VITAMIN C: EFFECTS ON COLLAGEN AND ELASTIN SYNTHESIS

Vitamin C is essential for collagen biosynthesis. Ascorbate serves as a cofactor for prolyl and lysyl hydroxylase, the enzymes responsible for stabilizing and cross-linking collagen. Ascorbate can also stimulate collagen synthesis directly by activating its transcription and stabilizing procollagen mRNA. Scurvy serves as prototype for the physiologic changes that occur when vitamin C is lacking and collagen biosynthesis is impaired.

In view of this, it is no surprise that topically applied vitamin C has been shown to enhance collagen production in human skin. Skin biopsies taken from postmenopausal women who applied 5% L-ascorbic acid to one forearm and vehicle to the other showed an increase in mRNA levels of collagen I and III. Additionally, levels of tissue inhibitor of MMP-1 were also increased, suggesting that topical vitamin C may mitigate collagen breakdown. Interestingly, mRNA levels of elastin, fibrillin, and tissue inhibitor of MMP-2 remained unchanged. The authors note that those most affected by topical vitamin C had low dietary intake of vitamin C and conclude that the functional activity of dermal cells can be improved by topically applied vitamin C.

L-ascorbic acid also appears to influence elastin biosynthesis. In vitro studies suggest elastin biosynthesis by fibroblasts may be inhibited by ascorbate. This may be helpful in reducing the elastin accumulation that is characteristic of photoaged skin.

PHOTOPROTECTION BY VITAMIN C

While sunscreens remain the mainstay for protecting skin against UV-induced changes, topical antioxidants are gaining favor. Recent studies suggest that while sunscreens reduce UV-induced erythema and thymine dimer formation, they do little to protect skin from free radicals. Sunscreens, even when applied properly, block only 55% of free radicals produced by UVA exposure. This is important in that UVA is believed to be significant in the pathogenesis of skin aging and possibly melanoma formation. These data suggest that in order to optimize UV protection, sunscreens should be used in conjunction with topical antioxidants.

L-ascorbic acid is known to have photoprotective effects on skin. Vitamin C does not act as a sunscreen per se as it does not absorb sunlight in the UV spectrum. Topical L-ascorbic acid has been shown to protect porcine skin from UVB-induced erythema and sunburn cell formation. Topical application of 10% vitamin C was shown to decrease UVB-induced erythema by 52% and the number of sunburn cells by 40–60%. Pre-treatment with topical vitamin C prior to PUVA mitigated phototoxic injury as measured by sunburn cells and resulted in a normal histology devoid of the usual PUVA-associated findings.

While vitamin C alone can confer photoprotection, it appears to function optimally in conjunction with vitamin E. In studies designed to evaluate this synergy, vitamins C and E were applied alone or in combination for 4 days to pig skin and then irradiated with a solar simulator (295 nm). On day 5, antioxidant protection factor was measured including erythema, sunburn cells, and thymine dimers. The combination of 15% L-ascorbic acid and 1% α-tocopherol provided superior photoprotective effects (fourfold) that were progressive over the 4-day period. Both antioxidants conferred photoprotection when applied alone but to a lesser degree than when used in combination.

More recently, it has been demonstrated that ferulic acid, a potent plant antioxidant, improves the chemical stability of vitamins C and E in combination. When stabilized with 0.5% ferulic acid, 15% vitamin C and 1% vitamin E offered an eightfold increase in photoprotection compared to fourfold with vitamins C and E alone. Additionally, inhibition of apoptosis and a reduction in thymine dimer formation were observed with the ferulic acid-stabilized vitamin formulation. In a comprehensive comparative study, the photoprotective effects of the vitamin C, vitamin E and ferulic acid (C+E+ferulic acid) combination was compared to 1.0% idebenone, 1.0% ubiquinone, and 0.5% kinetin formulations. While C+E+ferulic acid was protective against UV-induced erythema at up to 5 MEDs, ubiquinone, idebenone, and kinetin did not provide any protection against sunburn. Only C+E+ferulic acid was completely protective of thymine dimer formation at up to 4 MEDs while the other antioxidants provided no protection. This study suggests that topical application of C+E+ferulic acid may be useful for mitigating acute and chronic UV damage and may also be valuable for skin cancer prevention.

It is important to note that topically applied antioxidants must be applied prior to UV exposure in order to photoprotect. In a randomized, double-blinded, placebo-controlled human study, the short-term photoprotective effects of a variety of antioxidants was evaluated when applied after UV irradiation. Melatonin, vitamin C, and vitamin E were applied alone and in combination 30 minutes, 1 hour, and 2 hours after UV irradiation. No photoprotective effects were observed when these antioxidants were applied after UV irradiation.

In addition to topical vitamin C, some have advocated that oral supplementation may be useful for photoprotection and even skin cancer prevention. In mice, dietary supplementation with vitamin C reduced the incidence of UV light-induced skin neoplasms although similar effects have not been documented in humans. Patients taking vitamin C supplements demonstrated a significant rise in plasma and skin vitamin C content but the vitamins failed to confer any protective effect against sunburn threshold. In contrast, two studies have reported protection against UV-induced erythema following supplementation with both vitamin C and vitamin E. Thus, the benefits of oral supplementation may be enhanced by combination therapy. Further studies elucidating the role of vitamin supplements for photoprotection are clearly warranted.

VITAMIN C AS AN ANTI-INFLAMMATORY

Vitamin C is known to have anti-inflammatory activity and has been used by dermatologists to treat a variety of inflammatory dermatoses. Cultured human cells loaded with vitamin C show a significantly decreased activation of the transcription factor NF-κB. NF-κB is the transcription factor responsible for the production of a number of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1, IL-6, and IL-8. It is believed that this downregulation of NF-κB by vitamin C occurs by blocking TNF-α induced activation of NF-κB. This mechanism provides an explanation for the anti-inflammatory properties that are observed with vitamin C.

DELIVERY AND METABOLISM OF L-ASCORBIC ACID AND DERIVATIVES

While some believe that the ester derivatives are preferable in formulation, others remain committed to the use of L-ascorbic acid. Studies performed by Pinnell et al suggest that topical L-ascorbic acid can be formulated in a manner that ensures stabilization and enhances permeation. These studies demonstrated that L-ascorbic acid can be delivered across the stratum corneum as long as the ionic charge on the molecule is removed. This is achieved only at a pH of less than 3.5. The maximal concentration of L-ascorbic acid for percutaneous absorption was 20% and, curiously, higher levels failed to increase absorption. Daily application of 15% L-ascorbic acid at a pH of 3.2 increased skin L-ascorbic acid levels 20-fold and tissue levels were saturated after 3 days. The half life of L-ascorbic acid after tissue saturation was approximately 4 days. By contrast, topical 13% magnesium ascorbyl phosphate and 10% ascorbyl-6-palmitate failed to increase skin levels of L-ascorbic acid according to this study.

A recent study provides insight into the mechanisms involved in delivery of L-ascorbic acid and magnesium ascorbyl phosphate (MAP) across the stratum corneum. In vitro studies utilizing nude mice assessed the ability of lasers and microdermabrasion to enhance and control skin permeation and deposition of L-ascorbic acid and MAP. At baseline, L-ascorbic acid possessed very low passive permeability while MAP appeared to be more readily transported into the dermis where it was converted to L-ascorbic acid. This difference in permeability is likely due to the fact that L-ascorbic acid is hydrophilic whereas MAP is lipophilic. These studies demonstrated that microdermabrasion, erbium, and carbon dioxide lasers enhanced skin permeation of topically applied L-ascorbic acid, while there was no improvement in permeation of MAP by these treatments. These data suggest that the rate-determining step for topical delivery of MAP is not permeation across the skin, since it appears to traverse the stratum corneum readily, but instead diffusion from the vehicle. By contrast, L-ascorbic acid permeation was improved by treatments that disrupt the stratum corneum, thus breaking the barrier for its absorption. Studies such as these further elucidate biochemical difference between vitamin C and its derivatives as they relate to biologic activity.

CLINICAL STUDIES REGARDING TOPICAL VITAMIN C

Clinical studies have investigated the cosmeceutical effect of products containing L-ascorbic acid. A 3-month, double-blind, randomized, vehicle-controlled study was performed on 19 patients aged between 36 and 72 years with moderately photodamaged facial skin. Patients applied topical ascorbic acid 10% (Cellex-C high-potency serum, Cellex-C International, Toronto, Ontario) or vehicle serum to half the face for 3 months. Optical profilometry image analysis demonstrated a statistically significant improvement in the vitamin C-treated side when compared to control. Clinical assessment showed significant improvement in fine wrinkling, tactile roughness, coarse rhytides, skin laxity/tone, sallowness/yellowing, and overall features on the side treated with active. Photographic assessment showed a 57.9% improvement in the vitamin C-treated group compared to control. The patients shown in Figures 8.2 and 8.3 demonstrate the type of clinical improvement that can be expected with continued use of topical L-ascorbic acid. The patient in Figure 8.2 shows vastly improved periorbital wrinkles while the patient in Figure 8.3 demonstrates a significant lightening of actinically induced mottled hyperpigmentation.

Fig. 8.2 A patient with moderate photoaging. (A) Before with enlarged area (B) showing periorbital wrinkling. (C) One year after treatment, with enlarged area (D) showing a significant improvement in periorbital wrinkles.

(Courtesy of Sheldon R Pinnell, MD)

Fig. 8.3 A patient with actinically induced mottled hyperpigmentation. (A) Before with enlarged area (B) showing hyperpigmentation. (C) One year after treatment, with enlarged area (D) showing an improvement in sun-induced hyperpigmentation.

(Courtesy of Sheldon R Pinnell, MD)

More recently, Humbert et al reported a 6-month, double-blind, vehicle-controlled study of moderately photoaged patients applying 5% vitamin C cream to the neck and forearms. A highly significant decrease in deep furrows was observed and substantiated with silicon replicas on the vitamin C-treated side. Histology demonstrated ultrastructural evidence of elastic tissue repair. The authors suggest that topical vitamin C had a positive influence on all parameters of actinically damaged skin.

Fitzpatrick and Rostan reported a double blind, half face study of 10 patients treated with a new formulation containing 10% L-ascorbic acid and 7% tetrahexyldecyl ascorbate in an anhydrous polysilicone gel base. The inactive polysilicone gel base served as a control on the opposite side. Clinical evaluations were performed at 4, 8, and 12 weeks and punch biopsies were performed. There was overall improvement on the vitamin C-treated side that was statistically significant when compared to vehicle at 12 weeks. The vitamin C-treated side showed a decrease in photoaging score on the cheeks and perioral area. The periorbital areas improved on both sides, which the authors contribute to improved hydration. Skin biopsies after vitamin C treatment showed an increase in grenz zone collagen and increased staining for mRNA for type I collagen.

In addition to improving wrinkles, vitamin C may also be helpful for lightening hyperpigmentation. Studies conducted by Kameyama et al demonstrated that magnesium-L-ascorbyl-2-phosphate suppressed melanin formation by tyrosinase and melanoma cells. Additionally, topically applied 10% magnesium-L-ascorbyl-2-phosphate cream when applied to human skin caused a significant lightening of melasma and lentigines in 19 of 34 patients.

It has been suggested that topical vitamin C may be helpful in treating acne due to its anti-inflammatory properties. Sodium L-ascorbyl-2-phosphate (APS), a less frequently used derivative, has been shown to have beneficial effects on acne and acne scarring when used in conjunction with glycolic acid peels. Patients applied 5% sodium L-ascorbyl-2-phosphate or vehicle twice daily after 50% glycolic acid peels. The peels were applied between one and three times monthly at 10-day intervals. Seventy-nine percent of patients applying APS showed moderate to excellent improvement compared to 44% in the control group. The investigators conclude that topical sodium L-ascorbyl-2-phosphate may improve outcomes in patients treated for acne scarring. There are anecdotal reports that topical vitamin C may improve inflammatory forms of rosacea but objective clinical studies are lacking to date.

An innovative use for topical L-ascorbic was described by Alster and West who evaluated its efficacy for treating post-CO₂ laser resurfacing erythema. Split face studies showed a significant decrease in post-CO₂ laser resurfacing erythema by the eighth postoperative week in patients treated with an aqueous solution containing topical 10% L-ascorbic acid, 2% zinc sulphate, and 0.5% tyrosine. Interestingly, the same formulation in a cream base did not improve post-laser erythema.

CONCLUSION

Topically applied vitamins continue to be a mainstay of our antiaging armamentarium. There is now significant data confirming the benefits of topically applied vitamin C and supporting its use as a cosmeceutical. Its diverse biologic activity in the skin makes vitamin C a valuable agent for the practicing dermatologist.