Summary and Key Features
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L-ascorbic acid is a water-soluble antioxidant.
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Vitamin C serves as a cofactor for the enzymes that synthesize collagen.
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Topically applied vitamin C confers photoprotection.
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Vitamin C inhibits tyrosinase and is an effective skin lightener.
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Topical vitamin C rejuvenates photoaged skin.
See and .
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 ( Fig. 5.1 ) and leafy green vegetables. 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, to confer skin benefits, topically applied vitamin C is preferable.
Vitamin C is found in many cosmeceuticals and can be used alone or in combination with other active ingredients. The first vitamin C products contained 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. Pinnell et al. published the optimal parameters for stabilizing L-ascorbic acid in formulation including a pH of less than 3.5. To avoid these constraints, many cosmetic chemists turned to more stable and easier to formulate derivatives, such as ascorbyl-6-palmitate (A-6-P), magnesium ascorbyl phosphate (MAP), sodium ascorbyl phosphate (SAP), and tetrahexyldecyl ascorbate (THDA). In addition to being more stable, these derivatives are lipophilic and more easily transported across the stratum corneum than L-ascorbic acid.
Oxidative Stress, Aging Skin, and Vitamin C
Antiaging research has elucidated the role of reactive oxygen species (ROS) in the pathogenesis of skin aging. ROS, including superoxide anion, peroxide, and singlet oxygen, are generated when human skin is exposed to ultraviolet (UV) light, visible light (VL), and infrared (IR). In addition exposure to pollutants such as ozone, particulate matter, and cigarette smoke upregulate ROS in the skin. ROS mediate their deleterious effects by causing direct chemical alterations of DNA, cell membranes, and proteins including collagen.
Oxidative stress can also affect redox-sensitive transcription factors, thereby modulating cellular activity. ROS upregulate transcription factor activator protein-1 (AP-1). AP-1 increases matrix metalloproteinase (MMP) production, resulting in collagen breakdown, and downregulates transforming growth factor beta (TGF-β), reducing collagen production. It is this net loss in dermal collagen that causes the exaggerated wrinkling seen in extrinsic aging. Additionally, nuclear factor-kappa B (NF-κB) is also induced by oxidative stress and is responsible for the synthesis of cytokines and other inflammatory mediators that contribute to skin aging. Finally, 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 the 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 a 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 elastotic material that accumulates in 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 far less to protect skin from damaging free radicals. Sunscreens, even when applied properly, block only 55% of free radicals produced by UVA exposure. Sunscreens also do not protect adequately against longer wavelengths of light such as VL and IR and have no protective effects against pollutants. Thus the use of sunscreen in conjunction with topical antioxidants affords better overall photoprotection and protection against other environmental aggressors.
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%. Pretreatment 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% alpha-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 (CEF) offered an eightfold increase in photoprotection compared to fourfold with vitamins C and E alone. In a more recent study, the ferulic acid–stabilized vitamin formulation was shown to inhibit sunburn cell formation, thymine dimer formation, overexpression of p53 protein, and depletion of Langerhans cells after a single 5-MED dose of solar-simulated light in human subjects. It is important to note that topical antioxidants must be applied prior to UV exposure to confer photoprotection. In a randomized, double-blind, placebo-controlled human study, the short-term photoprotective effects of a variety of antioxidants were 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.
Topical vitamin C formulations have also been shown to confer protection against the damaging effects of pollutants. Antioxidant combinations including CEF and Phloretin CF have demonstrated protective effects against lipid peroxidation caused by ozone exposure in human skin. Similarly, these formulations also demonstrated protective effects on collagen degradation seen following ozone exposure. Thus topical antioxidants that contain vitamin C are well positioned to be part of a comprehensive strategy to protect skin against environmental aggressors.
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. Human subjects taking vitamin C supplements demonstrated a significant rise in plasma and skin vitamin C content, but the vitamin failed to confer any protective effect against sunburn threshold. In contrast, studies have demonstrated protection against UV-induced erythema following daily supplementation with 2 g of vitamin C and 1000 IU of vitamin E. Thus it may be that the photoprotective benefits of oral supplementation are enhanced by combination therapy in a similar fashion to what has been seen when these vitamins are applied topically.
Vitamin C as an Antiinflammatory
Vitamin C is known to have antiinflammatory activity and has been used by dermatologists to treat a variety of inflammatory conditions such as acne. Cultured human cells loaded with vitamin C show a significantly decreased activation of the transcription factor NF-κB. NF-κB is responsible for production of proinflammatory cytokines including 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 antiinflammatory properties that are observed with vitamin C.
Vitamin C Inhibits Melanogenesis
Skin lightening is desired for patients with sun-induced hyperpigmentation, melasma, and postinflammatory hyperpigmentation. Although 4% hydroquinone is still the gold standard, recent concerns about the safety of hydroquinone have fueled interest in alternative skin lightening compounds. Topical vitamin C is an effective skin lightener. It inhibits tyrosinase and acts as a reducing agent for melanin and melanin intermediates such as dopaquinone. A clinical study of patients who had melasma or lentigines demonstrated that applying 10% magnesium ascorbyl phosphate cream twice daily resulted in a significant lightening in 19 of 34 patients. More recently a split-face, randomized, double-blind study compared 5% L-ascorbic acid cream to 4% hydroquinone cream for treating patients with melasma. Subjective improvement was seen in 93% of patients using hydroquinone compared to 62.5% using vitamin C. The side treated with vitamin C experienced far fewer side effects (6.25%) compared to the hydroquinone-treated side (68%). THDC has also demonstrated effective skin lightening. In a 10-patient open-label, investigator-blind study, subjects with melasma applied 30% THDC serum twice daily in combination with a mineral-based tinted sunscreen SPF 45 for 12 weeks in the summer. Investigator grading using a modified Griffiths scale showed a mean improvement of hyperpigmentation from baseline of 33.7% with overall global aesthetic score improvement of 2.0 (very much improved).
Thus L-ascorbic acid and its lipid-soluble derivatives appear to confer benefits for treating hyperpigmentation.
Delivery and Metabolism of L-Ascorbic Acid and Derivatives
L -Ascorbic Acid
While some believe that the ester derivatives are preferable in formulation, others remain committed to the use of L-ascorbic acid. Ascorbic acid is hydrophilic and thus would not be expected to penetrate the skin readily. Studies performed by 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 if 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% A-6-P failed to increase skin levels of L-ascorbic acid according to this study.
Ascorbyl-6-Palmitate
Ascorby-6-palmitate (A-6-P) is a fat-soluble analog of L-ascorbic acid with a palmitic acid chain attached at the sixth position. As such, it readily penetrates the stratum corneum and cellular membranes. A-6-P is itself an antioxidant. When hydrolyzed, it yields ascorbic acid and palmitic acid, although some studies suggest that this conversion is limited. It is of interest that while the antioxidant effects of A-6-P are well documented, in vitro studies suggest that A-6-P promotes UVB-induced lipid peroxidation and keratinocyte cytotoxicity. The mechanisms behind these findings are unclear, but it is possible that once inside the cell membrane A-6-P becomes oxidized, thereby forming the ascorbyl free radical. This free radical is capable of irreversible damage to cell membranes and DNA.
Sodium Ascorbyl Phosphate and Magnesium Ascorbyl Phosphate
Sodium ascorbyl phosphate (SAP) and magnesium ascorbyl phosphate (MAP) are salts that are stable at neutral pH. Placement of the phosphate group in the second position of the cyclic ring protects these molecules from oxidation but prevents them from functioning as antioxidants. Thus their efficacy depends on their conversion to ascorbic acid. A well-designed study provides insight into the mechanisms involved in delivery of MAP compared to L-ascorbic acid across the stratum corneum. 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 because L-ascorbic acid is hydrophilic whereas MAP is lipophilic. These studies demonstrated that microdermabrasion along with erbium and CO 2 lasers enhanced skin permeation of topically applied L-ascorbic acid, while there was no improvement in permeation of MAP by these treatments. Thus it appears 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 differences between vitamin C and its derivatives as they relate to biologic activity.
In addition, studies have demonstrated that MAP protects against UVB-induced lipid peroxidation in hairless mice and confirm that it crosses the epidermis and is converted to ascorbic acid. In vitro studies utilizing human fibroblasts demonstrated that MAP is equivalent to ascorbic acid in its ability to stimulate collagen synthesis while SAP required at least a 10-fold greater concentration.
Tetrahexyldecyl Ascorbate
THDC is used widely in cosmeceutical formulations and favored by many cosmetic chemists. THDC is ascorbic acid esterized at four intramolecular hydroxyl groups with a hexyldecanoyl branched chain fatty acid group. This unique structure makes THDC a clear, colorless liquid that is lipid soluble, thermally stable, and pH tolerant. After penetrating the stratum corneum, THDC converts to L-ascorbic acid intracellularly via enzymatic conversion and is considered a precursor molecule to L-ascorbic acid.
Clinical Studies Confirming Antiaging Benefits of 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 Figs. 5.2 and 5.3 demonstrate the type of clinical improvement that can be expected with continued use of topical L-ascorbic acid. The patient in Fig. 5.2 shows vastly improved periorbital wrinkles while the patient in Fig. 5.3 demonstrates a significant lightening of actinically induced mottled hyperpigmentation.
