α-LIPOIC ACID

R-Alpha lipoic acid (αLA) is synthesized in the mitochondria of plants and animals, including humans. Natural αLA is covalently bound to protons via lysine; thus only minimal free αLA enters the circulation after biosynthesis or eating αLA-rich food. The lipoamide is a required cofactor for two enzymes in the citric acid cycle. It is also essential for the formation of a cofactor required in nucleic acid synthesis and for the metabolism of branched chain amino acids.

With oral supplements of free αLA, unbound αLA is transported to tissues. Free αLA is rapidly metabolized by the liver, so that the half-life in blood after absorption is only about 30 minutes, limiting the amount delivered. High tissue levels are short lived since most free αLA is rapidly reduced to dihydrolipoic acid (DHLA), as shown in Figure 18.1.

Fig. 18.1 The molecular structures of α-lipoic acid and dihydrolipoic acid

Notwithstanding this transient availability, free αLA has been shown to be therapeutic for autoimmune liver disease by binding autoantibodies, heavy metal intoxication by trapping circulating metals, diabetic polyneuropathy by preventing oxidative damage and mushroom poisoning. Although not normally found in significant amounts in the skin, αLA is a good candidate for topical application:

Indeed, αLA has been found to penetrate rapidly into murine and human skin to dermal and subcutaneous layers. Two hours after application of 5% αLA in propylene glycol, maximum levels of αLA were attained in the epidermis, dermis, and subcutaneous tissue. The stratum corneum concentration of αLA predicted the penetration and levels in the underlying skin. 5% of the αLA was converted to DHLA in both the epidermis and dermis, leading the researchers to conclude that both keratinocytes and fibroblasts reduce αLA.

Topical αLA with its metabolite DHLA could protect the skin from oxidative stress in several ways. Both αLA and DHLA are highly effective antioxidants as summarized in Table 18.1. DHLA is actually the more potent form. Both successfully scavenge reactive oxygen species (ROS) in vitro and in vivo. However, pro-oxidant activity has been observed. This occurs when an antioxidant reacts with a ROS scavenger, forming a product that is more harmful than the scavenged ROS. Fortunately, αLA can act as an antioxidant against the pro-oxidant activity of DHLA (Biewenga et al). Both αLA and DHLA further provide antioxidant activity by chelating Fe²⁺ and Cu²⁺ (αLA) and Cd²⁺ (DHLA).

Table 18.1 Antioxidant activity of α-lipoic acid and DHLA

+ activity; ++ greater activity; − no activity. DHLA, dihydrolipoic acid.

Reproduced with permission from Biewenga GP, Haenen GRMM, Bast A 1997 The pharmacology of the antioxidant lipoic acid. General Pharmacology 29:315–331.

DHLA, unlike αLA, has the capacity to regenerate the endogenous antioxidants vitamin E, vitamin C, glutathione, and ubiquinol, as illustrated in Figure 18.2. This is clearly of great importance for skin, since UV exposure directly depletes ubiquinone and vitamin E in particular, as well as vitamin C, thereby stressing the other linked antioxidants. Regeneration of these major membrane and cytosol antioxidants gives cascading protection. Increases in the other important antioxidants (intracellular glutathione and extracellular cysteine) are noted when αLA is added to cell cultures. Vitamin E-deficient animals do not show symptoms (weight loss, neuromuscular abnormalities) when supplemented with αLA.

Fig. 18.2 Interactions of low molecular weight antioxidants. The reactions which directly quench oxygen free radicals (RO•) are indicated by the red arrows (RO•→RO); the reactions regenerating these antioxidants are indicated by the green arrows. Reactions with arrows touching are directly linked. RO• generated in a cell membrane is reduced by tocopherol, forming a tocopheryoxyl free radical which can in turn be quenched within the membrane by ubiquinol or at the membrane–cytosol junction by ascorbate (vitamin C). RO• generated in cytosol is directly reduced by ascorbate. The oxidized dehydroascorbate is reconverted to ascorbate by glutathione (GSH). Both α-lipoic acid and DHLA directly reduce oxygen free radicals. Also DHLA is itself a potent reducing agent which regenerates the oxidized forms of vitamin C, vitamin E, and oxidized glutathione (GSSG); this linkage is indicated by an asterix.

(Adapted from diagram in Podda & Grundmann-Kollmann 2001, incorporating information from Biewenga et al 1997)

Although αLA is a potent antioxidant, it provides no effective protection against UV-induced erythema or cell damage measured as sunburn cells. However, αLA (but not DHLA) acts as an anti-inflammatory agent by reducing the production and inhibiting the binding of transcription factors such as nuclear factor kappa B (NF-κB), thereby indirectly affecting the gene expression of inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukins. DHLA (but not αLA) can repair oxidatively damaged proteins, which in turn regulate the activity of proteinase inhibitors such as α₁-AP, an inflammatory modulator. In vitro, both αLA and DHLA inhibit lipolysaccharide-induced nitric oxide (NO) and prostaglandin E₂ (PGE₂) formation and suppress inducible NO synthase (iNOS) but do not affect the expression of cyclooxygenase-2 (COX-2). In a mouse model, topical DHLA inhibits chemically induced activation of skin inflammation with a concomitant decrease in inflammatory modulators. Furthermore, topical DHLA (but not oral αLA) reduces chemically induced skin tumor incidence and multiplicity, and inhibits iNOS and COX-2 in a dose-dependent manner. As antioxidants, both αLA and DHLA are directly anti-inflammatory by virtue of their quenching oxidants secreted by leukocytes and macrophages at sites of inflammation.

αLA and DHLA have been shown to be effective depigmenting agents. Both depigment dark-skinned swine, inhibit tanning of light-skinned swine, and inhibit chemical and UVB-induced tyrosinase activity in melanocyte cultures. A recent new derivative of α-lipoic acid has been proven to be an effective depigmenting agent in melanoma cells in vitro. This depigmentation is achieved by formation of DAPA conjugate products.

αLA may prove to retard and correct both intrinsic and extrinsic aging of the skin as well as other organs. By damaging DNA, the ROS continuously formed in normal metabolism may be largely responsible for the functional deterioration of organs with aging. A decrease in cellular protein and DNA as well as in αLA levels has been measured in aged rat liver, kidney, and spleen. Supplementation with αLA increases nucleic acid and protein levels in the elderly organs. Similarly, the age-related decrease of mitochondrial function in cardiac and brain cells can be improved with αLA supplementation. Clearly, aging skin might similarly benefit.

To evaluate possible improvement to photodamage, a split face study was done on 33 women. Topical application twice daily of 5% lipoic acid cream for 12 weeks decreased skin roughness by 50.8% (as measured by laser profilometry) when compared with the placebo. Clinical and photographic evaluation showed reduction in lentigines and fine wrinkles. In another study, twice daily oral intake of αLA combined with other proteins, vitamins, and minerals improved wrinkles, roughness, and telangiectasias after 4–6 months in humans, as assessed clinically and by measurements of skin thickness and elasticity. Experiments with fibroblasts in vitro showed increased collagen synthesis when high concentrations of αLA were added to culture media. Clearly, topical αLA should be further studied by quantitative techniques to confirm these results and to elucidate mechanisms of action.

UBIQUINONE (COENZYME Q₁₀)

Ubiquinone (coenzyme Q₁₀, Fig. 18.3) is so named because it is ubiquitous in virtually all living cells, excluding some bacteria and fungi, although the level is quite variable. Since most human tissues synthesize ubiquinone, it is not considered to be a vitamin.

Fig. 18.3 The molecular structure of ubiquinone. The ‘head’ of the ubiquinone molecule is a fully substituted quinone ring which does not allow addition reactions with thiol groups in the cell (such as GSH). Ubiquinones vary by the length of the ‘tail’: Q₁₀ has 10 isoprene units. Humans can synthesize Q₁₀ out of the other coenzymes Q₁ to Q₉, though this ability decreases with age

Ubiquinone is primarily located in the inner mitochondrial membrane where it is essential for the production of the ATP required for all vital cellular functions. Until recently, ubiquinone was thought to function only in energy transduction; however, with the discovery that ubiquinone is also an antioxidant within subcellular membranes, new roles are now being recognized. Ubiquinone can regenerate reduced tocopherol, as depicted in Figure 18.2. In fact, within membranes the amount of ubiquinone is from three to thirty times that of tocopherol. Without ubiquinone, the regeneration of tocopherol would be very slow.

The concentration of ubiquinone is highest in organs with high rates of metabolism such as heart, kidney, and liver, where it functions as an energy transfer molecule. In skin, the level of ubiquinone is relatively low, with 10-fold higher levels in the epidermis than in the dermis. Thus, the epidermis might potentially benefit from topical ubiquinone. Indeed, it has been demonstrated that ubiquinone can be topically absorbed. Application of ubiquinone in ethanol to porcine skin achieved 20% penetration into the epidermis and 27% into the dermis. Oral supplementation increases serum and epidermal ubiquinone substantially in hairless mice, but does not increase levels in the dermis or other organs.

The fact that ubiquinone can serve not only as an energy generator but also as an antioxidant in the skin has been investigated. In cultured human keratinocytes exposed to hydrogen peroxide, the detrimental increase in the activity of phosphotyrosine kinase was suppressed and the loss of glutathione was prevented. Ubiquinone (0.3%) also suppressed the UVA-induced reduction of mitochondrial membrane potential in fibroblasts from both young and old donors. Finally, the UV-induced oxidative damage to DNA in keratinocytes in vitro was reduced significantly with ubiquinone.

Ubiquinone can retard loss of hyaluronic acid and slowdown of cell division—both manifestations of intrinsic aging. Aged human fibroblasts in vitro produce less glycosaminoglycan and proliferate more slowly than young cells. The addition of ubiquinone increased levels of glycosaminoglycan as well as rates of cell division.

Ubiquinone further protects from the UVA-induced degradation of collagen. Both ubiquinone and vitamin E were shown in vitro to suppress fibroblast production of UVA-induced collagenase, thereby markedly retarding collagen breakdown. Ubiquinone suppressed collagenase expression over a longer period of time than did vitamin E.

Recent research further demonstrated that ubiquinone can suppress the UV interleukin-1 (IL-1)-induced inflammatory response as well as the UV-induction of the matrix-eroding protein matrix metalloproteinase-1 (MMP-1) in dermal fibroblasts. Thus topical ubiquinone protects from inflammation and the appearance of premature aging from sun exposure. In addition, oral ubiquinone, pre- and post-treatment, was shown to enhance healing by accelerating re-epithelialization after laser resurfacing and chemical peels.

Ubiquinone’s antioxidant action in skin was confirmed in vitro by sophisticated ultra-weak photon emission (UPE). Increased antioxidants result in decreased UPE. Elderly volar skin demonstrated 33% reduction in antioxidant activity when compared with young skin. This was corrected after 1 week of twice daily topical application of 0.3% ubiquinone. After UVA irradiation, a decrease in antioxidant activity was noted; this loss was significantly corrected with topical 0.3% ubiquinone.

The efficacy of ubiquinol in reversing photoaging was further studied clinically. Ubiquinol cream (0.3%) was applied to one half of the face and placebo to the other once daily for 6 months. Casts were made of the periorbital rhytides. The improvement can be appreciated in the photographs shown in Figure 18.4. Quantitative microtopography demonstrated a 27% reduction in the mean wrinkle depth.

Fig. 18.4 Reduction of wrinkles with ubiquinone. Silicone replicas of the skin, analyzed by laser profilometry, show a significant reduction in the depth of periorbital fine lines and wrinkles in a 46-year-old female after 10 weeks of twice daily application of ubiquinone cream (Eucerin Q10 Anti-Wrinkle Sensitive Skin Crème).

Rights were not granted to include this figure in electronic media. Please refer to the printed book.

(From Wrinkle Reduction Study 2003 In: Eucerin Q10 Product Compendium 2003, Beiersdorf Inc., Wilton, CT, p 11)

Another clinical measure of photoaging is stratum corneum cell size. With deceased cell turnover time in aged skin, corneocytes become larger. Treatment once daily for 6 months with ubiquinone cream decreased corneocyte size equivalent to rejuvenation of 20 years. Thus, ubiquinone is an effective antioxidant protecting the dermal matrix from both intrinsic and extrinsic aging, making it a potentially important cosmeceutical.

A lower molecular weight analog of ubiquinone, idebenone, has also been shown clinically to repair photodamaged skin. In a nonvehicle controlled study, 41 females aged 30–65 years applied either 0.5% or 1.0% idebenone twice daily for 6 weeks. Use of 1.0% idebenone increased skin hydration by 37% (as measured by electrical conductance), reduced skin roughness/dryness by 26%, reduced fine wrinkles by 29%, and improved photoaged skin by 33% (as subjectively evaluated). The 0.5% idebenone was almost as effective as 1.0%. Immunofluorescence and staining of punch biopsies revealed a decrease in IL-1b, IL-6, and MMP-1 and an increase in collagen for both concentrations.

GENISTEIN

Genistein is an isoflavone cosmeceutical isolated from soy. Recent interest in genistein has been stimulated by epidemiologic studies which correlate diets high in soy with reduced incidence of cardiovascular disease, osteoporosis, and certain cancers in humans.

The direct anticarcinogenic action of genistein is documented. Animal studies demonstrate protection against bladder, breast, colon, liver, lung, prostate, and skin cancer with oral genistein, and dietary soy inhibits chemically induced skin cancer in mice. Growth of many in vitro cancer cell lines is inhibited by genistein. Genistein also arrests the growth and induces the differentiation of malignant melanoma cells in vitro and inhibits pulmonary metastases of malignant melanoma cells in vivo.

The mechanism by which genistein inhibits carcinogenesis may be through inhibition of tyrosine protein kinases (TPKs), the enzymes which phosphorylate proteins necessary for the regulation of cell division and transformation. Of particular importance is phosphorylation of TPK-dependent epidermal growth factor receptors (EGF-R), which are related to tumor promotion, including initiation of transcription factors, release of inflammatory mediators (as prostaglandins), and stimulation of cell proliferation. Genistein was found to downregulate both UVA- and UVB-induced EGF-R phosphorylation in human epidermoid carcinoma cells in vitro. In mouse skin, genistein also blocks the UVB-induced expression of the photo-oncogenes c-fos and c-jun which promote cell proliferation in oncogenesis. Similarly, genistein retards UV-induced apoptotic changes—including caspase-3 and p21-activated kinase 2 activation of human epidermal carcinoma cells and phosphokinase Cδ in human keratinocytes.

Genistein is also a potent antioxidant. Genistein scavenges peroxyl free radicals, thereby protecting against lipid peroxidation in vitro and in vivo. The decreased incidence of cardiovascular disease with high soy diets may be due to genistein’s inhibiting the oxidation of low density lipoprotein (LDL) cholesterol in both aqueous and lipophilic environments. Of direct importance in protection from UV-induced skin damage, genistein has been shown to inhibit in vitro chemical and UV-induced DNA oxidation as well as psoralen plus UVA (PUVA) DNA damage. The fact that genistein also reduces erythema and histologic inflammation caused by PUVA may have implications for PUVA therapy by reducing possible short- and long-term adverse reactions.

Topical genistein (10 μmol/cm²) protects against acute and chronic UV damage to the skin. After exposure of Skh:1 hairless mice to UVB, topical genistein blocked acute skin burns and inhibited UVB-induced cutaneous wrinkling, as demonstrated clinically in Figures 18.5 and 18.6. Histologic analysis confirmed that topical genistein blocks the signs of chronic photodamage—epidermal hyperplasia and reactive acanthosis with nuclear atypia (Fig. 18.7). At a molecular level, UV-induced damage to DNA (as measured by the biomarker 8-hydroxy-2′-deoxyguanosine) was reduced. Also, in Skh:1 mice, topical genistein inhibits UVB-induced pyrimidine dimer formation as well as UVB-generated suppression of repair of solar damage by proliferating cell nuclear antigen (PCNA). This photoprotection by genistein was further demonstrated in vitro with suppression of UVA-induced matrix metalloproteinases (MMPs) responsible for dermal destruction in photoaging. Inhibition of acute UV-induced erythema with topical genistein (5 μmol/cm²) was also demonstrated in humans: topical genistein (applied 30 minutes before UVB) inhibited by one minimal erythema dose (MED) the UVB-induced erythema as shown in Figure 18.8. Thus, topical genistein may protect human skin against photodamage.

Fig. 18.5 Effect of genistein on UVB-induced acute skin burns in mice. Skh:1 hairless mice were treated topically with 5 μmol genistein 60 minutes before UVB at a dose of 1.8 kJ/cm² for 10 days. Photographs were taken 24 hours after last UVB irradiation. (A) Negative control (sham irradiation). (B) Vehicle before UVB. (C) 5 μmol genistein before UVB.

(Reproduced with permission from Wei H, Saladi R, Lu Y, et al 2003 Isoflavone genistein: photoprotection and clinical implications in dermatology. Journal of Nutrition 133:3811S–3819S)

Fig. 18.6 Effect of genistein on UVB-induced chronic photodamage in mice. Skh:1 hairless mice were treated topically with 5 μmol genistein 60 minutes before or 5 minutes after twice weekly UVB at a dose of 0.3 kJ/cm² for 4 weeks. Photographs were taken 24 hours after last UVB irradiation. (A) Negative control (sham irradiation). (B) Vehicle plus UVB. (C) 5 μmol genistein before UVB. (D) 5 μmol genistein after UVB.

(Reproduced with permission from Wei H, Saladi R, Lu Y, et al 2003 Isoflavone genistein: photoprotection and clinical implications in dermatology. Journal of Nutrition 133:3811S–3819S)

Fig. 18.7 Effect of genistein on histologic alterations in mice exposed to UVB. Skh:1 hairless mice were treated topically with 5 μmol genistein 60 minutes before UVB at a dose of 0.3 kJ/cm² twice weekly for 4 weeks. Mice were killed 24 hours after the last UVB irradiation and skin specimens were taken for histology. (A) Negative control (sham irradiation). (B) Vehicle plus UVB. (C) 5 μmol genistein before UVB.

(Reproduced with permission from Wei H, Saladi R, Lu Y, et al 2003 Isoflavone genistein: photoprotection and clinical implications in dermatology. Journal of Nutrition 133:3811S–3819S)

Fig. 18.8 Effect of genistein on UVB-induced erythema in human skin. The study was performed in the phototherapy unit in the Department of Dermatology, Mount Sinai Hospital. UVB fluences used a range from 0 to 100 mJ/cm². Genistein was applied to dorsal skin either 60 minutes before or 5 minutes after UVB exposure. Photographs were taken 24 hours after UVB irradiation. A minimal erythema dose (MED) for this individual was 40 mJ/cm². Lane 1: Vehicle before UVB; lane 2: no treatment before or after UVB; lane 3: 1 μmol genistein/cm² of skin before UVB; lane 4: 1 μmol genistein/cm² of skin after UVB; and lane 5: dose–response of topical genistein applied before UVB (1 MED) at a dose ranging from 0.05 to 5 μmol/cm².

(Reproduced with permission from Wei H, Saladi R, Lu Y, et al 2003 Isoflavone genistein: photoprotection and clinical implications in dermatology. Journal of Nutrition 133:3811S–3819S)

Equally impressive is the fact that topical genistein also inhibits skin cancer, a consequence of chronic UVB damage. Both the incidence and the multiplicity of UVB-induced skin tumors in Skh:2 hairless mice were reduced by about 90% after 25 weeks of UVB exposure. Figure 18.9 shows protection from carcinogenesis of representative mice treated with genistein before UVB exposure. Also, after chemical induction and promotion of skin tumors, topical genistein inhibited tumor cell number by 60–75%.

Fig. 18.9 Representative photograph of inhibition of photocarcinogenesis in mice treated with genistein. (A) Hairless mice irradiated with 0.3 kJ/m² thrice weekly for 25 weeks. (B) Mice treated with 1 μmol genistein before UVB exposure. (C) Mice treated with 5 μmol genistein before UVB irradiation.

(Reproduced with permission from Wei H, Saladi R, Lu Y, et al 2003 Isoflavone genistein: photoprotection and clinical implications in dermatology. Journal of Nutrition 133:3811S–3819S)

Another possible dermatologic benefit of genistein is as a phytoestrogen. The skin has both α- and β-nuclear estrogen receptors through which estrogen binding can regulate linked genes of proliferation and differentiation. Genistein has a 30-fold higher affinity for ERβ than ERα, but a greater ERα agonist activity than ERβ. Although estradiol has 700-fold more ERα and 45-fold more ERβ activity than genistein, the possible biologic effect of genistein through dietary soy isoflavones may be important. This estrogen activity of genistein has been further confirmed in vitro using B16F1 melanoma cells.

Oral and topical estrogen increase the collagen content of skin which diminishes with aging. This effect is especially dramatic in women during and after menopause. Genistein may reduce the atrophic appearance of aging skin both by preventing photodamage through inhibition of metalloproteinases in human skin (independent of sunscreen effect) and by stimulating collagen synthesis. Thus, topical genistein shows promise not only in protecting the skin against acute and chronic photodamage but also in enhancing the diminished collagen synthesis of normal intrinsic aging.

SUMMARY

Nutritional antioxidants represent a novel category of cosmeceuticals. There is no doubt that higher levels are achieved in the skin through topical application than with oral supplementation, thus providing a protective antioxidant reservoir in the skin. Current research indicates that topical ubiquinone and genistein may provide UV photoprotection. In addition, they, as well as topical α-lipoic acid, may retard both intrinsic aging and photoaging. Topical antioxidants continue to be an important area of cosmeceutical research.