The free radical scavenging properties of retinyl ascorbate (RA-AsA) were determined by monitoring the decomposition of 2,2-diphenyl-1-picrylhydrazyl (DPPH) as a function of time and in comparison with ascorbic acid (AsA), ascorbic acid palmitate (AsA-Pal), retinoic acid (RA), retinol (ROL) and retinol palmitate (Rol-Pal). The rate constant of RA-AsA (mean3+/-SD) was 4.9+/-0.3 M(-1) s(-1), and indicated greater potency as an antioxidant compared to the rest of the test compounds (AsA 3.4+/-0.4 M(-1) s(-1), AsA-Pal, 2.9+/-0.2 M(-1) s(-1), RA 1.4+/-0.3 M(-1) s(-1), ROL 1.3+/-0.1 M(-1) s(-1), Rol-Pal exhibited insignificant activity). The decomposition rate constant of DPPH, 5+/-0.6 x 10(-8) M(-1) s(-1), in ethanol and BHA, 154+/-3 M(-1) s(-1) were both used as control. The compound RA-2-carboxy-2-hydroxy-ethanoate was isolated by prep-TLC and was identified, by 13C and 1HNMR spectroscopy, as the major by-product from the reaction of RA-AsA with DPPH, which was also found to be potent antioxidant, 2.1+/-0.2 M(-1) s(-1). This suggests that oxidation of AsA moiety did not lead to the production of erythrulose species, which could cause deleterious modifications of cellular proteins.

Retinyl ascorbate (RA-AsA), an ester co-drug of vitamins A (RA) and C (AsA), is proposed as a topical antioxidant/cell division regulator for reducing UV-induced generation of free radicals and disrupted dermal cell growth. The efficacy of dermatological agents is influenced by their retention within the skin, which is increased by the interaction with skin components. Keratin is the major protein (∼95%) in the skin, and this paper reports the binding of RA-AsA, RA, AsA, retinol, ascorbic acid palmitate and retinol palmitate to three tissues – human callus, pig ear skin and bovine horn keratin. Tissue samples were incubated with solutions of compounds and the uptake measured as the ratio of bound/free compound at equilibrium. Binding to keratin was assessed using delipidised tissue, and was much higher for the polar compounds, suggesting dipolar/H-bonding interaction. Binding strength was ranked as human > porcine > bovine, but there was no distinction for highly lipophilic compounds. The binding characteristic of native tissues was complicated by lipid content of the tissues. There seemed to be a dual effect. The binding of very lipophilic materials increased with lipid content, implying that a substantial amount is dissolved in the lipid matrix. For highly polar AsA, lipid content decreased the binding, suggesting that the lipid reduced the strong polar interactions with skin protein/keratin.

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Chemical and enzymatic hydrolysis of the co-drug of retinoic acid (vitamin A) and ascorbic acid (vitamin C) – retinyl ascorbate (RA-AsA) – have been studied. Firstly, the amount of protein and ester hydrolysis activity was determined in crude cellular extracts from freshly excised porcine ear skin (<3 h) and stored porcine ear skin (frozen >6 months) using ethyl butyrate as model substrate. The stability of RA-AsA was then determined in the crude cell extracts with and without additional antioxidants. Lastly, the enzymatic hydrolysis of RA-AsA and retinyl-2-carboxy-2-hydroxy-ethanoate were determined by incubating with porcine liver esterase – retinol palmitate and ascorbyl palmitate were included for comparison. Freshly excised skin contained higher amounts of active proteins than previously frozen skin. RA-AsA underwent hydrolytic reduction causing the AsA moiety to disintegrate due to the presence of free radicals in the media. An intermediate was produced that seemed to be cleaved by enzymes. Addition of ascorbic acid, as antioxidant, to the media of crude protein extracts decelerated the hydrolysis rate. This was supported when RA-AsA and retinyl-2-carboxy-2-hydroxy-ethanoate were incubated separately with pure esterase. There was ∼5-fold more soluble protein per ml of cytosol in the fresh skin compared to the stored skin. Therefore, the amount of protein present within ∼1.5 cm² of skin (average diffusion area in the Franz cells used in our skin penetration studies) was 0.06 mg cm^–2 and 0.01 mg cm^–2 for fresh and stored extracts, respectively.

This influence of skin stretching and hair follicle sealing on the delivery of retinyl ascorbate (RA-AsA) to the epidermis was probed in vitro. Porcine ear skin was subjected to stretching by 2 and 4 mm (3.3 and 6.7%, respectively); the hair follicles of other skin sections were located and painstakingly sealed using adhesive. After mounting in Franz cells the skin was dosed with 100 µl of 2.5 mM RA-AsA in methanol/PBS with water as receptor phase. After 24 h the diffused areas were subjected to tape stripping, and the amount of RA-AsA was determined in 5 groups of 9 strips. Statistical analysis of the resulting depth profiles showed that there was no statistical difference between unstretched skin and the skin that had the follicles sealed across the 5 depth bands. Between 0 and 2 mm stretch there were generally significant differences; between 0 and 4 mm p < 0.001 was obtained at each depth. The data from this limited exercise suggest that in native skin the follicular route does not contribute to dermal absorption, but when disturbed (as when stretched) follicular delivery can substantially increase drug delivery into the skin by up to approximately 20–40%. Skin stretching becomes difficult beyond about 7%.