(T1430-01-04) Exploration of Antioxidant Activity and Conversion Potential of Cosmeceutical Actives: Ascorbic Acid and Its Derivatives

Purpose: Vitamin C, also known as Ascorbic Acid (AA), is widely recognized as a cosmeceutical agent. It is a powerful antioxidant that neutralizes free radicals in the skin, protecting the layers from oxidative stress and cellular damage. It also plays a role in collagen synthesis, which is essential in maintaining the elasticity of the skin and preventing signs of aging. In addition, AA provides UV protection, and inhibits melanin production, leading to bright and even skin tone. The formulation development of AA faces a few major challenges. AA is sensitive to light, heat, and air; its rapid degradation severely impacts the efficacy of its topical formulations. Moreover, AA being a hydrophilic molecule, has poor passive permeation into the skin’s layers. To overcome these challenges, various derivatives of AA have been synthesized by researchers to retain the efficacy of AA while simultaneously improving stability and physicochemical properties. Although AA itself is an extensively researched molecule, the knowledge available regarding its derivatives is limited. We have attempted to provide a reliable, thorough comparison of AA and its six commercially available derivatives, namely, magnesium ascorbyl phosphate (MAP), sodium ascorbyl phosphate (SAP), ascorbic acid -2- glucoside (AA2G), 3-O-ethyl ascorbate (3OE), ascorbyl palmitate (AP), and tetrahexyldecyl ascorbate (THDA). We have explored the quantitative analysis, antioxidant potential, and the conversion of AA derivatives to AA due to the enzymatic activity in the skin layers.

Methods: We developed RP-HPLC/UPLC methods and validated the same for accuracy and precision for the quantitative analysis of AA and its derivatives. Further, we used the ferric reducing antioxidant power (FRAP) assay to rank order AA and its derivatives as per their antioxidant potential. The FRAP assay provides a quick, sensitive, and easy way to measure the antioxidant capacity of samples. It is a widely used method that uses antioxidants as reductants in a redox-linked colorimetric reaction. The antioxidant activity for aqueous derivatives was measured in two different buffers to check the dependence of antioxidant activity on the pH. For lipophilic derivatives, organic solvent isopropanol was used. Moreover, we tested the ability of AA derivatives to convert into AA because of the enzymatic activity of the skin. For this purpose, we used three different skin models: dermatomed cadaver human skin, full-thickness cadaver human skin, and in vitro cultured full-thickness human skin. For each model, equal amounts of skin were die-punched and minced in a bead mill homogenizer, in a 1 mg/mL solution of AA derivatives. This study was conducted in triplicate for all derivatives. Samples were taken at pre-determined timepoints (2, 5, 8, and 24 h) and analyzed for the amount of AA generated. From this, the percentage of derivative that converted to AA at each timepoint was calculated.

Results: HPLC/UPLC methods for AA and its derivatives were developed and validated (data in Figure 1). From the FRAP assay, we saw that among AA derivatives, 3-O-ethyl ascorbate (hydrophilic in nature) and ascorbyl palmitate (lipophilic in nature) were identified as the top two antioxidants, with ferrous concentrations of 5.46 and 5.07 respectively. As seen from the data in Figure 3, lower pH (citrate buffer, pH 3) resulted in higher antioxidant activity as compared to higher pH (phosphate buffer, pH 7.4) for hydrophilic derivatives. As per the conversion studies, three hydrophilic derivatives with the most structural similarities converted to AA, namely SAP, MAP, and AA2G. In addition, these derivatives were observed to have more easily hydrolyzable bonds allowing conversion to AA as compared to the other derivatives (Figure 2). As expected, the percentage of derivative converting to AA went on rising with increasing contact time with the homogenized skin. At the end of 24 h, SAP converted the most (35.38%), closely followed by MAP (32.91%), which was then followed by AA2G (27.93%). Across all skin models, SAP >MAP >AA2G was the order of conversion potential. The difference within the skin models can be attributed to the differences in the enzymatic activity of the models used. On the other hand, AP, THDA, and 3OE did not convert to AA at all. This can be attributed to the structural differences as well as the lipophilicity of these molecules.

Conclusion: Sensitive and robust analytical methods for AA and its six derivatives were developed and validated. Although AA is hydrophilic in nature, the antioxidant activity of the derivatives did not correlate with hydrophilicity. Based on the FRAP assay and conversion studies, some derivatives showed antioxidant activity by converting to AA, whereas others were good antioxidants by themselves. Overall, the quantitative analysis, comparison of antioxidant activity, and conversion potential of AA derivatives have been investigated.


Figure 1 Validated HPLC/UPLC analysis methods for ascorbic acid and its derivatives


Figure 2 Structures of ascorbic acid, its derivatives, and possible conversion pathways


Figure 3 Rank order of antioxidant activity and summary of conversion of MAP, SAP, AA2G into ascorbic acid using three skin models