3-O-Ethyl Ascorbic Acid: A Stable, Vitamin C-Derived Agent for Skin Whitening

Sep 1, 2013 | Contact Author | By: Jill Hsu, Corum Inc.
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Title: 3-O-Ethyl Ascorbic Acid: A Stable, Vitamin C-Derived Agent for Skin Whitening
3-O-ethyl ascorbic acidx whiteningx tyrosinasex TRP-1x TRP-2x melanin biosynthesisx
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Keywords: 3-O-ethyl ascorbic acid | whitening | tyrosinase | TRP-1 | TRP-2 | melanin biosynthesis

Abstract: Melanin-reducing agents are considered safer alternatives to melanocytotoxic compounds. Thus, the present work examines the effects of 3-O-ethyl ascorbic acid on enhancing skin tone by reducing melanin through the inhibition of tyrosinase-related proteins. Described studies include tyrosinase and TRP-2 inhibition, in vitro and ex vivo melanin inhibition, clinical whitening, DNA protection and collagen synthesis.

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J Hsu, 3-O-Ethyl Ascorbic Acid: A Stable, Vitamin C-Derived Agent for Skin Whitening, Cosm & Toil 128(9) 676 (2013)

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Aflawless complexion is without doubt a desirable attribute across many cultures. Especially in Asia, the obsession with whiter skin has greatly evolved, shifting from beauty to a sign of social class and representing sophistication, innocence, femininity and higher social stature. To achieve this lightening effect, product developers leverage active ingredients such as vitamin C, or L-ascorbic acid, which is not only a septicemia inhibitor, but also an effective reducing agent that can momentarily retard the melanin-biosynthesis pathway in skin. Vitamin C is also known to protect skin from ultraviolet B (UVB) damage and enhance collagen synthesis, therefore it is widely used in cosmetics as a whitening ingredient, especially in Asia. Due to its instability, however, several derivatives of it have been developed for cosmetic application; for example, ethyl ascorbic acid, magnesium L-ascorbyl-2-phosphate, L-ascorbic acid 2-glucoside and sodium ascorbyl phosphate.

3-O-Ethyl ascorbic acid, shown in Figure 1, is a derivative consisting of vitamin C with an ethyl group bound to the third carbon position. With its reducing ability, this structure helps to increase the stability of the compound when used in cosmetic products. The present article explores the function and efficacy of this material to determine its mechanism of action and potential for application against melanin biosynthesis.

Melanin Biosynthesis

To understand the potential mechanisms of skin lightening, it is helpful to review the melanin biosynthesis process. In brief, after exposure to sunlight, melanin is produced by melanocytes through melanogensis, a biological process that helps to protect human DNA from harmful UV rays. The amount of melanin produced by melanocytes embedded in the basal layer of the epidermis defines the skin color, but it is interesting to note that all individuals from different countries and climates tend to have the same number of melanocytes.

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Figure 1. Structure of 3-O-ethyl ascorbic acid

Figure 1. Structure of 3-O-ethyl ascorbic acid

3-O-Ethyl ascorbic acid, shown in Figure 1, is a derivative consisting of vitamin C with an ethyl group bound to the third carbon position.

Figure 2. Melanin biosynthesis schematic

Figure 2. Melanin biosynthesis schematic

Three enzymes are known to be involved in melanin biosynthesis in mammals—tyrosinase, tyrosinase-related protein-1 (TRP-1) and dopachrome tautomerase (DCT, also known as TRP-2) (see Figure 2).1

Figure 3. In vitro tyrosinase (II) inhibition; a) tyrosine g DOPA and b) DOPA g dopaquinone

Figure 3. In vitro tyrosinase (II) inhibition; a) tyrosine g DOPA and b) DOPA g dopaquinone

After the reaction, absorbance was measured at 475 nm by a UVC/Vis spectrophotometer. Results showed that 3-O-ethyl ascorbic acid inhibited tyrosinase activity (see Figure 3).

Figure 4. Western blotting detection: inhibitory effect of 3-O-ethyl ascorbic acid on tyrosinase (TYR)

Figure 4. Western blotting detection: inhibitory effect of 3-O-ethyl ascorbic acid on tyrosinase (TYR)

a) Western blotting bands, upper row = TYR, lower row = b-actin; b) optical band density analysis. TYR protein decreased significantly after 3-O-Ethyl ascorbic acid treatment; n = 3.

Figure 5. Western blotting detection: inhibitory effect of 3-O-ethyl ascorbic acid on tyrosinase (TRP-2)

Figure 5. Western blotting detection: inhibitory effect of 3-O-ethyl ascorbic acid on tyrosinase (TRP-2)

a) Western blotting bands, upper row = TRP-2, lower row = b-actin; b) optical band density analysis. TRP-2 protein decreased significantly after 3-O-ethyl ascorbic acid treatment; n = 4.

Figure 6. Western blotting detection: inhibitory effect of 3-O-ethyl ascorbic acid on tryrosinase (TRP-1)

Figure 6. Western blotting detection: inhibitory effect of 3-O-ethyl ascorbic acid on tryrosinase (TRP-1)

On the other hand, TRP-1 expression showed no significant change in this experiment (see Figure 6).

Figure 7. In vitro whitening activity study: melanin assessment

Figure 7. In vitro whitening activity study: melanin assessment

Results showed 3-O-ethyl ascorbic acid exhibited effective whitening, observed both at 15 mg/mL and 20 mg/mL concentrations (see Figure 7).

Figure 8. The result of ex vivo melanin assay for nine days; untreated (left) vs. treated (right)

Figure 8. The result of ex vivo melanin assay for nine days; untreated (left) vs. treated (right)

One set was used to determine changes in melanin content while the second was used assess changes in pigmentation via histology. The results are presented in Figure 8.

Figure 9. In vivo evaluationf of the efficacy of one whitening lotion in healthy Asian subjects by chromatography

Figure 9. In vivo evaluationf of the efficacy of one whitening lotion in healthy Asian subjects by chromatography

As shown in Figures 9 and 10, the ITA° angle increased by 4% after 28 days of formula application, and an obvious whitening effect could be observed with the naked eye.

Figure 10. Evolution of individual typological angle of the skin treated with 3-O-ethyl ascorbic acid

Figure 10. Evolution of individual typological angle of the skin treated with 3-O-ethyl ascorbic acid

As shown in Figures 9 and 10, the ITA° angle increased by 4% after 28 days of formula application, and an obvious whitening effect could be observed with the naked eye.

Figure 11. Stimulation of collagen synthesis

Figure 11. Stimulation of collagen synthesis

The test material, 3-O-ethyl ascorbic acid, presented a significant effect on collagen synthesis, as shown in Figure 11, similar to TGF-β1.

Figure 12. Comet assay shows: a) the appearance of undamaged/damaged DNA by electrophoresis, and b) the result of DNA protection

Figure 12. Comet assay shows: a) the appearance of undamaged/damaged DNA by electrophoresis, and b) the result of DNA protection

As a result, damaged DNA has a longer tail, as shown in Figure 12.

Figure 13. Heat stability

Figure 13. Heat stability

As shown in Figure 13, 3-O-ethyl ascorbic acid was more stable than the others, and its color remained unchanged.

Figure 14. Purity analysis

Figure 14. Purity analysis

Further, its purity also was analyzed vie HPLC, and as Figure 14 shows, the purity of 3-O-ethyl ascorbic acid crystalline powder remained stable at 45˚C for 60 days.

Footnotes (CT1309 Hsu)

a Corum 9515 (INCI: 3-O-Ethyl Ascorbic Acid) is a product of Corum, Inc., www.corum.com.tw.
b Mushroom Tyrosinase T3824 is a product of Sigma, www.sigmaaldrich.com.
c MelanoDerm tissues are manufactured by MatTek Corp., www.mattek.com.
d Sirius Direct Red 80 is a product of Sigma-Aldrich, www.sigma-aldrich.com.

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