The skin dryness inhibitory mechanism of GO-VC, an amphiphilic ascorbic-acid derivative

Since tight junctions are known to play an important role in skin barrier function, their functional deterioration is thought to induce skin dryness. Provitamin, represented locally by Afrisil Speciality Ingredients, has investigated the skin dryness-inhibitory effect of GO-VC with a focus on tight junctions.

Ascorbic-acid (AsA) derivatives are widely used in cosmetics for their skin-brightening, anti-wrinkle and anti-acne effects. However, since AsA and its derivatives are reported to suppress the secretion of sebum1, they have a possibility to promote skin dryness in some cases. In recent years, 2-O-glyceryl-3-O-octyl ascorbic acid (GO-VC), of which glycerine and octanol are bound to hydroxyl groups at the C-2 and C-3 positions of AsA respectively (see Figure 1), has been developed as an amphiphilic AsA derivative with excellent stability. Since glycerine and octanol are known to have humectant ability and antimicrobial activity, GO-VC is anticipated to have a moisturising effect and antimicrobial activity.

In a clinical study, GO-VC has been confirmed to have a suppressing effect on Propionibacterium acnes proliferation and to improve Acne vulgaris. Regarding the moisturising effect, a sensory evaluation confirmed that GO-VC provides skin with a moist feeling that is not sticky or greasy (see Figure 2).

However, the skin dryness-inhibitory effect and the mechanism of action of GO-VC were still unclear. Provitamin conducted various studies on human volunteers and epidermal keratinocytes in order to clarify the skin dryness-inhibitory effect and GO-VC mechanism of action. The results of this study were presented by Provitamin’s researchers at the 2020 IFSCC Congress in Yokohama, Japan.

Figure 1: Chemical structure of GO-VC

Figure 2: Sensory evaluation test of GO-VC

What are tight junctions?

Existing as an outermost layer, the skin is the organ responsible for barrier protection as it prevents water loss and protects the body from the invasion of external chemicals and microorganisms. The skin barrier function mainly originates from its lipid lamella structure consisting of ceramides, cholesterol and fatty acids that are found in the interstice of the stratum corneum (SC) and tight junctions (TJ), which exist at the second level of the granular layer. 2, 3, 4

TJ is one of the cell adhesion apparatuses consisting of several proteins such as claudin-1, occludin and zonula occludens-1 (ZO-1) (see Figure 3). Although the important function of TJ is recognised as its barrier protection property against the penetration of metal ions such as Ca2+ and Mg2+ between SC and the granular layer4, TJ is also known to function as a barrier against water. For instance, atopic dermatitis – a chronic inflammatory disease as a result of impaired barrier function – is reported to show the dysfunction of TJ due to the down-regulation of claudin-1 expression.5

Even in healthy skin, reduced barrier function can result in a rough texture and the start of skin dryness.6 Provitamin conducted a human volunteer test to evaluate the behaviour of the skin surface water content (SWC) and trans-epidermal water loss (TEWL) after an application of GO-VC. The researchers investigated the mechanism of action of GO-VC with a focus on TJ assembly.

Figure 3: Structure of tight junctions (TJ)

METHODS

1. Human volunteer test to determine SWC and TEWL

A group of 10 volunteers (five men and women between 24 to 57 years of age) participated in the test.

A sample solution containing 0.1% GO-VC and a placebo without the active ingredient were applied once a day to the inner sides of the left or right upper arm, respectively. SWC and TEWL were measured with a Corneometer CM825 (Courage and Khazaka, Köln) and a Tewameter TM300 (Courage and Khazaka) after treatment with the sample and the placebo solutions at six and 12 weeks, respectively. SWC and TEWL at both sites were also measured before starting the treatments as the baseline values.

2. Determination of the mRNA and protein expressions of TJ-member proteins

After being pre-cultured at 37°C in an environment of 5% CO2 for 24 hours, normal human epidermal keratinocytes (NHEKs) were treated with GO-VC for 72 hours. Thereafter the mRNA expressions of claudin-1, occludin, and ZO-1 in the cells were determined with real-time reverse transcription polymerase chain reaction (RT-PCR). Similarly, the protein expression of claudin-1 was also visualised with immunofluorescent staining.

RESULTS

1. The effect of GO-VC on SWC and TEWL

After the topical application of 0.1% GO-VC for 12 weeks on the inner side of the upper arm of the volunteers, the GO-VC-applied site was found to have a significant increase in SWC while TEWL significantly decreased at the site, compared to those of the placebo-applied site (see Figure 4).

2. The effect of GO-VC on TJ-member proteins

To clarify the impact of GO-VC on TJ assembly, the study investigated the mRNA expression levels of claudin-1, occludin, and ZO-1 in GO- VC-treated NHEKs. The results showed GO-VC significantly increased the mRNA expression levels of claudin-1, occludin, and ZO-1 (see Figure 5). In addition, the immunofluorescent staining of claudin-1 showed higher red fluorescence at a region of the cell membrane of GO-VC-treated NHEKs than that of the control or non-treated- NHEKs (see Figure 6), indicating that GO-VC increased the protein expression of claudin-1.

Discussion

Skin dryness, of which the typical characteristics are lower SWC and higher TEWL, is known to cause oxidative stress and inflammation in the skin, resulting in a probability of skin issues such as hyperpigmentation and wrinkles.

Figure 4: The effect of GO-VC on SWC and TEWL

Figure 6: The effect of GO-VC on the protein expression of claudin-1 in NHEKs

TJ, which consists of several proteins such as claudin-1, occludin, and ZO-1, exists at the second level of granular layer within the epidermis and is an important structure for maintaining hydration, because the TJ is known to act as a barrier preventing water loss from within the skin. 4, 5 Therefore, the reinforcement of TJ assembly is a promising approach to suppress skin dryness. This study investigated the potential of GO-VC to improve skin barrier function with a focus on TJ assembly.

Firstly, in the human volunteer test the site where GO-VC was applied showed a significant increase in SWC and a decrease in TEWL when compared to the placebo-applied site (see Figure 4).

An in vivo study was also conducted to investigate the mechanism of action of GO-VC on SWC and TEWL. GO-VC up-regulated the mRNA expressions of claudin-1, occludin, and ZO-1 in NHEKs (see Figure 5).

To confirm the results in RT-PCR, a cytohistological study was performed with a focus on the protein expression of claudin-1, which is a representative protein in TJ. GO-VC-treated NHEKs increased the protein expression of claudin-1 (see Figure 6).

In conclusion, GO-VC was effective in enhancing skin hydration by reinforcing the barrier function through an increase in the synthesis of TJ member proteins. The potential of GO-VC as a cosmetic ingredient was also shown in its ability to supress the effect of skin dryness. •

REFERENCES: 1. Y. Tokudome, Y. Takahashi, J Dermat Cosmetol, 1, 66 ~ 70 (2017) 2. M.J. Choi, H.I. Maibach, Am. J Clin Dermatol, 6, 215 ~ 223 (2005) 3. J.W. Fluhr et al., J Invest Dermatol, 117, 44 ~ 51 (2001) 4. N. Kirschner et al., J Invest Dermatol, 113, 1161 ~ 1169 (2013) 5. R. Gruber et al., Am J Pathol, 185, 2777 ~ 2789 (2015) 6. C.R. Harding et al., Int J Cosmet Sci, 22, 21 ~ 52 (2000) 7. H. Fujita, T. Hirao, M. Takahashi, Skin Res Technol, 13, 84 ~ 90 (2007) 8. Y. Kobayashi et al., Int J Cosmet Sci, 30, 35 ~ 40 (2008) 9. Y. Ashida, M. Ogo, M. Denda, Br J Dermatol, 144, 238 ~ 243 (2001)

Afrisil – www.afrisil.com Provitamin – ito-provitamin.co.jp/en