In recent years, "dullness" has been pointed out as one of the most common skin problems for both men and women1,2. According to our independent survey on 4,402 men and women in their 20s to 60s living in Japan, about 75% of women and about 50% of men feel that their skin is dull. Dullness is not only the problem of overall skin tone, but also leads to an uneven appearance due to an ununiform color tone. Dull skin tone negatively influences how you look, as it makes you look tired and older than your biological age.
However, if you think about what dullness is, there is no clear definition, and in fact it is a vague and broad idea. When we look at dullness more in detail by cause, there are, for example, gray tone caused by dryness or improper turnover, black tone caused by melanin, and brown tone caused by the accumulation of advanced glycation end-products (AGEs). On the other hand, as they overlap on the skin, it is extremely difficult to identify the cause of your dullness and treat it appropriately. In our survey, more than 40% of people who actually take some kind of care against dullness of their skin were dissatisfied with the current measures against dullness. This paper introduces that we focused on AGEs and their receptor RAGE (Receptor for AGEs), and found3 Peony Root Extract (PRE) as a cosmetic ingredient for comprehensively caring for dullness, and developed it as a cosmetic ingredient called PeoGlow.
Correlation Between Dullness and Glycation
AGEs, which we focused on, are a general term for compounds formed by non-enzymatic binding and glycation of proteins and lipids to reduce sugars. As glycation is a non-enzymatic reaction, it has been reported that AGEs are produced in the body significantly depending on blood glucose levels and accumulate in various organs, including the skin, and that their accumulation correlates with diabetes-related diseases4,5.
It has also been reported that an increase in reactive oxygen species enhances the late glycation reaction, leading to the accumulation of AGEs due to aging6, as well as a significant increase in the accumulation of AGEs in the exposed areas7. Glycated proteins denature and lose their functions. Glycation of the epidermis is also known to be the cause of "brown tone" due to AGEs themselves being brownish.
The accumulation of AGEs is not only involved in brown tone, but also in various elements of dullness. Glycation in the pericellular environment inhibits the differentiation of keratinocytes and causes improper turnover of the skin, leading to "gray tone". In addition, AGEs transmit various signals to cells through their receptors, RAGE.
RAGE is a transmembrane protein that exists on the cell surface and its expression has been confirmed in a wide variety of areas including epidermal keratinocytes, epidermal melanocytes, and dermal fibroblasts. The reactions attributed to RAGE are diverse, and it has been clarified that it promotes melanin production in melanocytes, causing "black tone"8. This suggests that brown, gray and black tones, which have been considered to be caused by independent factors, actually occur in combination in skin where AGEs have accumulated, making the skin look dull. Inhibiting the production of AGEs and controlling their action may be a comprehensive approach against skin dullness, but as far as we know, there are no cosmetic ingredients that can achieve this.
Peony Root Extract That Suppresses the Effect of AGEs
We investigated about 400 naturally-derived ingredients to suppress AGE signals via RAGE and found that PRE (derived from Paeonia albiflora) inhibits the interaction between AGEs and RAGE. Peonies are popular for ornamental purposes due to the beauty of their flowers, and there are various varieties. The root has been used as an herbal medicine since ancient times and is believed to have anti-convulsant and anti-inflammatory effects. In addition, it has been known for its AGE cross-link cleavage action. However, its effects on RAGE have not been reported.
The following section describes PRE’s effects on inhibiting AGEs-RAGE interaction, suppressing melanin production induced by AGEs, restoring keratinocytes’ differentiation potency reduced by glycation and combating glycation. Its effects on reducing AGEs and melanin, suppress age spots, and reduce dullness demonstrated in human clinical trials are also introduced.
Effect of PRE on Inhibiting AGEs-RAGE Interaction
We evaluated PRE to check whether it has AGEs-RAGE interaction inhibitory activity. Multi-well plates were coated with RAGE recombinant proteins and then washed with D-PBS containing 0.1% Tween20 after blocking. A blocking solution containing PRE or a solvent as the control was added to the wells and reacted at room temperature for 1 hour. After washing the wells, a blocking solution containing biotin-tagged AGEs was added and incubated at room temperature for 1 hour. The color from RAGE-bound biotin-tagged AGEs was detected by measuring absorbance at 450 nm. The AGEs-RAGE interaction rate was calculated with the absorption value of the control as 100% interaction rate and the absorbance value of the well without RAGE coating and with added solvent as 0% interaction rate.
Figure 1 - PRE inhibits AGEs-RAGE interaction.Courtesy of Ichimaru Pharcos
As a result, in the group where 1% PRE was added, AGEs-RAGE interaction was inhibited by approximately 90% compared to the untreated group (see Figure 1). As the wells were washed after PRE treatment and AGEs were added in this evaluation system, it is considered that PRE inhibits the AGEs-RAGE interaction by acting on RAGE.
Effect of PRE on Restoring Differentiation Potency Declined Due to Glycation
Next, we evaluated whether PRE restores the keratinocytes' differentiation potency that had been reduced due to glycation. Laminin 332, one of the components contained in the epidermal basal membrane and plays an important role in maintaining the homeostasis of keratinocytes in the basal layer, was used to coat 12-well plates. A PBS or glucose solution was added as a glycation treatment, and the plates were left at 37°C for 2 weeks.
The coated plates were washed with D-PBS and keratinocytes cultured using KGM Keratinocyte Growth Medium (KGM) were subcultured. After cultivation to confluence, the medium was replaced with a medium that contained a final concentration of 0, 0.2, or 0.4% of PRE and final concentration of 0.15 mM of calcium without any transferrin, hydrocortisone, and epinephrine.
After 6 hours of cultivation, the cells were collected, and mRNA expression of keratin 10 (K10) and transglutaminase 1 (TGM1), which are differentiation markers, was evaluated by RT-PCR. As a result, expression levels of K10 and TGM1 were low in the glycation treatment group (NC) despite calcium-induced differentiation (See Figure 2). On the other hand, in the group to which PRE was added, it was restored with a concentration-dependent tendency (see Figure 2). These results suggest that PRE is expected to restore the differentiation potency that has been reduced due to glycation.
Figure 2 - PRE restores the differentiation potency of keratinocytes delayed by glycation.Courtesy of Ichimaru Pharcos
Effect of PRE on Suppressing Melanin Production caused by AGEs
Normal human epidermal melanocytes were seeded in 12-well plates and cultured to 70% confluent. For the medium, MGM-4 Melanocyte Growth Medium-4 BulletKit (MGM) was used. MGM was replaced with a medium where FBS was removed, the cells were cultured overnight, then replaced with a medium containing 0, 0.2, or 0.4% of PRE.
After 1 hour of incubation, AGEs were added to achieve a final concentration of 400 μg/mL, the cells were cultured for 6 days, and the number of viable cells was evaluated using the WST method. Then, after washing with D-PBS, the cells were detached using trypsin and collected. An ethanol:ether = 3:1 solution was added to the obtained samples, they were centrifuged at 2000 rpm, then pellets of melanin were obtained as precipitate. The obtained pellets were dried overnight, a 25 μL of 1N NaOH / 10% DMSO solution was added, heated at 95 °C for dispersion, and the amount of melanin was evaluated by quantifying the absorption at 420 nm.
As a result, melanin production per cell significantly increased in the group to which AGEs were added (NC) compared to the group with no addition (n.t.) (see Figure 3). On the other hand, this was suppressed in the PRE treatment group with a concentration-dependent tendency (See Figure 3). These results suggest that PRE is expected to inhibit melanin production increased by AGEs.
Figure 3 - PRE suppresses melanin production caused by AGEs. Courtesy of Ichimaru Pharcos
Effect of PRE to Suppress AGEs Accumulation
We evaluated whether PRE can be used as a way to reduce brown tone by reducing the accumulation of AGEs taking advantage of conventional anti-glycation effects. The test was conducted with reference to the method in reference 9. Albumin and glucose were dissolved in 0.4 M phosphate buffer (pH 7.4) to 1 mg/mL and 10 mg/mL, respectively. PRE or 50% BG (control) was added to achieve a final concentration of 1% and filter sterilization was performed. After 4 weeks of incubation at 37°C, autofluorescence from AGEs produced by the reaction of albumin and glucose was measured (Ex: 370 nm, Em: 440 nm). As a result, the accumulation of AGEs significantly reduced in the group to which PRE was added (see Figure 4). These results suggest that PRE inhibits the accumulation of AGEs.
Figure 4 - PRE inhibits AGEs accumulation.Courtesy of Ichimaru Pharcos
Cosmetic Effects of PRE Demonstrated in Human Clinical Trials
We conducted human clinical trials to evaluate the effects of PRE on human skin. After obtaining written consent from 17 healthy men and women in their 40s to 60s, a lotion containing 1% PRE or a control lotion was applied to half of their face twice a day for two weeks. At the beginning of the trial and after two weeks, the following items were measured.
- Glycation levels on their cheeks using AGE Scanner
- Amount of melanin on their cheeks using Mexameter
- Age spots on their cheeks using VISIA
- Dullness score (out of ten) of their cheeks
Effect of PRE on Reducing Skin Glycation Level and Amount of Melanin
Figure 5 - PRE reduces the amount of AGEs and melanin in the skin. The amount of AGEs in the skin (a) was evaluated using AGE Scanner and the amount of melanin in the skin (b) was evaluated using Mexameter. (n = 17, mean ± SEM, *p < 0.05, vs 0w by Wilcoxon signed-rank test)Courtesy of Ichimaru Pharcos
We measured the amount of AGEs, which are the cause of brown tone, and melanin, which is the cause of black tone, in the skin. AGE Scanner is an instrument that measures the amount of AGEs deposited in approximately 1 mm from the skin surface using the autofluorescence of AGEs. It revealed that the amount of AGEs on the cheeks tended to decrease in the group that PRE-containing lotion was applied (See Figure 5a). There was also a significant decrease in the amount of melanin (See Figure 5b). None of these parameters showed significant changes in the group that the control lotion was applied.
Effect of PRE on Reducing the Wrinkle Score and Dullness Score
As an evaluation of appearance, we evaluated the age spots score, taking into account the number of age spots, their area values and their darkness using VISIA. The number of age spots tended to increase in the group that the control lotion was applied, while it tended to decrease in the group that a 1% PRE-containing lotion was applied (See Figure 6a). The age spot score significantly increased in the group that the control lotion was applied before and after the test. However, there was almost no change in the group that 1% PRE-containing lotion was applied (See Figure 6b).
Figure 6 - PRE suppresses age spots. Using VISIA, the number of age spots (a) and the age spot score (b) were evaluated. Both increased in the control group, but they tended to decrease or remain almost unchanged in the group that PRE was applied. (n = 17, mean ± SEM, †p < 0.05, vs 0w by Wilcoxon signed-rank test)Courtesy of Ichimaru Pharcos
We also evaluated the dullness score. In the dullness score, the amount of information is reduced while maintaining the impression of the image by SLIC processing the skin image. Therefore, it is possible to quantify the impression of the skin without the need for an evaluator. In addition to the overall lightness (L* value), uneven tone is taken into consideration, making it an indicator close to the impression that humans get when they look at other people's skin (see Figure 7a)10.
Figure 7 - PRE improves skin dullness.Courtesy of Ichimaru Pharcos
The dullness score significantly worsened before and after the test in the group that the control lotion was applied, while the dullness score significantly decreased in the group that the 1% PRE-containing lotion was applied (see Figure 7b). Also, compared to the group to which the control lotion was applied, the dullness score significantly decreased in the group that the 1% PRE-containing lotion was applied (see Figure 7b). The above results show that PRE suppresses the production of AGEs and melanin in human skin and helps to achieve even-toned beautiful skin by suppressing age spots and dullness.
Conclusion
In this paper, we introduced that peony root extract has an effect on inhibiting the AGEs-RAGE interaction, improving improper differentiation caused by glycation processes, suppressing melanin production caused by AGEs and suppressing the accumulation of AGEs as confirmed via in vitro tests. In human trials as well, it was confirmed that the amount of AGEs and melanin that cause dullness was reduced after two weeks, and that the age spots and dullness scores were lowered in the group that the 1% PRE-containing lotion was applied.
These results showed that PRE has a possibility to efficiently improve overall skin tone and uneven tone. By focusing not only on melanin, which is a representative product of photoaging, but also on AGEs, we hope that PRE will help develop more effective and comprehensive products for caring for skin dullness.
References
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- Over 60% of men have beauty concerns! Approximately 60% also engage in skincare - Survey by Kufu Consumer Research Institute https://www.commercepick.com/archives/65996
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