Results & Discussion
Promotion of HAS2 gene expression and hyaluronic acid synthesis
The effect of RSE on hyaluronic acid (HA) promotion was investigated. HA is widely present in the skin and maintains its hydrophilicity. In this study, we confirmed the expression of the HAS2 gene, which is an enzyme that synthesizes HA from fibroblasts. The change in the expression of the HAS2 gene by RSE was verified by RT-qPCR. HAS2 expression increased in a concentration-dependent manner with the addition of 0.01% or 0.1% RSE (Fig. 1A). We also examined the quantitative changes of hyaluronic acid produced in the culture medium using ELISA. Hyaluronic acid production in the medium was greatly increased by the addition of 0.01% RSE, and the concentration was approximately 10 times higher than that in the steady state (Fig. 1B). The amount of hyaluronic acid synthesized increased substantially with respect to the degree of enhancement of the HAS2 gene. HAS2, a hyaluronic acid synthase, has been mainly expressed in fibroblasts, but the presence of HAS1 and HAS3 has also been confirmed (Siiskonen et al ., 2015). It has been speculated that RSE may also increase the gene expression of hyaluronan synthases other than HAS2.
Promotion of COL3 gene expression and type III collagen synthesis
The effect of RSE on the promotion of type III collagen expression was investigated. RSE increased the expression of the COL3 gene in a concentration-dependent manner (Fig. 1C). We also verified the quantitative change in type III collagen produced in the medium using ELISA. With the addition of 0.01% RSE, the amount of type III collagen produced in the medium greatly increased (Fig. 1D). The dermis is mainly composed of an extracellular matrix, and collagen is one of its main components. There are approximately 30 types of human collagen, and type I collagen is widely distributed in adult skin. Type III collagen, which is thought to be involved in wound healing, is also widely distributed in the skin, especially in newborns, but its presence gradually decreases with age. The reduction of collagen leads to the loss of the structure-maintaining function of the skin (Murakami et al. , 2013). The fatty acid composition of RSE derived from rice bran oil is 40% oleic acid (Takagi et al ., 1984). Oleic acid and linoleic acid are important factors that promote wound healing, and have been confirmed to enhance the expression of type III collagen (Cardosoet al. , 2011). It was suggested that RSE treatment increased the amount of type III collagen and contributed to the improvement of sagging and wrinkles.
Result of subject grouping
The two groups were assigned to equalize the measured mean values, ​​such as the skin viscoelasticity R2, trans-epidermal water loss (TEWL), and wrinkle score (Fig. 2). There was no significant difference between the initial values on the RSE-applied and placebo-applied sides for all measurement items. The study design for topical application of RSE is shown in Fig. 3. Twenty-five subjects were assessed for eligibility; only one subject did not meet the inclusion criteria. Twenty-four subjects were divided into two groups: one group applied RSE cream on the right side of the face and the other group applied RSE cream on the left side. Two subjects dropped out during the test period, and the final analysis was conducted on 22 subjects with an average age of 38 ± 13.2 years, of which 10 women in their 40s and 50s had an average age of 51.5 ± 2.46 years.
Effect on skin elasticity
The effect of RSE on the human face was verified using a placebo-controlled half-face application test. The R2 value was used as the index of elasticity. The R2 value is determined by the recovery rate (%) of the skin height after stretch/involution, and values closer it is to 100%, indicate greater elasticity. The viscoelasticity of the skin decreased from the initial measurement value, with a smaller decline in the RSE group than in the control group, and there was no significant difference between the measurement values and the groups (Fig. 4A). As type III collagen is promoted in fibroblasts, it was expected that the elasticity of the cheeks would be improved, but no significant contribution to viscoelasticity was observed. Type I collagen accounts for the majority of adult dermal collagen, and the contribution of the increase in type III collagen to skin elasticity may be low.
Effect on barrier function
The amount of trans-epidermal water loss (TEWL), which is an index of skin barrier function, was measured. The TEWL amounts in both groups changed from the 0th week and increased slightly at the 4th week, but were suppressed at weeks 8 and 12. The TEWL of the RSE topical side decreased by -2.68 ± 0.93 at week 8 and by -2.31 ± 1.04 at week 12, and a significant suppression was observed between before and after treatment (Fig. 4B). We considered that topical application of RSE to subjects increased the production of hyaluronic acid in the skin. Hyaluronic acid, a glycosaminoglycan, is abundant in the dermis and is involved in water retention. The water retention of human skin is enhanced by increasing the production of hyaluronic acid. Another factor is the increase in type III collagen due to topical application of RSE. Type III collagen is involved in wound healing, and when collagen production is enhanced in the dermis, the repair of the epidermis is accelerated. In mice, an increase in collagen suppresses TEWL and increases the water content of the stratum corneum (Lee et al. , 2020). Taken together, the enhancement of type III collagen maintains the dermis structure, contributes to the normalization of turnover of the stratum corneum, and improves the barrier function of the skin.
Effect on wrinkle score
The facial wrinkle score was calculated using the image analyzer VISIA evolution. Although the wrinkle score decreased on both face sides of the subjects, there was no significant difference in measurements between the initial and later stages, and between the RSE topical side and placebo side (Fig. 4C). Although RSE enhanced type III collagen along with skin elasticity, it was suggested that the actual contribution to human skin wrinkles was small.
Effect on facial redness
The redness of the face was analyzed using VISIA evolution. The redness score in the 4th week of the RSE group decreased -1.15 ± 0.31 points, and significant suppression was observed (Fig. 4D). It has been reported that rice bran components such as the plant sterol γ-oryzanol, which is an ester of triterpene alcohol, and ferulic acid, have various anti-inflammatory effects (Saenjum et al. , 2012). Anti-inflammatory effects, such as suppression of IL-6 and COX-2, have been reported in plant sterols such as hydrolyzate sitosterol and stigmasterol, as well as with triterpene alcohol (Loizou et al , 2009, Pereda et al , 2018). Oleic acid, the main fatty acid in rice bran, has also been confirmed to be involved in skin wound healing and have an anti-inflammatory effect (Cardoso et al ., 2011). However, the anti-inflammatory action and pharmacokinetics of oleic acid and sterols in the ester form remain unclear. It is also possible that free phytosterols and free oleic acid in the RSE suppress inflammation. Dry skin, due to a decrease in barrier function, also induces chronic inflammation, such as with atopic dermatitis and dry diseases. The application of RSE improved the barrier function of the skin and made it possible to retain water, which improved the dry condition and suppressed the chronicity of the inflammatory reaction.
Improvement of pore issues
The subjects were asked about pore troubles using the VAS questionnaire. The score was significantly increased for the side of the face treated with RSE by 1.39 ± 0.45 at 4 wk, 1.35 ± 0.39 at 8 wk, and 1.7 ± 0.36 at 12 wk compared to the values at 0 wk. At 12 wk, an increase of 1.43 ± 0.38 was observed on the placebo side, but the rate of increase was higher in those with the RSE applications (Fig. 5). It is thought that the problems with pores are caused by an increase in sebum amount, an increase in hole volume due to clogging of keratin plugs, and a decrease in elasticity around the pores (Laneri et al. , 2021). In our study, it appears that the improvement in pore issues was not caused by repairing the skin structure and improving elasticity around pores by collagen production (Schütz et al ., 2019), but by promoting turnover by improving TEWL. In addition, the suppression of chronic inflammation confirmed in the improvement of redness was also considered to reduce the problem of pores.
Wrinkle improvement
The subjects were asked about wrinkles using the VAS questionnaire. On the RSE application side, the score increased from week 4, but there was no significant difference in the multiple comparison test with week 0 (Fig. 6A). Increased expression of type III collagen in culture fibroblasts had been suggested to have the effect of suppressing wrinkle formation in human skin, but the effect of RSE was not clear. However, in the VAS questionnaire, the average score for wrinkles was 4.53 ± 0.39, but for those subjects in their 40s and 50s , the score decreased to 2.32 ± 0.24. It has been reported that wrinkles on the face are only slightly observed in women in their 20s and 30s, and increase in women in their 40s and 50s (Hayashi et al. , 1993). For the wrinkle score in VISIA, the average value for subjects in their 20s and 30s was 10.33 ± 1.76, and the average value for subjects in their 40s and 50s was 15.86 ± 1.66, which is a difference of more than 5 points. Therefore, we re-analyzed the wrinkle score of the skin image analysis of subjects in their 40s and 50s (total number 10) (Fig. 6B), and it was confirmed that the wrinkle score was significantly reduced to -2.82 ± 1.53 at 4 weeks. This indicates that RSE effectively improved wrinkles in higher-aged women. It is considered that the application of RSE promoted the production of type III collagen in skin parts with problems, repaired the support structure, and improved wrinkles. The effect was weaker in the subjects with fewer skin problems and those who were younger.