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.