Experimental Procedures
Samples
The RSE used in this study was provided by Tsuno Food Industry Co., Ltd.
and was dissolved in DMSO before being added to the medium.
Cell culture
Hs68, a normal human fibroblast, was purchased from the JCRB cell bank.
The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM;
Fuji Film Wako Pure Chemical Corporation, Osaka, Japan) containing 10%
fetal bovine serum (FBS, Biosera, Nuaille, France) and 1% penicillin
streptomycin (Fuji Film Wako Pure Chemical Corporation, Osaka, Japan).
After 24 h pre-cultivation, the cells were suspended and moved on to a
24-well plate (WATOSON, Tokyo, Japan) with 5.0 × 104cells/well, and pre-cultured overnight at 37 °C under 5%
CO2 atmosphere.
mRNA expression level
The RSE was dissolved in DMEM containing 1% FBS to a final
concentration of 0.1 or 0.01% (w/v). Pre-cultured cells were treated
with the RSE medium and cultured under 5% CO2 at 37 ℃
for 24 h. Total RNA was extracted from the cells using ISOGEN II (Nippon
Gene, Tokyo, Japan). Total RNA dissolved in Milli-Q water was
fluorescently labeled using the Qbit RNA BR Assay kit (Thermo Fisher
Scientific, Tokyo, Japan), and RNA concentration was calculated using
Qbit (Thermo Fisher Scientific, Waltham, MA, USA). Each RNA solution was
adjusted to 50 μg/μL. Reverse transcription was performed in a thermal
cycler (Thermo Fisher Scientific, Waltham, MA, USA) using the Prime
Script RT Reagent kit (Takara Bio, Shiga, Japan) according to the
manufacturer’s instructions. The cDNA obtained was used as a template.
Real-time PCR was performed using the StepOne system (Thermo Fisher
Scientific, Waltham, MA, USA) with specific primers. Real-time PCR
detected the glyceraldehydes 3-phosphatedehydrogenase (GAPDH),
hyaluronan synthase 2 (HAS2), and type III collagen (COL3) genes using
the Power Up SYBR Green Master Mix (Takara Bio, Shiga, Japan). The
expression levels of the target genes were calculated by comparing with
that of the housekeeping gene GAPDH using the ΔΔCT method. Primer
sequences of HAS2, COL3, and GAPDH were as follows: HAS2,
F5ʹ-GTGGATTATGTACAGGTTTGTGA-3ʹ and R5ʹ-TCCAACCATGGGATCTTCTT-3ʹ; COL3,
F5ʹ-TGGTGCCCCTGGTCCTTGCT-3ʹ and R5ʹ-TACGGGGCAAAACCGCCAGC-3ʹ; and GAPDH,
F5ʹ-CTCCTGTTCGACAGTCAGCC-3ʹ and R5ʹ-TCGCCCCACTTGATTTTGGA-3ʹ (Seoet al., 2019, Tong et al ., 2019).
ELISA
RSE dissolved in DMSO was added to DMEM containing 10% FBS and the RSE
concentration was adjusted to 0.01% (w/v). The RSE medium was poured
onto pre-cultured cells, and the cells were cultured under 5%
CO2 at 37 ℃ for 48 h. The amounts of hyaluronic acid and
type III collagen in the treated culture supernatants were quantified
using enzyme-linked immunosorbent assay (ELISA). The examinations were
performed using ELISA kits for hyaluronic acid (HA) and collagen type
III (COL3) (Cloud-Clone, Houston, TX, USA), respectively. Specifically,
RSE-reacted media were added to the wells of a specific antibody-coated
96-well plate to capture hyaluronic acid and type III collagen. Then,
the trapped targets were reacted with a specific adsorbent bound to
western wasabi peroxidase.
The detection was performed by the colorimetric method using
tetra-hydroxy benzidine.
Clinical trial subjects
This study was conducted according to the guidelines of the Declaration
of Helsinki and the Ethical Guidelines for Medical and Health Research
Involving Human Subjects proposed by the Japan Ministry of Health,
Labor, and Welfare. All subjects provided written informed consent
before the commencement of the study, which was approved by the Ethics
Committees at Tsuno Food Industry Co., Ltd. (approval number: Rin
21-002). In addition, we provided sufficient explanation to test
participants during the briefing session. The participants in this study
confirmed the registration/selection criteria and did not violate the
exclusion criteria. In addition, we selected subjects who confirmed that
they were healthy, did not have skin diseases by questionnaire, and did
not correspond to the statistical outliers in the measurement items.
Test cream product
The composition of the creams used in this study is shown in Table 1.
The RSE cream contained 0.5% RSE, and the placebo cream contained the
same amount of water instead of RSE.
Clinical trial design
This clinical trial was performed
as a placebo-controlled, single-blind test. Application to half of the
face was continued for 12 wk. Each cream was applied twice daily in the
morning and evening. The subjects were assigned so that the group
applying the RSE-containing cream (RSE cream) on the right side and the
group applying the placebo cream on the right side were evenly
distributed.
Measurements
Measurements were performed every 4 wk after the start of the
application (4th, 8th, and 12th weeks). Before the measurement, the
subjects washed their faces and remained for 20 min or more in a room
with constant temperature and humidity (22 ± 2 °C, RH% = 40 ± 5). The
cheeks of the subjects were measured using a Cutometer DUAL MPA580
(Courage + Khazaka, Cologne, Germany) for skin viscoelasticity, and a
Tewameter TM Hex (Courage + Khazaka, Cologne, Germany) for transdermal
water evaporation (TEWL), which is one of the indicators of skin barrier
function. In addition, wrinkles and redness were analyzed using VISIA
evolution (Canfield Scientific, Parsippany, NJ, USA) for facial image
analysis. Subjects responded to a VAS questionnaire at the time of
measurement (Grant et al ., 1999).
Statistical processing
In this study, the values are shown as mean ± standard error, and only
the age of the subjects is shown as mean ± standard deviation. The
results of the cell experiments and the VAS questionnaire were analyzed
using Dunnett’s test, and if a risk rate of 5% or less occurred, it was
considered a significant difference. The clinical test results were
compared between before and after the application of test creams, and
were analyzed by Paired t-test. Cases with a risk rate of 5% or less
were determined to have a significant difference. MEPHAS, a statistical
analysis program for pharmaceutical data provided by the Center for
Genetic Information Experiments at Osaka University, was used.