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.