The human rights of individuals participating in this study were protected throughout the study. The study was conducted in accordance with the Declaration of Helsinki and the Japanese Ethical Guidelines for Medical and Health Research Involving Human Subjects. Written informed consent was obtained from all participants. The study protocol was approved by the clinical trial ethics review committee of Chiyoda Paramedical Care Clinic (approval date: 21 October 2022) and is publicly registered at UMIN-CTR (trial number: UMIN000049702).

Among 192 healthy women who completed baseline measurements, 80 participants were selected based on the following inclusion criteria: (i) healthy women aged 20 to 64 years at the time of providing informed consent; (ii) individuals aware of dry or rough skin; and (iii) individuals who understood the study procedures and provided written informed consent to participate prior to the study. The exclusion criteria were as follows: (i) consumption of foods for specified health uses, foods with functional claims, supplements, and/or health foods that could affect skin condition more than 2 times per week; (ii) consumption of drugs or quasi-drugs that could affect skin condition more than 2 times per week; (iii) excessive sunburn or risk of excessive sunburn (e.g., due to travel or events); (iv) plans to change cosmetics; (v) plans to begin a new skin care routine; (vi) skin diseases (e.g., atopic dermatitis); (vii) presence of a bruise and/or scar near measurement sites; (viii) use of specific treatments near measurement sites (e.g., electrical facial treatment, peeling, or laser therapy); (ix) excessive alcohol consumption; (x) consumption of mushrooms more than 4 days per week; (xi) consumption of liver more than 4 days per week; (xii) dislike of mushrooms; (xiii) rough skin caused by pollinosis; (xiv) aware of menstruation-related skin deterioration; (xv) irregular life style (e.g., night shift work); (xvi) enrollment in other clinical trials involving medicine or health foods from the start of this trial until 4 weeks after its completion; (xvii) plans to change the shape of eyelashes or eyebrows; (xviii) past/current medical history of severe cardiac, hepatic, renal, or digestive diseases; (xix) pregnancy, lactation, or plans to become pregnant; (xx) risk of medicine/food allergy; (xxi) donation of whole blood and/or blood components (200 mL) within 1 month prior to this trial; (xxii) donation of whole blood (400 mL) within 4 months prior to this trial; (xxiii) blood collection (800 mL) within the past 12 months, including this trial; and (xxiv) ineligibility for the study, as determined by the investigator.

This randomized, double-blind, placebo-controlled, parallel-group trial enrolled Japanese individuals at Chiyoda Paramedical Care Clinic (Tokyo, Japan) from December 2022 to April 2023. To our knowledge, there are no available data regarding the distribution of EGT in the skin after oral ingestion, although daily EGT administration for 1 week led to maximal EGT concentrations in whole blood approximately 4 weeks later in humans (13). Therefore, we hypothesized that daily ingestion of EGT would also lead to a gradual increase in EGT concentrations within the skin. Based on previous data regarding skin turnover rates (20), we planned to measure skin parameters at baseline, 8 weeks, and 12 weeks during the dietary intervention period. Additionally, we collected blood samples at baseline and 12 weeks. The primary outcome measured was skin moisturizing function (skin moisture content and transepidermal water loss (TEWL) on the left temple and arm). Facial conditions were analyzed as the secondary efficacy outcome. The sample size was determined based on previous studies that identified significant differences in skin moisturizing functions after oral supplementation (21, 22). Eighty participants were randomly assigned to two groups according to age, skin moisture content, and TEWL. Randomization was performed in a confidential manner by the participant assignment manager. Group assignments were disclosed to the participants, investigators, and analysts after data fixation.

Hiratake tablets were used as the test food. One tablet of the test food was 240 mg, and each participant ingested 21 tablets daily (25 mg of EGT/day). In the placebo tablets, hiratake powder was replaced by glucose and caramel. Fresh fruiting bodies of hiratake were cultivated by Hokuto Corporation (Nagano, Japan), then processed into dried powder. The compositions and nutrient contents of the test and placebo foods are shown in Supplementary Tables 1A, B.

At baseline, 8 weeks, and 12 weeks, participants were instructed to wash their faces and hands and to remain in a room at a temperature of 21 ± 1°C with a humidity level of 50 ± 5% for at least 30 min. After this acclimation period, skin moisturizing function was assessed on the left temple and on the left forearm. Skin moisture content was measured five times using a Corneometer CM825 (Courage + Khazaka Electronic GmbH, Cologne, Germany); after exclusion of the maximum and minimum values, the mean of the remaining values was calculated. TEWL from skin was measured with a Tewameter TM300 (Courage + Khazaka Electronic GmbH, Cologne, Germany). Facial scores regarding wrinkles, texture, spots, UV spots, and pores were measured using the VISIA Evolution and its internal software (Canfield Inc., Fairfield, NJ, USA).

Blood samples were mixed with heparin sodium and centrifuged to separate the plasma. Each plasma sample was then mixed with eight volumes (v/v) of acetonitrile and an equal volume of 10 μM d9-ergothioneine (d₉-EGT; internal standard) in water. These mixtures were centrifuged, and the supernatants were analyzed using a triple quadruple mass spectrometer with electrospray ionization coupled to a liquid chromatography system (LCMS-8040; Shimadzu, Kyoto, Japan). The EGT standard and d₉-EGT were provided by Tetrahedron (Paris, France). Chromatography was performed via step-gradient elution (10 mM ammonium acetate, 0.1% formic acid in 5% H₂O and 95% acetonitrile in the initial step; 10 mM ammonium acetate, 0.1% formic acid in 20% H₂O and 80% acetonitrile in the final step) on an ACQUITY UPLC BEH Amide Column (130 Å, 1.7 μm, 2.1 × 150 mm; Waters Corporation, Milford, MA, USA) at 40°C. The multiple reaction monitoring settings were 230.00 to 127.10 m/z for EGT and 239.15 to 127.00 m/z for d₉-EGT.

Within-group changes from baseline were assessed using the Wilcoxon signed-rank test with Bonferroni correction. Between-group differences at each time point were evaluated using the Wilcoxon rank-sum test. Correlations were evaluated by Pearson correlation analysis. Statistical analyses were conducted using Microsoft Excel 2021 (Microsoft, Redmond, WA, USA) and IBM SPSS Statistics version 26 (IBM, Armonk, NY, USA). Differences with p-values < 0.05 were considered statistically significant.

The flow diagram for the trial is presented in Figure 1. Table 1 shows the participants’ baseline characteristics. Overall, 77 participants (39 in the hiratake group and 38 in the placebo group) were included in the analysis. No participants withdrew from the study due to adverse effects related to the test foods.

The effects of EGT-rich mushrooms on skin moisturizing functions were assessed by measuring the skin moisture content and TEWL on the left temple and arm. On the temple at 8 weeks, skin moisture content was significantly higher in the hiratake group than in the placebo group (Figure 2A). In the hiratake group, skin moisture content tended to increase (Figure 2A, p = 0.099) and TEWL tended to decrease (Figure 2C, p = 0.083) on the temple at 8 weeks compared with baseline. Furthermore, significant improvements in skin moisture content on the arm were observed in the hiratake group at both 8 and 12 weeks compared with baseline (Figure 2B), whereas no significant difference was observed in the placebo group. Although there was no significant difference in TEWL between the groups, a significant time-dependent decrease in TEWL on the arm was observed in both groups (Figure 2D).

Facial scores regarding wrinkles, texture, spots, UV spots, and pores were measured using the VISIA skin analysis system (Table 2). At 12 weeks, wrinkle and texture scores were significantly lower in the hiratake group than in the placebo group, suggesting improvements in wrinkles and texture upon ingestion of hiratake (Table 2). Additionally, the hiratake group exhibited significantly lower UV spot scores at 8 weeks than those at baseline (Table 2). The placebo group demonstrated significantly higher pore scores at 8 weeks and texture scores at 12 weeks than those scores at baseline, whereas the hiratake group did not exhibit significant changes in these scores (Table 2).

There was no significant difference in the plasma EGT concentration at baseline between the two groups, and the mean concentration across all participants was 3.3 μM (Figure 3). At 12 weeks, the plasma EGT concentration was significantly higher in the hiratake group than in the placebo group, and a significant increase (from 3.4 μM at baseline to 15.9 μM at 12 weeks) was observed in the hiratake group but not the placebo group (Figure 3).

To determine the contribution of EGT to the improvements in skin moisturizing functions during hiratake supplementation, we investigated correlations between skin parameters and plasma EGT concentrations by Pearson correlation analysis. Analyses of all data from both groups at baseline and 12 weeks revealed a significant positive correlation between skin moisture content on the arm and plasma EGT concentration (Figure 4B, r = 0.168, p = 0.038), whereas no significant correlation involving skin moisture content on the temple was observed (Figure 4A). Moreover, there was a significant negative correlation between TEWL on the arm and plasma EGT concentration (Figure 4D, r = −0.188, p = 0.019) and a tendency toward a negative correlation involving TEWL on the temple (Figure 4C, r = −0.137, p = 0.091).

To evaluate the effects of hiratake in participants with limited variability in baseline EGT concentrations, a stratified analysis was conducted using data from participants with plasma EGT concentrations below the mean level at baseline (< 3.3 μM). The characteristics of participants with a low baseline EGT are presented in Supplementary Table 2. The analysis included 19 and 25 participants in the hiratake and placebo groups, respectively. Skin moisture content on the temple at 8 and 12 weeks was significantly higher in the hiratake group than in the placebo group (Figure 5A). Additionally, skin moisture content on the arm in the hiratake group was significantly higher at 8 and 12 weeks than at baseline (Figure 5B). Finally, skin moisture content on the arm at 12 weeks tended to be higher (p = 0.074) in the hiratake group than in the placebo group (Figure 5B).

Throughout the trial, 24 mild adverse events were reported (11 and 13 in the hiratake and placebo groups, respectively). The principal investigator determined that none of these mild adverse events were related to the intake of either the hiratake or placebo tablets. No significant changes were observed in blood biochemistry analyses at 12 weeks (Supplementary Table 3). All participants who completed the study were included in the safety analysis set.