Fruiting bodies of A. confluens and A. dispansus were collected in Japan (Mie, Tottori, Okayama, and Hiroshima Prefectures, 2019) and authenticated by mycologists. Additional mushroom specimens (212 species) collected in Hokkaido were cleaned, authenticated, and deposited in the Natural Product Library of Hokkaido University. Samples were extracted with organic solvent, concentrated under reduced pressure, and stored at −20 °C. Crude extracts were dissolved in DMSO (10 mg/mL) for screening, resulting in a mushroom extract library of 212 species.

Mouse embryonic fibroblasts lacking SMS1 and SMS2 (ZS2), and stable ZS2 lines expressing SMS1 or SMS2, were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C in a 5% CO₂ incubator. Human hepatoma HepG2 cells were used for cytotoxicity and lipid uptake assays.

SMS activity was measured in cell lysates (0.1 μg/μL protein). Lysates were preincubated with test compounds at 37 °C for 30 min, followed by addition of C6-NBD-ceramide (NBD-Cer; N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]sphingosine, 5 μM) and C6-NBD-phosphatidylcholine (NBD-PC; 1-acyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl]-sn-glycero-3-phosphocholine, 5 μM) as substrates. The reaction converts ceramide (Cer) and phosphatidylcholine (PC) into sphingomyelin (SM) and diacylglycerol (DAG). Reactions were terminated with methanol/chloroform (1:2, v/v). The fluorescent products C6-NBD–diacylglycerol (C6-NBD-DAG) and C6-NBD–sphingomyelin (C6-NBD-SM) were separated and quantified by HPLC with fluorescence detection (λ_ex = 470 nm, λ_em = 530 nm). Assay linearity was verified with respect to both protein concentration and reaction time. Enzyme activity was calculated from product formation relative to control samples without inhibitor.

Cell viability was determined by the CCK-8 assay after 24 h incubation with 0–20 μM grifolin or grifolic acid. For lipid droplet accumulation experiments, HepG2 cells were treated with 1 mM oleic acid in the presence or absence of 20 μM compounds for 24 h. Cells were stained with Nile Red (λ_ex = 545 nm, λ_em = 605 nm) to visualize neutral lipids and with DAPI (λ_ex = 360 nm, λ_em = 460 nm) to label nuclei. Images were acquired using a Keyence BZ-X70 fluorescence microscope (Plan Apochromat 40x objective, fixed exposure times) and analyzed in ImageJ. Lipid droplet content was quantified by Otsu thresholding, expressed as Nile Red fluorescence intensity normalized to DAPI counts, yielding lipid content per cell in arbitrary units (AU/cell). Data are presented from three independent experiments, each including multiple fields of view, and statistical significance was assessed by one-way ANOVA with Tukey’s post hoc test. Fatty acid uptake assays such as BODIPY-C16 transport were not performed in this study, and our lipid accumulation measurements therefore reflect neutral lipid storage but not uptake kinetics.

The acetone extract of A. confluens fruiting bodies (4.96 kg fresh weight) exhibited strong SMS inhibitory activity. Sequential liquid–liquid extraction with hexane, diethyl ether, and ethyl acetate was followed by silica gel chromatography. The ether fraction showed the greatest activity and yielded two phenolic compounds, grifolin (10.4 g) and grifolic acid (5.8 g). Structural identities were confirmed by NMR spectroscopy and high-resolution mass spectrometry, consistent with reported values.

All animal experiments were approved by the Animal Research Committee of Hokkaido University and performed in accordance with institutional guidelines. Male C57BL/6J mice (5 weeks old, Japan SLC Inc., Shizuoka, Japan) were housed under controlled environmental conditions (24 °C, 50% ± 10% humidity, 12 h light/dark cycle) with ad libitum access to food and water. After a one-week acclimatization period, animals were randomly assigned to four groups (n = 6 per group):

Grifolin and grifolic acid were incorporated directly into the diet (AIN-93G, Oriental Yeast Co, Tokyo, Japan) and provided as oral dietary supplementation for 9 weeks. Body weight and food intake were monitored weekly.

Glucose tolerance was assessed after overnight fasting by oral gavage of glucose (1 g/kg body weight), and blood glucose was measured at 0, 30, 60, and 120 min. At study termination blood was collected by cardiac puncture under 2% isoflurane inhalation anesthesia.² Liver and kidney tissues were excised, snap-frozen in liquid nitrogen, and stored at −80 °C for molecular analyses or processed for histological evaluation (hematoxylin and eosin, Oil Red O staining). Hepatic triglyceride content was measured enzymatically. Lipidomic profiling of ceramide and sphingomyelin species was conducted by Liquid chromatography–tandem mass spectrometry (LC–MS/MS). Serum 25(OH)D₃ levels were quantified using a commercial enzyme-linked immunosorbent assay (ELISA) kit, and hepatic and renal expression of CYP24A1, CYP27B1, and CYP2R1 was determined by quantitative reverse transcription PCR (RT–PCR).

Randomization was applied at group assignment, and investigators performing histological and biochemical analyses were blinded to treatment groups.

All experiments were performed at least in triplicate unless otherwise specified. Data are presented as mean ± standard deviation (SD). Statistical comparisons were performed using Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test (GraphPad Prism 9). Differences were considered significant at p < 0.05.

To identify SMS inhibitors, we screened methanol and acetone extracts of the fruit bodies, leaves, stems, and roots of approximately 650 medicinal plants and 212 mushrooms from the Hokkaido University natural product library using a cell-based SMS assay (Supplementary Figures 1A,B). The full list of species screened is provided in Supplementary Table S2. We found that the fruiting body extracts of A. dispansus and A. confluens showed marked inhibitory activity against SMS (Supplementary Figures 1A,B). A. dispansus and A. confluens are edible and have been used as natural medicines, functional foods, and dietary supplements in China for thousands of years. Bioassay-guided fractionation of the A. confluens extract resulted in the identification of grifolin and grifolic acid as inhibitor compounds (Figure 1A). Each was confirmed by NMR spectroscopy, HRMS, and spectroscopic data, similar to previous reports (Supplementary Figure 1D). The dose-dependent inhibitory effects of grifolin and grifolic acid on SMS1 and SMS2 were validated using a cell-based assay, with both compounds exhibiting substantial inhibition across isoforms (Figure 1B).

Grifolin and grifolic acid were identified as major components of Albatrellus species. These compounds exhibited strong inhibitory activity against SMS using both PC and Cer substrates. SMS inhibitors were identified medicinal mushroom and plant extracts library of Hokkaido university, screening assays by using both fluorescence substrates, C6-NBD (N-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl])-Cer and C6-NBD-PC. The inhibitory activities of the extraction were quantified as follows: against the C6-NBD-PC substrate, both SMS1 and SMS2 showed inhibition at 4.5 μg/mL; against the C6-NBD-Cer substrate, SMS1 was inhibited at 0.5 μg/mL, and SMS2 at 0.3 μg/mL. The inhibitory effects of grifolin and grifolic acid are among the strongest (Supplementary Table 1) natural SMS inhibitors reported to date, targeting both substrates (21–23). The half maximal inhibitory concentration (IC₅₀) was defined as the compound concentration required to reduce enzyme activity by 50%. The estimated IC₅₀ values for grifolin were: 15.2 μM (SMS1, C6-NBD-PC), 18.3 μM (SMS2, C6-NBD-PC), 0.6 μM (SMS1, C6-NBD-Cer), and 3.0 μM (SMS2, C6-NBD-Cer). For grifolic acid, the IC₅₀ values were 3.0 μM (SMS1, C6-NBD-PC), 4.0 μM (SMS2, C6-NBD-PC), 3.0 μM (SMS1, C6-NBD-Cer), and 2.5 μM (SMS2, C6-NBD-Cer) (Table 1).

We examined cytotoxicity in human hepatoma HepG2 cells. Neither compound showed significant cytotoxicity up to 20 μM (Figure 2A). SMS-deficient cells and mice have previously been reported to exhibit impaired fatty acid uptake and lipid droplet formation (16, 24). To investigate whether the identified SMS inhibitors affected lipid accumulation, we assessed lipid droplet formation in HepG2 cells following treatment with oleic acid (OA). Lipid droplets, which were stained with Nile Red, were remarkably reduced by both grifolin and grifolic acid treatment in a dose-dependent manner (grifolin treated 43% and grifolic acid treated 48% of lipid reduction at 20 μM, Figures 2B,C). Building on a previous report that oral administration of grifolin at 2,000 mg/kg was safe in mammals (25), we conducted an in vivo study using a high-fat diet-induced obesity mouse model.

Oral dietary supplementation with grifolin or grifolic acid (0.1% in HFD, corresponding to ~100 mg/kg/day) for 9 weeks markedly reduced body weight gain in male C57BL/6J mice (n = 6 per group). The mean final body weights were: ND – 27.0 g, HFD – 31.2 g, HFD + grifolin – 26.7 g, and HFD + grifolic acid – 25.8 g. When compared to the HFD group, this corresponds to a relative reduction of 14.6% for grifolin and 17.4% for grifolic acid (Figures 3A,C; representative images in Figure 3B).

Feed intake was recorded weekly and differed significantly among groups (Supplementary Figure 2). HFD-fed mice consumed significantly less food (3.02 ± 0.54 g/day) compared with HFD + Gri (4.68 ± 1.24 g/day, p < 0.05) and HFD + GA (4.82 ± 1.06 g/day, p < 0.05), while intake in the treated groups did not differ significantly from ND controls (4.55 ± 0.8 g/day). Thus, the reduced body weight observed in the treated groups was not attributable to reduced caloric intake but rather to the metabolic effects of grifolin and grifolic acid.

We investigated the effects of these inhibitors on glucose tolerance, hepatic TG levels, and lipid droplet accumulation. As expected, after 9 weeks an oral glucose tolerance test (1 g/kg glucose by gavage following overnight fasting) showed that both grifolin- and grifolic acid–treated mice exhibited significantly improved glucose tolerance compared to HFD-group (Figure 3D).

In agreement with the results obtained in HepG2 cells, the histological study of the Oil Red O staining demonstrated that the treatment with grifolin and grifolic acid prevented lipid accumulation in the liver of HFD-fed mice (Figure 4A). Conversely, the untreated HFD-fed mice showed substantial lipid droplet accumulation, indicative of liver steatosis. In contrast, the treated groups showed improved lipid metabolism and minimal lipid deposition in the liver (Figure 4A). Hepatic triglyceride (TG levels in the HFD group were 30.5 and 38.4% higher than those in the grifolin-treated and grifolic acid-treated groups, respectively Figure 4B).

Subsequently, we measured SM levels in liver tissues collected from control and grifolin-treated HFD mice. As expected, in the liver tissues of grifolin-treated mice, SM levels were significantly reduced, suggesting suppressed SMS activity (Supplementary Figure 3A). Consistent with the SM measurement results, analysis of SMS activity in liver tissues from control and grifolin-treated HFD mice showed that DAG and SM synthesized from PC and Cer were significantly reduced in the grifolin-treated group (Supplementary Figures 3B,C). Grifolic acid treatment also showed a trend toward reduced DAG and SM levels, although the differences were not statistically significant. Taken together, these results demonstrate that grifolin and grifolic acid suppress weight gain, prevent hepatic lipid droplet formation, and improve glucose tolerance, likely by suppressing SMS activity in HFD-induced obese mice.

We selected three genes based on a previous study and performed qRT-PCR on mRNA extracted from liver and kidney tissues of DIO mice after 9 weeks of treatment. The selected CYP24A1, CYP27B1, and CYP2R1 genes showed involvement in 25(OH)D₃ regulation. CYP27B1 is involved in the synthesis of active vitamin D, whereas CYP24A1 and CYP2R1 are involved in its catabolism. Serum 25(OH)D₃ concentration significantly decreased in the HFD-fed group. In contrast, 25(OH)D₃ levels were approximately 2.5-fold higher in the grifolin-fed group (p < 0.05) and approximately 6.3-fold higher in the grifolic acid-fed group (p < 0.001) than in the HFD-fed group (Figure 5A). The mRNA expression of CYP24A1 was significantly upregulated in the HFD-fed group (Figure 5B). Conversely, no significant differences in the CYP27B1 and CYP2R1 expression levels were observed between the groups (Figures 5C,D). These findings suggest that SMS inhibition by grifolin and grifolic acid may downregulate CYP24A1 expression, thereby reducing the active vitamin D catabolism and stabilizing the physiological levels of active 25(OH)D₃.