Hispidin-enriched S. sanghuang mycelia (GKSS) were grown under optimal conditions according to the previous study (20) to enhance the production of hispidin, which was quantified by high-performance liquid chromatography (HPLC) to be 3 mg/g. Following fermentation, the mycelial biomass is separated from the fluid media, dried, and ground to a powder. Proximate analysis of GKSS including crude protein, crude fat, crude fiber, ash, moisture, and carbohydrate were then examined according to the Association of Official Analytical Chemists methods (24). The energy value was calculated by multiplying proteins by 4, carbohydrates by 4, and fats by 9.

According to a previous study, ethanol was the best solvent for obtaining extracts rich in hispidin (25) and hence was selected as the extraction solvent for subsequent experiments. In brief, 50 g of GKSS powder was added to 1000 ml of absolute alcohol, extracted for 1 h in an ultrasonic bath, filtered through Whatman filter paper No. 4, and concentrated through a rotary evaporator (R-220; Büchi Labortechnik AG, Flawil, Switzerland). Dried ethanolic extract was then further reconstituted in ethanol to obtain a final concentration of 100 mg/mL and filtered through a sterilized 0.22 μm syringe before exposure to the animals.

Hispidin was isolated according to a previous study with slight modification (26). Solvents such as methanol, formic acid, acetonitrile, and n-hexane used for extraction and chromatographic separation were analytical grade (Merck, Darmstadt, Germany). In brief, the ethanolic extract was further partitioned with Hexane −90% MeOH (2:1) to afford a hexane and a 90% MeOH layer. The hexane layer was re-extract with hexane, vortex and centrifuge at 3,600 × g for 5 min. After the upper layer was discarded, the extraction steps were repeated two additional times and concentrated through a rotary evaporator to afford hispidin. Ultra-Performance Liquid Chromatography (UPLC) analysis of hispidin was executed with a Kinetex Core-Shell C₁₈ analytical column (3 × 100 mm, particle size 1.7 μm; Phenomenex, CA, USA) on an Agilent 1,290 system (Agilent Technologies, Santa Clara, CA, USA). Separation was performed at 40 °C with a mobile phase gradient of 0.1 % formic acid (A) and acetonitrile (B). The gradient elution had the following profile: 0–10 min, 15–40 % (B); 10–10.1 min, 40–100 % (B); 10.1–12 min, 100–100 % (B); 12–12.5 min, 100–15% (B); 12.5–15 min, 15–15 % (B). The retention time of hispidin was 3.36 min at a flow rate of 1.0 mL/min with a scanning UV wavelength at 380 nm.

Sprague Dawley rats procured from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan) were acclimated and quarantined for 1 week prior to the initiation of the study. The animals were housed in pairs in polypropylene cages in a well-ventilated room (10–15 air changes/h) under an ambient temperature of 22 ± 3°C and 55 ± 15% relative humidity, with a 12:12 h light regime (lights on at 6:00 AM, lights off at 6:00 PM). Standard rodent diet (MGF; Oriental Yeast Co., Ltd., Tokyo, Japan) and purified water were available ad libitum.

This animal study was implemented in accordance with guidelines of the Organization for Economic Cooperation and Development Guideline 407 “Repeated Dose 28-day Oral Toxicity Study in Rodents” (27) and approved by the Institutional Animal Care and Use Committee (IACUC No. 102-9n). As our earlier study on acute oral toxicity indicated that acute oral LD₅₀ of GKSS was greater than 12 g/kg in Sprague-Dawley rats (20), three treatment doses of 1 g/kg (low dose), 2 g/kg (mid-dose) and 5 g/kg (high-dose) were thus selected for this study. After 80 healthy 5 weeks-old rats of both genders with an average body weight ranging from 180 to 265 g had their health status evaluated, they were randomly assigned into one vehicle control group and three treatment groups. GKSS was orally administered by gavage. Throughout the experiment, signs of toxicity, mortality and morbidity were monitored daily. Detailed clinical signs including changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activities were recorded. The body weight and feed intake were recorded weekly while ophthalmological examination was performed prior to initiation of dosing and before sacrifice. Before the scheduled sacrifice, urine samples were collected from surviving rats for 16 h and the following parameters were evaluated with a urine analyzer (PU-4010, ARKRAY, Japan): specific gravity (SG), color, protein, urobilinogen, pH, ketone, bilirubin, glucose, nitrite, and occult blood. Urine samples were then centrifuged to obtain sediments, which were examined microscopically for the presence of white blood cell (WBC), red blood cell (RBC), epithelial cell (EP), crystals, and microbes.

At the end of the stipulated experimental period, rats were fasted overnight and anesthetized by inhalation of carbon dioxide. Blood was drawn from the abdominal aorta into EDTA-containing and non-anticoagulant-containing collection tubes for hematological and biochemistry analyses, respectively. Hematology was measured using an automatic blood analyzer (Sysmex CA-1500, Kobe, Japan) with the following parameters including RBC, WBC, hematocrit, hemoglobin, platelet count, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), lymphocyte, neutrophil, monocyte, eosinophil, basophil and prothrombin time (PT). On the other hand, serum biochemistry parameters such as alkaline phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, total protein, total bilirubin, creatinine, blood urea nitrogen (BUN), glucose, cholesterol, triglyceride, phosphorus, calcium, chloride, potassium and sodium, were examined using an automated biochemistry analyzer (7070, Hitachi Ltd., Tokyo, Japan).

After sacrifice, a complete gross necropsy including examination of external surfaces, orifices, cranial cavities, carcass, and organs was performed. Selected organs such as brain, heart, kidneys, livers, spleen, adrenal gland and ovaries/testes were isolated, weighed, fixed and preserved in 10% neutral buffered formalin for histology. 2 μm paraffin wax tissue sections from fixed organs were then stained with hematoxylin-eosin and examined with a microscope (BX51, Olympus, Tokyo, Japan) to detect any lesions.

The protocol for sedative activity was approved by the Institutional Animal Care and Use Committee (IACUC approval: NTU107-EL-00182) of National Taiwan University. Following acclimatization, eight-week-old male Sprague Dawley rats weighing 250–300 g were anesthetized with Zoletil (50 mg/kg; Virbac, Carros, France) and xylazine (14.8 mg/kg; Sigma-Aldrich, MO, USA) before being surgically implanted with electroencephalography (EEG) and electromyography (EMG) electrodes as well as a microinjection cannula for polysomnographic recordings. Precisely, two and one EEG screw electrodes were implanted in the frontal and parietal lobes of the right hemisphere and the occipital lobe of the left hemisphere, respectively while two EMG electrodes were implanted in the neck muscle. Once the signals from the EEG and EMG electrodes had been detected, they were fed into an amplifier (Colbourn Instruments, Lehigh Valley, PA, USA; model V75-01), filtered from 0.1 and 40 Hz and digitized (NI PCI-6033E; National Instruments, Austin, TX, USA) at 128 Hz. Following 7 days of postsurgical recovery, rats were randomly assigned one control and two treatment groups, each comprising six rats. Control group (5.5% ethanol) and two doses (75 and 150 mg/kg; suspended in 5.5% ethanol) of GKSS ethanolic extract were administered orally 20 min before the onset of the dark period. To easily observe the hypnotic effect, the substance was administered prior to the dark period as rats are generally active during the dark period and somnolent during the light period. The sleep-wake activity patterns were then recorded consecutively for 24 h in 12-s epochs to determine different sleep stages (wakefulness, non-REM (NREM) sleep, REM sleep) based on previously defined criteria (28).

Cells were transfected with Nrf2 pathway responsive gene, antioxidant-responsive element (ARE) pGL3 vector and internal control pRL-TK (Renilla luciferase) plasmids according to our previous study (29). In brief, stable ARE-driven luciferase reporter cells HSC3-ARE9 were cultured in MEM (GibcoBRL, NY, USA), supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, penicillin (100 U/mL), streptomycin (100 μg/mL), and 2 mM L-glutamine, at 37°C in a humidified incubator containing 5% CO₂. For cell viability assay, 2.5 × 10⁴ cells in 100 μL of culturing medium were seeded onto 96-well plates for 24 h before treatments. Cells were then treated with appropriated concentrations (12.5, 25, 50, and 100 μg/mL) of hispidin and GKSS ethanolic extract (37.5, 75, 150, and 300 μg/mL) for 24 h. 20 μL of MTT (5 mg/mL) was added into each sample and incubated for 4 h, under 5% CO₂ and 37°C. 200 μL of DMSO was subsequently added into each sample and gently shaked for 10 min at room temperature. Absorbance was measured at 560 nm using a Multiskan™ GO Microplate Spectrophotometer (Thermo Scientific, MA, USA). For luciferase activity assay, HSC3-ARE9 cells at a density of 2.5 × 10⁴ per well were seeded in 96-well plates and incubated at 37°C for 16 h. Cells were treated with various concentrations of hispidin, GKSS ethanolic extract and tert-butylhydroquinone (t-BHQ, #112941, Sigma, MO, USA; positive control) for 24 h, and then cell lysates were prepared for assessment of luciferase activity. Luciferase activities were quantified using Steady-Glo® Luciferase Assay System (Promega, WI, USA), according to the manufacturer's protocol.

RAW 264.7 macrophages (ATCC TIB-71™, VA, USA) were grown at a density of 2.5 × 10⁵ cells/mL in a 96-well plate, pretreated with various doses of isolated hispidin (50, 75, and 100 μg/mL) and GKSS (75, 150 and 300 μg/mL) for 1 h before stimulated with 100 ng/mL of LPS for 24 h. After incubation, the cells were centrifuged and the supernatant was harvested. Cell viability was examined using MTT assay. The interleukin-6 (#88-7064-88, Thermo Fisher Scientific, MA, USA) and tumor necrosis factor-alpha (#88-7324, Thermo Fisher Scientific, MA, USA) concentrations in cell culture supernatants were then measured via ELISA kit according to the manufacture's protocol. As nitric oxide (NO) is unstable, the quantitative of NO was indirectly determined through the measurement of nitrite (NO₂) levels. Briefly, 100 μL of supernatant was mixed with an equal volume of Griess reagent for 10 min and the absorbance at 550 nm was measured using a microplate spectrophotometer (Bio-RAD Laboratories, CA, USA). The amount of NO concentration was then calculated by referring to a sodium nitrite standard curve.

In the 28-day oral toxicity study and Nrf2 luciferase reporter assay, data are given as mean ± standard deviation (SD). For 28-day oral toxicity study, data were analyzed using a one-way analysis of variance (ANOVA) method, followed by Duncan's multiple range test (SPSS, version 12.0, Illinois, Chicago, USA). For Nrf2 luciferase reporter assay and anti-inflammatory activities, one-way ANOVA was performed, followed by pairwise Tukey test. For the sedative activity study, data are given as mean ± standard error of the mean (SEM), respectively and were analyzed using ANOVA. In all cases, p-value below 0.05 is considered statistically significant.

The proximate composition of 100 g GKSS powder is shown in Table 1. Parameters studied in the proximate analysis include moisture, ash, crude lipid, crude protein, carbohydrate, and energy, which were found to be 2.4 ± 0.3%, 8.0 ± 2.5%, 1.7 ± 0.3%, 22.9 ± 1.2%, 65.1 ± 3.1%, and 367.1 ± 10.2 kcal/100 g respectively.

After ingesting GKSS for 28 days, all the animals were in good condition and showed no symptoms of toxicity, morbidity or mortality in both sexes of rats (Supplementary Table 1). There were no abnormal urinalysis findings in any of the animals (data not shown). Except for male rats in the high dose group, which consumed significantly less (25.5 ± 1.8) than the control group (28.3 ± 1.1) during the third week of the study (p < 0.05), GKSS did not cause significant changes in the body weight and feed intake during the observation period as depicted in Figure 1.

Blood hematology values in male and female rats are shown in Table 2. There were statistically significant decreases (p < 0.05) in the absolute value of platelet counts in the mid- and high-dose test male groups when compared to the control group (Table 2). However, no statistically significant alteration was found in other parameters investigated.

Table 3 shows the mean biochemical data in male and female rats across the group. In low-dose treated males, GKSS significantly decreased the serum sodium and chloride values (p < 0.05) when compared to the control group. In addition, there was a significant decrease (p < 0.05) in serum cholesterol for female groups treated with 2 g/kg of GKSS. No significant alterations were observed, however, in other male or female rat biochemical parameters.

As shown in Table 4, no significant differences in the absolute organ and relative organ weights were noted between the treatment and control groups in both male and female rats throughout the study period. Furthermore, no abnormal gross findings were identified in any of the animals at necropsy (data not shown).

To evaluate the effects of GKSS ethanolic extract on spontaneous sleep-wake activity, two doses, 75 mg/kg and 150 mg/kg, were administered 20 min prior to the dark period of the 12:12h L:D cycle. Results indicated that 75 mg/kg of GKSS ethanolic extract does not affect wakefulness, NREM sleep, and REM sleep during both the dark period and the light period (Figure 2A). However, high dose GKSS ethanolic extract (150 mg/kg) significantly enhanced NREM sleep during hours 13–24 during the 12-h light period (p < 0.05; Figure 2A). Moreover, REM sleep was also increased after administration of 150 mg/kg of GKSS ethanolic extract during hours 13–21 of the light period (p < 0.05; Figure 2A). Alternatively, the awake was mirrored in response to the summation of NREM sleep and REM sleep (p < 0.05; Figure 2A). The percentage of time spent in awake decreased from 43.6 ± 3.6% obtained from vehicle to 27.4 ± 2.8% during hours 13–24 (p < 0.05; Figure 2B). Likewise, the percentage of time spent in NREM and REM sleep increased from 42.8 ± 2.8% obtained from vehicle to 53.5 ± 2.4% during hours 13–24 and from 13.7 ± 1.2% obtained after vehicle to 19.1 ± 1.3% during hours 13–24, respectively (p < 0.05; Figure 2B).

Hispidin was first determined by comparison of UPLC retention time with that of the authentic compound purchased from Sigma (H5257, Sigma, MO, USA) as shown in Figure 3. Various concentrations of hispidin (12.5, 25, 50, and 100 μg/mL) and GKSS (37.5, 75, 150, and 300 μg/mL) were applied to the ARE-luciferase reporter system to investigate their Nrf2/ARE activation abilities (Figure 4). Parallel cell viability assays revealed no obvious cytotoxic effects from these treatments. After the cells were treated with test samples at the indicated concentration for 24 h, both hispidin and GKSS significantly induced ARE-luciferase activity in a dose-dependent manner (p < 0.05). Hispidin and GKSS treatment can reach as high as 4.23 and 3.02-fold increases in ARE-driven luciferase activity, respectively, suggesting that they are potential Nrf2 activators, with effects even greater than that of positive control t-BHQ (1.35 ± 0.07 and 1.71 ± 0.14 at 25 and 50 μM, respectively).

For hispidin concentrations ranging from 25–100 μg/mL and GKSS concentrations ranging from 75–300 μg/mL, RAW267.4 cell viability was not significantly changed after 24 h treatment with 100 ng/mL LPS compared to control group cells (data not shown). To confirm the effect of hispidin and GKSS on IL-6 (Figure 5A) and TNF-α (Figure 5B) production in LPS-induced RAW 267.4 cells, enzyme-linked immunosorbent assays (ELISA) were applied. LPS treatment significantly increased (p < 0.05) the IL-6 and TNF-α levels by 202.4- and 5.2-fold, respectively. However, the LPS-induced increases were dose-dependently reversed by hispidin and GKSS treatments. Results showed that 50–100 μg/mL hispidin and 150–300 μg/mL GKSS significantly suppressed the production of IL-6 while 100 μg/mL hispidin and 300 μg/mL GKSS significantly suppressed the release of TNF-α when compared to LPS-stimulated control cells. Next, the effects of hispidin and GKSS on NO production in the supernatant of LPS-treated RAW 264.7 cells were measured via Griess reagent assay. Treatment with hispidin and GKSS increased NO production in a dose-dependent manner, suggesting their color may potentially interfere with the detecting assays (Supplementary Figure 1). Despite color interference, both hispidin at 25–100 μg/mL and GKSS at 75–300 μg/mL significantly inhibited NO production in RAW 264.7 cells stimulated with LPS (p < 0.05; Figure 5C).