Galgeun-tang (GGT) granules were obtained from Hanpoong Pharm and Foods Co., Ltd. (Jeonju, Republic of Korea) that followed Good Manufacturing Practice (GMP) procedures. GGT is composed of seven botanical drugs, all of which are listed in the Korean Pharmacopoeia (KP).

All botanical drugs were authenticated by the manufacturer using morphological identification and pharmacopoeial criteria, and confirmed by Prof. Park, Sun-dong, a pharmacognosist at Dept. of Formula Science in Korean Medicine Dongguk University. Voucher specimens (Specimen Nos. GGT-001–002) have been deposited in the Herbarium of Dongguk University, Republic of Korea.

The botanical drugs were identified as: P. montana var. lobata (Willd.) Sanjappa and Pradeep [Fabaceae; Puerariae Radix], E. sinica Stapf [Ephedraceae; Ephedrae Herba], Z. jujuba Mill [Rhamnaceae; Zizyphi Fructus], Neolitsea cassia (L.). Kosterm. [Lauraceae; Cinnamomi Ramulus], P. lactiflora Pall. [Paeoniaceae; Paeoniae Radix], G. uralensis Fisch. [Fabaceae; Glycyrrhizae Radix], and Z. officinale Roscoe [Zingiberaceae; Zingiberis Rhizoma] (Supplementary Table S1).

The combined botanical drugs were extracted with 10 volumes (v/w) of distilled water at 100 °C for 3 h. The aqueous extract was then filtered and concentrated under vacuum below 60 °C, followed by drying to yield a powdered extract. The extraction yield was approximately 23.2%, corresponding to a drug–extract ratio (DER) of approximately 4.5:1 (w/w).

Quantitative analysis of ephedrine, glycyrrhizic acid, paeoniflorin, and puerarin was performed by HPLC-UV at Hanpoong Pharm & Foods, with compound-specific analytical methods optimized for each metabolite (Supplementary Table S2). The HPLC–UV analysis was conducted as a targeted quality-control method based on pharmacopoeial marker metabolites to ensure batch consistency of the GMP-manufactured extract, rather than as a comprehensive metabolomic fingerprint.

Murine C2C12 myoblasts (American Type Culture Collection, ATCC, Manassas, VA, United States) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Welgene, Daegu, Republic of Korea) supplemented with 10% fetal bovine serum (Gibco, CA, United States) and 1% penicillin-streptomycin (Invitrogen, CA, United States) at 37 °C, 95% relative humidity, and 5% CO₂. For differentiation, cells were cultured in DMEM containing 2% horse serum (Invitrogen) for 4–6 days until ≥90% of myoblasts had formed myotubes (Kislinger et al., 2005).

C2C12 myotubes were seeded in 96-well plates (1 × 10⁵ cells/well) and treated with GGT (0.4–2.0 mg/mL) for 24 h. Cell viability was determined using the EZ-Cytox assay kit (Daeil Lab Service, Seoul, Republic of Korea) according to the manufacturer’s instructions. Absorbance was measured at 450 nm with a microplate reader (Tecan Spark^Ⓡ; Tecan Group Ltd., Männedorf, Switzerland). The viability of the control cells, in terms of absorbance, was set to 100%.

Glucose uptake was measured using 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-d-glucose (2-NBDG; Invitrogen), a fluorescent derivative of glucose. Differentiated C2C12 myotubes were incubated in DMEM containing 2.5 mM glucose for 2 h, then treated with metformin (0.75 mM, Sigma-Aldrich, MO, United States) or GGT (1.0 mg/mL) for 12 h. Cells were exposed to 2-NBDG (50 μg/mL, Invitrogen) for 1 h, washed twice with Dulbecco’s phosphate-buffered saline (DPBS; Welgene), and fluorescence was measured at 485/535 nm using a microplate reader (Tecan Spark^Ⓡ). Fluorescence images of C2C12 myotubes were examined under an epifluorescence optical microscope (BX53; Olympus, Tokyo, Japan) equipped with a digital camera device (DP73; Olympus) and a mercury lamp (U-RFL-T; Olympus). Images were captured using a ×10 objective lens. Image acquisition was performed using cellSens Standard software (version 1.7.1; Olympus) under identical exposure and gain settings for all experimental groups. The fluorescence intensity was quantified using ImageJ software (version 1.43; NIH, Bethesda, MD, United States).

Forty-eight male C57BL/6J mice (6 weeks old, 18 ± 1 g; Daehan Biolink Co., Ltd., Eumseong, Republic of Korea) were acclimatized for 2 weeks under a 12 h light/dark cycle at a constant temperature of 25 °C and humidity levels of 50%–60%. The animals were provided with free access to a standard chow diet (38057; Purina Korea Inc., Seoul, Republic of Korea) and water. After acclimatization under specific pathogen-free (SPF) conditions, mice were randomly assigned to six groups (n = 8 per group): normal diet (ND), high-fat diet (HFD), metformin (MET; 100 mg/kg), and GGT at low (GGT-L; 504 mg/kg), medium (GGT-M; 612 mg/kg), or high (GGT-H; 720 mg/kg) doses.

The high dose of GGT (720 mg/kg) was determined by converting the clinically prescribed human dose to a mouse-equivalent dose based on body surface area normalization. The conversion was performed according to the FDA-recommended dose translation guidelines (Nair et al., 2018), using a clinical human dose of 12 mg/kg/day. The medium and low doses were set at approximately 85% (612 mg/kg) and 70% (504 mg/kg) of the high dose, respectively, to evaluate dose-dependent effects within a clinically relevant range.

Oral administration of distilled water (vehicle), MET, or GGT was initiated immediately after group allocation and continued once daily by oral gavage for a total experimental period of 10 weeks. During the initial 2-week pretreatment phase, all mice were maintained on a chow diet to allow stabilization of drug exposure before dietary challenge. This was followed by an 8-week dietary intervention phase, during which mice in the ND group were fed a normal diet (10% kcal fat; D12450B, Research Diets, NJ, United States) with regular drinking water, whereas mice in the HFD and treatment groups were fed a high-fat diet (45% kcal fat; D12451, Research Diets) supplemented with 10% fructose in drinking water.

The combination of a high-fat diet with fructose-supplemented drinking water was employed to exacerbate insulin resistance and metabolic dysfunction, as this dietary paradigm more closely mimics Western dietary patterns and has been widely used to induce obesity, glucose intolerance, and hepatic steatosis in rodents (Lim et al., 2010; Softic et al., 2019; Radhakrishnan et al., 2021).

This experimental design allowed assessment of the preventive and modulatory effects of GGT on the development of diet-induced obesity and metabolic dysfunction under high-fat/high-fructose conditions. Body weight was recorded weekly, and food intake was measured twice per week.

Following a 16 h overnight fast, mice were administered a sterilized glucose solution (2 g/kg; Sigma-Aldrich) via oral gavage. Blood glucose levels were measured from the tail vein at 0, 30, 60, 90, and 120 min using a handheld glucometer (Accu-Chek Active; Roche Diagnostics, Switzerland). The area under the curve (AUC) was calculated from glucose–time plots (Choi et al., 2021).

Serum total cholesterol (TC), triglycerides (TG), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), and high-density lipoprotein cholesterol (HDL) were measured colorimetrically using commercial assay kits from Asan Pharmaceutical (Seoul, Republic of Korea), as described previously (Yun et al., 2024). Serum insulin and HbA1c were determined by ELISA kit (Morinaga, Yokohama, Japan; MyBioSource Inc., CA, United States).

Liver and epididymal fat tissue samples from mice were fixed in 4% formalin and embedded in paraffin (FFPE, formalin-fixed and paraffin-embedded). The tissues were sectioned at a 4 µm thickness using a rotary microtome (RM2235; Leica, Nussloch, Germany). Sections were deparaffinized and rehydrated via serial passages through xylene and graded ethanol for subsequent hematoxylin and eosin (H&E) staining, as described previously (Choi et al., 2021). Images were acquired using a light microscope (BX53; Olympus) equipped with a digital camera under identical illumination and exposure settings for all experimental groups. Images were captured at a fixed magnification.

Adipocyte size in epididymal adipose tissue sections was quantified using ImageJ software as previous described (Hu et al., 2021). Briefly, H&E-stained images were converted to 8-bit grayscale, and adipocytes were segmented using an automatic thresholding method (Otsu’s algorithm). Individual adipocytes were identified using the “Analyze Particles” function after watershed separation to distinguish adjacent cells. Cells that were incomplete, distorted, or located at the image borders were excluded from the analysis. For each mouse, adipocyte size was quantified from five randomly selected microscopic fields, and at least 100 adipocytes were analyzed per section. The mean adipocyte area was calculated and used for statistical analysis.

Liver tissues (50–100 mg) were homogenized in EzRIPA lysis buffer (WSE-7420; ATTO, Tokyo, Japan) supplemented with 1% Xpert duo inhibitor cocktail solution (GenDEPOT, TX, United States). The homogenates were centrifuged at 12,000x g for 10 min at 4 °C, and the supernatant was collected. Total protein concentrations were determined using a Bradford assay (Bio-Rad, Hercules, CA, United States) according to the manufacturer’s instructions.

Equal amounts of protein were mixed with Laemmli sample buffer (Bio-Rad, Hercules, CA, United States) containing 5% β-mercaptoethanol, and denatured by heating at 95 °C for 5 min. Proteins were separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (IPVH00010; Millipore, Billerica, MA, United States) using a Mini Trans-Blot^® electrophoretic transfer system (Bio-Rad).

Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% skim milk (Becton Dickinson, Sparks, MD, United States). After washing with TBST, membranes were incubated overnight at 4 °C in TBST containing 5% bovine serum albumin (MP Biomedicals, Irvine, CA, United States) with primary antibodies against phosphorylated AKT (p-AKT), total AKT, phosphorylated AMPK (p-AMPK), and total AMPK (Cell Signaling Technology, Danvers, MA, United States). Following primary antibody incubation, membranes were washed with TBST and incubated for 1 h at room temperature with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, United States), as appropriate. Immunoreactive protein bands were visualized using enhanced chemiluminescence (ECL) reagent (34075; Thermo Scientific, Waltham, MA, United States) and detected with a ChemiDoc XRS imaging system (Bio-Rad).

Densitometric analysis was performed using ImageJ software after background subtraction. Phosphorylated AKT and AMPK levels were quantified by normalization to their corresponding total AKT or total AMPK levels (p-AKT/AKT and p-AMPK/AMPK) to assess pathway activation. All densitometric analyses were conducted using identical settings across all samples.

Wild-type N2 C. elegans (Bristol) strain and Escherichia coli (E. coli) OP50 were obtained from the C. elegans and Nematode Bank (CeNBank, Republic of Korea) and maintained on nematode growth medium (NGM) at 20 °C. The High-Glucose Diet (HGD) C. elegans model was established by exposing N2 worms to NGM supplemented with 2% glucose (Alcántar-Fernández et al., 2018). Age synchronization was achieved by treating worms with hypochlorite to dissolve the adult bodies and isolate the eggs. The collected eggs were cultured at 20 °C until they reached the L4 stage, either in the presence or absence of glucose. Experimental groups included a normal control group without glucose, a high-glucose group (2% glucose), treatment groups exposed to 2% glucose supplemented with GGT (0.5, 1.0, or 2.0 mg/mL), and a metformin-treated group (50 mM) under high-glucose conditions, which served as a pharmacological reference control (Mejia-Martinez et al., 2017). Synchronized L1-stage worms were grown to the L4 stage in media supplemented with the respective compounds to assess body size. Worm images were acquired using an Olympus BX53 microscope equipped with a digital camera and cellSens acquisition software under identical imaging settings for all groups. Images were captured at a fixed magnification and analyzed using ImageJ software.

After synchronization, L4 stage worms were cultured on NGM plates containing 50 μM floxuridine (FUDR) for 3 days. Fifty L4 stage worms were transferred to plates containing the respective compounds, and the number of dead worms was counted every 48 h. Surviving worms were transferred to fresh NGM plates every 2 days. Worms were considered dead if they did not respond to gentle probing with a platinum wire. Survival data were analyzed by generating Kaplan–Meier curves.

Oil Red O (ORO; Sigma-Aldrich) and Nile Red (NR; Sigma-Aldrich) staining methods, with minor modifications based on a previously described protocol (Escorcia et al., 2018). Fresh ORO staining solution was prepared by diluting ORO stock solution (0.5% in isopropanol) with distilled water at a 6:4 ratio, followed by filtration. To prepare the NR working solution, 5 mg/mL NR stock was dissolved in 100% acetone. L4 stage worms were collected, washed, and fixed in 60% or 40% isopropanol for 3 min. After removing isopropanol, worms were stained with the prepared ORO or NR solution for 2 h. Following staining, worms were washed with phosphate-buffered saline containing 0.01% Triton X-100 (PBST) to remove excess dye. For ORO staining, worms were mounted on microscope slides (HSU-0810001; Marienfeld Superior, Germany) and observed using an Olympus BX53 Microscope. For NR staining, worms were observed using a fluorescence microscope (Olympus U-RFL-T) attached to an Olympus BX53 Microscope. All images were captured using identical microscope settings, including exposure time and gain, within each staining modality. Quantification of ORO and NR fluorescence intensity was performed using ImageJ by measuring mean pixel intensity after background subtraction. At least 30 worms per group were analyzed, and all image analysis parameters were kept constant across groups.

Triglyceride (TG) levels were measured using the PicoSens™ TG Assay Kit (Biomax, Republic of Korea), following the manufacturer’s protocol. Absorbance was read at 570 nm using a Tecan Spark 10M Multimode Plate Reader with SPARKCONTROL software (Version 2.1).

After synchronization, C. elegans at the L4 or adult-stage were collected and incubated at 20 °C for 2 h in M9 buffer containing 0.5 mM 2-deoxyglucose (2-DG) (provided with the kit). Glucose uptake was assessed using a commercial assay kit (ab136955; Abcam, United Kingdom), following the manufacturer’s instructions. The absorbance was measured at 412 nm using a TECAN Spark 10M Multimode Plate Reader and SPARKCONTROL software.

Quantitative Real-time PCR was performed using the TRIzol reagent kit (Invitrogen) according to the manufacturer’s protocol. For qPCR analysis, SYBR^® Green Real-time PCR Master Mix (TOYOBO, Japan) using the LightCycler^® 96 platform (Roche, Germany). The ΔC_t value was determined as the difference between the Ct values of the target and housekeeping genes. Relative gene expression was quantified using the 2^−ΔΔCT method (Livak and Schmittgen, 2001), where ΔΔC_t represents the difference between the ΔC_t values of the experimental and control groups. Data was analyzed using LightCycler software (version 1.1; Roche Applied Science). The primer sequences are listed in Supplementary Table S3.

Data from individual experiments are presented as mean ± SD. Group comparisons were made using one-way ANOVA, followed by Dunnett’s multiple comparison test to assess the effects of the treatments. Statistical analyses were performed using GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA, United States). Statistical significance was set at p < 0.05.

The effect of GGT on cell viability was also analyzed. Compared to the NC group, 0.4, 0.6, and 0.8 mg/mL GGT did not induce a statistically significant difference in cell viability. However, the 1, 1.5, and 2 mg/mL GGT groups showed a significant reduction in cell viability compared to the NC group (p < 0.001, p < 0.0001, and p < 0.0001, respectively) (Figure 1A). In differentiated myotubes, both GGT and MET groups significantly enhanced glucose uptake compared with the NC group (p < 0.05 and p < 0.0001, respectively) (Figure 1B). After qPCR analysis in C2C12 myotubes, the MET and GGT groups showed significant upregulation of GLUT4, GK, Tfam, CPT1α, and PGC1α, while NRF expression was significantly increased only by MET (Figure 1C).