According to the drug usage standards of the 2020 edition of the Chinese Pharmacopeia, the daily formula dosage of CEHG is as follows: Ganoderma lucidum 6 g, Paeonia lactiflora 6 g and Glycyrrhiza glabra 3 g. Preparation of the extract: Weigh the Chinese herbal slices in proportion, place them in a stainless steel pot, and add water to soak (preliminary experiments show that the water absorption rate of the herbal slices is approximately twice their own weight, so an additional twice the amount of water needs to be added for subsequent extraction). After soaking, heat and decoct the mixture; the filtrate is filtered through 8 layers of medical gauze to obtain the extract. Concentrate the extract in a material-to-liquid ratio of 1:1 using a rotary evaporator, freeze-dry it, then crush it in a mortar and pass through an 80-mesh pharmaceutical sieve to obtain the extract powder.

An orthogonal experimental design with three factors and three levels was adopted to select the levels of the water addition ratio (A), the number of extraction times (B), and the extraction duration (C). The experimental combinations of these factors were arranged according to the orthogonal table, as detailed in Table 1.

The critical quality indicators of the Compound Liver-protecting Effervescent Granules, determined by the functions of the product, were identified as the dry extract ratio and the content of albiflorin, paeoniflorin, liquiritin, glycyrrhizic acid and ganoderic acid A. The dry extract ratio was measured by the loss on drying method and the contents of albiflorin, paeoniflorin, liquiritin, glycyrrhizic acid and ganoderic acid A were determined by high-performance liquid chromatography (HPLC). Chromatographic column: caprisil C18-AQ (5 μm, 250 mm × 4.6 mm), mobile phase: 0.1% formic acid water (A) acetonitrile (B); gradient elution: 0–8 min, 90%−86%A; 8–16 min, 86%−75%A; 16–21 min, 75%−74%A; 21–26 min, 74%-68%A; 26–48 min, 68%−42%A; 48–53 min, 42%−12%A; 53–54 min, 12%−90%A; 54–55 min, 90%A; flow rate: 0.8 ml/min; detection wavelength: 254 nm; column temperature: 30 °C; injection volume: 10 μl.

Accurately weigh appropriate amounts of paeoniflorin lactone, paeoniflorin, liquiritin, ammonium glycyrrhizinate, and ganoderic acid A, dissolve them in 80% methanol to prepare reference standard stock solutions with mass concentrations of 1.25, 2.66, 1.38, 1.29, and 0.10 mg/ml respectively. Pipette different volumes of the above reference standard solutions into 5 ml volumetric flasks, dilute to the mark, and obtain a mixed reference standard solution with mass concentrations of 62.50, 532.0, 69.00, 129.0, and 25.00 μg/ml respectively.

Accurately weigh 0.2 g of the main drug into a 50 ml volumetric flask, dilute to the mark with 80% methanol, and weigh the mass. Subject the flask to ultrasonic treatment for 30 min (40 kHz, 200 W), then allow it to cool to room temperature. Replenish the lost mass with 80% methanol, shake well, take 1 ml of the solution, centrifuge it at 12,000 r/min for 10 min, and collect the supernatant to obtain the test sample solution.

According to Table 2, paeoniflorin and albiflorin, act as the main hepatoprotective components with equal importance, both scoring 1 point, but slightly more important than liquiritin and glycyrrhizic acid (both scoring 3 points), and significantly higher than ganoderic acid A and the dry extract ratio (both scoring 5 points); AHP hierarchical analysis, subjective and objective entropy weighting of information were carried out using the SPSSAU online analysis tool (https://spssau.com/indexs.html), in which the combined weights were determined according to formula 1, orthogonal experimental data were standardized according to formula 2. A comprehensive score was calculated by comprehensive weighting according to formula 3, and finally the best process was defined through variance and range hierarchical analysis of AHP.

Take the mixed reference standard solution, inject it continuously for 6 times under the chromatographic conditions specified in “Section 2.4.3”, record the peak areas and retention times, and calculate the relative standard deviation (RSD) percentage.

Take the main drug from the same batch, prepare 6 parallel test sample solutions according to the method specified in “Section 2.4.3”, and perform injection analysis. Record the peak areas and retention times, then calculate the relative standard deviation (RSD) percentage.

Accurately pipette the same test sample solution, let it stand at room temperature, and perform injection analysis at 0, 2, 4, 8, 12, 16, and 24 h under the chromatographic conditions specified in “Section 2.4.3”. Record the peak areas and retention times, then calculate the relative standard deviation (RSD) percentage.

The extract powder was prepared according to the optimal process parameters. For the preparation of CEHG, the extract powder was first mixed with a diluent (mannitol: maltodextrin = 1:1) and divided into two equal parts. Anhydrous citric acid was added to one part, and sodium bicarbonate was added to the other. The ethanol solution was used as the binder for wet granulation of each part separately; after drying, the granules were mixed uniformly and sized. The amount of extract was fixed at 10 g, whereas the amounts of diluent and binder and the volume fraction of ethanol were varied, with the molding rate used as an index to optimize the process parameters. Based on preliminary experiments, the amount of diluent, the volume fraction of ethanol, and the amount of binder were selected as optimization factors for the Box–Behnken experimental design, with 3 levels set for each. The response surface analysis was performed using Design-Expert v.13.0 software, including 12 factorial points and 5 center points, resulting in a total of 17 experiments. The design is shown in Table 3.

After determining the optimal process, the validation experiments were conducted in triplicate. The prepared granules were subjected to quality inspection in accordance with the requirements for the granule agents (0104) specified in Volume III of the 2020 Chinese Pharmacopeia. The tests included measurements of particle size, moisture content, dissolving properties, and angle of repose.

Literature was reviewed to collect active components of Paeonia lactiflora, Glycyrrhiza uralensis, and Ganoderma lucidum. Their SDF (2D) structure files were downloaded from the PubChem (https://pubchem.ncbi.nlm.nih.gov/) platform and imported into the PharmMapper (https://lilab-ecust.cn/pharmmapper/submitfile.html) database to predict potential targets. Data with a normalized fit score ≥0.6 were selected to obtain the UniProt IDs of the targets for each component. These IDs were consolidated and duplicates were removed. Finally, the IDs were converted to gene names via the UniProt (https://www.uniprot.org/) website (21–23).

Targets related to liver injury were collected by searching the GeneCards (https://www.genecards.org/) database using the keyword “Liver injury” (24). The exported data were filtered in Excel to select target information with a relevance score of at least twice the median value of the relevance scores.

Online tool Venny 2.1.0 (https://bioinfogp.cnb.csic.es/tools/venny/index.html) was used to find the intersection of active component targets and liver injury target genes, and to create a Venn diagram. These intersectional genes (or “genes from the intersection”), regarded as potential targets for the granules against liver injury, were used for further analysis.

Cytoscape 3.9.1 was used to construct the target network of active components and visualize the relationships between active components, targets, and diseases in the form of an “Active Component–Target–Disease” network diagram. A network topology analysis was performed, and the network style was set based on node degrees. The importance of nodes in the network was reflected by their degree values: the higher the degree value, the larger the node and the darker its color.

The intersecting targets were imported into the STRING (http://string-db.org) platform to construct a PPI network, with the organism set to Homo sapiens and a confidence score >0.7 (25). After removing isolated nodes, the network was imported into Cytoscape 3.9.1. Network topology analysis was performed using the CentiScape 2.2 plugin, and key targets were identified based on Degree, Betweenness Centrality (BC), and Closeness Centrality (CC) thresholds.

The intersecting targets were imported into the DAVID (https://davidbioinformatics.nih.gov/) database for GO and KEGG pathway enrichment analyses, with the species set to Homo sapiens and a significance threshold of P < 0.01 (26). The top 20 entries were selected and visualized as bar and bubble charts using the WeBioinformatics (http://www.bioinformatics.com.cn/) platform, and the results were analyzed (27).

Obtain the SDF files of the 2D structures of the screened core components from the PubChem database, then import the SDF files into Chem3D software for energy optimization and conversion into 3D structures; Download the 3D structures of key targets from the PDB database, perform molecular docking using AutoDock software and related plugins, and plot the results using PyMOL software.

After 7 days of adaptive feeding in an SPF-grade environment (temperature 23 °C ± 2 °C, 12 h light-dark cycle), 60 rats were randomly divided into 6 groups (10 rats per group). The doses were determined based on the conversion of the human body surface to the rat body area, with low, medium, and high doses set at 5, 10, and 20-fold the human recommended dose, respectively. Carbon tetrachloride (CCl4) (160 mg/kg·BW, dissolved in olive oil) was administered by gavage. Silymarin served as a positive control drug. The experimental groups were as follows: control group (A), model group (B), low-dose group (2.7 g/kg) (C), medium-dose group (5.4 g/kg) (D), high-dose group (10.8 g/kg) (E), and silymarin group (1 g/kg) (F).

The test groups received the samples by gavage daily, whereas the control group and the model group received distilled water. The rats were weighed twice a week to adjust the dose. On day 30, after 16 h of fasting, the model group and the test groups received a single gavage of CCl4, the control group received olive oil, and the test groups continued their respective treatments until the end of the experiment (with a 4-h interval after the gavage with CCl₄). Twenty-four hours after CCl4 administration, rats were anesthetized with 20% urethane (0.5 mL/100 g), blood was collected for serum separation, and the rats were euthanized by cervical dislocation before the livers were harvested for further analysis.

Pathological examination of liver tissue was performed using HE staining of paraffin sections: Paraffin-embedded liver sections were dewaxed with xylene three times (15 min each), then sequentially hydrated with 100%, 95%, and 80% ethanol (5 min each); rinsed under running water until no alcohol remained and the sections were clean and transparent. Sampless were stained with hematoxylin for 3–5 min, rinsed with water, differentiated in 1% hydrochloric acid alcohol for several seconds, then rinsed with water again; treated with dilute lithium carbonate solution for 30 s to restore blue color, rinsed with water, dehydrated with 80% ethanol, and stained with alcohol-soluble eosin for 20 s. The sections were placed in 95% ethanol I and II for about 10 s each for color adjustment, dehydrated in anhydrous ethanol I and II for 1–2 min each, and finally cleared in xylene I and II for 1–2 min each, mounted, and observed under a microscope.

Rat blood was collected and allowed to stand for 30 min to 1 h for coagulation. The blood was then centrifuged at 4,000 rpm for 10 min to separate the serum, and the supernatant was collected for analysis. Commercial kits were used to measure the levels of ALT, AST, TG, TC, LDH, TP, TBIL, TNF-α, IL-1β, and IL-6 in rat serum.

Liver tissue was homogenized using PBS (0.01 M, pH 7.4). The homogenate was centrifuged at 10,000 rpm for 10 min at 4 °C and the supernatant was collected on ice. Commercial kits were used to measure the levels of SOD, MDA, GSH, and GSH-Px in the liver homogenate.

The tissue was homogenized using the homogenate solution according to the ROS kit instructions. After centrifugation, 200 μl of supernatant was retrieved, mixed with 2 μl of probe, and incubated at 37 °C for 30 min. During incubation, cells were mixed to ensure full probe binding. Part of the supernatant was used for protein content determination. After protein measurement, the sample was filtered through a 200-mesh filter and tested using a flow cytometer. The results were analyzed using NovoExpress software.

Liver tissue sections were dewaxed with xylene three times (15 min each), then sequentially hydrated with 100%, 95%, and 80% ethanol (5 min each); and rinsed under running water until clean and transparent. The sections were treated with 20 μg/ml proteinase K (without DNase) at 37 °C for 20 min, then washed with PBS three times. The TUNEL detection solution (50 μl; TdT enzyme + Biotin-dUTP=5 μl + 45 μl) was added to each sample, and incubated at 37 °C for 60 min. After washing with PBS three times, 100 μl of reaction termination solution was added and the samples were incubated at room temperature for 10 min. Following 3 washes with PBS, 100 μl of Streptavidin-HRP working solution (0.5 μl + 99.5 μl) was added and the samples were incubated at room temperature for 30 min. After the final three washes with PBS, color was developed using DAB at room temperature for 8 min and the sections were rinsed three times with PBS, counterstained with hematoxylin, to differentiate and restore blue color. Finally, the sections were dehydrated and cleared, mounted with neutral gum, and observed under a microscope.

Liver tissue samples were ground on ice, centrifuged at 3,000 rpm for 3 min at 4 °C, and the supernatant was collected. Cells were collected at a density of 1–2 × 10⁶ cells, centrifuged at 1,500 rpm for 3 min, and the supernatant was discarded. The cell suspension was then mixed with 2.5 μl of Annexin V-FITC Reagent and 2.5 μl of PI Reagent (50 μg/ml). After gentle vortexing, the mixture was incubated at room temperature in the dark for 15–20 min. Next, 400 μl of diluted 1 × Annexin V Binding Buffer was added and thoroughly mixed. The sample was filtered through a 200-mesh filter and analyzed using a flow cytometer. The flow cytometry images of the apoptotic cells were analyzed using NovoExpress software.

Liver tissue processing: 50–100 mg of liver tissue was ground to powder in liquid nitrogen, and 1 mL of TRIzol was added to lyse the cells. After the addition of 0.2 ml of chloroform, the samples were vortexed for 15 s, and incubated on ice for 5 min; and centrifuged at 12,000 rpm for 10 min at 4 °C.

RNA extraction and cDNA synthesis: the supernatant was collected and the RNA was precipitated with isopropanol. The precipitate was washed with 75% ethanol before resuspending in 20–50 μl of DEPC-treated water. RNA was reverse-transcribed into cDNA using one-step first-strand synthesis.

Gene expression: The reaction system contained 10 μl of Taq SYBR Green qPCR Premix (Universal), 0.4 μl each of forward/reverse primers (10 μm), 3 μl of cDNA, and 6.2 μl of RNase-free water, with a total volume of 20 μl. The cycling conditions were as follows: pre-denaturation at 95 °C for 30 s; 40 cycles (95 °C for 15 s, 60 °C for 30 s). Relative quantification was performed using the 2^−ΔΔCt method, and primer sequences are listed in Table 4.

Liver tissue samples of 50–100 mg were homogenized in RIPA lysis buffer (containing 1 mM PMSF) at a 1:100 ratio and incubated on ice for 30 min. Samples were centrifuge at 12,000 rpm for 15 min at 4 °C to collect the supernatant as total protein. Protein concentration was determined using the BCA assay kit according to the instructions. 5 × SDS-PAGE loading buffer was added to protein samples at a 1:4 ratio. Samples were heated in boiling water for 10 min to denature the proteins and then loaded onto a 10% SDS-PAGE gel for electrophoresis for 1 h. The proteins were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk for 2 h, and after discarding the blocking solution, the membranes were washed with TBST. Ther membrane was incubated overnight at 4 °C with primary antibodies diluted as follows: p53 (1:1,000), Bax (1:1,000), Bcl-2 (1:1,000), HIF-1α (1:500), and β-actin (1:1,000). After washing, the secondary antibody (1:10,000) was incubated for 2 h. Proteins were detected using an ECL hypersensitive luminescent reagent kit and the grayscale values of the bands were analyzed with ImageJ software. The relative protein expression levels were determined based on by the grayscale densitometric values of β-actin bands.

The dry extract ratio and the contents of albiflorin, paeoniflorin, liquiritin, glycyrrhizic acid, and ganoderic acid A in the extract were determined, with the results presented in Table 5 and Figure 1.

The subjective weight coefficients based on AHP, the objective weight coefficients based on entropy, and the combined weight coefficients for the ratio of paeoniflorin, albiflorin, glycyrrhizic acid, liquiritin, ganoderic acid A and dry extract are shown in Table 6.

After standardization of orthogonal experimental data, the significance of the influence of factors on the results was determined through a range and variance analysis. The intuitive analysis of the R range showed that the primary and secondary factors that affected the comprehensive extraction score were A > C > B (the solid-liquid ratio had the greatest influence, followed by the extraction time, and finally the extraction times). As shown in Tables 7A–C all had significant effects on the complete score (P < 0.05). The optimal scheme was A3B1C1 (water addition ratio 1:20, extracted once, 1 h each time). The results of the range analysis and variance analysis are shown in Tables 7, 8, respectively.

For the five components (paeoniflorin lactone, paeoniflorin, liquiritin, ammonium glycyrrhizinate, and ganoderic acid A), in the precision investigation, the peak area RSD% values were all in the range of 0.30%−1.63% (all < 2%), and the retention time RSD% values were all in the range of 0.01%−0.09% (all < 0.10%), indicating good precision of the instrument; in the repeatability investigation, the peak area RSD% values were all in the range of 0.24%−1.24% (all < 2%), and the retention time RSD% values were all in the range of 0.03%−0.11% (all < 0.20%), indicating good repeatability of the method; in the stability investigation, within 24 h, the peak area RSD% values were all in the range of 0.67%−1.71% (all < 2%), and the retention time RSD% values were all in the range of 0.02%−0.30% (all < 0.30%), indicating good stability of the test sample within 24 h.

The results of the validation testing are presented in Table 9. Three batches of parallel tests showed similar index component content and dry extract rates, with RSD values below 5%. This indicated that the extraction process was stable and feasible, with excellent reproducibility.

The results and analysis of the Box–Behnken experiment are shown in Tables 10, 11. The Box–Behnken experiment yielded a second order polynomial regression equation for the molding rate Y = 84.03 – 0.94a – 10.26b + 1.83c – 1.33ab + 0.098ac + 2.13bc – 1.03a² – 0.17b² – 1.85c². As shown in the table below, the model's F value was 67.36 and P < 0.05, indicating that the model terms were statistically significant. The lack of fit item had a P-value > 0.05, indicating a good fit of the regression equation. R² = 0.9886 and Radj² = 0.9739, which indicated that the model had a good fit. Independent variables effectively explained the changes in the response variable. The similar R² and Radj² values suggested that the independent variables introduced were valid. Based on the F value, the significant effects on the formation rate were in the order of b > c > a. In the regression model, the linear terms b and c, the quadratic term c², and the interaction term bc were significant, whereas the linear term a, the quadratic terms a² and b², and the interaction terms ab and ac were not significant. The optimized forming process parameters were as follows: 11 parts of diluent, 12% binder, and 80% ethanol volume fraction.

Three tests showed a particle formation rate of 95.42%, 96.33%, and 94.76%. The moisture of the CEHG was 5.44%, below 8%. The dissolving time was within 5 min, meeting the Chinese Pharmacopeia requirements. With a resting angle of < 40°, the flow ability of the particles satisfied basic production needs.

HE staining showed that in the control group, rat liver cells were normal in size and shape, with large round nuclei and a clear structure. The hepatocyte cords were arranged radially around the central vein. In the model group of CCl4-induced acute liver injury, significant necrosis, steatosis, inflammatory infiltration, and hemorrhage was observed in liver tissue. As the dose of the granules increased, the liver damage eased and the morphology of the liver improved visibly. Silybin, a reference drug, also improved CCl4-induced liver injury, nearly restoring the tissue to its normal state (Figure 5).

Compared with the control group, the model group showed significant increases in ALT and AST, indicating the successful establishment of liver injury models. Compared with the model group, the low, medium, and high dose groups had notable reductions in ALT and AST levels, demonstrating that the effervescent protective granules of compound liver could improve liver function indicators in rats with liver injury in a dose-dependent manner. The positive drug group also had much lower ALT and AST levels than the model group. Compared with the control group, the model group presented higher levels of TC, TG, TBIL, and LDH and significantly lower levels of TP. These changes confirmed the successful establishment of the liver injury model, reflecting lipid metabolic disorders, impaired hepatic synthetic function, and abnormal bilirubin metabolism due to liver injury. Compared with the model group, except for the low-dose group, which showed no significant changes in TP and LDH, all other dosing groups exhibited significantly reduced levels of TC, TG, TBIL, and LDH and significantly increased TP levels. After treatment, TC, TG, TBIL and LDH decreased with increasing doses, whereas TP increased with higher doses. Compared with the control group, the model group showed significantly increased levels of IL-6, IL-1β, and TNF-α. Compared with the model group, all treatment groups, except the low-dose group with non-significant changes in IL-6 levels, exhibited significantly reduced levels of IL-6, IL-1β, and TNF-α. Overall, the liver-protecting effervescent granules of the compound suppressed the release of IL-6, IL-1β, and TNF-α, thus alleviating the inflammatory response (Figure 6).

Compared with the control group, the model group showed significantly reduced SOD and GSH-Px activity, increased MDA levels, and decreased GSH levels. This indicates a weakening of hepatic antioxidant capacity, increased oxidative stress, and severe damage to stem cell membranes in the liver injury model. Compared with the model group, all treatment groups (except for GSH content without significant difference) showed significantly increased SOD and GSH-Px activity and significantly decreased MDA levels and increased GSH levels. Thus, CEHG can protect the liver by enhancing antioxidant capacity and reducing oxidative stress (Figure 7a). Compared with the control group, the model group showed a significant increase in ROS levels in rat liver tissue. Compared with the model group, all treatment groups had markedly lower ROS levels. This indicated that the liver-protecting effervescent granules of CEHG reduced ROS in liver tissue from rats with chemical-induced liver injury, thus decreasing damage due to oxidative stress (Figures 7b, c).

The results of the TUNEL assay revealed a significantly higher positivity rate in the hepatocytes of the model group than in the control group, indicative of intensified apoptosis. Compared with the model group, the low dose group showed a decrease in positivity rate, albeit not significant, whereas other medicated groups presented markedly lower positivity rates, signaling a substantial reduction in hepatocyte apoptosis. Flow cytometry apoptosis images were analyzed using NovoExpress software as follows: quadrant 1: necrotic cells; quadrant 2: late apoptotic cells; quadrant 3: normal cells; quadrant 4: early apoptotic cells. The combined percentage of cells in quadrants 2 and 4 (late and early apoptotic cells) was calculated. The flow cytometry results showed a higher rate of apoptosis in the model group than in the control group (Figure 8). In contrast, the apoptosis rate was significantly lower in the treatment groups than in the model group, which was consistent with the findings of the TUNEL assay. Overall, these results demonstrate that CEHG can inhibit hepatocyte apoptosis in rats with liver injury, fulfilling their liver-protecting function.

The results of RT-qPCR showed that, compared with the control group, the model group had a significant higher mRNA expression of P53, Bax, and HIF-1α, and a lower expression of Bcl-2 mRNA. Compared with the model group, the treatment groups had a significant decrease in P53, Bax, and HIF-1α mRNA levels and increased Bcl-2 mRNA levels.

The WB results showed that, compared with the control group, the model group had significantly higher expression of P53, Bax and HIF-1α protein, and decreased expression of protein Bcl-2. Compared with the model group, the dosed groups had significantly lower protein levels of P53, Bax, and HIF-1α protein levels but higher Bcl-2 levels (Figure 9).