Erythropalumscandens Bl. was provided by Guangxi Erythropalumscandens Biotechnology Co., Ltd.

Fructose and potassium oxonate were purchased from Shanghai Adamas Reagent Co., Ltd.; 60% high-fat diet was obtained from Hunan Syno Biological Technology Co., Ltd.; ESP was prepared in the laboratory; Urethane was supplied by Shanghai Hengyuan Biotechnology Co., Ltd.; Detection kits for serum FBG and UA were purchased from Suzhou Grace Bio-Technology Co., Ltd.; Blood glucose meter (Sinocare EA-19) was purchased from Sinocare Inc.; Tissue clearing and deparaffinizing agent as well as alcoholic Eosin Y staining solution were obtained from Zhuhai Baso Biotechnology Co., Ltd.; Hematoxylin bluing solution was purchased from Wuhan Servicebio Biotechnology Co., Ltd. The Motic BA210 digital biological microscope and Motic BA600 virtual slide scanning system, used for the histological analysis of liver, kidney and pancreatic tissues, were both provided by Motic China Group Co., Ltd. (Xiamen, China). The LB941 multifunctional microplate reader (Berthold Technologies GmbH & Co. KG, Germany) was purchased from Hunan Xiangyi Laboratory Instrument Development Co., Ltd. The B-100 rotary evaporator was a product of BUCHI Labortechnik AG (Switzerland). The FA1204B electronic analytical balance was manufactured by Shanghai Techcomp Precision Instruments Co., Ltd. Both the SHB-III circulating water multi-purpose vacuum pump and DLSB-5/10 low-temperature cooling liquid circulation pump were products of Zhengzhou Greatwall Scientific Industrial and Trade Co., Ltd.

Forty SPF-grade male Sprague-Dawley (SD) rats, aged 6 - 8 weeks, were purchased from Guangdong Weitong Lihua Experimental Animal Technology Co., Ltd. and raised in the Animal Experiment Center of Youjiang Medical University for Nationalities. The animal production license number was SCXK (Yue) 2022-0063, and the experimental animal use license number was SYXK (Gui) 2022-0004. The rats were housed under the controlled conditions of constant temperature (24˚C ± 2˚C) and relative humidity (50% - 70%), which complied with the requirements for medical experimental animal facilities. The basal diet was the special feed for experimental animals. The ethical review number of the animal experiment was 2024102501.

1 - 2 kg of Erythropalumscandens Bl. powder was weighed and mixed with 95% ethanol at a solid-liquid ratio of 1:20 (g/mL). Reflux extraction was performed once at 70˚C for 1 hour to remove pigments and lipids. The mixture was suction-filtered, and the residue was dried for subsequent use. The treated residue was mixed with ultrapure water at a solid-liquid ratio of 1:20 (g/mL) and soaked for 2 hours, followed by extraction twice at 80˚C for 1 hour each time. The extract was filtered, and the filtrate was subjected to reflux evaporation and concentrated to 1/4 of its original volume. Alcohol precipitation was then conducted by adding anhydrous ethanol to the concentrated solution to adjust the anhydrous ethanol concentration to 50%. After thorough stirring, the mixture was statically placed at a low temperature (4˚C) overnight, then centrifuged at 3000 r/min for 20 minutes. The precipitate was collected and freeze-dried to obtain crude polysaccharides. The crude polysaccharides were dissolved in a small amount of water and subjected to decolorization and deproteinization using a macroporous adsorption resin, with 50% ethanol as the eluent. The eluates were collected, combined, concentrated and freeze-dried for subsequent use.

3.1.1. ESP Content Determination Method

Glucose was used as the standard solution. Precisely weigh 10 mg of anhydrous dried glucose, dissolve it in pure water and dilute to the mark in a 10 mL volumetric flask, and shake well to obtain a 1 mg/mL glucose standard stock solution. For standard curve preparation, the glucose standard solution was diluted into five concentration gradients (0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL), with three parallel experiments set up and each determination repeated three times per group. The detailed operation steps were as follows: pipette each gradient solution into 5 mL stoppered test tubes, add phenol reagent and cap the test tubes, then slowly add concentrated sulfuric acid along the tube wall and shake well. The mixture was placed at room temperature for 30 minutes, then heated in a constant temperature water bath at 90˚C for 15 minutes and cooled to room temperature. Using ultrapure water as the blank control, the absorbance value at 490 nm (A_490nm) was measured with a microplate reader. The glucose concentration was set as the X-axis and the absorbance value as the Y-axis to plot the standard curve.

Polysaccharide content (%) = [(C × D × V)/(m × 10³)] × 100%

where: C = glucose concentration in the sample assay solution obtained from the standard curve after blank correction (mg/mL); V = total volume of the sample extract after constant volume (mL); D = dilution multiple before determination; m = initial sample weight (g); 10³ = unit conversion factor (converting mg to g for consistency with the unit of initial sample weight).

3.1.2. Determination of ESP Protein Content

After the crude polysaccharides were deproteinized by macroporous adsorption resin, the Coomassie Brilliant Blue method was used to determine the protein content. The detailed operation was as follows: prepare bovine serum albumin (BSA) standard solutions with five concentration gradients (0.1, 0.2, 0.3, 0.4 and 0.5 mg/mL). Pipette each standard solution into glass test tubes, add Coomassie Brilliant Blue solution to each tube and dilute to the mark, mix thoroughly and let stand at room temperature for 5 minutes. The absorbance value was measured at 595 nm within 20 minutes. The absorbance value was set as the ordinate and the BSA content (mg) as the abscissa to plot the protein standard curve. An aliquot of the crude polysaccharide solution was also treated and assayed according to the above steps and conditions, and its absorbance value at 595 nm was measured and substituted into the standard curve to calculate the protein content in the crude polysaccharides.

Protein removal rate (%) = [(m_{Beforedeproteinization} − m_{Afterdeproteinization})/m_{Beforedeproteinization}] × 100%

A total of 40 male Sprague-Dawley (SD) rats aged 6 - 8 weeks were housed in separate cages based on their body weight and acclimatized for one week; the housing environment was maintained under a 12 h light/12 h dark cycle, with free access to food and water, and the rats were fed with standard SPF-grade basal feed. After one week of acclimatization, the rats were rehoused for model establishment. The grouping was as follows: the Control group was fed with a basal diet; the Model group was fed with a high-fat diet, provided with drinking water containing 30% - 40% fructose ad libitum, and intraperitoneally injected with potassium oxonate daily for 20 weeks; the ESP-administered groups included the ESP high-dose (ESP-H, 3 g/kg/d) and ESP low-dose (ESP-L, 3 g/kg/d) groups, which were given intragastric gavage for 4 weeks. For model establishment, 30 rats were gradually fed with SPF-grade high-fat feed; their body weight was measured weekly, and fasting blood glucose was detected in the morning after 12 hours of fasting. The remaining 10 rats served as the blank Control group, which continued to be fed with SPF-grade basal feed, and changes in their body weight and blood glucose were recorded simultaneously. During the modeling period, blood glucose was measured by tail-tip blood sampling; combined with the rats’ body weight, dietary intake, and general physical condition, rats with blood glucose levels close to 11.1 mmol/L that remained stable for approximately two weeks were selected as ideal hyperuricemia (HUA) rat models with glucose metabolism disorders. These rats were then regrouped according to their blood glucose levels to ensure the randomness and scientificity of grouping, with 10 rats in each group. At the end of the 4-week administration period, all rats were sacrificed, and samples were collected for subsequent experiments.

Liver, pancreas, and kidney tissues were harvested and cut into 1 cm × 1 cm square pieces, which were then placed into embedding cassettes. After fixation in 4% paraformaldehyde for 24 hours, the tissues were subjected to dehydration, paraffin embedding, sectioning (4 μm), hematoxylin-eosin (HE) staining, and mounting. The morphological changes of the tissues were observed under a light microscope.

The NAFLD Activity Score (NAS) was independently evaluated in a blinded manner by two experienced pathologists. For each group, five liver tissue sections were randomly selected. Each section was systematically observed in 10 fields at 400× magnification. Three parameters were assessed in each field: steatosis, hepatocyte ballooning, and lobular inflammation.

Each parameter was scored based on the percentage of the affected area using a 4-point scale as follows:

Score 0: No steatosis, no hepatocyte ballooning, and no lobular inflammation;Score 1: Mild steatosis (5% - 33%), a small number of ballooned cells, and 1 - 2 foci of necrosis;Score 2: Moderate steatosis (34% - 66%), numerous ballooned cells, and 2 - 4 foci of necrosis;Score 3: Severe steatosis (>66%), no ballooning, and >4 foci of necrosis.

The total score for each field was the sum of the scores from the three parameters.

Statistical analysis was conducted using IBM SPSS Statistics 26 software. Quantitative data were expressed as mean ± standard deviation ( x ¯ ± s ). One-way analysis of variance (ANOVA) was used to compare the differences among multiple groups, with a P-value < 0.05 considered statistically significant. GraphPad Prism 8 and Origin software were employed for the construction of experimental figures and tables.

In this study, the crude extract yield of ESP obtained from 500 g of Erythropalumscandens Bl. was 3.189 g; as shown in Figure 1(A), regression analysis was performed using the least squares method, and the glucose standard curve equation was obtained as: y₁ = 1.0459x₁ + 0.0445, R² = 0.9952; where x₁ represents concentration (mg/mL) and y₁ represents absorbance. As shown in Figure 1(B), the protein standard curve was: y₂ = 1.441x₂ + 0.0747, R² = 0.9912; where x₂ represents concentration (mg/mL) and y₂ represents absorbance. According to the formula, the protein removal rate reached 80.83%. These results demonstrated that after deproteinization by macroporous adsorption resin, the protein content in the polysaccharide sample was significantly reduced, which effectively improved the purity, enhanced the biological activity and stability of ESP, and laid a solid foundation for the subsequent experiments.

(A) (B)

Note: Panel A shows the standard curve of glucose; Panel B shows the standard curve of protein.

Figure 1. Determination of Erythropalumscandens Bl. Polysaccharides.

4.2.1. Body Weight of Rats

Glucose and lipid metabolism disorders induced by diabetes are core pathological features of metabolic diseases. Rats induced by a high-sugar and high-fat diet exhibited a significant increase in body weight accompanied by abdominal obesity at the initial stage of modeling, which was closely related to increased fat synthesis and imbalanced energy metabolism caused by decreased insulin sensitivity. During the experiment, the body weight of rats in the Model group was significantly higher than that in the Control group (as shown in Figure 2). After 4 weeks of intervention, compared with the Model group, diabetic symptoms in the ESP-H and ESP-L groups were alleviated, and body weight was decreased with an obvious recovery trend, indicating that ESP exerts an improving effect on glucose metabolism.

Note: Figure 2 records the body weight of rats during modeling and treatment.

Figure 2.Body weight of rats.

4.2.2. ESP Improves Blood Glucose Indexes in HUA Rats

To validate the successful establishment of a glucose metabolism disorder model in HUA rats, this study evaluated key blood glucose parameters. As shown in Figure 3(A), rats subjected to high-fat diet combined with fructose and OXO administration exhibited significantly elevated blood glucose concentrations compared to the Control group (P < 0.0001), confirming successful model induction. Further pharmacodynamic analysis, Figure 3(B) revealed that following 4 weeks of continuous intervention with high- and low-dose ESP (ESP-H and ESP-L), fasting blood glucose levels showed a significant decreasing trend relative to the Model group (P < 0.05).

(A) (B)

Note: Panel A shows the blood glucose levels in the Control and Model groups; Panel B shows the fasting blood glucose levels of rats in each group after 4 weeks of ESP administration. ****P < 0.0001, ***P < 0.001, *P < 0.05.

Figure 3. Effects of Erythropalumscandens Bl. polysaccharides on blood glucose in rats induced by a high-fat and high-sugar diet combined with potassium oxonate.

Elevated serum uric acid (UA) is the hallmark pathological feature of hyperuricemia (HUA) and serves as both a critical diagnostic biomarker and a key metric for assessing therapeutic efficacy. As illustrated in Figure 4, the Control group exhibited a mean serum UA level of 391.2 μmol/L, whereas the HUA model group showed a marked elevation to 1058.3 μmol/L (P < 0.01)—representing an approximately 2.7-fold increase. This substantial elevation confirms the successful establishment of the HUA model and demonstrates significant disruption of uric acid metabolism in the model rats.

Following ESP intervention, serum UA levels were significantly reduced compared to the Model group, decreasing to 700.1 μmol/L in the ESP-L group and further to 531.6 μmol/L in the ESP-H group (P < 0.05). Notably, the uric acid-lowering effect in the ESP-H group was significantly superior to that observed in the ESP-L group. This dose-dependent response substantiates the role of ESP in modulating uric acid metabolism in HUA rats, providing compelling experimental evidence for non-pharmacological interventions in hyperuricemia management.

Note: Figure 4 shows serum UA levels in rats of each experimental group. ***P < 0.001, **P < 0.01, *P < 0.05.

Figure 4. Effects of ESP on serum uric acid (UA) in HUA rats.

4.4.1. Liver

HE staining results of liver tissues are shown in Figure 5(A). In the Control group, hepatic lobule architecture remained intact, with hepatocytes displaying their characteristic polygonal shape and arranged in an orderly radial pattern; cells showed abundant cytoplasm and portal vein structures were clearly visible. By contrast, the Model group exhibited extensive fatty and granular degeneration throughout the hepatocytes, with fatty degeneration covering 16.4% ± 0.66% of the area; cell arrangement was highly disordered, indicating severe hepatocyte injury. In the ESP-L group, both fatty and granular degeneration were markedly reduced, and the previously disordered hepatocyte arrangement showed initial signs of improvement. The ESP-H group demonstrated even more pronounced benefits: fatty degeneration dropped to just 2.3% ± 0.21%, with hepatocyte arrangement and morphology approaching normal levels.

Note: Panel A shows HE staining images of rat liver tissues in each experimental group; Panel B shows the percentage of fatty area; Panel C shows NAFLD activity score. Scale bar = 50 μm.

Figure 5. Representative photomicrographs of rat liver tissue sections (×400).

4.4.2. Pancreas

HE staining results of pancreatic tissues are shown in Figure 6; in the Control group, islet architecture remained intact (black arrows), with abundant β-cells and well-defined acinar cells (red arrows) showing plentiful cytoplasm. The Model group, however, presented a starkly different picture: islets were markedly reduced in number or completely absent (black arrows), β-cell counts dropped sharply, extensive acinar cell necrosis occurred with disorganized arrangement (red arrows), and islet cytoplasm showed clear degenerative changes. Both ESP-L and ESP-H groups demonstrated substantial recovery—islet and β-cell numbers increased significantly, acinar cell necrosis was markedly reduced, and overall pancreatic tissue damage showed remarkable improvement compared to the Model group.

Note: Panel 6 shows HE staining images of pancreatic tissues in rats of each experimental group; Scale bar = 50 μm.

Figure 6. Representative photomicrographs of rat islet tissue sections (×400).

4.4.3. Kidney

HE staining results of kidney tissues are shown in Figure 7; in the Control group, glomerular and tubular structures appeared clear and intact, with regular capsular spaces and well-defined boundaries. The Model group showed considerable damage: glomeruli displayed obvious atrophy, tubular epithelial cells underwent necrosis, and various pathological injuries including fatty degeneration were evident. The ESP-L group showed partial improvement—fatty degeneration and epithelial cell necrosis in the tubules were reduced, though glomerular structure remained incompletely restored. In the ESP-H group, recovery was nearly complete: glomerular architecture was essentially returned to normal, fatty degeneration and epithelial cell necrosis were markedly improved, and overall kidney tissue morphology approached healthy levels.

Note: Panel 7 shows HE staining images of kidney tissues in rats of each experimental group; Scale bar = 50 μm.

Figure 7. Representative photomicrographs of rat kidney tissue sections (×400).