Structural Characterization and Hypoglycemic Mechanism of Pumpkin Seed Polysaccharides

1. Introduction

The development of bioactive compounds derived from natural products as supplements or alternatives to pharmaceuticals for diabetes management has emerged as a significant trend in the field of functional food development. Among these compounds, polysaccharides are regarded as having substantial potential for applications in diabetes regulation. Currently, numerous natural polysaccharides have been found to exhibit favorable hypoglycemic effects, such as polysaccharides from Finger citron (Yang et al., 2025), Coix seed (Xia et al., 2021), Ganoderma lucidum (Shao et al., 2022), and Polygonatum cyrtonema Hua (Xie et al., 2020).

Pumpkin seed, derived as a primary by-product from the processing of pumpkin (Cucurbita moschata (Duchesne ex Lam.) Duchesne ex Poir.), are recognized for their edible and medicinal attributes. They are abundant in various nutritive and functional constituents, including oil, proteins, phenols, flavonoids, and polysaccharides (Cai et al., 2025; Fatima et al., 2025). Pumpkin seed oil is the main product from processing pumpkin seeds, but the residue left after oil extraction remains underutilized. Notably, polysaccharides constitute one of the effective functional components within this oil-extracted pumpkin seed residue, yet research on pumpkin seed polysaccharides remains limited both domestically and internationally. Recently, our research group isolated and purified three previously unreported pumpkin seed polysaccharides (PSP-1-1, PSP-1-2, and PSP-1-3) from pumpkin seed meal, all of which exhibited good in vitro hypoglycemic activity (Cai et al., 2025). Monosaccharide composition analysis revealed a high degree of structural homology among them, as they were all composed of galactose, arabinose, glucose, and xylose. Specifically, the relative molar ratios of these four monosaccharides in PSP-1-1, PSP-1-2, and PSP-1-3 were 61.5:24.4:5.9:0.5, 38.2:14.8:28.6:8.9, and 38.8:14.2:31.7:7.3, respectively.

Polysaccharides, as a class of biologically active macromolecules, have difficulty in penetrating the intestinal mucosal barrier to enter the bloodstream and reach target organs. Consequently, their biological effects are predominantly manifested within the gut (Makki et al., 2018). Nevertheless, the precise mechanisms through which polysaccharides modulate glucose metabolism and regulate appetite remain inadequately understood. Current research suggests that the gut-brain axis is instrumental in the transmission of hypoglycemic signaling molecules (Feng et al., 2022). The gut microbiota and the human host exist in a dynamic equilibrium characterized by mutual dependence and constraint, forming a symbiotic system. Furthermore, the gut maintains intricate connections with the brain, relaying various signals via the vagus nerve, spinal neurons, and other pathways. These signals, which may include gut hormones or cytokines secreted by immune cells, constitute the gut-brain axis through their interactions between the digestive tract and the central nervous system, thereby playing a role in and influencing the body’s energy metabolism.

In the gut-brain axis, glucagon-like peptide-1 (GLP-1), secreted by enteroendocrine L cells, serves as a crucial signaling molecule through which polysaccharides regulate glucose metabolism (Luo et al., 2018). Its receptors are widely distributed in the brain, gastrointestinal tract, and pancreas, where they stimulate insulin secretion, enhance satiety, slow gastric emptying, and promote pancreatic islet cell regeneration, making GLP-1 a key therapeutic target for type 2 diabetes. There are two pathways by which polysaccharides promote GLP-1 secretion from L cells: First, the fermentation of polysaccharides by gut microbiota produces short-chain fatty acids (SCFAs), which in turn stimulate L cells to secrete GLP-1 (Chambers et al., 2015). For instance, polysaccharides from Shiitake (Chakraborty et al., 2023), Ganoderma lucidum (Shao et al., 2022), White hyacinth bean (Chen et al., 2023), Achyranthes bidentata (Xia et al., 2024), and Lycium barbarum (Ma et al., 2022) have been reported to modulate the composition and abundance of gut microbiota, particularly by increasing bacteria that produce SCFAs. The elevation of SCFAs induces the release of more downstream target signaling molecule GLP-1, thereby regulating gut-brain axis signaling, suppressing appetite (Medawar et al., 2021), and exerting hypoglycemic effects. Second, research has demonstrated that sweet taste receptors located on L cells play a role in the signaling pathway by which monosaccharides, polysaccharides, oligosaccharides, or carbohydrates stimulate GLP-1 secretion. Polysaccharides can enhance GLP-1 synthesis via sweet taste receptor-mediated pathways (Xie et al., 2020; Yang et al., 2021). Consequently, it is reasonable to hypothesize that pumpkin seed polysaccharides could activate the GLP-1 signaling pathway within the gut-brain axis through these two mechanisms, thereby playing a role in the regulation of hypoglycemic activity. However, it is important to note that empirical studies supporting this hypothesis have not yet been published.

Building upon the preceding analysis, this study initially characterized the structural properties of pumpkin seed polysaccharide fractions PSP-1-1, PSP-1-2, and PSP-1-3 through molecular weight determination, Fourier-transform infrared spectroscopy (FT-IR), and methylation analysis. Subsequently, utilizing the intestinal endocrine L-cell model NCI-H716 as the focal point of investigation, the study examined the effects of pumpkin seed polysaccharides on GLP-1 secretion and the expression of genes associated with the sweet taste receptor signaling pathway. Furthermore, through the implementation of animal experiments, the hypoglycemic activity of pumpkin seed polysaccharides was assessed. From the perspective of hypoglycemic signal transduction, and in conjunction with alterations in serum GLP-1 levels, gut microbiota composition, SCFAs levels, and changes in hypothalamic GLP-1 receptor (GLP-1R) protein and appetite-related gene expression, the hypoglycemic mechanism of pumpkin seed polysaccharides via the “gut-brain” axis was ultimately elucidated.

2. Materials and Methods

2.1. Chemicals and Materials

Pumpkin seeds were provided by Xinjiang Aiyouwei Feed Food Co., Ltd. (Urumqi, China). NCI-H716 cells were supplied by American Type Culture Collection (ATCC, Manassas, USA). Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS) were obtained from Gibco (Grand Island, New York, USA). Methyl thiazolyl tetrazolium (MTT) was purchased from Sigma-Aldrich (St Louis, MO, USA). The RC112, R223, and Q711 kits were supplied by Nanjing Vazyme Biotech Co., Ltd. (Nanjing, China). Male C57BL/6N mice (NO. SCXK (ZHE) 2024-0011) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Streptozotocin (STZ) and metformin were bought from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Hematoxylin-eosin (HE) was obtained from Solarbio (Beijing, China). The commercial kits for the determination of TC, TG, LDL-C, HDL-C, glycated serum protein (GSP), insulin, and GLP-1 were provided by Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Radio immunoprecipitation assay Lysis Buffer (RIPA) and polyvinylidene difluoride (PVDF) membranes were acquired from Merck KGaA (Darmstadt, Germany). Mouse monoclonal antibodies for β-actin and GLP-1R, as well as horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody were supplied by Proteintech. Group Inc. (Chicago, USA). Enhanced chemiluminescence (ECL) kit was bought from Thermo Fisher Scientific (Carlsbad, USA). All other reagents in this study were analytical grade.

2.2. Preparation of Pumpkin Seed Polysaccharides

The isolation and purification of pumpkin seed polysaccharides were carried out based on our previous study (Cai et al., 2025). In brief, the crude polysaccharide from pumpkin seeds was obtained through continuous phase-transition extraction and deproteinized using the Sevag method and papain. The resulting crude polysaccharide was dissolved in deionized water to a concentration of 15 mg/mL. After filtration through a 0.45 μm membrane, the solution was loaded onto a DEAE-52 cellulose column and eluted with water and NaCl solutions of varying concentrations (0.1, 0.3, 0.5, and 0.7 mol/L) in a gradient manner. The eluates were collected using an automated fraction collector, and their absorbance was determined by the phenol-sulfuric acid method, yielding a single symmetrical elution peak, which was designated as PSP-1 (Figure S1A). PSP-1 was further separated on a Sephadex-75 gel column, and three elution peaks were observed after water elution, which were named PSP-1-1, PSP-1-2, and PSP-1-3, respectively (Figure S1B). Additionally, the three polysaccharide fractions (PSP-1-1, PSP-1-2, and PSP-1-3) obtained after purification of PSP-1 through a Sephadex-75 gel column were pooled and designated as PSP-1s. Table S1 presents the physicochemical properties and yields of each pumpkin seed polysaccharide fraction.

2.3. Molecular Weight Detection

The molecular weights of PSP-1-1, PSP-1-2, and PSP-1-3 were determined by high-performance gel permeation chromatography (HPGPC) (Wang et al., 2023). Accurately weighed 2 mg of each sample (PSP-1-1, PSP-1-2, and PSP-1-3) and dextran standards of varying molecular weights (5×10³, 1×10⁴, 2×10⁴, 5×10⁴, 1×10⁵, 2×10⁵, 4×10⁵, and 8×10⁵ Da) were dissolved in 1 mL of mobile phase to prepare 2 mg/mL solutions. After vortexing for 10 min, the solutions were centrifuged at 12,000 r/min for 10 min. The supernatant was collected and passed through a 0.22 µm filter membrane prior to injection. Analysis was performed on a high-performance liquid chromatography system equipped with a differential refractive index detector (Shimadzu, Japan) connected in series with TSKgel G5000 PWxl and TSKgel G3000 PWxl gel columns. A 20 µL aliquot was injected and eluted with pure water at a flow rate of 0.6 mL/min, with the column compartment maintained at 40 ℃. A standard curve of lg(molecular weight) versus retention time (RT) was constructed to obtain the molecular weight calculation equation. The chromatograms and retention times were recorded, and the retention times were substituted into the equation to calculate the molecular weights of the polysaccharides.

2.4. FT-IR Analysis

The FT-IR analysis was performed with reference to the method of Wang et al. (2023). Accurately weigh 1.3 mg each of PSP-1-1, PSP-1-2, and PSP-1-3, together with 100 mg of potassium bromide, into a dried mortar and grind thoroughly. The mixture was then uniformly ground under an infrared lamp until no crystalline particles were observed, after which it was pressed into a thin pellet and placed in a desiccator. The sample was scanned using a Vertex 70 FT-IR spectrometer (Bruker, USA) at a resolution of 4 cm^-1 over the range of 4000-400 cm^-1.

2.5. Methylation Analysis

Based on the method reported by Ji et al. (2019) with slight modifications, place 2 mg of the polysaccharide sample in a glass reaction vial. Briefly, 2 mg of sample was dissolved in 1.0 mL anhydrous dimethyl sulfoxide before the addition of dried NaOH powder (10 mg). React the mixture in a 30 ℃ magnetic stirring water bath for 60 min. Subsequently, add 2 mL of ultrapure water to terminate the reaction. Following the sequential processes of hydrolysis of the methylated polysaccharide using trifluoroacetic acid, reduction with sodium borodeuteride, and subsequent acetylation with acetic anhydride, the resultant acetylated product sample was subjected to analysis via gas chromatography-mass spectrometry (GC-MS). This analysis was conducted utilizing the Ultra instrument (Shimadzu GCMS-QP2010 Ultra, Shimadzu Co., Ltd., Kyoto, Japan), which was equipped with an RXI-5 SIL MS column (dimensions: 30 m × 0.25 mm × 0.25 μm) and a flame-ionization detector (FID). The injection port temperature was 250 ℃. The initial temperature of column was set at 120 ℃, then increased to 280 ℃ at a heating rate of 4 ℃/min and held for 5 min. The mobile phase was helium at a flow rate of 1 mL/min.

2.6. Cytotoxicity Assessment by MTT Assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed based on the methods of Sung et al. (2021) and Xu et al. (2018) with some modifications. In brief, NCI-H716 cells were cultured at 37 °C in a humidified incubator supplied with 5% CO₂ in RPMI 1640 medium containing 10% FBS, 100 IU/mL penicillin, and 100 μg/mL streptomycin. Then, 5 × 10⁴ NCI-H716 cells per well were incubated for 2 h on 96-well plates with different concentrations of pumpkin seed polysaccharide (final concentrations of 12.5, 25, 50, 100, 200, 400 μg/mL) dissolved in 1640 medium (without FBS). PSP-1, PSP-1-1, PSP-1-2, PSP-1-3, and PSP-1s were tested separately, with the Blank group containing only culture medium. After gently mixing the liquid in the wells, the cells were continuously cultured in a CO₂ incubator for 24 h. At 16 h of culture, 20 μL of 5 mg/mL MTT solution was added to each well, and incubation was continued until the end of the culture period. Thereafter, the MTT solution was discarded, and 200 μL of dimethyl sulfoxide was added to each well. Absorbance was measured at 570 nm using a multi-function microplate reader (Perkin-Elmer, Waltham, Massachusetts, USA).

2.7. Detection of GLP-1 Secretion in NCI-H716 Cells

The experiment was performed with slight modifications according to the method of Okimura et al. (2019). NCI-H716 cells in the logarithmic growth phase were collected and resuspended in DMEM complete medium. The cell suspension was seeded into 24-well plates at a density of 3×10⁵ cells per well and incubated in a humidified incubator at 37 °C with 5% CO₂ for 48 h. PSP-1, PSP-1-1, PSP-1-2, PSP-1-3, and PSP-1s were individually dissolved in PBS to prepare working solutions at a concentration of 200 μg/mL, with the pH adjusted to 7.0. The old medium in the 24-well plates was discarded, and the prepared sample solutions were added. After 2 h of incubation, the supernatants were collected into 1.5 mL centrifuge tubes and immediately measured according to the kit instructions. A Blank group without polysaccharide treatment was set as the control.

2.8. Determination of Sweet Taste Receptor Signaling Pathway-Related Gene Expression in NCI-H716 Cells

Real-time quantitative polymerase chain reaction (RT-qPCR) was performed following the methods described by Bai et al. (2005), Kim et al. (2015), and Wang et al. (2019). Total RNA was extracted with an RC112 kit. The extracted cellular total RNA was then reverse-transcribed into cDNA according to the instructions of the reverse transcription kit (R223). The obtained cDNA was amplified using a qPCR kit (Q711) and the designed primers, followed by real-time fluorescent quantitative detection (CFX Connect Real-Time Fluorescent Quantitative PCR Detection System, Bio-Rad Laboratories, Inc., Hercules, USA). Using β-actin as the internal reference gene, the relative expression levels of the T1R2, T1R3, α-gustducin, phospholipase Cβ2 (PLCβ2), inositol 1,4,5-triphate receptor (IP3R), transient receptor potential cation channel subfamily M member 5 (TRPM5), glucose transporter 2 (GLUT2), and sodium-glucose cotransporter 1 (SGLT1) genes were calculated using the relative quantification method of 2^-△△Ct. The primers designed are shown in Table S2.

2.9. Animal Experiment

All animal experimental procedures were approved by the Ethics Committee for Experimental Animals at South China Agricultural University (Animal Experiment Ethics Review No. 2024b151). All animal experiments were conducted in accordance with the ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). A total of 36 male C57BL/6N mice (12-17 g), were housed in a specific pathogen-free laboratory at the Animal Center of South China Agricultural University (Certificate Number SYXK 2024-0136). The housing conditions were maintained at a temperature range of 22 to 28 ℃ and humidity levels of 50 to 60%, with a light/dark cycle of 12 h each. The experimental methods were adapted and enhanced from Sun et al. (2022) and Yang et al. (2025). The mice were randomly assigned to either a control group (Con group, n=6), which received a standard diet (2530 kcal/kg), or a high-glucose/high-fat diet group (HFD group, n=30), which received a high-fat diet (5240 kcal/kg). After 4 weeks of dietary intervention, the HFD group received intraperitoneal injections of low-dose STZ at 50 mg/kg, while the Con group was administered citrate buffer (pH=4.5) following an overnight fast. Post-STZ injection, fasting blood glucose (FBG) levels were measured, and mice with FBG levels exceeding 14 mmol/L were further randomized into five subgroups (n=6 per group): the model group (Model group), the metformin group (Met group, 100 mg/kg/day), the low-dose PSP-1s group (L-PSP-1s group, 50 mg/kg/day), the medium-dose PSP-1s group (M-PSP-1s group, 100 mg/kg/day), and the high-dose PSP-1s group (H-PSP-1s group, 200 mg/kg/day). After establishing the model, mice in the Con and Model groups received normal saline via oral gavage for 4 weeks, while the mice in the other experimental groups were administered the respective substances through gavage. Throughout the study, the body weight, food consumption, and water intake of the mice in each group were monitored bi-daily. Additionally, FBG levels were assessed on a weekly basis, and oral glucose tolerance tests (OGTT) were administered one day prior to the conclusion of the experiment. Upon conclusion of the trial, following an overnight fasting period, the mice were euthanized, and samples of blood, pancreas, hypothalamus, and colonic contents were collected for analysis.

2.10. Determination of FBG Level

Following a 12 h fasting period, blood samples were obtained from the tail vein of the mice in each experimental group. The FBG levels were subsequently measured utilizing a glucometer (ACCU-CHEK Guide Me, Roche; Shanghai, China) in conjunction with glucose testing strips, in accordance with the manufacturer’s protocol.

2.11. OGTT

The methods developed by Sun et al. (2022) were referenced. Following a 12 h fasting period, the mice in each group received an intragastric administration of glucose at a dosage of 2 g/kg body weight. Subsequently, blood glucose levels were measured at 0, 30, 60, and 120 min post-glucose administration, utilizing the methodology outlined in Section 2.4. The corresponding area under the curve (AUC) was then calculated as follows:

AUC[mmol/(L·h)]=(BG₀+BG₃₀)/2×0.5+(BG₃₀+BG₆₀)/2×0.5+(BG₆₀+BG₁₂₀)/2×1

(1)

where BG₀, BG₃₀, BG₆₀, and BG₁₂₀ represented the blood glucose levels determined at 0, 30, 60, and 120 min, respectively.

2.12. Pancreatic Pathology Analysis

Referring to the method of Wang et al. (2024), the mice pancreas was fixed in 10% neutral formalin for 24 h, then washed with phosphate-buffered saline, dehydrated with gradient ethanol, cleared with xylene, embedded in paraffin, sectioned, and mounted on slides. After drying, the samples were dewaxed, rehydrated, stained with HE, dehydrated again, cleared, sealed with neutral balsam, and examined under a microscope.

2.13. Serum Biochemical Analysis

The blood samples collected were subjected to centrifugation at 3,000 r/min for 10 min at 4 ℃. The resultant serum was subsequently stored at -80 ℃ until further analysis. Serum concentrations of TC, TG, LDL-C, HDL-C, GSP, insulin, and GLP-1 were quantified using commercial kits in accordance with the manufacturer’s protocols. The homeostasis model assessment of insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI) were calculated as follows:

HOMA-IR=[insulin (μIU/mL)×FBG (mmol/L)]/22.5

(2)

QUICKI=1/{log [insulin (μIU/mL)]+log [FBG (mg/dL)]}

(3)

2.14. Determination of SCFAs Level in Colonic Contents

In reference to the methodology employed by Zhu et al. (2022), GC-MS was employed to measure the levels of six SCFAs—acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid—in the colon contents of mice. Weigh 50 mg of intestinal content, add 10 μL of 50% phosphoric acid. Add 600 μL of anhydrous diethyl ether, shake for 30 min, and centrifuge at 12,000 r/min for 10 min at 4 ℃. Transfer the diethyl ether layer to a tube with 10 mg of anhydrous sodium sulfate to remove water. Filter through a 0.22 μm membrane, and the sample is ready for GC-MS analysis. The SCFAs were quantified using an Agilent HP6890-5973 gas chromatograph (Agilent 122-7062, Agilent Technologies, Santa Clara, USA) equipped with a flame ionization detector. The analysis was conducted with a 60 m × 0.25 mm × 0.25 µm gas chromatography column (Agilent 122-7062, Agilent Technologies, Santa Clara, USA).

2.15. 16S rRNA Gut Microbiota Analysis

The analysis of gut microbiota was performed following the methodology outlined by Chen et al. (2023). Microbial genomes were isolated from the colonic contents of mice utilizing a DNA extraction kit (Qiagen, Inc., Shanghai, China). Subsequent to quality assessment, amplification, and purification of the extracted genomic DNA, the samples were submitted to Oebiotech Co., Ltd. (Shanghai, China) for sequencing. Utilizing the QIIME 2 software, representative sequences for each Amplicon Sequence Variant (ASV) were initially selected. Subsequently, these sequences were aligned and annotated using the Silva database (version 138) with the q2-feature-classifier plugin’s default settings in QIIME 2.

2.16. Determination of Hypothalamic GLP-1R Protein Expression in Mice

Western Blotting (WB) analysis was performed in accordance with the protocol established by Chen et al. (2018) to assess the protein expression levels of GLP-1R in the hypothalamus. In brief, murine hypothalamic tissues were homogenized in cold RIPA lysis buffer supplemented with protease inhibitors. The extracted proteins were resolved by 10% SDS-PAGE and subsequently transferred onto a PVDF membrane. The membrane was blocked with 5% non-fat milk and then incubated overnight at 4 °C with anti-GLP-1R primary antibodies. After thorough washing, the membrane was incubated with HRP-conjugated goat anti-mouse secondary antibody. Target protein expression was detected using an ECL kit, with β-actin serving as the loading control.

2.17. Determination of Appetite-Related Gene Expression in Mice

RT-qPCR was conducted with minor modifications based on the methodologies outlined by Bai et al. (2005) and Lin et al. (2023). Briefly, hypothalamus tissue was collected and transferred to a mortar pre-cooled with liquid nitrogen, then ground into a fine powder. An appropriate amount of the powder was then transferred to a 1.5 mL centrifuge tube containing lysis buffer, and immediately vortexed at high speed or pipetted repeatedly until no obvious precipitate remained in the lysis buffer. The lysate was placed on ice for 2 min, then centrifuged at 12,000 r/min at 4 °C for 5 min. The supernatant was carefully aspirated into a new 1.5 mL centrifuge tube, and the subsequent procedure was carried out according to the method described in Section 2.6. Using β-actin as the internal reference gene, the relative expression levels of the Proopiomelanocortin (POMC), Cocaine and Amphetamine-Regulated Transcript (CART), Neuropeptide Y (NPY), and Agouti-Related Protein (AgRP) gene were calculated using the relative quantification method of 2^-△△Ct. The primers designed are shown in Table S3.

2.18. Statistical Analysis

Each experiment was conducted in triplicate or more, and all data were presented as the mean ± standard deviation (SD). One-way Analysis of Variance (ANOVA) was implemented using SPSS 25.0 software (SPSS Inc., Chicago, USA). P<0.05 represented a statistical difference, while P<0.01 indicated a more significant statistical difference.

3. Results and Discussion

3.1. Molecular Weight of Pumpkin Seed Polysaccharides

As shown in Figure 1A-C, PSP-1-1, PSP-1-2, and PSP-1-3 all exhibited single symmetrical peaks with peak areas of 97%, 93%, and 89%, respectively, indicating that all three polysaccharides are homogeneous with good purity. The retention times obtained from gel permeation chromatography were 17.39 min, 23.11 min, and 28.91 min, respectively. Using the standard curve derived from dextran standards, the weight-average molecular weights were calculated to be 29,797 Da, 11,209 Da, and 4,346 Da, respectively.

3.2. FT-IR Analysis of Pumpkin Seed Polysaccharides

As shown in Figure 1D-F, all three pumpkin seed polysaccharide fractions exhibited a broad and intense absorption peak around 3360-3400 cm^-1, which is a characteristic absorption peak of polysaccharides, arising from the stretching vibration of O-H groups in the polysaccharide chains. This also indicates the presence of hydrogen bonds caused by O-H groups within or between polysaccharide molecules, resulting in peak broadening (Yan et al., 2021). Meanwhile, a characteristic absorption peak was observed around 2930 cm^-1, attributed to the stretching vibration of alkane C-H bonds, further confirming the polysaccharide nature (Zhou et al., 2022). An absorption peak appeared around 1658 cm^-1, caused by the asymmetric and symmetric stretching vibrations of C=O, indicating the presence of C=O functional groups. Additionally, the peak around 1410 cm^-1 is associated with C-O stretching vibrations, suggesting the existence of uronic acids (Wang et al., 2023). Weak absorption peaks around 1570 cm^-1 for PSP-1-2 and PSP-1-3 indicate the presence of N-H functional groups, suggesting a small amount of protein bound to the polysaccharides (Wang et al., 2023). Absorption peaks in the range of 1200-1000 cm^-1 are attributable to pyranose rings, resulting from the stretching vibration of C-O-H in the ether linkage C-O-C, indicating that all three fractions contain pyranose sugars (Rozi et al., 2020). The peak near 920 cm^-1 corresponds to D-pyranoglucosyl groups, which is consistent with the monosaccharide composition results showing that PSP-1-2 and PSP-1-3 have higher glucose content (Tang et al., 2023). Furthermore, PSP-1-1 showed absorption peaks at 874 cm^-1 and 769 cm^-1, assigned to α- and β-glycosidic linkages, respectively. In addition, PSP-1-1 displayed two peaks in the range of 1100-1010 cm^-1, suggesting the possible presence of furanose residues. In other words, PSP-1-1 may simultaneously contain α- and β-glycosidic linkages as well as both pyranose and furanose ring forms. For PSP-1-2 and PSP-1-3, peaks near 860 cm^-1 and 937 cm^-1 were also observed, indicating the possible presence of α- and β-glycosidic linkages as well (Zhong et al., 2022).

3.3. Methylation Analysis of Pumpkin Seed Polysaccharides

According to our group’s previous study (Cai et al., 2025), pumpkin seed polysaccharides were mainly composed of galactose (Gal), arabinose (Ara), glucose (Glc), and xylose (Xyl). The relative molar ratios of these four monosaccharides were 61.5 : 24.4 : 5.9 : 0.5 for PSP-1-1, 38.2 : 14.8 : 28.6 : 8.9 for PSP-1-2, and 38.8 : 14.2 : 31.7 : 7.3 for PSP-1-3. Methylation analysis was further performed to verify their monosaccharide compositions and elucidate the configurations of their glycosidic linkages.

As shown in Figure 1G and Table S4, PSP-1-1 was composed of 11 types of glycosidic linkages: Ara(f)-(1→, Gal(p)-(1→, Glc(p)-(1→, →5)-Ara(f)-(1→, →2,3)-Rha(p)-(1→, →3)-Gal(p)-(1→, →4)-Gal(p)-(1→, →6)-Gal(p)-(1→, →4,6)-Gal(p)-(1→, →3,6)-Gal(p)-(1→, and →1,3,6)-Gal(p)-(1→, with molar percentages of 20.82%, 13.04%, 5.56%, 9.93%, 1.56%, 10.70%, 8.75%, 3.75%, 2.61%, 20.85%, and 2.43%, respectively. The main constituent monosaccharide residues were arabinose and galactose, whose combined proportion was close to the monosaccharide composition, thereby verifying the monosaccharide composition results. Among the arabinose residues, the predominant form was terminal residues, indicating that most branches of this polysaccharide were terminated by arabinose. Additionally, approximately 10% of →5)-Ara(f)-(1→ was present, which was speculated to constitute part of the branches. For the other major constituent monosaccharide, galactose, the main residues were Gal(p)-(1→, →3)-Gal(p)-(1→, →4)-Gal(p)-(1→, and →3,6)-Gal(p)-(1→, indicating that galactose exhibited diverse glycosidic linkage patterns and may constitute the main chain.

As shown in Figure 1H and Table S5, PSP-1-2 was composed of 14 types of glycosidic linkages: Ara(f)-(1→, Xyl(p)-(1→, Gal(p)-(1→, Glc(p)-(1→, →5)-Ara(f)-(1→, →3)-Gal(p)-(1→, →4)-Glc(p)-(1→, →4)-Gal(p)-(1→, →6)-Gal(p)-(1→, →3,4)-Glc(p)-(1→, →2,4)-Glc(p)-(1→, →4,6)-Glc(p)-(1→, →4,6)-Gal(p)-(1→, and →3,6)-Gal(p)-(1→, with molar percentages of 8.22%, 7.69%, 1.97%, 14.63%, 8.65%, 8.07%, 15.20%, 3.18%, 2.09%, 1.92%, 1.77%, 1.36%, 1.94%, and 23.31%, respectively. The main monosaccharide residues constituting the polysaccharide were arabinose, xylose, galactose, and glucose, and their combined proportion was close to the monosaccharide composition, thereby verifying the monosaccharide composition results. In PSP-1-2, arabinose mainly existed as Ara(f)-(1→ and →5)-Ara(f)-(1→; xylose mainly existed as Xyl(p)-(1→; galactose mainly existed as →3)-Gal(p)-(1→ and →3,6)-Gal(p)-(1→; and glucose mainly existed as Glc(p)-(1→ and →4)-Glc(p)-(1→. Accordingly, it was inferred that the main terminal residues of PSP-1-2 were Ara(f)-(1→, Xyl(p)-(1→, and Glc(p)-(1→, and the main chain was likely composed of →5)-Ara(f)-(1→, →3)-Gal(p)-(1→, →4)-Glc(p)-(1→, and →3,6)-Gal(p)-(1→.

As shown in Figure 1I and Table S6, PSP-1-3 was composed of 14 types of glycosidic linkages: Ara(f)-(1→, Xyl(p)-(1→, Gal(p)-(1→, Glc(p)-(1→, →5)-Ara(f)-(1→, →3)-Gal(p)-(1→, →4)-Glc(p)-(1→, →4)-Gal(p)-(1→, →6)-Gal(p)-(1→, →3,4)-Glc(p)-(1→, →2,4)-Glc(p)-(1→, →4,6)-Glc(p)-(1→, →4,6)-Gal(p)-(1→, and →3,6)-Gal(p)-(1→, with molar percentages of 7.38%, 11.57%, 4.70%, 8.21%, 8.80%, 16.78%, 2.86%, 3.69%, 4.94%, 1.97%, 1.52%, 19.33%, 3.79%, and 4.46%, respectively. The main monosaccharide residues constituting the polysaccharide were arabinose, xylose, galactose, and glucose, and their combined proportion was close to the monosaccharide composition, thereby verifying the monosaccharide composition results. In PSP-1-3, the glycosidic linkage types of arabinose were similar to those of PSP-1-2, mainly existing as Ara(f)-(1→ and →5)-Ara(f)-(1→; xylose was also similar to PSP-1-2, mainly existing as Xyl(p)-(1→; galactose mainly existed as →3)-Gal(p)-(1→, and the →3,6)-Gal(p)-(1→ linkage that had a relatively high molar percentage in PSP-1-2 was present at a very low proportion in PSP-1-3; whereas glucose mainly existed as Glc(p)-(1→ and →4,6)-Glc(p)-(1→, which differed from PSP-1-2. Thus, it could be inferred that the main terminal residues of PSP-1-3 were Ara(f)-(1→, Xyl(p)-(1→, and Glc(p)-(1→, and the main chain was likely composed of →5)-Ara(f)-(1→, →3)-Gal(p)-(1→, and →4,6)-Glc(p)-(1→.

It was found that the three pumpkin seed polysaccharides shared certain structural similarities: their main residue compositions all contained →5)-Ara(f)-(1→ and →3)-Gal(p)-(1→, and all possessed terminal residues composed of Ara(f)-(1→ and Glc(p)-(1→. For instance, the arabinogalactan with hypoglycemic activity isolated from the tuberous cortex of white-skinned sweet potato by Ozaki et al. (2010) also contained →5)-Ara(f)-(1→ and Ara(f)-(1→ structures. PSP-1-1 additionally contained Gal(p)-(1→, whereas PSP-1-2 and PSP-1-3 had low levels of Gal(p)-(1→ and exhibited Xyl(p)-(1→ terminal residues. The main chains of PSP-1-1 and PSP-1-2 may contain →3,6)-Gal(p)-(1→ structures; similarly, He et al. (2024) found that the main chain of the hypoglycemic polysaccharide from the peel of wampee was composed of →3,6)-Gal(p)-(1→. The main chain of PSP-1-3 may be composed of →4,6)-Glc(p)-(1→, a structure also found in the hypoglycemic polysaccharide from Polygonum cuspidatum (Zhang et al., 2025). Furthermore, the →4)-Glc(p)-(1→ structure present in PSP-1-2 is also an important backbone in the hypoglycemic polysaccharides from Lanzhou lily (Hui et al., 2024) and Polygonum cuspidatum (Zhang et al., 2025).

3.4. Cytotoxic Effects of Pumpkin Seed Polysaccharides on NCI-H716 Cells

As shown in Figure 2A, within the concentration range of 12.5-200 μg/mL, none of the pumpkin seed polysaccharide fractions exhibited cytotoxicity after 24 h of treatment; cell viability was not significantly lower than that of the Blank group (P>0.05). However, when the concentration was increased to 400 μg/mL, the cell viability of the PSP-1-1 group dropped to 90.56%, which was significantly lower than that of the Blank group (P<0.05). Based on these cytotoxicity assessment results, the upper concentration limit for subsequent experiments was set at 200 μg/mL to ensure that the experimental system remained within the non-toxic range.

3.5. Effects of Pumpkin Seed Polysaccharides on GLP-1 Secretion in NCI-H716 Cells

As shown in Figure 2B, PSP-1, PSP-1-1, PSP-1-2, PSP-1-3, and PSP-1s all began to exhibit GLP-1 secretion-promoting activity at a concentration of 12.5 μg/mL. Within the concentration range of 12.5-200 μg/mL, the stimulatory effects of all pumpkin seed polysaccharide fractions on GLP-1 secretion from NCI-H716 cells were dose-dependent. Specifically, at a concentration of 200 μg/mL, compared with the Blank group, the GLP-1 secretion levels of the above polysaccharide groups were significantly increased by 35.45%, 44.41%, 47.17%, 42.61%, and 47.10%, respectively (P<0.05). Meanwhile, there were no significant differences in GLP-1 secretion among the PSP-1-1, PSP-1-2, PSP-1-3, and PSP-1s groups (P>0.05); however, all of these groups were significantly higher than the PSP-1 group (P<0.05), indicating that the biological activity of pumpkin seed polysaccharide fractions was enhanced after purification. A similar finding was reported by Wang et al. (2020), in which two purified polysaccharide fractions from Inonotus obliquus polysaccharides exhibited significantly higher α-glucosidase inhibitory activity than the unpurified fraction.

3.6. Effects of Pumpkin Seed Polysaccharides on the Gene Expression in NCI-H716 Cells

Sweet taste receptor signaling enhances glucose absorption and promotes the synthesis and secretion of GLP-1 in intestinal endocrine L cells (Smith et al., 2018). As shown in Figure 2C, all pumpkin seed polysaccharide fractions increased the gene expression levels of the sweet taste receptors T1R2 and T1R3 in NCI-H716 cells. Compared with the Blank group, the T1R2 expression levels in the PSP-1, PSP-1-1, PSP-1-2, PSP-1-3, and PSP-1s groups were increased by 6.32-, 11.11-, 15.25-, 9.14-, and 18.95-fold, respectively (P<0.01), while the T1R3 expression levels were increased by 1.28-, 2.30-, 4.63-, 1.98-, and 4.57-fold, respectively (P<0.01). These results confirm that all pumpkin seed polysaccharide fractions promoted the gene expression of sweet taste receptors on L cells, and further demonstrate that the biological activity of the polysaccharides was enhanced after purification. A similar finding was reported by Yang et al. (2021), who showed that the gene expression levels of intestinal sweet taste receptors T1R2 and T1R3 were decreased in diabetic rats, whereas Astragalus polysaccharides could increase the expression of T1R2 and T1R3, which was identified as a key mechanism underlying the hypoglycemic effect of this polysaccharide.

Figure 2C also showed that, compared with the Blank group, the α-gustducin expression levels in the PSP-1, PSP-1-1, PSP-1-2, PSP-1-3, and PSP-1s groups were increased by 2.45-, 3.79-, 4.40-, 3.36-, and 5.04-fold, respectively (P<0.01); the PLCβ2 expression levels were increased by 1.83-, 3.60-, 1.96-, 2.44-, and 4.37-fold, respectively (P<0.01); the IP3R expression levels were increased by 0.61-, 1.55-, 1.85-, 1.40-, and 1.96-fold, respectively (P<0.01); and the TRPM5 expression levels were increased by 1.02-, 3.75-, 3.74-, 3.21-, and 4.47-fold, respectively (P<0.01). These results indicate that all pumpkin seed polysaccharide fractions significantly upregulated the gene expression levels of α-gustducin, PLCβ2, IP3R, and TRPM5 in NCI-H716 cells (P<0.01), suggesting that the α-gustducin/PLCβ2/IP3R/TRPM5 signaling cascade was activated. α-Gustducin is a G protein that serves as a downstream signaling molecule of the sweet taste receptors T1R2 and T1R3, and is involved in sweet, umami, and bitter taste receptor signal transduction (Xie et al., 2020; Yang et al., 2021). Jang et al. (2007) observed in α-gustducin gene knockout mice that GLP-1 secretion was significantly reduced after oral glucose administration, and the regulation of plasma insulin and glucose levels was impaired. Further studies on isolated mouse intestines confirmed that the absence of α-gustducin severely impaired the intestinal GLP-1 secretory response to glucose stimulation. Upon activation, α-gustducin further activates PLCβ2 (Macrae et al., 2025), which generates IP3. IP3 binds to the Ca²⁺ channel IP3R on the endoplasmic reticulum, leading to the release of Ca²⁺ from the endoplasmic reticulum into the cytoplasm (Diepeveen et al., 2022; Palmer, 2007). The elevated intracellular Ca²⁺ concentration directly activates the Ca²⁺-sensitive monovalent cation channel TRPM5 (primarily permeable to Na⁺ and K⁺), causing Na⁺ influx and generating a depolarizing receptor potential (Ohtsu et al., 2014; Wang et al., 2018). When depolarization reaches a certain threshold, voltage-gated sodium channels (VGSCs) open, generating an action potential. The action potential propagates along the cell membrane, causing voltage-gated calcium channels (VGCCs) to open, through which extracellular Ca²⁺ rapidly flows into the cell, bringing the cytoplasmic Ca²⁺ concentration to its peak. The sharply elevated cytoplasmic Ca²⁺ then acts as the final messenger, triggering GLP-1-containing secretory vesicles to translocate toward the cell membrane. Therefore, it is speculated that all pumpkin seed polysaccharide fractions promote GLP-1 secretion by activating the sweet taste receptor and its associated signaling molecules (α-gustducin, PLCβ2, IP3R, TRPM5) in L cells, a mechanism closely related to the cascade activation of the aforementioned signaling pathway.

GLUT2 and SGLT1 are important glucose transporters in the intestine. SGLT1 forms a ternary complex with Na⁺ and glucose, utilizing the Na⁺ electrochemical gradient to transport glucose into the cell against its concentration gradient via secondary active transport, whereas GLUT2 facilitates glucose entry into the cell through passive transport. Together, they maintain glucose metabolic homeostasis (Koepsell, 2020). Diabetes has been shown to cause abnormally increased expression of GLUT2 and SGLT1 in the intestine (Wemelle et al., 2022). Lin et al. (2021) found that in vitro fecal bacterial fermentation of flaxseed polysaccharides downregulated the mRNA and protein expression levels of GLUT2 and SGLT1 in Caco-2 cells, thereby effectively inhibiting glucose uptake in this cell model. In the present study, Figure 2D showed that, compared with the Blank group, the GLUT2 gene expression levels in the PSP-1-1 and PSP-1s groups were significantly decreased by 20.26% and 17.56%, respectively (P<0.05), while the SGLT1 gene expression levels were significantly decreased by 18.80% and 18.92%, respectively (P<0.05). These results suggest that these two pumpkin seed polysaccharide fractions may also influence cellular metabolic function by suppressing the expression of glucose transporter-related genes. In contrast, PSP-1, PSP-1-2, and PSP-1-3 had no significant effects on the gene expression levels of GLUT2 and SGLT1 in NCI-H716 cells (P>0.05). A similar phenomenon was reported by Yang et al. (2023), who found that Polygonatum odoratum polysaccharides, which promoted GLP-1 secretion, had no significant effect on the mRNA expression of intestinal GLUT2 and SGLT1 in diabetic rats.

Taken together, all pumpkin seed polysaccharide fractions upregulated the gene expression levels of sweet taste receptors, α-gustducin, PLCβ2, IP3R, and TRPM5 in NCI-H716 cells. These findings suggest that pumpkin seed polysaccharides may promote GLP-1 secretion from L cells by activating the sweet taste receptor/α-gustducin/PLCβ2/IP3R/TRPM5/GLP-1 signaling pathway, thereby exerting hypoglycemic effects. Meanwhile, PSP-1-1 and PSP-1s may also influence cellular glucose metabolic balance by suppressing the expression of glucose transporter-related genes.

Furthermore, the combined collected fraction PSP-1s induced significantly higher T1R2 gene expression than all other treatment groups (P<0.05), and exhibited the highest gene expression levels of α-gustducin, PLCβ2, IP3R, and TRPM5 among all groups, with no significant difference from the purified fraction PSP-1-1 (P>0.05). Additionally, the GLUT2 and SGLT1 gene expression levels in the PSP-1s group were comparable to those in the PSP-1-1 group (P>0.05) and were significantly lower than those in all other treatment groups (P<0.05). These results indicate that the combined collected fraction PSP-1s may also exhibit synergistic effects among its components. A similar finding was reported by Hu et al. (2022) in their study on Osmunda japonica polysaccharide (WOJP), in which the two fractions WOJP-N and WOJP-A, which were responsible for the antioxidant activity, exhibited synergistic antioxidant effects. Therefore, the combined collected fraction PSP-1s was selected for subsequent animal experiments.

3.7. Effects of PSP-1s on Body Weight, Food Consumption, and Water Intake in Diabetic Mice

As depicted in Figure 3A, over the course of the 4-week period, the body weight trajectory of the Con group demonstrated an overall upward trend. Conversely, the Model group mice experienced a weekly reduction in body weight, which was significantly lower than the pre-intervention level (week 0) by week 4 (P<0.01), and also significantly lower than that of mice in all experimental groups except the Con group (P<0.05). Figure 3B and 3C demonstrate that the diabetic mice exhibited a marked increase in both food consumption and water intake compared to the mice in the Con group. (P<0.05), indicating that STZ-induced modeling had already impacted the physiological activities of the mice. At week 4, the food consumption and water intake of mice in the Model group were significantly higher than those in all other groups (P<0.05). This pattern aligns with findings from pertinent studies. In diabetic mice, accelerated lipid degradation and enhanced protein catabolism contribute to muscle atrophy and weight loss (Castillo et al., 2025). Regarding food and water intake, diminished glucose uptake by peripheral tissues, such as muscle and adipose tissue, results in an inadequate cellular energy supply, thereby stimulating appetite through hypothalamic feeding centers, such as the activation of NPY/AgRP neurons (Alkhalidy et al., 2018). Additionally, osmotic diuresis caused by hyperglycemia exacerbates polydipsia (Talbot et al., 2024).

Following the administration of PSP-1s at doses of 100 or 200 mg/kg, the body weight of the diabetic mice initially decreased during the first week but subsequently recovered, ultimately surpassing the pre-intervention levels by week 4 (P<0.05 and <0.01, respectively), while their food consumption decreased markedly (P<0.05 and <0.01, respectively). Moreover, the water intake of mice across all groups receiving varying doses of PSP-1s demonstrated an initial increase, followed by a decrease over the 4-week period. These findings suggest that PSP-1s can mitigate weight loss and ameliorate symptoms of polyphagia and polydipsia in diabetic mice.

3.8. Effects of PSP-1s on FBG and Glucose Tolerance in Diabetic Mice

As depicted in Figure 3D, the FBG levels of mice in the Con group remained within the normal physiological range, while those on a high-glucose/high-fat diet with STZ had FBG levels above 14 mmol/L, meeting the criteria for experimental research. Beginning in week 2, the FBG levels in mice from the Model group were significantly elevated compared to all other groups (P<0.05) and a marked increase from pre-intervention levels by week 4 (P<0.01). By week 4, the FBG levels of mice in the Met group, as well as those receiving various doses of PSP-1s, exhibited a significant decrease compared to pre-intervention levels (all P<0.01). Metformin demonstrated the most pronounced effect, followed sequentially by PSP-1s at doses of 200 mg/kg, 100 mg/kg, and 50 mg/kg. Among them, intervention with 200 mg/kg PSP-1s resulted in a reduction of FBG in diabetic mice from 18.46 ± 1.58 mmol/L pre-intervention to 12.60 ± 1.67 mmol/L. These findings suggest that PSP-1s effectively reduces FBG in type 2 diabetic mice, with the efficacy of FBG improvement becoming more pronounced with increasing doses of PSP-1s. A similar phenomenon was reported by Wang et al. (2020). They discovered that a polysaccharide from the Gynostemma pentaphyllum herb reduced FBG levels in a dose-dependent manner over 4 weeks.

Glucose tolerance pertains to the body’s capacity to regulate blood glucose levels following the ingestion of external glucose sources. A greater degree of impaired glucose tolerance is associated with an increased AUC (Gileles-Hillel et al., 2022). As illustrated in Figure 3E and 3F, the AUC value for mice in the Model group was significantly elevated compared to each of the experimental groups (P<0.05). The AUC values were reduced in both the Met group and the groups administered varying doses of PSP-1s (P<0.05). Within the PSP-1s groups, the mice in the H-PSP-1s group exhibited the most pronounced improvement in glucose tolerance, with their AUC value being 36.93% lower than that of the Model group. This was followed by the M-PSP-1s and L-PSP-1s groups, between which no significant difference was observed (P>0.05). These findings suggest that PSP-1s has the potential to ameliorate glucose intolerance in diabetic mice to a certain extent. Similar findings were also observed in the study by Lin et al. (2023), where glucose tolerance was severely impaired in diabetic mice. After intervention with Acanthopanax trifoliatus (L.) Merr polysaccharides, the AUC of the mice decreased significantly.

3.9. Effects of PSP-1s on Pancreatic Pathology in Diabetic Mice

As illustrated in Figure 4, the pancreatic tissue of mice in the Con group exhibits no pathological damage, characterized by clearly defined islet edges and a relatively high density of uniformly distributed islet cells. Compared with mice in the Model group, the atrophy of islet tissue is improved to some extent after intervention with metformin or different doses of PSP-1s. Specifically, there is an increase in islet volume to varying extents, clearer delineation of boundaries, and an augmented number of islet cells. These findings suggest that PSP-1s intervention effectively mitigates pancreatic tissue damage in diabetic mice. A comparable phenomenon of islet repair resembles that reported by Tong et al. (2023) regarding low molecular weight polysaccharides obtained from Laminaria japonica, which exhibit hypoglycemic activity.

3.10. Effects of PSP-1s on Serum Biochemical in Diabetic Mice

The study by Diamond et al. (2021) indicated that hyperglycemia is closely associated with dyslipidemia, and that type 2 diabetes complications are frequently accompanied by abnormal lipid profiles. As depicted in Figure 5A-D, the serum levels of TC, TG, and LDL-C were significantly elevated in type 2 diabetic mice compared to the Con group, while the HDL-C level was significantly reduced (P<0.05). These findings suggest that the diabetic mice exhibited pronounced lipid metabolism abnormalities. The experimental data demonstrated that PSP-1s ameliorated the abnormalities in the four serum lipid markers in diabetic mice, with the ameliorative effect becoming more pronounced with increasing concentrations of PSP-1s. Specifically, in the H-PSP-1s group, the serum levels of TC, TG, and LDL-C were 26.06%, 44.12%, and 22.71% lower, respectively, than those in the Model group mice (P<0.05). The HDL-C level in the H-PSP-1s group was 60.93% higher than that in the Model group mice, and the improvement effects on TG and HDL-C were comparable to those of 100 mg/kg metformin (P>0.05). This improvement may be due to enhanced glucose utilization and reduced lipolysis, which decrease free fatty acid production and increase fatty acid absorption in tissues (Tsujita, 2006).

GSP level provide an assessment of average blood glucose concentrations over the preceding 2 to 3 weeks, making them a reliable marker for short-term glycemic monitoring (Chen et al., 2019). As shown in Figure 5E, the GSP level in the Model group mice was 150.74% higher than that in the Con group (P<0.05). The GSP levels in the Met group, M-PSP-1s group, and H-PSP-1s group were significantly reduced in comparison to the Model group (P<0.05). The GSP level in mice of the H-PSP-1s group was 25.02% lower than that in the model group.

In type 2 diabetes, the body exhibits insulin resistance, resulting in a compensatory hypersecretion of insulin (Xu et al., 2012). As shown in Figure 5F-H, the serum insulin level in the Model group mice was significantly higher than that in the Con group, indicating enhanced insulin resistance and reduced insulin sensitivity (P<0.05). The serum insulin levels in the Met group, M-PSP-1s group, and H-PSP-1s group mice were significantly lower (P<0.05). In addition, the HOMA-IR in the H-PSP-1s group was 49.95% lower than that in the Model group (P<0.05), and the QUICKI was 9.17% higher (P<0.05). The administration of 200 mg/kg PSP-1s resulted in a reduction of HOMA-IR that was 81.98% as effective as the reduction achieved with 100 mg/kg metformin, and an increase in QUICKI that was 71.19% as effective as that achieved with 100 mg/kg metformin. Similar to the study by Song et al. (2024), which discovered that after 4 weeks of intervention with Artemisia argyi polysaccharide in diabetic mice, insulin levels decreased and the QUICKI improved in a dose-dependent manner, the PSP-1s in our present study also exhibited dose-dependent effects in reducing insulin levels and the Homeostatic Model Assessment of HOMA-IR, while enhancing QUICKI.

GLP-1, a pivotal incretin hormone, is essential for the regulation of glucose homeostasis and the preservation of pancreatic β-cell functionality (Boer et al., 2020). As a significant mediator of the gut-brain axis, GLP-1 influences this axis through the hypothalamic appetite regulation center, thereby exerting further advantageous effects on blood glucose management by enhancing satiety and suppressing appetite (Richards et al., 2021). In diabetic murine models, intestinal dysbiosis and diminished production of SCFAs impede the secretion of GLP-1 from intestinal L cells. Additionally, the cytotoxic effects of STZ on β-cells exacerbate metabolic disturbances, resulting in a further decline in GLP-1 secretion (Yoshimura et al., 2021). As illustrated in Figure 5I, the GLP-1 concentration in the Model group mice was reduced by 21.02% compared to the Con group. The GLP-1 level in mice from the H-PSP-1s group was 106.06 pg/mL, which was 16.01% higher than that in mice from the Model group and 14.21% higher than that in mice from the Met group (P<0.05). Comparable findings were documented in the studies conducted by Wang et al. (2024) and Yang et al. (2025), demonstrating that polysaccharides exhibited greater efficacy than metformin in enhancing GLP-1 secretion. The results suggest that PSP-1s can promote GLP-1 secretion in diabetic mice, and the serum GLP-1 level increases with the increase in PSP-1s concentration, indicating that PSP-1s is a potential GLP-1 agonist.

3.11. Effects of PSP-1s on SCFAs Level in the Colonic Contents of Diabetic Mice

SCFAs are the terminal products of polysaccharide metabolism by the gut microbiota, serving as chemical mediators that facilitate communication between the microbiota and the host (Nie et al., 2021; Zhang et al., 2019). As modulators of food intake and energy metabolism, an elevation in SCFA levels triggers the release of the downstream signaling molecule GLP-1, thereby exerting an appetite-suppressing effect (Medawar et al., 2021). More precisely, SCFAs activate the cyclic adenosine monophosphate (cAMP) signaling pathway by targeting G protein-coupled receptors (GPR41 and GPR43) located on L cells, which in turn promotes the transcription and expression of the GLP-1 gene. This process results in enhanced synthesis and secretion of GLP-1 in the intestine, ultimately improving metabolism via the gut-brain axis (Dalile et al., 2019).

Based on the findings discussed above, PSP-1s at a concentration of 200 mg/kg demonstrated significantly enhanced hypoglycemic activity relative to the other doses assessed. Therefore, the 200 mg/kg dosage was chosen for further exploration of PSP-1s’s effects in subsequent studies. As shown in Table 1, compared with the Con group, the contents of acetic acid, propionic acid, butyric acid, valeric acid, and isovaleric acid in mice of the Model group all decreased significantly. This indicates that diabetes induced by a high-glucose/high-fat diet combined with STZ reduces the levels of SCFAs in the intestinal tract of mice. Intervention with 200 mg/kg PSP-1s increased the contents of acetic acid, propionic acid, butyric acid, valeric acid, and isovaleric acid in the colon contents of diabetic mice (P<0.05), and the effect showed no significant difference from that of 100 mg/kg metformin (P>0.05). Similar findings have been reported, such as the study by Zhao et al. (2025), which revealed that patients with diabetes exhibited lower levels of total SCFAs in their feces, and following intervention with Scrophularia ningpoensis polysaccharide, the levels of acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid increased.

Acetic acid and propionic acid are SCFAs with relatively high contents in the intestinal tract of mice. Acetic acid serves as a crucial signaling molecule in glucose metabolism, capable of improving glucose tolerance and inhibiting weight gain in diabetic and obese rats (Yamashita et al., 2007). Propionic acid participates in the gluconeogenesis pathway, suppresses the production of fat and cholesterol, increases postprandial GLP-1 levels, and enhances insulin sensitivity (Chambers et al., 2015). Under PSP-1s intervention, the contents of acetic acid and propionic acid in the colon contents of mice increased by 87.32% and 57.14% respectively compared with those in mice of the Model group. The results indicate that PSP-1s can restore the content of intestinal SCFAs.

3.12. Effects of PSP-1s on the Gut Microbiota in Diabetic Mice

The gut microbiota modulates host energy metabolism and feeding behavior. A growing consensus in the scientific community recognizes these microbial communities as critical determinants of both health and disease states. For example, Yao et al. (2020) demonstrated that Cyclocarya paliurus polysaccharides alleviate type 2 diabetes symptoms in rats by lowering blood glucose and insulin levels and modifying gut microbiota. The proposed mechanism involves an increase in SCFAs-producing bacteria, which enhances the production of SCFAs.

From Figure 6A, it can be seen that as the sample size increases, the species accumulation curve gradually flattens out, indicating that the total number of ASVs in the microbiota will no longer increase significantly with the addition of new samples. This suggests that the sample size well reflects the richness of the microbial community. As shown in Figure 6B, a total of 1046 ASVs were detected in the gut microbiota of mice in the Con group, Model group, and H-PSP-1s group. Among them, 398, 144, and 163 ASVs were unique to the Con group, Model group, and H-PSP-1s group, respectively. The Model group had the fewest unique ASVs and the lowest total number of ASVs, suggesting that the composition and diversity of its gut microbiota were disrupted. This phenomenon may be attributed to elevated blood glucose levels, which stimulate the cells of the intestinal epithelium to produce substantial quantities of serotonin, thereby disrupting the gut microbiota (Xiu et al., 2023). In addition, there were 153 ASVs shared between the Con group and the Model group, 171 ASVs shared between the Con group and the H-PSP-1s group, and 283 ASVs shared between the Model group and the H-PSP-1s group. The results showed that although the gut microbiota of the three groups of mice shared a common microbial community, each group still had its own unique microbial characteristics, and PSP-1s intervention might have promoted the formation of characteristic gut microbiota while increasing microbial diversity.

As shown in Figure 6D and 6G, the dominant phyla in the gut microbiota of mice in each group were Bacteroidota, Firmicutes, Desulfobacterota, Verrucomicrobiota, Proteobacteria, Actinobacteriota, Deferribacterota, etc. Among them, Firmicutes and Bacteroidota were the dominant phyla, with a relative abundance of >85%. Diabetes can disrupt the balance of these two phyla in the gut microbiota, leading to an increase in the Firmicutes/Bacteroidota (F/B) ratio (Han et al., 2023). As shown in Figure 6C, the F/B ratio in the Model group was significantly higher than that in the Con group (P<0.05). After PSP-1s intervention, the F/B ratio in diabetic mice significantly decreased compared to the Model group (P<0.05), an effect also observed with Lycium barbarum polysaccharide (Ma et al., 2022), which exhibit hypoglycemic activity. In addition, the abundance of Desulfobacterota in the gut microbiota of the Model group was lower than that in the Con group, while the abundance of Desulfobacterota in the gut microbiota of the H-PSP-1s group was higher than that in the Model group. The H-PSP-1s group had an increased abundance of Verrucomicrobiota compared to both the Con group and the Model group. Similar findings were reported in the study by Hu et al. (2021), which demonstrated that extracellular polysaccharides produced by Sporidiobolus pararoseus could enhance the relative abundance of Desulfobacterota and Verrucomicrobiota, promote the proliferation of beneficial bacteria in the human and mouse gut, and stimulate the production of SCFAs and amino acid metabolism, thereby improving intestinal health.

As shown in Figure 6E and 6H, the dominant families in the gut microbiota of mice in each group were Muribaculaceae, Rikenellaceae, Lachnospiraceae, Bacteroidaceae, Prevotellaceae, Oscillospiraceae, Desulfovibrionaceae, Marinifilaceae, Akkermansiaceae, Ruminococcaceae, etc. The Model group had increased abundances of Rikenellaceae, Oscillospiraceae, and Ruminococcaceae in the gut microbiota compared to the Con group, while the H-PSP-1s group had decreased abundances of these bacteria compared to the Model group. The Muribaculaceae family is recognized as a beneficial group of bacteria that contributes to maintaining intestinal homeostasis, modulating the composition of gut microbiota, and enhancing the secretion of SCFAs in diabetic mice (Zhu et al., 2024). Song et al. (2024) identified that Artemisia argyi polysaccharide can increase the abundance of Muribaculaceae. In the present study, the abundance of Muribaculaceae in the Model group mice was lower than that in the Con group mice, and PSP-1s intervention led to an upregulation of Muribaculaceae abundance.

As shown in Figure 6F and 6I, at the genus level, the dominant bacterial genera in the intestinal tracts of mice across different groups are Muribaculaceae, Bacteroides, Alistipes, Alloprevotella, Rikenellaceae RC9 gut group, Lachnospiraceae NK4A136 group, Akkermansia, Colidextribacter, Clostridia vadinBB60 group, Blautia, etc. In comparison to the Model group, the abundance of Muribaculaceae increased in mice following PSP-1s intervention. Furthermore, relative to both the Con and Model groups, the abundances of Bacteroides, Akkermansia, Clostridia vadinBB60 group, and Blautia in the gut microbiota of mice treated with PSP-1s were elevated. Previous research has indicated that Muribaculaceae (Murakami et al., 2023), Bacteroides (Wu et al., 2019), Akkermansia (Chu et al., 2022), and Blautia (Holmberg et al., 2024) are capable of producing SCFAs. Notably, Akkermansia muciniphila, a species within the Akkermansia genus, has attracted significant attention in recent studies focused on mitigating the risks associated with diabetes and obesity. Evidence suggests that this bacterium can enhance glucose homeostasis in mice and reduce the risks of conditions such as diabetes, obesity, inflammatory bowel disease, and colorectal cancer. Additionally, a novel protein (P9) secreted by Akkermansia muciniphila has been shown to stimulate the secretion of GLP-1 (Cani et al., 2021; Yoon et al., 2021).

The results of the α-diversity analysis are shown in Figure 7A-D and Table S7. The Good’s Coverage index of all groups was close to 1, indicating a high reliability of the sequencing results. The Chao1, Observed Species, and ACE indices in the Model group were significantly lower than those in the Con group and the H-PSP-1s group (P<0.05), while there was no significant difference between the Con group and the H-PSP-1s group (P>0.05). This suggests that the richness of the gut microbiota in the Model group was reduced, and PSP-1s intervention could restore the richness of the gut microbiota. The results of the β-diversity analysis are shown in Figure 7E and 7F. The PCoA results showed that the microbial community compositions of samples within each group were similar, but there were differences between groups. This indicates that while PSP-1s alleviated the abnormal changes in the gut microbiota of diabetic mice, it also promoted the formation of a microbial characteristic pattern different from that of the Con group and the Model group.

Utilizing linear discriminant analysis (LDA) effect size (LEfSe) analysis (LDA>3, P<0.05), we identified distinct species exhibiting statistically significant differences across groups. The results are shown in Figure 8. Among the differential species in the gut microbiota of the H-PSP-1s group, Bacteroidaceae, Bacteroides (Wu et al., 2019), Verrucomicrobiae, Verrucomicrobiales, Akkermansiaceae, Akkermansia (Chu et al., 2022), Blautia (Holmberg et al., 2024), Odoribacter (Wan et al., 2023), Bifidobacteriales, Bifidobacteriaceae, and Bifidobacterium (Usta-Gorgun et al., 2020) have all been reported as beneficial bacteria capable of producing SCFAs. The Clostridia vadinBB60 group also belongs to beneficial bacteria (Song et al., 2023). In a human study, Sanmiguel et al. (2017) found that Akkermansia was significantly associated with a reduction in hedonic eating, while Bilophila showed a significant association with weight loss. The above results indicate that PSP-1s promotes the increase of beneficial bacteria, especially those microbial communities capable of producing SCFAs. Some polysaccharides with hypoglycemic effects exhibit similar outcomes. For instance, the study by Pan et al. (2022) demonstrated that Morchella importuna polysaccharide can reverse intestinal diseases caused by type 2 diabetes by increasing the abundances of Akkermansia and Blautia, and the research by Ni et al. (2025) showed that Ramaria botrytoides polysaccharide can improve the gut microbiota of diabetic mice and also increase the abundances of Bacteroides, Odoribacter, and Akkermansia.

3.13. Effects of PSP-1s on the Protein Expression in the Hypothalamus of Diabetic Mice

GLP-1 exerts its effects on the GLP-1R located within the arcuate nucleus (ARC) of the hypothalamus, a region recognized as a critical center for the regulation of feeding behavior (Li et al., 2025). Activation of the GLP-1R facilitates alterations in feeding behavior and stimulates insulin secretion, thereby influencing abnormal blood glucose fluctuations associated with dietary intake (Darsalia et al., 2014). Prior studies have demonstrated that the GLP-1/GLP-1R system within the brain-gut axis plays a mediating and beneficial role in maintaining glucose homeostasis in patients with type 2 diabetes (Li et al., 2021). The study by Yang et al. (2025) found that Finger citron polysaccharide could increase the serum GLP-1 level in diabetic mice and activate the expression of GLP-1R in the hypothalamus. As shown in Figure 9, the protein expression level of GLP-1R in the hypothalamus of mice in the Model group was 16.35% lower than that in the Con group, showing a significant decrease (P<0.01). After intervention with PSP-1s, the protein expression level of GLP-1R in the hypothalamus was significantly increased (P<0.01), reaching 72.96% of that in the Con group. Our data demonstrated that the expression of GLP-1R protein in the hypothalamus was significantly upregulated in the H-PSP-1s group, suggesting that GLP-1 in the central nervous system exerted an anorexigenic effect (Turton et al., 1996).

3.14. Effects of PSP-1s on the Gene Expression in the Hypothalamus of Diabetic Mice

POMC/CART neurons and NPY/AgRP neurons are two independent neuronal populations in the ARC of the hypothalamus, playing a pivotal role in regulating appetite (Li et al., 2025). Two of these genes, POMC and CART, are anorexigenic neuropeptides whereas the other two, NPY and AgRP are orexigenic (Bai et al., 2005). For instance, saponins are believed to suppress appetite and subsequently reduce energy intake by regulating POMC/CART neurons and NPY/AgRP neurons in the hypothalamus, the appetite control center (Cao et al., 2024). The research conducted by Li et al. (2025) revealed that the administration of highly-expanded konjac fiber and konjac powder resulted in increased plasma GLP-1 levels in rats, accompanied by enhanced protein expression of GLP-1R in the hypothalamus. Simultaneously, there was a downregulation of NPY and AgRP gene expression in the hypothalamus, which collectively contributed to a decrease in food consumption and mitigated weight gain. Nevertheless, there is a lack of reported studies examining polysaccharides in a similar context.

As shown in Figure 10, in the hypothalamus of mice in the H-PSP-1s group, the expression level of the POMC gene was significantly increased by 241.70% compared to that in the Model group mice (P<0.05). Compared with both the Con group and the Model group mice, the expression level of the CART gene was significantly increased by 159.61% and 3020.35%, respectively (P<0.05). This indicates that PSP-1s enhances the expression of appetite suppression-related genes in the hypothalamus. Conversely, in the hypothalamus of mice in the H-PSP-1s group, the expression level of the NPY gene was significantly decreased by 30.93% and 82.99%, respectively, compared to the Con group and the Model group mice (P<0.05). The expression level of the AgRP gene was significantly decreased by 79.04% compared to the Model group mice (P<0.05). This suggests that PSP-1s inhibits the expression of appetite-stimulating genes in the hypothalamus. These results indicate that PSP-1s exerts an appetite-suppressing effect by regulating the expression of appetite-related genes in the hypothalamus.

4. Conclusions

This study revealed that the three pumpkin seed polysaccharide fractions, PSP-1-1, PSP-1-2, and PSP-1-3, had weight-average molecular weights of 29,797 Da, 11,209 Da, and 4,346 Da, respectively, and all were likely to contain both α- and β-glycosidic linkages. PSP-1-1 was composed of 11 types of glycosidic linkages, with arabinose, galactose, and glucose as the predominant sugar residues. Both PSP-1-2 and PSP-1-3 consisted of 14 types of glycosidic linkages, with arabinose, xylose, galactose, and glucose as the main sugar residues. All three fractions shared common structural units, including Araf-(1→, Glcp-(1→ as terminal residues, as well as →5)-Araf-(1→ and →3)-Galp-(1→ linkages.

Pumpkin seed polysaccharides may directly stimulate the sweet taste receptors on the surface of L cells, thereby enhancing the biosynthesis and release of GLP-1. Each fraction promoted GLP-1 secretion from L cells in a dose-dependent manner and significantly upregulated the gene expression of sweet taste receptors (T1R2, T1R3), α-gustducin, PLCβ2, IP3R, and TRPM5, suggesting activation of the sweet taste receptor/α-gustducin/PLCβ2/IP3R/TRPM5/GLP-1 signaling pathway to enhance GLP-1 release. Moreover, PSP-1-1 and PSP-1s may additionally influence cellular glucose metabolic balance by suppressing the expression of glucose transporter-related genes. Notably, the pooled fraction PSP-1s exhibited the highest expression levels across multiple key genes compared with any individual fraction, indicating potential synergistic effects among the three components. Consequently, PSP-1s was selected for subsequent animal experiments.

In diabetic mice, PSP-1s alleviated weight loss, reduced food and water intake, and lowered blood glucose levels. It also improved glucose tolerance, repaired pancreatic islet damage, ameliorated dyslipidemia, and decreased serum levels of GSP, insulin, and HOMA-IR while increasing QUICKI. These beneficial effects exhibited a clear dose-dependent relationship. Mechanistically, PSP-1s facilitated GLP-1 secretion, elevated SCFAs concentrations in colonic contents, and restructured the gut microbiota in diabetic mice. Specifically, it increased the relative abundance of beneficial bacteria capable of producing SCFAs favorable to host health. Following PSP-1s intervention, GLP-1R protein and appetite-suppressing gene expression were elevated in the hypothalamus, whereas appetite-stimulating gene expression was reduced.

In summary, PSP-1s may modulate GLP-1 secretion through two distinct mechanisms to exert hypoglycemic effects. On one hand, PSP-1s reshapes gut microbiota composition and increases SCFA levels derived from microbial metabolism, thereby promoting GLP-1 secretion by intestinal L cells. On the other hand, PSP-1s may directly activate sweet taste receptors on the surface of L cells, enhancing GLP-1 biosynthesis and release. Upon recognition of these GLP-1 signals by hypothalamic GLP-1R, blood glucose regulation is ultimately achieved. In other words, PSP-1s is highly likely to regulate hypoglycemic activity via the GLP-1 signaling pathway of the gut-brain axis. Overall, these findings highlight the potential of PSP-1s in ameliorating type 2 diabetes and provide novel mechanistic insights into its mode of action.