Separation, Purification, Structural Characterization and Hypoglycemic Effect Study of Homogeneous Mori fructus Polysaccharide

TL;DR

This study isolates and characterizes a homogeneous polysaccharide from Mori fructus and shows it has strong hypoglycemic effects.

Contribution

The study provides the first detailed structural and functional analysis of a homogeneous Mori fructus polysaccharide.

Findings

MFP-III has a molecular weight of 6.83 kDa and is composed of rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid.

MFP-III strongly inhibits α-glucosidase and α-amylase, showing better activity than acarbose at the same concentration.

The structural backbone of MFP-III is →2,4)-α-L-Rhap-(1 → 4)-α-D-GalpA-(1→ with potential branching units.

Abstract

Background/Objectives: Mori fructus polysaccharides are key bioactive components with diverse activities, but structural characterization of homogeneous fractions remains limited, hindering insights into structure–activity relationships. This study addresses this gap by isolating and characterizing a homogeneous polysaccharide (MFP-III) from M. fructus. Methods: MFP-III, representing the final gel-filtration homogeneous fraction, was purified using defined procedures: DEAE-52 cellulose chromatography followed by Sephadex G-100 gel filtration. Purity and homogeneity were validated by high-performance liquid chromatography (HPLC). Structural characteristics were analyzed via HPLC, GC-MS, FTIR, and NMR spectroscopy. Meanwhile, hypoglycemic activity of MFP-III was evaluated. Results: MFP-III (94.2 ± 2.6%) has a molecular weight of approximately 6.83 kDa, primarily composed of rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid. Its backbone structure is presumed to be →2,4)-α-L-Rhap-(1 → 4)-α-D-GalpA-(1→, with branching units potentially attached to O-4. MFP-III demonstrated significant inhibitory activity against α-glucosidase (IC50 = 1.56 mg/mL) and α-amylase (IC50 = 2.07 mg/mL), stronger than acarbose at equivalent concentrations. Conclusions: The findings provide preliminary insights into the hypoglycemic structure–activity relationship of MFP-III, providing data support for the development of blood glucose-lowering natural inhibitors, and offering a theoretical foundation for advancing the application of polysaccharides from other sources.

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Full text

1. Introduction

Diabetes, a global health challenge characterized by chronic hyperglycemia leading to multi-organ damage. Projections from the International Diabetes Federation (IDF) indicate that over 780 million individuals (>90.0% having type II diabetes (T2DM)) worldwide will develop diabetes by 2045 [1,2]. Controlling postprandial glucose excursions is a central therapeutic goal in T2DM management. α-Amylase and α-glucosidase are dual therapeutic targets, yet first-line inhibitors like acarbose cause dose-dependent gastrointestinal adverse effects [3,4]. These limitations have spurred increased investigation into natural products to identify novel candidates with targeted inhibitory efficacy and improved intestinal tolerance.

Plant-derived polysaccharides exhibit hypoglycemic potential with superior biocompatibility and lower toxicity [5]. They exert hypoglycemic effects through mechanisms such as modulating intestinal enzymes and enhancing insulin sensitivity, among others. Importantly, their bioactivity correlates significantly with structural parameters, including molecular weight, monosaccharide profile, glycosidic bond type, and conformational architecture [6,7]. Notably, acidic polysaccharides containing uronic acids demonstrate enhanced α-glucosidase inhibition via electrostatic interactions [4].

Morus alba L., a traditional medicinal-edible fruit, has been used for millennia across Asia [8]. Rich in polysaccharides, polyphenols, and alkaloids, its crude extracts demonstrate diverse bioactivities, including antioxidant and anti-inflammatory effects, as well as regulation of glucose and lipid metabolism [9]. Our group identified that the crude polysaccharide of mulberry extracted by ultrasonic extraction with 50% ethanol (crude-MFP-III) exhibited an α-glucosidase inhibitory capacity of 83.6% (at 10 mg/mL), the highest polysaccharide yield (13.3 ± 1.0%), and high total sugar content (92.2 ± 0.7%) [10]. Despite extensive research on MFPs, structural characterization of homogeneous fractions from M. fructus remains limited.

In this study, the crude polysaccharide (crude MFP-III) was extracted from mulberry using the same method as in previous research [10]. MFP-crude was further purified by DEAE-52 cellulose chromatography and Sephadex G-100 gel filtration, resulting in a homogeneous component, MFP-III. We systematically characterized MFP-III’s physicochemical properties and structural features, using techniques including HPAEC, GC-MS, and 2D-NMR, and evaluated its hypoglycemic efficacy. Structure–activity relationships were elucidated to establish a mechanistic basis for MFP-III’s bioactivity, suggesting its application in functional foods and pharmaceuticals.

2. Materials and Methods

2.1. Materials and Reagents

M. fructus were procured from Sichuan Borun Pharmaceutical Co., Ltd. (Chengdu, China, Batch No. 200802) in October 2020. Dextran molecular weight standards (1–200 kDa) and monosaccharide standards were obtained from Sigma-Aldrich (St. Louis, MO, USA). Methylation analysis kits were sourced from Bioray Sugar Biotechnology Co., Ltd. (Shanghai, China). α-amylase, soluble starch, p-nitrophenyl-α-D-glucopyranoside, α-glucosidase, and acarbose were purchased from Solarbio Science & Technology Co., Ltd. (Beijing, China). All other chemicals and solvents, including ethanol, sodium hydroxide, and hydrochloric acid, were of analytical grade (≥99.0%) and purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Ultrapure water (18.2 MΩ) was prepared using a Milli-Q Integral Water Purification System (Millipore, Bedford, MA, USA).

2.2. Extraction and Purification of Polysaccharides from Mulberry [10]

Dried M. fructus underwent sequential drying, grinding, and sieving. Defatting was achieved through two cycles of petroleum ether reflux (boiling range of 40–60 °C; material-to-solvent ratio of 1:2 g/mL; 1 kg of material per 2 L of solvent per cycle; 2 h/cycle under condenser), followed by filtration and drying. Crude polysaccharides were extracted from the defatted powder using ultrasound-assisted extraction (540 W, 60 kHz, 20–25 °C, 30 min; solid–liquid ratio of 1:15 g/mL; two cycles). Deproteinization used 3% trichloroacetic acid (sample: TCA 1:1 v/v; 4 °C, 30 min mixing; two cycles) with centrifugation (4000× g, 10 min) to remove precipitated protein. The supernatant underwent dialysis (8000–14,000 Da) and ethanol precipitation (4:1 v/v), with recovery by vacuum freeze-drying (yielding crude polysaccharides, designated MFP-crude) [10]. Crude MFP-III was then separated and purified sequentially using DEAE-52 cellulose and Sephadex G-100. The final result is a pure polysaccharide (MFP-III) free of pigments and polyphenols. The process technology roadmap is shown in Figure 1. The extraction rate (R) was computed by the following formula:

$$[eqn]$$

where M_1_ is the dry weight of MFP-III, and M is the dry weight of mulberry fruits.

DEAE-52 cellulose columns (2.0 cm × 25 cm; bed volume of 80 mL) were preconditioned prior to use. A total of 5 mg/mL MFP-crude solution in ultrapure water was centrifuged and membrane-filtered (0.45 μm) before column loading. The filtrate was loaded onto a DEAE-52 cellulose column and eluted sequentially with 400 mL ultrapure water or NaCl solutions of varying concentrations (0.2, 0.5, 1.0 M) at a flow rate of 1.5 mL/min. The phenol–sulfuric acid method was used for monitoring, and the absorbance was measured at 490 nm using a microplate reader. Based on the data, scatter plots were generated, and fractions corresponding to target peaks were collected. These fractions were dialyzed (retentate collected; 3500 Da; 500 mL of external water; 4 °C; water changed every 4 h until conductivity < 10 μS/cm), concentrated, and freeze-dried.

The dominant fractions were further purified using Sephadex G-100 gel filtration chromatography (1.6 cm × 100 cm) with a gel permeation chromatography (GPC) auto-purifier system equipped with a RI-502 SHODEX refractive index detector for online monitoring (eluent: 0.1 M NaCl; flow rate: 0.5 mL/min, injection volume: 2 mL). Symmetrical peaks were collected and combined. The pooled fractions were concentrated under reduced pressure and dialyzed in flowing water for 48 h, and the dialysis solution was collected, concentrated, and freeze-dried under a vacuum. The MFP-III obtained from gel filtration chromatography was sealed and stored for subsequent analyses.

2.3. Structural Characterization of Mulberry Polysaccharides

2.3.1. Homogeneity and Molecular Weight Determination

The molecular weight of MFP-III was determined by high-performance gel permeation chromatography (HPGPC). A 5 mg sample was dissolved in 1 mL of ultrapure water, centrifuged at 12,000 r/min for 10 min, and filtered through a 0.22 µm membrane. The analysis used a high-performance liquid chromatography system (Shimadzu LC-10A, Shimadzu Corporation, Kyoto, Japan) with a refractive index detector (Shimadzu RI-10A, Shimadzu Corporation, Kyoto, Japan). A series of gel filtration columns (BRT105-104-102, 8 × 300 mm) was used. The mobile phase was a 0.05 M NaCl solution at a flow rate of 0.6 mL/min, with an elution time of 60 min. The column temperature was maintained at 40 °C, and the injection volume was 25 µL.

The weight-average molecular weight (Mw) and number-average molecular weight were derived from the RI signal using a dextran calibration curve established with dextran standards of known molecular weights (5, 11.6, 23.8, 48.6, 80.9, 148, 273, 409.8, and 667.8 kDa). The calibration curve was established via least-squares linear regression of logM versus retention time. The reported value corresponds to the weight-average molecular weight (Mw). The calibration curve, validated with a 5 kDa dextran standard near the target molecular weight, showed excellent linearity (R^2^ > 0.99), ensuring accurate determination of MFP-III’s Mw at 6.83 kDa.

2.3.2. Chemical Composition Analysis

The total sugar content was quantified using the phenol–sulfuric acid method, with D-glucose as the standard, following the procedure described in reference [11].

The uronic acid content was determined using the sulfuric acid–carbazole method [12]. Briefly, 100 µL of the polysaccharide solution (3 mg/mL) was mixed with 500 µL of sodium tetraborate–sulfuric acid solution (10 mg/mL) in an ice water bath, vortexed to mix, and then heated in a boiling water bath for 20 min. After the time elapsed, it was immediately cooled to room temperature, and 20 µL of carazole solution (1.5 mg/mL) was added, vortexed to mix, and reacted at room temperature for 1 h. D-glucuronic acid served as the standard. The uronic acid content of MFP-III was calculated to be x% (where x is the actual value obtained from the assay).

Protein content was determined using the Coomassie Brilliant Blue G-250 method, with bovine serum albumin (BSA) as the standard to establish a standard curve (Figure S3) [13]. In total, 3 mg/mL polysaccharide sample solution was prepared in ultrapure water and transferred to a test tube. The detection limit of the assay was 4.8 μg/mL. The protein content in MFP-III was found to be <1.6 μg/mg, indicating negligible protein contamination.

Using an Epoch Biotek spectrophotometer (BioTek, Winooski, VT, USA), the sample was subjected to full-wavelength scanning in the range of 200–700 nm. Ultrapure water was used as the blank control. Absorbance peaks at 260 nm and 280 nm were observed. The peak at 260 nm is characteristic of nucleic acids, while the peak at 280 nm is indicative of aromatic amino acids and proteins. No significant absorbance at 260 nm suggested the absence of nucleic acid contamination, and minimal absorbance at 280 nm confirmed the low protein content (<LOD).

2.3.3. Monosaccharide Composition Analysis

The composition of MFP-III monosaccharides was determined by high -performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) [14]. A precisely weighed 5 mg sample was placed in an ampoule, and 2 mL of 3 M trifluoroacetic acid (TFA) was added. The sample was hydrolyzed at 120 °C for 3 h to completely hydrolyze the polysaccharides. After hydrolysis, the solution was transferred to a tube and dried under a nitrogen stream. The residue was dissolved in 5 mL of ultrapure water and vortexed thoroughly. Subsequently, 400 µL of the solution was diluted with 600 µL of ultrapure water, centrifuged at 12,000 rpm for 5 min, and filtered through a 0.22 µm membrane. The filtrate was used for ion chromatography analysis. The analysis was conducted using an ion chromatography system (Thermo Fisher, Waltham, MA, USA, ICS5000) equipped with a Dionex Carbopac™ PA20 column (3 × 150 mm). The eluents (carbonate-free NaOH and degassed eluents) were: A: ultrapure water; B: 15 mM NaOH solution; and C: 15 mM NaOH and 100 mM NaOAc. The flow rate was set to 0.3 mL/min, the injection volume was 25 µL, and the column temperature was maintained at 30 °C. Detection was performed using an electrochemical detector, and elution conditions are detailed in Table 1.

2.3.4. Fourier Transform Infrared (FT-IR) Spectroscopy Analysis

Polysaccharide samples (2 mg) were precisely weighed and mixed with 200 mg of dried, constant-weight potassium bromide (KBr) powder in a dry agate mortar. The mixture was ground thoroughly and pressed into a pellet. A blank control pellet was prepared using KBr powder alone. The samples were analyzed using a Fourier transform infrared spectrometer (FT-IR 650, Tianjin Gangdong Technology Development Co., Ltd., Tianjin, China) over a wavenumber range of 400–4000 cm^−1^. The acquisition parameters included a resolution of 4 cm^−1^, 32 scans per sample, and automatic baseline correction to ensure spectral accuracy and comparability.

2.3.5. Methylation Analysis

The glycosidic bonds of MFP-III were determined by gas chromatography–mass spectrometry (GC-MS) and compared with the standard mass spectrometry library. Following established methodologies [15], 2 mg polysaccharide sample was dissolved in 1 mL anhydrous dimethyl sulfoxide (DMSO) under a nitrogen atmosphere. Following the methylation kit’s instructions, the methylation reagent was added to the solution and reacted with the polysaccharide for 60 min at 30 °C.

To ensure complete methylation, the reaction mixture was analyzed by FTIR spectroscopy (FT-IR 650, Tianjin Gangdong Technology Development Co., Ltd.). The disappearance of characteristic hydroxyl (OH) absorption bands (3400–3200 cm^−1^) confirmed full methylation, as incomplete methylation would introduce errors in linkage inference.

After hydrolysis of 2 M TFA for 90 min at 100 °C, for uronic acids, the carboxyl groups were reduced by adding sodium borohydride (NaBH_4_) for 8 h at room temperature to convert them into neutral sugars, followed by neutralization with acetic acid.

To ensure complete removal of borate residues (critical for high-quality partially methylated glycol acetate (PMAA) formation by preventing interference in acetylation), the solution was treated with 1% (v/v) acetic acid in methanol and evaporated to dryness under a nitrogen stream; this borate removal step was repeated three times. Following a 20% (v/v) glacial acetic acid neutralization, the solution was reacted with acetic anhydride for one hour at 100 °C. Excess acetic anhydride was removed 4–5 times with 3 mL of toluene. The resulting monosaccharides were extracted 4 times with CH_2_Cl_2_ and dried over anhydrous Na_2_SO_4_ to give PMAAs.

The acetylated product samples were analyzed using an Agilent GC-MS 6890–5973 gas chromatography–mass spectrometry instrument. RXI-5 SIL MS column: 30 m × 0.25 mm × 0.25 µm; temperature program: initial temperature of 120 °C, increased to 250 °C at 3 °C/min and held for 5 min, injector temperature of 250 °C, and detector temperature of 250 °C; carrier: gas helium; flow rate: 1 mL/min.

2.3.6. Nuclear Magnetic Resonance (NMR) Spectroscopy Analysis

A total of 50 mg polysaccharide was weighed, dissolved in 0.5 mL of D_2_O, and then freeze-dried. To completely exchange active hydrogen, the lyophilized powder was then mixed once again in 0.5 mL of D_2_O, freeze-dried, and the previous procedure was repeated. The sample was dissolved in 0.5 mL of D_2_O and kept at room temperature (25 °C) for NMR analysis. NMR experimental parameters included a final sample concentration of 100 mg/mL, 64 scans for ^1^H NMR, 1024 scans for ^13^C NMR, and 256 scans for DEPT135 experiments, with relaxation delays of 2 s for ^1^H NMR and 1.5 s for ^13^C NMR. A nuclear magnetic resonance (NMR) machine (Bruker, Germany) was used to determine the ^1^H and ^13^C NMR spectra, as well as the DEPT135 one-dimensional map and two-dimensional map. The ^1^H NMR spectra were recorded at 600 MHz (^1^H frequency), with the ^13^C frequency set to 150 MHz.

2.3.7. Triple-Helical Structure Analysis

The conformational structure of the polysaccharide sample was analyzed following the method of Deng et al. [16]. A 5 mg polysaccharide sample was dissolved in 10 mL ultrapure water and combined with 10 mL of 80 µM Congo red solution. The final concentration of the polysaccharide in the mixture was 0.25 mg/mL, and the final Congo red concentration was 40 µM. The mixture was homogenized and subsequently adjusted with varying concentrations of NaOH (0.1 M to 0.5 M). To maintain consistent final volumes, the total volume of each solution was normalized to 20 mL by adding ultrapure water as needed. The maximum absorption wavelength of the resulting solutions at each NaOH concentration was measured using a UV-Vis spectrophotometer. A blank control was prepared by replacing the polysaccharide solution with ultrapure water. A plot was constructed with the NaOH concentration as the x-axis and the maximum absorption wavelength as the y-axis to evaluate the conformational properties of the polysaccharide sample.

2.3.8. Thermal Stability Analysis

The thermal stability of the purified MFP-III sample was analyzed using a thermogravimetric analyzer (Netzsch STA 449C, Selb, Germany). A precisely weighed 3 mg sample was placed in a crucible and compressed gently. The experiments were conducted in a constant-temperature and -humidity environment filled with high-purity nitrogen atmosphere (flow rate: 40 mL/min) to ensure that no additional thermal effects caused by oxidation reactions would interfere with the results during the heating process. The heating rate was set to 10 °C/min, rising smoothly from an initial temperature of 25 °C to a maximum temperature of 800 °C. The entire heating process was strictly controlled by a precision temperature control system to guarantee uniformity of the temperature gradient. Temperature and heat flow calibrations were performed using standard reference materials (indium and zinc) prior to analysis. Baseline correction was applied using empty crucible measurements under identical heating conditions to ensure accurate differential scanning calorimetry (DSC) signal interpretation. Thermogravimetric (TG) and DSC signals were recorded simultaneously throughout the heating process to monitor mass loss and thermal transitions, respectively.

2.3.9. Scanning Electron Microscopy Analysis

Approximately 5 mg of dried polysaccharide sample was precisely weighed and adhered to a conductive carbon tape with a double-sided adhesive surface. The sample was then placed in the chamber of an ion sputter coater for gold sputtering for approximately 40 s. After sputtering, the sample was transferred to the observation chamber of an ultra-high-resolution scanning electron microscope (Nova NanoSEM 450, FEI, Hillsboro, OR, USA). The analysis was conducted using secondary electron detectors (ETD and TLD, FEI,) at an accelerating voltage of 3 kV. The sample surface morphology was observed at magnifications of 1000×, 2000×, 4000×, 8000×, 12,000×, 24,000×.

2.4. Hypoglycemic Activity Analysis

2.4.1. α-Amylase Inhibition Activity Analysis

The α-amylase inhibition assay was conducted based on a previously reported method [17]. A total of 50 µL of sample solution at each concentration (2–10 mg/mL) was mixed thoroughly with 50 µL of α-amylase solution (1 U/mL in sodium phosphate buffer, pH 6.9) and incubated at 37 °C for 10 min. Afterward, 50 µL of 1.0% (w/v) soluble starch solution was added, and the mixture was incubated again at 37 °C for 10 min. At the end of the incubation, 100 µL of 3,5-Dinitrosalicylic acid (DNS) reagent was added to terminate the reaction, followed by boiling the mixture for 5 min. The solution was then cooled to room temperature and diluted to 10 mL with distilled water, and the absorbance was measured at 540 nm using a microplate reader. The formula for α-amylase inhibition rate is as follows:

$$[eqn]$$

where A_b_, A_s_, and A_c_ are the absorbances of the blank (without sample), sample or acarbose (with enzyme and sample), and control (without enzyme), respectively.

2.4.2. α-Glucosidase Inhibitory Activity Analysis

The α-glucosidase inhibitory activity of polysaccharide samples at different concentrations (2–10 mg/mL) was determined according to the previously described method with slight modifications [18]. Polysaccharide sample solutions at different concentrations (50 µL) were mixed with 100 µL of α-glucosidase solution (0.3 U/mL in sodium phosphate buffer, pH 6.9) and incubated at 37 °C for 20 min. Then, 100 µL of p-nitrophenyl-α-D-glucopyranoside solution (PNPG, 1.5 mM) was added, and the mixture was further incubated at 37 °C for 10 min. The reaction was terminated by adding 1 mL of Na_2_CO_3_ (1 M), and the absorbance was measured at 400 nm using a microplate reader, with acarbose as the positive control. Acarbose served as the positive control, and the inhibition rate was calculated using Formula (1).

2.5. Statistical Analysis

All experimental data were expressed as mean ± standard deviation (with n = 5 biological replicates, each measured in triplicate technical replicates). Statistical significance was analyzed using analysis of variance (ANOVA), followed by multiple comparisons between groups using the Least Significant Difference (LSD) method. A value of p < 0.05 was considered to indicate significant differences between groups. All calculations were performed using Origin 2024 and GraphPad Prism v9 software.

3. Results

3.1. Extracting and Purifying Crude Polysaccharides

A polysaccharide was extracted from mulberries using 50% ethanol-assisted ultrasound and subsequently precipitated with ethanol. Ion exchange chromatography is a common technique for separating charged and polar compounds based on their ionic properties. Neutral and acidic polysaccharides were separated by gradient elution using anionic resins with eluents of different ionic strengths [19]. As shown in Figure 2A, four peaks were obtained by elution and separation of crude polysaccharides with DEAE-52 cellulose. The yield of each group was as follows: 17.0 ± 0.01% (ultrapure water), 10.5 ± 0.02% (0.2 M), 7.0 ± 0.01% (0.5 M), and 1.8 ± 0.04% (1.0 M). The remaining material likely retained on the column due to incomplete elution or increased binding strength at higher NaCl concentrations, leading to recovery losses. The absorbance values decreased in the following order: 0.2 M, 0.5 M, ultrapure water, and finally 1.0 M. Among these, the 0.2 M NaCl-eluted component showed the highest sugar content. A relatively low level of NaCl increases the ionic strength, thereby facilitating polysaccharide elution. However, higher NaCl concentrations may lead to increased binding of polysaccharides to the resin, reducing the elution efficiency and yield [14].

Gel column chromatography separates polysaccharides based on their molecular size and conformation, following the molecular sieving principle. It is usually found in the last step of polysaccharide purification. Currently, gel filtration chromatography integrated with refractive index detection has seen extensive application in polysaccharide analysis [20]. The component eluted at 0.2 M was purified using the Sephadex G-100 automatic gel filtration system. As illustrated in Figure 2B, a relatively homogeneous elution peak was obtained. Based on the gel purification chromatogram, the symmetrical portion of the peak, corresponding to the 70–85 min range, was collected. This selection was based on the peak’s symmetry and stability, ensuring the highest purity and minimal contamination from trailing or leading edge components. The collected fraction underwent rotary evaporation concentration, dialysis, and subsequent freeze-drying, resulting in a purified mulberry polysaccharide with an 11.0% yield.

3.2. Evaluation of Sample Homogeneity and Molecular Weight

Figure 2C displays the high-performance gel permeation chromatography results, where MFP-III showed a distinct symmetrical peak, confirming its homogenous nature. This observation aligns with the elution profile obtained during the Sephadex G-100 purification step (Figure 2B), validating the consistency of the isolation protocol. Using dextran standards, a calibration curve was established, log_10_M_p_ = −0.1787x + 11.404 (R^2^ = 0.9987), where x represents the retention time (in minutes) (Figure S1). The retention time of MFP-III used for molecular weight calculation was 42.2 min to estimate molecular weights. The M_w_ and Mn of MFP-III were found to be 6.83 kDa and 6.577 kDa, respectively. The polydispersity index (M_w_/M_n_) of MFP-III was 1.038, further verifying structural uniformity.

The ability of polysaccharides to reduce blood glucose levels is strongly associated with their molecular weight [21]. Excessively high M_w_ (>100 kDa) may impede cellular uptake due to steric hindrance, thereby reducing bioavailability and attenuating glucose-lowering effects [22]. On the other hand, excessively low M_w_ (<3 kDa) often fails to maintain the tertiary conformations essential for target engagement, such as α-glucosidase active site occlusion [17]. Notably, the M_w_ of MFP-III (6.83 kDa) resides within this theorized optimal zone, suggesting favorable bioactivity potential.

3.3. Analysis of Chemical Composition

Following purification using DEAE-52 cellulose and Sephadex G-100 columns, the total carbohydrate content of MFP-III was measured at 94.2 ± 2.6%. Protein in the sample is below the detection limit, suggesting that the purification method efficiently eliminated protein impurities, thereby minimizing potential interference in downstream structural analysis. As illustrated in Figure 2D, the UV absorbance spectrum of MFP-III lacked peaks at both 260 nm and 280 nm, which further supports the absence of nucleic acid and protein/aromatic components [23].

3.4. Monosaccharide Composition of MFP-III

The types and ratios of monosaccharides significantly influence the blood glucose-lowering potential of polysaccharides [24]. Accumulating evidence indicates that bioactive polysaccharides with glucose-lowering activity typically comprise heterogeneous monosaccharide profiles, including arabinose, galactose, glucose, and rhamnose, among others [25,26]. Notably, diversity in monosaccharide species and specific molar ratios synergistically enhance hypoglycemic activity. HPAEC analysis of MFP-III (Figure 2E–F and Figure S2) revealed MFP-III’s composition: Rha (28.5% ± 0.3%) > Glc (25.6% ± 0.2%) > Gal (18.7% ± 0.4%) > Ara (16.4%± 0.5%) > GalA (7.3% ± 0.1%) > Man (3.5% ± 0.2%). Recent studies have demonstrated that polysaccharides with elevated galacturonic acid content significantly enhance enzyme binding affinity through electrostatic interactions, thereby augmenting hypoglycemic efficacy [27]. Based on these pieces of evidence, our research indicates that MFP-III has structural advantages in terms of hypoglycemic activity.

3.5. FT-IR Spectral Characterization

As presented in Figure 2G, the infrared (IR) spectrum of MFP-III within the 4000–500 cm^−1^ range displayed distinct absorption bands typically associated with polysaccharide compounds. A strong, wide signal observed at 3396 cm^−1^ was attributed to O-H bond stretching, where the peak broadening suggests the presence of intra- and intermolecular hydrogen bonding—an essential factor in maintaining the stability of polysaccharide tertiary configurations [28]. The signal observed at 2927 cm^−1^ corresponds to C-H bond stretching in methyl and methylene moieties [29]. These absorption signals are typical for saccharides. Prominent bands at 1635 cm^−1^ and 1384 cm^−1^ were linked to asymmetric and symmetric stretching vibrations of carboxyl groups, respectively, suggesting the presence of galacturonic acid units [30]. A strong absorption peak at 1087 cm^−1^ indicates pyranose ring ether bonds and hydroxyl groups, confirming that MFP-III is a pyranose sugar [31]. Moreover, the signal near 983 cm^−1^ corresponds to C-H deformation vibrations associated with β-anomeric pyranose rings, while the band at 873 cm^−1^ indicates similar vibrations for α-anomeric ring configurations [32]. These results demonstrate that MFP-III contains both β-glycosidic and α-glycosidic linkages and is classified as a pyranose sugar. Such findings correspond to those previously described by Liu et al. [33].

3.6. Methylation Analysis Results

The glucose-lowering effect of polysaccharides is largely determined by both the types and the spatial distribution of their glycosidic bonds, as these features influence molecular rigidity, solubility in aqueous environments, and interactions with metabolic enzymes [34]. As shown in Figure 2H and Figure S4, and Table 2, a total of nine different glycosidic linkages were identified in MFP-III, each associated with specific bond types and corresponding relative molar percentages, detailed as follows: Araf-(1 → (8.2%), →5)-Araf-(1 → (16.6%), Glcp-(1 → (7.5%), Galp-(1 → (4.5%), →2,4)-Rhap-(1 → (10.9%), →3)-Galp-(1 → (20.3%), →4)-Manp-(1 → (16.5%),→4)-Galp-(1 → (9.4%), →6)-Galp-(1 → (6.1%).

Before methylation analysis, an additional reduction reaction using NaBH_4_ was performed to convert GalpA into Galp. However, the substitution of NaBD_4_ with NaBH_4_ in the reduction step does not distinguish 4-GalpA from 4- Galp [35]. Therefore, the assignment of 2,3,6-Me3-Galp in Table 2 corresponds to →4)-Galp-(1→, which represents the combined content of →4)-GalpA-(1→ and →4)-Galp-(1→, with →4)-GalpA-(1→ being predominant [36]. FT-IR and monosaccharide composition content analyses confirm the presence of uronic acids in MFP-III.

In previously studied polysaccharides with hypoglycemic potential, 1 → 3, 1 → 4, and 1 → 6 glycosidic linkages were always observed, with 1 → 3 glycosidic linkages occurring more frequently [37]. In the present study, the →3)-Galp-(1→ content constitutes the highest among all linkages in MFP-III. →4)-Manp-(1→ and →4)-Galp-(1→, altogether representing the 1 → 4 linkage, account for a molar ratio of 25.9%.

3.7. NMR Analysis

The 1D (^1^H and ^13^C), DEPT135, and 2D NMR (HSQC, ^1^H-^1^HCOSY, NOESY, and HMBC) spectra of MFP-III are shown in the figures.

In Figure 3A, the 1H NMR spectrum shows that most signals corresponding to the polysaccharide are distributed between 3.0 and 5.5 ppm. The solvent peak (D_2_O) appears at approximately 4.7 ppm, while the region of 3.2–4.0 ppm corresponds to ring protons of sugars [18]. The region from 4.3 to 5.5 ppm primarily contains signals attributed to terminal protons. Twelve anomeric proton peaks are observed at 5.73, 5.26, 5.20, 5.14, 5.04, 5.00, 4.95, 4.60, 4.55, 4.42, 4.37, and 4.34 ppm, which correspond to 12 different types of glycosidic linkages. Typically, signals for β-glycosidic linkages are found in the range of δ4.4–4.8 ppm, whereas those for α-glycosidic linkages are primarily observed in the range of δ4.8–5.8 ppm [35]. This indicates that MFP-III contains residues with both α- and β-configurations. Additionally, the 1.21 ppm signal in the ^1^H NMR spectrum is attributed to the Rhap residue [16]. However, because of signal overlap, the chemical shift assignments for protons H2 through H6 in the saccharide units require further clarification using HCOSY and HSQC spectra.

In the ^13^C NMR spectrum (Figure 3B), most of the carbon resonances attributable to the polysaccharide are distributed within the δ60–120 ppm region. The anomeric carbon signals of MFP-III are primarily found between 93 and 110 ppm. Twelve anomeric carbon signals are identified at δ107.61, 103.43, 103.17, 101.06, 100.98, 100.51, 100.14, 98.36, 97.78, 97.50, 96.70, and 92.52 ppm, corresponding to the C1 of 12 terminal residues. A ^13^C chemical shift at δ175.22 ppm is attributed to the carboxyl carbon, consistent with the presence of GalpA [38]. Additionally, the δ16.59 ppm chemical shift is assigned to the C6 of rhamnose [39]. The DEPT135 spectrum (Figure 3C) reveals inverted signals at δ62.44, 62.52, 70.76, 70.63, and 68.09 ppm, indicating that these peaks correspond to C6 chemical shifts [40].

The HSQC spectrum (Figure 3D) displays cross-peaks linking 1H and 13C signals within the anomeric proton and carbon regions, highlighting distinct resonances in these areas. The chemical shifts in the HSQC spectrum are observed at δ4.95/97.50 ppm, 5.04/97.78, 4.42/96.70, 4.55/103.17, 4.34/101.06, 4.60/103.43, 5.20/100.51, 5.00/98.36, 5.73/107.61, 5.14/92.52, 4.37/100.98, and 5.26/100.14 ppm. These signals correspond to residues denoted by the letters A through L, as detailed in Table 3.

In the HSQC spectrum, the anomeric carbon signal at 104.70 ppm corresponds to the anomeric proton signal at δ4.37 ppm. Through ^1^H-^1^HCOSY analysis (Figure 3E), the correlations between the protons are observed as follows: H1–2 at 4.37/3.44 ppm, H2–3 at 3.44/3.58 ppm, H3–4 at 3.58/3.86 ppm, and H4–5 at 3.86/4.14 ppm. From the data, the proton resonances H1 through H5 were identified at 4.34, 3.45, 3.58, 3.86, and 4.14 ppm, respectively. The carbons C2 to C5 exhibited resonances at 72.16, 73.93, 74.96, and 69.79 ppm, while C6 appeared at 67.88 ppm. This particular signal was assigned to the →6)-β-D-Galp-(1→ glycosidic linkage [41]. Following a similar approach and referencing the relevant literature, the ^1^H and ^13^C signals of all glycosidic linkages were assigned based on 2D NMR data (^1^H-^1^HCOSY, HSQC, NOESY, and HMBC), as summarized in Table 3.

Using both one-dimensional and two-dimensional NMR data, the glycosidic linkage signals of the polysaccharide were identified in the NOESY spectrum (Figure 3F). The anomeric proton of →2,4)-α-L-Rhap-(1 → (G H1, 5.20 ppm) displayed a correlation with its own H2 proton (G H1–G H2, 5.20/4.04 ppm), an intra-residue interaction. However, the presence of the →2,4)-α-L-Rhap-(1 → 2,4)-α-L-Rhap-(1→ linkage was confirmed by the inter-residue NOESY correlation between G H1 and the H2 proton of the subsequent →2,4)-α-L-Rhap-(1→ residue (G’ H2, 4.04 ppm). Additionally, the anomeric proton of →2,4)-α-L-Rhap-(1→ exhibited a correlation peak with the H4 of →4)-α-D-GalpA-(1→ (G H1–H H4, 5.20/4.35 ppm), suggesting the linkage →2,4)-α-L-Rhap-(1 → 4)-α-D-GalpA-(1→. In the NOESY spectrum, the anomeric proton of β-D-Galp-(1 → (F H1, 4.60 ppm) showed a correlation peak with the H6 of →6)-β-D-Galp-(1→ (F H1-E H6, 4.60/3.8 ppm), indicating the linkage β-D-Galp-(1→6)-β-D-Galp-(1→. The anomeric proton of →6)-β-D-Galp-(1→ exhibited a correlation peak with the H4 of →4)-β-D-Galp-(1→ (E H1-C H4, 4.34/3.86 ppm), suggesting the linkage →6)-β-D-Galp-(1 → 4)-β-D-Galp-(1→. Similarly, the anomeric proton of →4)-β-D-Galp-(1→(C H1, 4.42 ppm) showed a correlation peak with the H3 of →3)-β-D-Galp-(1→ (C H1-D H3, 4.42/3.78 ppm), confirming the presence of the linkage →4)-β-D-Galp-(1→3)-β-D-Galp-(1→. For α-L-Araf-(1→, the anomeric proton exhibited a correlation peak with the H5 of →5)-α-L-Araf-(1→ (A H1–B H5, 4.95/3.8 ppm), indicating the linkage α-L-Araf-(1→5)-α-L-Araf-(1→.

In the HMBC spectrum (Figure 3G), the anomeric proton of →5)-α-L-Araf-(1→ correlated with the C4 of →4)-β-D-Manp-(1→ (B H1–L C4, 5.04/70.71 ppm), suggesting the linkage →5)-α-L-Araf-(1 → 4)-β-D-Manp-(1→. The anomeric carbon of →4)-β-D-Manp-(1→ correlated with the H4 of →4)-β-D-Galp-(1→ (C H4-L C1, 3.86/100.14 ppm), indicating the presence of →4)-β-D-Manp-(1→4)-β-D-Galp-(1→. Finally, in the NOESY spectrum, the anomeric proton of →4)-β-D-Galp-(1→ correlated with the H6 of →6)-β-D-Galp-(1→ (C H1-E H6, 4.42/3.8 ppm), further confirming the linkage →4)-β-D-Galp-(1→6)-β-D-Galp-(1→.

In the HMBC spectrum, a correlation peak between the anomeric hydrogen of α-D-Glcp-1→ and the C4 of →2,4)-α-L-Rhap-(1→ is observed (L H1-G C4, 5.26/76.3 ppm), indicating the presence of α-D-Glcp-1→2,4)-α-L-Rhap-(1→. In the NOESY spectrum, a correlation peak between the anomeric hydrogen of →3)-β-D-Galp-(1→ and the H4 of →2,4)-α-L-Rhap-(1→ is observed (D H1-G H4, 4.55/3.61 ppm), indicating the presence of →3)-β-D-Galp-(1→2,4)-α-L-Rhap-(1→. Additionally, in the NOESY spectrum, a correlation peak between the anomeric hydrogen of →6)-β-D-Galp-(1→ and the H4 of →2,4)-α-L-Rhap-(1→ is observed (E H1-G H4, 4.34/3.61 ppm), confirming the presence of →6)-β-D-Galp-(1→2,4)-α-L-Rhap-(1→.

Therefore, we propose that the main chain of this polysaccharide is composed of →2,4)-α-L-Rhap-(1→4)-α-D-GalpA-(1→, characteristic of an RG-I–like pectic polysaccharide motif found in mulberry and other fruit sources [42]. The branch chains, including α-D-Glcp-1→, β-D-Galp-(1 → 6)-β-D-Galp-(1 → 4)-β-D-Galp-(1 → 3)-β-D-Galp-(1→, and α-L-Araf-(1→5)-α-L-Araf-(1 → 4)-β-D-Manp-(1 → 4)-β-D-Galp-(1→6)-β-D-Galp-(1→, are linked to the main chain through the O-4 of →2,4)-α-L-Rhap-(1→. The proposed structure is shown in Figure 4. The proposed structure is tentative, pending further verification of the repeating unit definition, degree of branching, and mass balance between monosaccharide composition and linkage percentages.

3.8. Characterization of Triple-Helical Conformation

Figure 5A shows the maximum absorption wavelengths of Congo red and its complex with MFP-III at varying NaOH concentrations. The results show that, compared to the blank Congo red solution, the sample’s maximum absorption wavelength exhibits a redshift, suggesting potential interaction with an ordered conformation in the polysaccharide [22]. However, as the NaOH concentration increases, the absorption wavelength decreases, indicating disruption of the polysaccharide structure and a weakening of the redshift. In summary, we preliminarily infer that MFP-III may possess an ordered conformation, which could be further confirmed in future studies using orthogonal techniques such as atomic force microscopy or X-ray diffraction. Previous studies have shown that such helical structures in polysaccharides are linked to various biological activities, including antidiabetic effects [43,44]. Therefore, we next sought to determine the hypoglycemic activity of MFP-III.

3.9. Thermal Stability Analysis

The thermal stability of MFP-III, a critical parameter for industrial applications such as thermal sterilization, was evaluated using thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) (Figure 5B,C).

The TGA curve (Figure 5B) revealed four distinct degradation stages: Stage I (25–97 °C) showed a 4.0% weight loss attributed to evaporation of adsorbed and free water; Stage II (98–362 °C) exhibited a 20.0% loss from decomposition of labile functional groups (e.g., hydroxyl, carboxyl) and low-molecular-weight chains; Stage III (363–700 °C) displayed a 15.0% loss due to pyrolytic cleavage of the polysaccharide backbone (C-O-C and C-C bonds); and Stage IV (700–800 °C) displayed a 19.0% mass loss, resulting in a final residual mass of 54.0% at 800 °C, corresponding to carbonaceous residue formation [45,46].

Concurrently, the DSC profile demonstrated an exothermic phase (25–213 °C), likely associated with the reorganization and decomposition of labile surface components, followed by an endothermic phase (213–800 °C) linked to dehydration of hydrophilic domains and progressive depolymerization.

3.10. SEM Analysis

The microstructural characteristics of MFP-III were examined by ultrahigh SEM. As shown in Figure 5D, the samples were in the form of dense, tightly structured lamellae with a sense of compaction at 1000× and 2000×; continued magnification to 4000 and 8000 times showed that the individual forms were irregular and of different sizes. This structural formation may be related to the molecular weight of the polysaccharide and the arrangement of different monosaccharide molecules.

3.11. Results of Hypoglycemic Activity Analysis

3.11.1. Inhibition Effect Analysis of α-Amylase

α-Amylase can efficiently hydrolyze the α-1,4 glycosidic bonds in starch molecules. This enzyme acts on complex carbohydrates such as starch (including amylose and amylopectin), breaking them down into smaller oligosaccharide products, such as maltose, maltotriose, and α-limit dextrin [3]. Its metabolic process is closely related to blood glucose regulation and holds significant reference value in diabetes research. Therefore, by reducing α-amylase activity, delaying carbohydrate digestion, and reducing intestinal glucose absorption, it is essential to control postprandial hyperglycemia in T2DM patients. As depicted in Figure 6A, crude MFP-III, purified MFP-III, and the positive control (acarbose) all exhibited concentration-dependent α-amylase inhibition between 2 and 10 mg/mL. At the highest tested concentration (10 mg/mL), the inhibition rates were 86.3 ± 0.9%, 94.8 ±1.0%, and 97.7 ± 0.4% for crude MFP-III, purified MFP-III, and acarbose, respectively. Statistical analysis demonstrated that purified MFP-III achieved significantly more potent α-amylase inhibition compared to its crude form (p < 0.001). Figure 6B presents the IC_50_ values for α-amylase inhibition by crude MFP-III polysaccharide, purified MFP-III, and acarbose, which were calculated using a four-parameter logistic nonlinear regression model in GraphPad Prism 9.0, with no weighting applied. The IC_50_ values are reported with 95% confidence intervals: 2.98 mg/mL, 2.07 mg/mL, and 0.50 mg/mL, respectively. The IC_50_ of purified MFP-III was significantly lower than that of the crude polysaccharide (p < 0.001), demonstrating that purified MFP-III exhibits stronger α-amylase inhibitory activity and plays a dominant role in this effect.

3.11.2. Inhibition Effect Analysis of α-Glucosidase

α-Glucosidase is a membrane-bound intestinal enzyme that hydrolyzes oligosaccharides into absorbable monosaccharides, thereby causing postprandial hyperglycemia. Its isoenzymes, such as maltase-glucosidase and sucrase-isomaltase, are present in the microvilli of the small intestine [3]. Inhibiting alpha-glucosidase activity can delay glucose absorption, reduce postprandial blood glucose peaks, and assist in the management of T2DM by controlling hyperglycemia [4]. Consequently, selective inhibition of this enzyme is a cornerstone therapeutic strategy for T2DM, as it directly attenuates post-meal glycemic spikes without affecting upstream starch digestion. Figure 6C illustrates that crude MFP-III, purified MFP-III, and the positive control (acarbose) inhibited α-glucosidase activity in a concentration-dependent manner (2–10 mg/mL). At the highest concentration tested (10 mg/mL), inhibition rates were 66.8 ± 0.6%, 74.2 ± 0.8%, and 91.2 ± 1.0% for crude MFP-III, purified MFP-III, and acarbose, respectively. Statistical analysis showed that purified MFP-III had significantly stronger inhibitory activity compared to the crude form (p < 0.001). Figure 6D shows that the IC_50_ values for α-glucosidase inhibition were 3.56 mg/mL for crude MFP-III, 1.56 mg/mL for purified MFP-III, and 1.23 mg/mL for acarbose. The significantly lower IC_50_ of purified MFP-III compared to its crude form (p < 0.001) suggests that MFP-III is the primary active component responsible for α-glucosidase inhibition.

In this study, although MFP-III exhibited stronger hypoglycemic activity than the crude polysaccharide, its efficacy remained lower than that of acarbose. However, the strong enzyme inhibitory activity of acarbose may cause fermentation of undigested starch, leading to adverse effects like flatulence and abdominal bloating [18]. Therefore, MFP-III may be a potentially natural and safe hypoglycemic agent, which is expected to assist in improving T2DM with weaker enzyme inhibition and fewer side effects than acarbose.

3.12. Structure-Activity Relationship

The hypoglycemic effect of MFP-III arises from its unique structure, which synergistically enhances its dual inhibitory activity against α-amylase and α-glucosidase. With a molecular weight of 6.83 kDa, MFP-III balances bioavailability and structural rigidity, enabling deep penetration into enzyme active sites while resisting gastrointestinal degradation—a critical advantage over high-molecular-weight polysaccharides (>100 kDa) with limited membrane permeability. Its heterogeneous monosaccharide profile, dominated by rhamnose (28.5%), arabinose (16.4%), and galacturonic acid (7.3%), facilitates multi-modal enzyme interactions: galacturonic acid mediates electrostatic binding to positively charged residues, while rhamnose-rich branches (→2,4)-Rhap-(1→) introduce structural flexibility for adaptive binding to α-amylase’s substrate channel. Methylation and NMR analyses further reveal a pyranose-rich backbone comprising →2,4)-α-l-Rhap-(1 → 4)-α-D-GalpA-(1→, stabilized by ordered conformations (Congo red assay), and branched side chains (e.g., α-D-Glcp-1→, β-D-Galp-(1→6)) that enhance steric hindrance and hydrophobic interactions. The prevalence of →3)-Galp-(1 → (20.3%) and →5)-Araf-(1→ (16.6%) linkages further stabilize the triple-helix conformation, creating a physical barrier to substrate-enzyme binding. This distinctive structure, characterized by intermediate molecular weight, uronic acid-enhanced electrostatic properties, and conformation-driven steric resistance, indicates that MFP-III functions as an effective natural dual-target inhibitor for blood glucose regulation (Figure 7). It has been shown that polysaccharides can reduce blood glucose concentration by inhibiting glucosidase and amylase activities [18].

4. Discussion

In this research, a homogeneous polysaccharide named MFP-III was successfully extracted from mulberry fruits through a series of optimized purification steps, ensuring high purity and structural integrity. Its structural features and in vitro hypoglycemic effects were thoroughly examined using advanced analytical techniques such as HPLC, GC-MS, FTIR, and NMR spectroscopy. Structural analysis of MFP-III revealed a molecular weight of approximately 6.83 kDa and a monosaccharide composition of rhamnose, arabinose, galactose, glucose, mannose, and galacturonic acid. The main chain is linked by →2,4)-α-L-Rhap-(1 → 4)-α-D-GalpA-(1→, with branching units attached to the O-4 linkage. The structure–activity relationship analysis further demonstrated that MFP-III, featuring multiple glycosidic bonds (1 → 3, 1 → 4, 1 → 6), exhibits a dual inhibitory effect on α-glucosidase (IC_50_ = 1.56 mg/mL) and α-amylase (IC_50_ = 2.07 mg/mL). Its inhibitory potency is approximately 2.3 times greater than that of the crude mulberry fruit polysaccharide extract, highlighting the significance of structural homogeneity in enhancing biological activity. This enhanced activity may be attributed to structural homogeneity and specific glycosidic bonds (e.g., 1 → 3, 1 → 4, 1 → 6). However, mechanistic causality requires further validation. Compared to other mulberry polysaccharides [47], MFP-III’s unique RG-I-like backbone suggests potential for superior hypoglycemic effects, emphasizing its value in functional food development.

5. Conclusions

This work elucidates the structure–function correlation of MFP-III and offers a promising strategy to valorize mulberry, fostering advances in the nutraceutical field through the development of functional food ingredients for diabetes management. Future studies should focus on in vivo validation using animal models of T2DM to assess systemic efficacy and safety, as well as scalable extraction processes to improve yield and reduce production costs, thereby translating these findings into clinical and industrial applications.