Optimization of compound enzyme-assisted ultrasound extraction for Polygonatum sibiricum polysaccharides with characterization and bioactivity evaluation

2.2.1. Extraction of PSP

3 g of PS powder was accurately weighed and mixed with distilled water. The extraction was performed under varying conditions, including enzyme ratio (cellulase: papain), enzyme concentration, extraction temperature, extraction duration, and pH value. Following ultrasonication (Kunshan Hechuang Ultrasonic Instrument, KH-250DB), the mixture was heated in a boiling water bath for 10 min, filtered, and the filtrate was concentrated under reduced pressure to 100 mL. Subsequently, 400 mL of anhydrous ethanol was slowly added to the concentrate, which was then maintained at 4°C overnight. The precipitated polysaccharides were collected by centrifugation at 3,500 rpm for 10 min, followed by freeze-drying for 48 h to obtain the final PSP product.

2.2.2. Single-factor experimental design

Critical parameters affecting polysaccharide yield were systematically evaluated through single-factor experiments: Enzyme ratio (cellulase: papain), Solid-to-liquid ratio (1:10-1:30, w/v), Enzyme concentration (1,500–4,500 U g^-1), Extraction temperature (30-70°C), Extraction time (30–150 min), pH range (4.0-6.0).

2.2.3. Response surface methodology optimization

Building upon the single-factor results, the extraction process was further optimized using a Box-Behnken experimental design with four independent variables at three levels each.

2.2.4. Determination of PSP

The polysaccharide content was determined spectrophotometrically at 490 nm using the phenol-sulfuric acid method with D-glucose as the standard reference. The extraction yield was calculated according to the following equation: Yield (%) = (Mass of extracted polysaccharides / Mass of raw material) × 100%.

2.3.1. Scanning electron microscopy (SEM)

PSP samples were mounted on adhesive stages and sputter-coated with gold to enhance conductivity prior to SEM(Hitachi, S-3000N) observation. The specimens were secured on copper plates for microscopic examination.

2.3.2. X-ray diffraction analysis (XRD)

The sample was compressed into a tablet and placed in the XRD (Shimadzu, 6100) for analysis using a Cu Kα radiation source (40 kV, 40 mA). The parameters were set to step-scan mode with a step size of 0.02°, a 2θ range of 10-80°, and a scan rate of 2°/min.

2.3.3. Fourier transform infrared spectroscopy (FT-IR)

The sample was placed in FT-IR(Thermo Fisher Scientific, Nicolet iS50) using the KBr pellet method under the following conditions: spectral resolution of 4 cm⁻¹, 16 cumulative scans, and a wavenumber range of 4000-450 cm⁻¹.

2.3.4. Monosaccharide composition analysis

Standard solutions were prepared from 16 monosaccharide references after acid hydrolysis. PSP samples (5 mg) were hydrolyzed with 3 M trifluoroacetic acid (TFA) at 110°C for 3 h, evaporated under nitrogen, reconstituted in deionized water, and filtered (0.22 μm). Analysis was performed on a Dionex Carbopac™ PA20 column (3×150 mm) with isocratic elution (0.3 mL min^-1) at 30°C.

2.3.5. Molecular weight determination

Samples (5 mg) were dissolved in 0.2 M NaCl (mobile phase), filtered (0.45 μm), and analyzed using gel permeation chromatography (GPC) with the following conditions: column temperature 40°C, flow rate 0.7 mL min^-1, refractive index detection (RID-20A), and injection volume 50 μL.

2.4.1. Behavioral assessments

2.4.2. Wet weight of organs

After the last administration to the mice, they were fasted (with free access to water) for 12 h, weighed, and then sacrificed to collect the testes, which were also weighed.

2.4.3. Sperm quality

2.4.4. Serum testosterone

Serum testosterone levels were measured using the ELISA method.

2.4.5. Transcriptome sequencing

Total RNA was extracted from the testicular tissues of mice in each group using the TRIzol method, and the concentration and purity of the RNA were measured using the Nanodrop 2000. mRNA was enriched using Oligo (dT) magnetic beads and fragmented by ion disruption. The fragmented mRNA served as a template for cDNA synthesis, followed by PCR amplification to enrich the library fragments. The PCR products were then purified to construct the sequencing library. Sequencing was performed on the NovaSeq X Plus platform. The raw sequencing data underwent quality control processing to remove adapter-contaminated sequences and low-quality reads, resulting in high-quality clean reads. Subsequently, the clean reads were aligned to the mouse reference genome, and the gene expression levels of each sample were quantified. Using |log₂Fold Change| > 1 and p < 0.05 as the screening criteria, differentially expressed genes (DEGs) between different groups were identified. The screened DEGs were further subjected to gene ontology (GO) functional enrichment analysis and kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis to explore the biological functions of the differential genes and the potential signaling pathways they may be involved in.

2.4.6. Immunohistochemistry

Take 4μm paraffin sections of mouse testis, observe the expression of PCNA in the testes under the microscope after antigen retrieval (microwave boiling), inactivation of endogenous catalase (3% hydrogen peroxide for 15 min), blocking with 5% BSA for 2 h, adding primary antibody and incubating at 4°C overnight, secondary antibody incubation, DAB color development, hematoxylin staining, ethanol dehydration, and neutral resin mounting.

2.4.7. Immunofluorescence

Take 4μm paraffin sections of mouse testes, perform antigen retrieval, blocking, incubate with primary antibody at 4°C overnight, incubate with secondary antibody, DAPI nuclear staining, and observe the expression of HSD3β2 in the testes under a fluorescence upright microscope.

2.4.8. Western blot

Take the testicular tissue of mice and add RIPA lysis buffer. Determine the protein concentration using the Eva3200 ultra-micro nucleic acid protein detector, followed by denaturation, SDS-PAGE electrophoresis, membrane transfer, and blocking. Then, add HSD3β2 and CYP17A1 antibodies, respectively and incubate overnight at 4°C. After washing three times with PBST, add the corresponding secondary antibody and incubate on a shaker at room temperature for 2 h. After washing three times with PBST, perform ECL color development and exposure, and analyze the results using ImageJ software.

3.1.1. The influence of pH value on the extraction rate of PSP

As shown in Figure 1(a), the extraction rate of PSP remained stable within the pH range of 4.0–5.5 and significantly decreased when the pH increased to 6.0. This phenomenon indicates that an acidic environment is more conducive to the dissolution of polysaccharides, which may be related to the optimal pH range (pH 4.5–6.0) of cellulase and papain. Taking into account both extraction efficiency and cost-effectiveness, pH 5.5 was selected as the optimal extraction pH.

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Figure 1.

Effects of different extraction parameters on the yield of polysaccharides: (a) effect of pH; (b) effect of cellulase/papain ratio; (c) effect of extraction time; (d) effect of enzyme dosage; (e) effect of solid-to-liquid ratio; (f) effect of extraction temperature.

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3.1.2. The influence of enzyme ratio on the extraction rate of PSP

As shown in Figure 1(b), the ratio of the compound enzymes has a significant impact on the yield of PSP. When the mass ratio of cellulase to papain (mcellulase:mpapain) is 3:7, the yield of PSP reaches the highest value of 21.39%, indicating that in this compound enzyme system, papain plays a more significant role in promoting polysaccharide extraction. Therefore, the enzyme ratio of 3:7 was selected as the optimized condition for subsequent experiments.

3.1.3. The influence of extraction time on the extraction rate of PSP

As shown in Figure 1(c), the yield of PSP initially increased and then decreased with the extension of enzymatic hydrolysis time. When the enzymatic hydrolysis time was 120 min, the PSP yield reached its peak value of 23.39%. This phenomenon may be attributed to the gradual release of polysaccharides through enzymatic reactions in the early stages of hydrolysis, allowing them to fully dissolve [18], thereby increasing the yield. However, as the time further extended, the polysaccharide structure may have been compromised due to decreased enzyme activity or adverse conditions such as localized temperature increases in the system[19,20], leading to a reduction in yield. Therefore, 120 min was selected as the optimized condition for subsequent experiments.

3.1.4. The influence of enzyme dosage on the extraction rate of PSP

As shown in Figure 1(d), the yield of PSP significantly increased as the enzyme dosage rose from 1500 U g^-1 to 3000 U g^-1, reaching a maximum of 22.09% at an enzyme dosage of 3000 U g^-1. Further increases in enzyme dosage showed no significant difference compared to the 3000 U g^-1 group (p > 0.05). The possible reasons for this are as follows: at lower enzyme concentrations, the amount of enzyme is insufficient to effectively degrade the cell wall or release polysaccharides; as the enzyme amount increases, the efficiency of enzyme-substrate binding improves, accelerating the reaction rate and thereby promoting polysaccharide release. However, when the enzyme concentration exceeds a certain critical value, the substrate is fully bound, and the excess enzyme remains in a free state due to insufficient substrate, making it difficult to enhance the reaction rate further [21,22]. Therefore, taking into comprehensive consideration the yield performance and cost control in actual production, the enzyme dosage of 3000 U g^-1 was selected as the optimized condition for subsequent experiments.

3.1.5. The influence of solid-liquid ratio on the extraction rate of PSP

As shown in Figure 1(e), the yield of PSP initially increases and then decreases with the increase in the solid-to-liquid ratio (g mL^-1) during the enzymatic hydrolysis process. The yield of PSP reaches its highest value of 21.78% at a solid-to-liquid ratio of 1:25. The possible reason for this is that at lower solid-to-liquid ratios, the insufficient amount of solvent leads to inadequate soaking of the raw material, resulting in a higher diffusion resistance of polysaccharides from the cells to the solvent, thus leading to a lower extraction yield. As the solid-to-liquid ratio increases, the solvent can fully penetrate the raw material, creating a larger concentration gradient and enhancing the mass transfer efficiency, thereby promoting the release of polysaccharides [23,24]. However, when the liquid ratio is further increased, the system becomes excessively diluted, leading to a decrease in polysaccharide concentration, a reduction in the driving force for diffusion, and a diminished probability of active components within a unit volume coming into contact with factors such as enzymes or ultrasound, resulting in a decline in extraction efficiency [25]. Therefore, a solid-to-liquid ratio of 1:25 (g mL^-1) was determined as the optimized condition for subsequent experiments.

3.1.6. The influence of temperature on the extraction rate of PSP

As shown in Figure 1(f), the yield of PSP gradually increases as the extraction temperature rises from 30°C to 60°C, reaching a maximum of 21.55%. However, when the temperature continues to rise, the PSP yield begins to decline. The reason for this may be that within the optimal temperature range for enzymatic reactions, enzyme activity is enhanced, accelerating the degradation of cell walls and facilitating the release of polysaccharides. However, when the temperature exceeds the enzyme’s optimal range, the enzyme protein denatures and inactivates, significantly reducing the efficiency of enzymatic hydrolysis and affecting the extraction of polysaccharides [26,27]. Additionally, high temperatures may directly disrupt the glycosidic bonds of polysaccharides or alter their molecular structure, particularly for heat-sensitive polysaccharides, which can lead to a decrease in molecular weight or solubility, further inhibiting the increase in yield [28]. Therefore, 60°C was selected as the optimized condition for subsequent experiments.

3.3.1. Scanning electron microscope analysis

The three-dimensional structure of polysaccharides is generally more complex than that of proteins and nucleic acids, exhibiting a high degree of conformational diversity. To further investigate the microscopic morphological structure of PSP, SEM was employed to observe its surface characteristics, as shown in Figure 3. From the figure, it can be observed that PSP primarily presents an irregular flake-like structure with a relatively smooth surface, and cotton-like aggregated substances are visible in local areas. The overall structure is relatively dense, with unevenly distributed pore structures inside, indicating that aggregation and collapse phenomena may have occurred during the extraction and drying processes of this polysaccharide. Such microscopic pores may facilitate its dissolution in water and binding with active sites, thereby influencing its biological activity.

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Figure 3.

SEM images showing the microstructural morphology of polysaccharide samples under different extraction conditions: (a) polysaccharide sample obtained under condition A, showing a loose and porous surface structure; (b) polysaccharide sample obtained under condition B, exhibiting a compact structure with irregular cavities; (c) polysaccharide sample obtained under condition C, characterized by relatively smooth and lamellar fragments; (d) polysaccharide sample obtained under condition D, displaying an aggregated and rough surface morphology.

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3.3.2. Infrared spectroscopy analysis

Figure 4(a) illustrates the infrared spectral characteristics of PSP, which exhibit typical polysaccharide absorption peaks. The broad peak at 3425.01 cm⁻¹ corresponds to the (O–H) stretching vibration absorption peak, indicating the presence of intermolecular hydrogen bonding; the weak peak at 2939.07 cm⁻¹ is attributed to the asymmetric stretching vibration of (CH₂). The absorption band at 1607.40 cm⁻¹ indicates the presence of the carbonyl group (C=O) in uronic acid; the peak at 1350.91 cm⁻¹ arises from the in-plane bending vibration of (C–O–H) in the sugar ring; the peak at 1035.60 cm⁻¹ corresponds to the stretching vibration of (C–O–C) in the furanose or pyranose ring; the absorption peak at 784.90 cm⁻¹ suggests the possible existence of an α-type glycosidic bond structure in the sample.

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Figure 4.

Structural characterization of the polysaccharide by spectroscopic analysis: (a) FTIR spectrum of the polysaccharide, showing the characteristic absorption bands at approximately 3425 cm⁻¹ (O–H stretching vibration), 2939 cm⁻¹ (C–H stretching vibration), 1607 cm⁻¹ (bound water bending vibration), 1359 cm⁻¹ (C–H bending vibration), 1036 cm⁻¹ (C–O–C and C–O stretching vibrations of polysaccharides), and 785–620 cm⁻¹ (fingerprint region); (b) XRD pattern of the polysaccharide, exhibiting a broad diffraction peak around 2θ ≈ 20°, indicating an amorphous structure.

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3.3.3. X-ray diffraction analysis

The structural characterization of PSP samples was conducted using an X-ray diffractometer, and the diffraction pattern has been shown in Figure 4(b). Within the 2θ range of 10° to 100°, the PSP exhibited only a few weak diffraction peaks, with no distinct characteristic crystalline diffraction peaks observed. These results indicate that, under the testing conditions, the PSP did not form a highly ordered crystalline structure, existing primarily in an amorphous form, which may be either an amorphous structure or a partially ordered metastable structure.

3.3.4. Monosaccharide composition and molecular weight analysis

As shown in Figure 5, PSP contained arabinose (1.7 μg mg^-1), galactose (7.61 μg mg^-1), glucose (571.47 μg mg^-1), mannose (17.4 μg mg^-1), and fructose (25.83 μg mg^-1)..The molecular weight standard curve equation is y = -0.172x + 11.729 (R² = 0.992), and the calculated molecular weight is 25,444 Da(within the medium molecular weight range of 10-100 kDa). No uronic acids or amino sugars were detected, indicating that the obtained polysaccharides are neutral medium-molecular-weight polysaccharides with a relatively simple and pure structure. Recent studies have shown that the biological activity of polysaccharides is closely related to their monosaccharide composition and molecular weight. Polysaccharides with high glucose content often exhibit good antioxidant and immunomodulatory functions, while the presence of fructose, galactose, and mannose can enhance the structural diversity and biological effects of polysaccharides. PSP have been reported in the literature to possess various pharmacological activities such as hormone regulation and anti-fatigue, providing a theoretical basis for their application in andrological diseases[29-32].

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Figure 5.

Chromatographic analysis of the polysaccharide: (a) HPLC chromatogram of monosaccharide standards; (b) HPLC chromatogram of the monosaccharide composition of the polysaccharide after hydrolysis, indicating that glucose is the predominant monosaccharide, with minor amounts of arabinose, galactose, mannose, and fructose;(c) GPC profile of the polysaccharide, showing its molecular weight distribution.

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3.4.1. Behavioral indicators

Compared to the normal group, the model group mice exhibited obvious sub-healthy conditions, including reduced huddling, sluggish movement, and dull fur, indicating that their physiological state was significantly affected. In comparison with the model group, the mice in the PSP administration group showed significant improvement, with active mental states and gradually restored fur luster. Behavioral and physiological indicators, as shown in Figure 6(a-d), revealed that the model group mice had significantly lower spontaneous activity counts, grip strength, anal temperature, and urine volume than the normal group (p < 0.01); whereas in the high-dose PSP group, these indicators were significantly higher than those in the model group (p < 0.05). The above results suggest that PSP has specific physiological regulatory effects and can effectively alleviate behavioral and physiological functional abnormalities in model mice.

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Figure 6.

Effects of polysaccharide treatment on sexual behavior, reproductive organ indices, and sperm quality in mice. (a) Self-directed activities; (b) grip force; (c) anal temperature; (d) urine volume; (e) testicular index; (f) sperm motility; (g) sperm concentration; (h) sperm deformity rate; (i) sperm survival rate; (j) serum testosterone (T) level. NG, normal group; MG, model group; PSP-H, high-dose polysaccharide group; PSP-L, low-dose polysaccharide group. Data are presented as mean ± standard deviation (SD). Statistical significance: ##p < 0.01 vs. NG; *p < 0.05 and **p < 0.01 vs. MG.

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3.4.2. Effect on testicular wet weight

As shown in Figure 6(e), compared to the model group, the testicular wet weight of mice in the PSP high-dose group was significantly increased (p < 0.05), suggesting that PSP may have a certain protective effect on testicular tissue.

3.4.3. Sperm Quality

As shown in Figure 6(f-i) compared with the normal group, the sperm density, sperm motility, and sperm survival rate in the model group were significantly decreased (p < 0.01), and the sperm deformity rate was significantly increased (p < 0.01). Compared with the model group, the sperm density, sperm motility, and sperm survival rate in the high-dose PSP group were significantly increased (p < 0.01), and the sperm deformity rate was significantly decreased (p < 0.01). Compared with the model group, the sperm density and sperm motility in the low-dose PSP group were significantly increased (p < 0.01), and the sperm deformity rate was significantly decreased (p < 0.01). Compared to the model group, the PSP group exhibited an increase in sperm density by more than 216.50%, an enhancement in sperm motility by 145.79%, an improvement in sperm viability by 18.46%, and a reduction in sperm deformity rate by 24.29%.These results suggest that PSP can significantly improve the sperm quality in model mice, demonstrating a beneficial promoting effect, and these improvements exhibit dose dependency.

3.4.4. Effect on Serum Testosterone Levels in Mice

As shown in Figure 6(j), compared with the normal group, the testosterone content in the model group mice significantly decreased (p < 0.01); compared with the model group, the testosterone level in the PSP high-dose group mice significantly increased (p < 0.05), suggesting that PSP has a promoting effect on the testosterone synthesis system.

3.4.5. Differential gene screening and analysis

Principal component analysis (PCA) revealed that the samples from the normal group, model group, and drug administration group formed independent clusters in three-dimensional space, with no overlap between groups, indicating that the experimental grouping was reasonable and the data reproducibility was good. Meanwhile, the model group deviated from the normal group along the PC3 axis, while the administration group moved closer to the normal group, suggesting that the administration group delayed the disease progression by regulating the organism, as shown in Figure 7(a). Through differential analysis between the model group and administration group, a total of 179 (DEGs) were identified, including 96 upregulated genes and 83 downregulated genes. As shown in Figure 7(b). The heatmap analysis results further revealed that the samples from each group exhibited distinct clustering characteristics in the differential gene expression patterns, with clear boundaries between high and low expression genes, demonstrating good expression consistency. Moreover, compared to the model group, the drug-administered group and the normal group showed greater similarity in their differential gene expression profiles. These findings are consistent with the PCA results, further corroborating that the drug-administered group has delayed the disease progression, as shown in Figure 7(c).

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Figure 7.

Transcriptomic profiling and functional enrichment analysis of the MG, PSP, and NG groups. (a) Principal component analysis (PCA) score plot showing the overall distribution and clustering of samples from different groups. (b) Volcano plot of differentially expressed genes (DEGs) between groups. Red dots represent significantly upregulated genes, blue dots represent significantly downregulated genes, and gray dots represent non-significant genes. (c) Hierarchical clustering heatmap of DEGs among MG, PSP, and NG groups, illustrating distinct gene expression patterns. (d) Gene Ontology (GO) enrichment analysis of DEGs, including biological process (BP), cellular component (CC), and molecular function (MF) categories. (e) KEGG pathway enrichment analysis of DEGs. The x-axis represents the Rich factor, and the y-axis represents enriched KEGG pathways. The color of the dots indicates the P value, and the size of the dots represents the number of enriched genes. The steroid biosynthesis pathway was significantly enriched. (NG, normal group; MG, model group; PSP, polysaccharide-treated group).

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3.4.6. GO functional and KEGG pathway enrichment analysis of differentially expressed genes in the transcriptome

GO functional analysis revealed that the differentially expressed genes were primarily involved in biological processes such as developmental process, anatomical structure development, and cellular developmental process; in cellular components such as membrane-bounded organelle, cell projection, and intracellular membrane-bounded organelle; and in molecular functions such as protein binding, cytoskeletal protein binding, and actin binding. As shown in Figure 7(d). KEGG pathway enrichment analysis revealed that the differentially expressed genes were primarily involved in regulating key pathways such as Steroid biosynthesis, Citrate cycle (TCA cycle), and Pentose phosphate pathway. This suggests that disturbances in steroid metabolism and abnormal cellular development may be important pathological mechanisms underlying OAS. The prominent enrichment of the steroid biosynthesis pathway, in particular, is closely related to cholesterol metabolism during spermatogenesis, testosterone synthesis, and cell membrane stability, as shown in Figure 7(e).

3.4.7. Testicular tissue morphology and protein expression of PCNA, HSD3β2, and CYP17A1

As shown in Figures 8 and 9, compared with the normal group, the number of spermatogonial stem cells decreased, and the expression of PCNA was reduced in the model group. After PSP administration, the number of spermatogonial stem cells increased and the expression level of PCNA elevated, suggesting that the proliferation of spermatogonial cells slowed down in the model group, which could be improved after administration. Compared with the normal group, the CYP17A1 protein in the testicular tissue of mice in the model group significantly decreased (p < 0.05). Compared with the model group, the CYP17A1 protein in the testicular tissue of mice in the PSP high-dose group significantly increased (p < 0.05). Compared with the model group, the HSD3β2 protein in the testicular tissue of mice in the PSP administration group significantly increased (p < 0.05). The results indicate that PSP may promote the conversion of cholesterol to testosterone by upregulating the expression of key rate-limiting enzymes CYP17A1 and HSD3β2 in the steroid synthesis process in testicular tissue. The increase in testosterone levels can provide the necessary hormonal environment for the proliferation of spermatogonia[33-35], thereby ensuring the normal growth and development of sperm, improving OAS, and exerting its therapeutic effects.

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Figure 8.

Immunohistochemical and immunofluorescence analysis of testicular tissues from different groups. (a) Immunohistochemical staining of proliferating cell nuclear antigen (PCNA) in testicular tissues from the NG, MG, PSP-L, and PSP-H groups. Brown staining indicates PCNA-positive cells. (b) Immunofluorescence staining of 3β-hydroxysteroid dehydrogenase (HSD3β2) in testicular tissues. HSD3β2-positive signals are shown in green, and cell nuclei are counterstained with DAPI (blue). Merged images show the colocalization of HSD3β2 and nuclei. (NG: normal group; MG: model group; PSP-L: low-dose PSP-treated group; PSP-H: high-dose PSP-treated group. Scale bar = 50 µm).

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Figure 9.

Representative Western blot images and quantitative analysis of steroidogenic enzyme expression in testicular tissues. (a) Representative Western blot bands showing the protein expression levels of cytochrome P450 17A1 (CYP17A1) and 3β-hydroxysteroid dehydrogenase (HSD3β2). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal loading control. (b) Relative protein expression of CYP17A1 normalized to GAPDH. (c) Relative protein expression of HSD3β2 normalized to GAPDH.NG, normal group; MG, model group; PSP-L, low-dose PSP-treated group; PSP-H, high-dose PSP-treated group. Data are presented as mean ± SD. # p < 0.05 vs. NG group; * p < 0.05 vs. MG group.

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