Polygonatum cyrtonema polysaccharide and application thereof

Abstract

The invention relates to polygonatum cyrtonema polysaccharide and application thereof. The novel polysaccharide PRP is extracted and purified from polygonatum cyrtonema and is neutral inulin type polysaccharide. The PRP takes a node alpha-D-glucose-(22)-beta-D-fructose similar to cane sugar as a center. The C6 site of glucose has an additional beta-fructose (2-> 6) substituent, and two chains extend out of the core through a (2-> 1)-beta-fructose bond to form a Y-type moderate branch structure. Compared with other selenium nanoparticles with stable polysaccharides, the selenium nanoparticles coated with polygonatum polysaccharide provided by the invention have smaller initial particle size, more negative zeta potential, stable batch repeatability and excellent long-term stability.

Description

Technical Field

[0001] This invention relates to a polysaccharide of Polygonatum odoratum and its applications. Background Technology

[0002] Spinal cord injury (SCI) is a highly destructive neurological disorder that leads to permanent motor and sensory dysfunction, severely reducing patients' quality of life and imposing a heavy socioeconomic burden globally. Currently, there is no effective cure for SCI. One of the greatest challenges is preventing and mitigating the damage caused by secondary injury processes following the initial trauma. These secondary cascades, characterized by oxidative stress, ferroptosis, and neuroinflammation, create an unfavorable microenvironment that exacerbates neuronal loss and hinders functional recovery. In SCI, the acute phase involves significant local hemorrhage and a marked increase in iron ion levels. The high levels of polyunsaturated fatty acids in the spinal cord tissue exacerbate the iron-mediated Fenton reaction, leading to lipid peroxidation and triggering ferroptosis in nerve cells. This not only results in neuronal loss but also damages the myelin sheath following the spinal cord injury. Therefore, inhibiting ferroptosis has been shown to protect neurons, maintain myelin sheath integrity, mitigate secondary damage, and improve motor function recovery.

[0003] Selenium (Se), an essential trace element for the human body, can exert antioxidant, anti-apoptotic, and immune response-regulating effects by integrating into various selenium enzymes (such as GPX4 and TrxR). Current research shows that selenium supplementation after spinal cord injury can reduce iron-mediated oxidative stress, decrease lipid peroxidation products such as malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), and reduce ferroptosis in neurons and oligodendrocytes. However, traditional selenium supplements have the following drawbacks: inorganic selenium (such as Na2SeO3) is easily oxidized or reduced, resulting in low bioavailability; selenium has a narrow therapeutic window, and excessively high doses can lead to cytotoxicity and systemic side effects; and it is difficult to stably accumulate in the lesion area, limiting its clinical translation.

[0004] In recent years, selenium nanoparticles (SeNPs), a nanoscale form of elemental selenium, have gradually become an important form of selenium delivery due to their good biocompatibility, water solubility, and controllable release characteristics. However, exposed SeNPs still suffer from problems such as unstable particle size, easy aggregation, and easy loss of activity. Therefore, the scientific community generally uses polysaccharides, proteins, or surface modifiers as stabilizers to improve the physicochemical properties of SeNPs. It has been reported that natural polysaccharides such as lentinan (LNT) and chitosan (CS) can be used for surface modification of SeNPs, which can improve their stability and certain biological activities. However, there are still the following shortcomings: although commonly used chitosan and lentinan can improve the dispersibility of nanoparticles, their effects on regulating inflammation, ferroptosis, and nerve repair are limited; most polysaccharides reported only play a "stabilizing" role and lack obvious synergistic pharmacological activities; existing research on polysaccharide-modified SeNPs is mostly limited to antibacterial or tumor fields, and there is insufficient research on spinal cord injury, remyelination, and neuroprotection.

[0005] Polygonatum contains abundant polysaccharides with complex chemical structures. The content, structure, and activity of polysaccharides vary considerably among different sources and varieties. (Polygonatum multiflorum...) Polygonatum *Polygonatum cyrtonema* (PCH) is a perennial herb belonging to the genus *Polygonatum* in the family Liliaceae. It possesses kidney-tonifying, essence-boosting, yin-nourishing, and dryness-moistening effects. It is one of the three *Polygonatum* species listed in the 2020 edition of the *Chinese Pharmacopoeia*, and is widely distributed in southern my country. *Polygonatum cyrtonema* polysaccharides are the main active ingredient in *Polygonatum cyrtonema*, and have been proven to possess pharmacological activities such as antioxidant, hypoglycemic, hypolipidemic, anti-atherosclerotic, anti-tumor, and anti-fatigue effects. However, there are no reports of *Polygonatum cyrtonema* polysaccharides being used to treat spinal cord injury (SCI) or ferroptosis. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings and deficiencies of the existing technology and to provide a Polygonatum multiflorum polysaccharide and its applications.

[0007] The technical solution adopted in this invention is as follows: a polysaccharide of Polygonatum multiflorum, the chemical formula of which is shown in formula (I): (I); m + n = 22 - 24, m ≠ 0, n ≠ 0.

[0008] This invention further provides a method for preparing Polygonatum multiflorum polysaccharide as described above, comprising the following steps: 1. Take the rhizome of Polygonatum multiflorum, dry it until the moisture content is ≤10%, pulverize it and pass it through a 20-40 mesh sieve to obtain Polygonatum multiflorum rhizome powder; 2. Mix the powdered rhizome of Polygonatum multiflorum with deionized water or buffer solution, and extract by stirring under conditions of 55–60 °C and pH 5.5–6.5 using inert gas headspace or low dissolved oxygen. Then, obtain the filtrate by hot filtration at 55–60 °C. 3. An anion-exchange cellulose column was used to elute and collect the neutral polysaccharide main peak with deionized water, followed by elution of acidic heteropolysaccharides with 0.1–0.5 M NaCl. The collected neutral polysaccharide main peak was then polished by Sephadex G-100 gel filtration to collect symmetrical single peaks. The aqueous eluent was desalted by ultrafiltration. IV. Freeze-drying yields Polygonatum polysaccharides.

[0009] The Polygonatum multiflorum root and stem powder in step two is obtained by pre-treating the Polygonatum multiflorum root and stem powder obtained in step one. The pre-treating includes the following steps: mixing the Polygonatum multiflorum root and stem powder obtained in step one with 60-80% ethanol at a mass-volume ratio of 1:8-1:12, stirring, filtering, taking the filter residue, and removing the residual ethanol from the filter residue.

[0010] In step two, the stirring extraction involves gentle stirring for 60–120 minutes, repeated twice, and the filtrates are combined.

[0011] In step four, the sample is pre-frozen at –40 °C for 6–12 h and then sublimated and dried for 24–36 h.

[0012] The present invention further provides the application of the polysaccharide of Polygonatum odoratum as described above in the preparation of products that improve glucose and lipid metabolism or insulin resistance.

[0013] The present invention further provides selenium nanoparticles, comprising selenium nanoparticles and a coating layer modified on the outside of the selenium nanoparticles, wherein the coating layer is Polygonatum polysaccharide as described above.

[0014] The present invention further provides a method for preparing selenium nanoparticles as described above, comprising the following steps: (1) Disperse the polysaccharide of Polygonatum odoratum as described above in water to prepare an aqueous solution of polygonatum odoratum polysaccharide; (2) Mix the selenium precursor solution with the polysaccharide aqueous solution of Polygonatum odoratum prepared in step (1) under magnetic stirring until homogeneous. Add the reducing agent solution dropwise while stirring and react. (3) Dialysis removes free small molecules to obtain purified selenium nanoparticles.

[0015] In step (3), dialysis is performed using a dialysis bag with a molecular weight cutoff of 6000–8000 kDa.

[0016] The selenium nanoparticles described above are used in the preparation of selenium supplements.

[0017] The beneficial effects of this invention are as follows: 1. This invention extracts and purifies a novel polysaccharide PRP from Polygonatum cyrtonema. PRP is a neutral inulin-type polysaccharide with a molecular weight of 4.8 kDa. Monosaccharide composition analysis shows that PRP is composed of fructose and glucose. Structural characterization shows that PRP has sucrose-like nodes. α -D-glucose-(1↔2)- β -D-fructose is the central component. Glucose has an extra one at the C6 position. β -Fructose (2→6) substituents, while both chains pass through (2→1)- fructose- β -Fructose bonds extend from the core (chain lengths are m and n, m+n≈23, and m and n are not 0), forming a Y-shaped moderately branched structure.

[0018] 2. The novel Polygonatum polysaccharide PRP has antioxidant and anti-inflammatory activities. In vivo studies have shown that PRP can significantly reduce weight loss, hyperglycemia, and liver and kidney damage in type 2 diabetic mice induced by high-fat diet / streptozotocin (HFD / STZ).

[0019] 3. This invention provides selenium nanoparticles coated with Polygonatum sibiricum polysaccharide (PRP@SeNPs). The PRP topology of Polygonatum sibiricum polysaccharide provides dense hydroxyl clusters that can be anchored at multiple Se–OH / Se–O⁻ sites, and constructs a non-ionic, highly hydrated spatial brush layer (“segment-loop-tail” canopy) on the slip surface. This brush layer resists ion shielding, protein-mediated bridging, and Ca²⁺-induced flocculation through steric hindrance / hydration repulsion, resulting in PRP@SeNPs exhibiting smaller initial particle size, more negative zeta potential, stable batch reproducibility, and excellent long-term stability compared to other polysaccharide-stabilized selenium nanoparticles. Unlike chitosan or lentinan, which only provide physical stabilization, PRP in PRP@SeNPs not only provides stability but also synergistically exerts pharmacological effects, such as scavenging ROS, inhibiting inflammatory factors, and promoting immune balance. Therefore, in the treatment of diseases such as spinal cord injury, PRP@SeNPs achieves more significant therapeutic effects through “polysaccharide + selenium synergy.” Furthermore, the PRP coating imparts a negative charge to the surface of SeNPs, resulting in a zeta potential of approximately -36 mV. This significantly enhances stability in the blood and tissue environments, preventing rapid sedimentation and clearance. This allows PRP@SeNPs nanoparticles to penetrate, distribute, and remain more effectively at the site of spinal cord injury, thereby increasing the effective local concentration of Se. Simultaneously, the anti-inflammatory properties of PRP improve the local microenvironment, enabling SeNPs to better exert their anti-ferroptosis and neuroprotective effects. Additionally, the PRP coating slows the release rate of Se and enhances the stability of SeNPs in vivo, significantly broadening the safe dosage window for Se. This means that PRP@SeNPs require lower doses to achieve the desired therapeutic effect compared to traditional SeNPs or inorganic Se supplements, thus reducing the risk of hepatotoxicity, nephrotoxicity, and systemic side effects. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, obtaining other drawings based on these drawings without creative effort still falls within the scope of the present invention.

[0021] Figure 1 The image shows the Fourier transform infrared (FTIR) spectrum of the Polygonatum polysaccharide PRP-W-1 prepared in Example 1. Figure 2 The methylation determination diagram is shown for the hydrolysis product of Polygonatum odoratum polysaccharide PRP-W-1 prepared in Example 1. Figure 3 The liquid chromatograms of the polysaccharide PRP-W-1 prepared in Example 1 after hydrolysis are compared with those of the standard monosaccharide. Figure 4 The 1H spectrum of the Polygonatum polysaccharide PRP-W-1 prepared in Example 1; Figure 5 The 13C spectrum of PRP-W-1; Figure 6 The HSQC spectrum of PRP-W-1; Figure 7 The HMBC spectrum of PRP-W-1; Figure 8 The COSY spectrum of PRP-W-1; Figure 9 DEPT 135 spectra of PRP-W-1; Figure 10 A is the chemical structural formula of Polygonatum multiflorum polysaccharide PRP-W-1. Figure 10 B is a simplified symbolic diagram of the PRP-W-1 structure; Figure 11 A is a schematic diagram of the process for preparing PRP@SeNPs in Example 2; Figure 11 B shows transmission electron microscopy images of CS@SeNPs, LNT@SeNPs, and PRP@SeNPs. Figure 11 C represents the average particle size of CS@SeNPs, LNT@SeNPs, and PRP@SeNPs; Figure 11 D is the infrared spectrum of PRP@SeNPs; Figure 11 E represents the zeta potential of CS@SeNPs, LNT@SeNPs, and PRP@SeNPs; Figure 11 F represents the stability test results for CS@SeNPs, LNT@SeNPs, and PRP@SeNPs; Figure 12 A represents the results of the live / dead cell staining experiment; Figure 12 B represents the CCK-8 test result; Figure 12 C represents the DCFH-DA imaging result; Figure 12 D represents the results of flow cytometry experiments; Figure 12 E represents the speciation results from HPLC–ICP-MS analysis. Figure 12 F represents the effects of CS@SeNPs, LNT@SeNPs, and PRP@SeNPs on the expression of GPX4 and TFR1; Figure 12 G represents the quantitative analysis results of the effects of CS@SeNPs, LNT@SeNPs, and PRP@SeNPs on the expression of GPX4 and TFR1; Figure 13 The effects of different treatments on hindlimb motor function after spinal cord injury (SCI); Figure 13A represents the scores of five anatomical landmarks (toes, ankles, knees, hips, and iliac crests) for each experimental group; Figure 13 B represents the results of SCI mouse footprint analysis using CS@SeNPs, LNT@SeNPs, and PRP@SeNPs. Figure 13 C represents the trajectory of mice in each experimental group based on DeepLabCut; Figure 13 D represents the quantitative analysis results of the swing percentage and joint angle range of motion (hip, knee, ankle) of mice in each experimental group; Figure 14 The effects of different treatments on muscle electrophysiology, tissue morphology and neuronal function after spinal cord injury (SCI); Figure 14 A represents the electrophysiological waveforms of each muscle group; Figure 14 B represents the statistical results of the amplitude of the electrophysiological waveform; Figure 14 C is a radar chart integrating multiple indicators (hip joint angle amplitude, knee joint angle amplitude, sensory function, motor function, and toe amplitude); Figure 14 D represents the HE staining result; Figure 14 E represents the Nissl staining result; Figure 14 F represents the quantitative analysis results of the lesion area in each experimental group of mice; Figure 15 The effects of different treatments on microtubule stability, glial scar formation, and neuronal axonal integrity following spinal cord injury (SCI); Figure 15 A represents the results of Tyr Tubulin / Ace Tubulin / DAPI staining; Figure 15 B represents the GFAP / NF200 / DAPI staining result; Figure 15 C represents the quantitative analysis results of the microtubule stable region; Figure 15 D represents the fluorescence intensity ratio analysis of acetylated microtubules (AceTubulin) / tyrosineized microtubules (TyrTubulin); Figure 15 E represents the quantification result of GFAP fluorescence intensity; Figure 15 F represents the quantification result of NF200 fluorescence intensity; Figure 16 The effects of different treatments on neuronal survival, myelin repair, and ferroptosis after spinal cord injury (SCI); Figure 16 A represents the results of NeuN / 4-HNE / GFAP trichromatic immunofluorescence. Figure 16 B represents the results of whole spinal cord MBP / NF200 / DAPI staining; Figure 16 C and 11E represent the results of Western blotting and quantitative analysis of proteins; Figure 16 D and 11F show the results of Luxol Solid Blue (LFB) staining and quantitative analysis. Figure 17 A represents the results of the BV2 cell proliferation experiment; Figure 17 B represents the results of total superoxide dismutase (SOD) activity analysis; Figure 17 C represents the detection result of polarization markers; Figure 17 D represents the results of the CD86 / CD206 / DAPI immunofluorescence assay; Figure 18 Results of different treatments on the regulation of macrophage polarization after spinal cord injury (SCI). Figure 18 A represents the results of CD86 / CD206 / DAPI immunofluorescence analysis; Figure 18 B represents the Western blot results for polarization-related proteins. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings.

[0023] Example 1: Preparation and identification of Polygonatum multiflorum polysaccharide PRP: In this embodiment, polysaccharide PRP of Polygonatum odoratum was extracted and purified from the rhizome of Polygonatum odoratum. The specific process is as follows: 1. Crushing and sieving: Dry the dried rhizomes of Polygonatum odoratum at ≤40 °C until the moisture content is ≤10%, crush them and pass them through a 20–40 mesh sieve (preferably 30 mesh), and store them in an airtight container to prevent moisture absorption; II. Degreasing / Small Molecule Removal Pretreatment: Mix the powder with 60–80% (v / v) ethanol at a ratio of 1:8–1:12 (w / v), magnetically stir at room temperature for 2 × 30 min, and filter to recover ethanol to remove pigments, oligosaccharides, free polyphenols, and small molecules; remove residual ethanol (residual ethanol < 0.5%) from the wet residue under aeration or rotary evaporation, and set aside. This step is optional and is used to improve the clarity and color of subsequent solutions; III. Extraction Medium and Conditions: Mix the pretreated plant powder with deionized water or a weak buffer solution (e.g., 10–20 mM phosphate, pH 6.0±0.2) at a solid-liquid ratio of 1:10–1:20 (w / v), and gently stir at a constant temperature of 55–60 °C for 60–120 min. The extraction can be repeated twice, and the filtrates should be combined. The pH should be maintained at 5.5–6.5 throughout the process to avoid strong acid inducing fructan hydrolysis. Use an inert gas (nitrogen) headspace or low dissolved oxygen conditions to reduce Maillard reaction and browning. IV. Coarse Filtration and Clarification: Remove coarse residue by hot filtration (60 °C). If necessary, add 0.05–0.10% (w / v) diatomaceous earth as a filter aid, or use a 0.45 µm microfiltration membrane for clarification to obtain a light-colored and transparent extract. V. Purification: An anion-exchange cellulose (DEAE) column (diameter:height ≈ 1:10) was used. The neutral polysaccharide main peak was collected by elution with deionized water (inulin-type polysaccharides are mostly neutral components and can be eluted with water). Acidic heteropolysaccharides were then eluted with 0.1–0.5 M NaCl. The main peak was then polished by Sephadex G-100 gel filtration to collect symmetrical single peaks. The eluent was always aqueous and subsequently desalted by ultrafiltration. VI. Freeze-drying (research grade): Pre-freeze at –40 °C for 6–12 h, sublime dry for 24–36 h, the finished product is loose, designated as PRP-W-1.

[0024] The above process was carried out under mild conditions of 55–60 °C and pH 5.5–6.5 and a low-oxygen environment for water extraction. Combined with fractional alcohol precipitation and membrane separation, it significantly reduced fructan chain breakage and depolymerization, resulting in stable molecular weight and distribution of the main peak and clear FT-IR inulin fingerprint. At the same time, the target component was obtained through one-pot fractionation, taking into account both yield and preservation of active structure.

[0025] Figure 1 The Fourier Transform Infrared (FTIR) spectrum of the prepared Polygonatum multiflorum polysaccharide PRP-W-1 is shown, indicating that it is located at 3200-3600 cm⁻¹. -1 The -OH peak is at 2800-3000 cm⁻¹. -1 The -CH peak at 1700 cm⁻¹ -1 The -COO-R peaks are around 1000-1200 cm⁻¹. -1 The C−O−C peak.

[0026] Figure 2 The total ion chromatogram for methylated sugar alcohol acetates of Polygonatum polysaccharide PRP-W-1 is shown. Methylation analysis was performed to determine the glycosidic bond pattern of PRP-W-1. The major residues are 1,2-linked fructose (1,2-Fru), accounting for more than 53%, and were identified as the main component of the backbone. Terminal glucose (t-Glc) was identified as the initiating glucose residue of the inulin structure, while 1,2,6-linked fructose (1,2,6-Fru) is part of the branching backbone, and 1,6-linked glucose (1,6-Glc) was found to be part of the side chain. 2-Fru residues were identified as terminal fructose units in the polysaccharide.

[0027] Figure 3The image shows a comparison of the liquid chromatograms of hydrolyzed Polygonatum polysaccharide PRP-W-1 with standard monosaccharides. The standard monosaccharides include fucose, arabinose, galactose, glucose, fructose, ribose, galacturonic acid (Gal-UA), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), and mannuronic acid (Man-UA). The comparison shows that the hydrolyzed Polygonatum polysaccharide PRP-W-1 mainly consists of glucose (Glc) and fructose (Fru).

[0028] Figure 4 The 1H spectrum of the Polygonatum polysaccharide PRP-W-1 prepared in Example 1; Figure 5 The 13C spectrum of PRP-W-1; Figure 6 The HSQC spectrum of PRP-W-1; Figure 7 The HMBC spectrum of PRP-W-1; Figure 8 The COSY spectrum of PRP-W-1; Figure 9 The DEPT 135 spectrum of PRP-W-1. (By...) Figures 4-9 A comprehensive characterization of the composition and structure of PRP-W-1 confirms that PRP-W-1 is... Figure 10 The chemical structure shown in A, Figure 10 B is a simplified symbolic diagram of the PRP-W-1 structure, with the blue circle representing... α - Glucose ( α -Glc), the green pentagon represents β - Fructose ( β -Fru), with numbers (such as "1", "2", "6") indicating the positions of glycosidic bonds, can show that PRP uses sucrose-like nodes. α -D-glucose-(1↔2)- β -D-fructose is the central component. Glucose has an extra one at the C6 position. β -Fructose (2→6) substituents, while both chains pass through (2→1)- fructose- β -Fructose bonds extend from the core (chain lengths are m and n, m+n≈23, and m and n are not 0), forming a Y-shaped moderately branched structure.

[0029] In vivo studies have shown that PRP-W-1 treatment significantly reduced weight loss, hyperglycemia, and liver and kidney damage in high-fat diet / streptozotocin (HFD / STZ)-induced type 2 diabetes mice. Furthermore, the study elucidated that PRP-W-1 exerts its regulatory role through multiple signaling pathways, indicating a multifaceted mechanism of action. These findings highlight the potential of PRP-W-1 as a novel bioactive polysaccharide in antidiabetic therapy.

[0030] Example 2: Preparation of selenium nanoparticles (PRP@SeNPs) stabilized by Polygonatum polysaccharide PRP-W-1: like Figure 11 As shown in Figure A, this embodiment uses sodium selenite (Na2SeO3) as a selenium precursor, vitamin C (Vc) as a reducing agent, and the Polygonatum polysaccharide PRP-W-1 prepared in Example 1 as a stabilizer. A one-pot reduction method is used to synthesize Polygonatum polysaccharide-stabilized selenium nanoparticles (PRP@SeNPs). The specific preparation steps are as follows: (1) Prepare 5–20 mM Na2SeO3 aqueous solution, 20–50 mM Vc aqueous solution, and 1–10 mg / mL Polygonatum polysaccharide PRP-W-1 aqueous solution; (2) Mix 2 mL of Na2SeO3 aqueous solution with 5 mL of PRP-W-1 aqueous solution under magnetic stirring until homogeneous; (3) Slowly add 1 mL of deionized water to dilute and keep the reaction system homogeneous; (4) While stirring, add the vitamin C aqueous solution dropwise, so that Se 4 ⁺ is reduced to elemental Se; (5) Continue stirring for 24 h to form an orange transparent PRP@SeNPs solution at room temperature; (6) Use a dialysis bag with a molecular weight cutoff of 6000–8000 kDa to remove free small molecules by dialysis to obtain purified PRP@SeNPs.

[0031] Comparative Example 1: Preparation of chitosan-stabilized selenium nanoparticles (CS@SeNPs): Chitosan was used as a stabilizer, and chitosan-stabilized selenium nanoparticles (CS@SeNPs) were prepared under the same conditions as in Example 2.

[0032] Comparative Example 2: Preparation of lentinan-stabilized selenium nanoparticles (LNT@SeNPs): Using lentinan as a stabilizer, chitosan-stabilized selenium nanoparticles (CS@SeNPs) were prepared under the same conditions as in Example 2.

[0033] Transmission electron microscopy showed that all coated particles were uniformly dispersed, mainly spherical, with PRP@SeNPs being smaller and more uniformly dispersed than CS and LNT-modified nanoparticles. Figure 11B). The dynamic light scattering results are consistent with this: in the first batch of samples, the average diameters of CS@SeNPs, LNT@SeNPs, and PRP@SeNPs were 121.68±1.94 nm, 104.18±1.06 nm, and 87.86±2.14 nm, respectively, and this order remained consistent in the repeated batches. Figure 11 C). Infrared spectroscopy confirmed successful modification of the polysaccharide canopy: PRP@SeNPs retained the characteristic O–H stretching vibration (~3320 cm⁻¹) and C–O ring vibration (~1010 cm⁻¹) of PRP, indicating the presence of a hydrogen-bonded non-covalent coating. Figure 11 D). Zeta potential measurements further confirmed its excellent colloidal stability: in the first batch of samples, the zeta potentials of CS@SeNPs, LNT@SeNPs, and PRP@SeNPs were −15.42 mV, −27.93 mV, and −36.40 mV, respectively, and the values ​​were similar in repeated experiments. Figure 11 E). Long-term stability tests in PBS (4 weeks) showed that the particle size of CS@SeNPs continued to increase and aggregate (to approximately 400 nm by week 4), while the particle size distribution of LNT@SeNPs and PRP@SeNPs remained relatively stable; notably, the particle size of PRP@SeNPs changed the least over time. Figure 11 F). These results—smaller initial particle size, more negative zeta potential, stable batch repeatability, and excellent long-term stability—demonstrate that PRP is an ideal coating material for preparing compact, stable SeNPs.

[0034] Example 3: Application of PRP@SeNPs: (1) PRP@SeNPs can promote selenium uptake in PC12 cells and inhibit the ferroptosis signaling pathway: When the selenium concentration was between 0 and 100 ng / mL (in the form of CS@SeNPs, LNT@SeNPs, or PRP@SeNPs), the results of live / dead cell staining and CCK-8 assays were not significantly different from the control group; however, when the concentration reached 200 ng / mL, all coated nanoparticles reduced cellular metabolic activity and increased cell death. Figure 12 (A–B). This “trace element window” supports the use of a concentration of 100 ng / mL in mechanistic studies to ensure that differences between formulations stem from variations in how cells deliver and process selenium.

[0035] Under ferroptosis stress (FINO2 treatment) conditions, DCFH-DA imaging and flow cytometry showed that the inhibitory effect of the nanoparticle coating on reactive oxygen species (ROS) increased in a stepwise manner. Figure 12(C–D). The proportions of ROS-positive cells were: 5.51% in the control group, 35.9% in the FINO2 treatment group, 24.3% in the CS@SeNPs treatment group, 20.9% in the LNT@SeNPs treatment group, and 13.9% in the PRP@SeNPs treatment group. This is consistent with the microscopic observation results, indicating that PRP@SeNPs are most effective in reducing lipid peroxidation stress. Mechanistically, early control of ROS is important in ferroptosis because it can block the positive feedback loop of lipid peroxide amplifying iron uptake / signal transduction.

[0036] HPLC-ICP-MS speciation analysis showed that, compared with PC12 cells treated with LNT@SeNPs or CS@SeNPs, cells treated with PRP@SeNPs formed selenocysteine ​​(SeCys2) faster and reached a higher peak at 24 hours, which is consistent with the kinetic advantage in the selenium metabolic pathway. Figure 12 E). Since SeCys2 is the direct source of selenocysteine ​​biosynthesis, this result indicates that PRP can accelerate the metabolic flux of selenium into selenoprotein synthesis. Consistent with this, all three formulations increased GPX4 expression at 24 hours (100 ng / mL), but transferrin receptor 1 (TFR1, a signaling pathway for iron uptake driven by ferroptosis) was significantly reduced only in the LNT@SeNPs and PRP@SeNPs treatment groups, with the largest reduction observed in the PRP@SeNPs treatment group. Figure 12 F).

[0037] In summary, PRP@SeNPs not only inhibit FINO2-induced ROS production but also accelerate selenium uptake (in the form of SeCys2), thereby promptly meeting the synthesis requirements of GPX4 and restoring iron uptake signals to normal. This kinetic / mechanistic advantage at the neuronal level provides a reasonable basis for the excellent in vivo experimental results observed subsequently.

[0038] (2) PRP@SeNPs can promote the recovery of motor function and protect the integrity of the spinal cord in vivo: To assess functional recovery and tissue protection after spinal cord injury (SCI), the study was conducted over 35 days using longitudinal behavioral / kinematic tracking, electrophysiological testing, and endpoint histopathological examination.

[0039] For hindlimb kinematics, five anatomical landmarks (toes, ankles, knees, hips, and iliac crests) were marked and tracked. As the first functional indicator, compared with the SCI group, the Basso mouse score (BMS) in the selenium nanoparticle (SeNPs) treatment group gradually improved, and the PRP@SeNPs group showed the most significant improvement throughout the observation period. Figure 13 A).

[0040] Footprint analysis confirmed these findings: the gait trajectories of SCI mice were irregular and asymmetrical, while the gait patterns of mice treated with PRP@SeNPs were more continuous and coordinated. Figure 13 B). Trajectory plots based on DeepLabCut further differentiated the differences between groups: the trajectory of the injured mice was shortened and unstable, while PRP@SeNPs recovered a trajectory close to the physiological state to some extent. Figure 13 C). Quantitative analysis of swing percentage and range of motion of joint angles (hip, knee, ankle) showed that the gait dynamics of the PRP@SeNPs group were significantly improved compared with the LNT@SeNPs group and the SCI group. Figure 13 D).

[0041] Next, neuromuscular electrophysiological parameters were assessed as independent functionally relevant indicators. The motor / compound muscle action potentials were significantly reduced and delayed in the SCI group, partially recovered in the LNT@SeNPs group, and significantly improved in the PRP@SeNPs group, exhibiting higher amplitude and shorter latency—indicating better axonal conduction and neuromuscular connectivity. Figure 14 A–B). To integrate multi-parameter gait characteristics, stride length and joint indices were summarized in the radar plot (hexagon). The results showed that compared with the SCI group and the LNT@SeNPs group, the PRP@SeNPs group had a wider range of motion and was closer to normal. Figure 14 C). Finally, endpoint histological examination confirmed the functional results: HE staining showed extensive cavity formation and parenchyma loss in the SCI group, which was alleviated in the LNT@SeNPs group, while the PRP@SeNPs group showed the most significant improvement. Figure 14 D); Nissl staining showed a significant reduction in neurons after SCI, but neuronal cell bodies were preserved in the spinal cord treated with PRP@SeNPs. Figure 14 E). Quantitative analysis confirmed that, compared with the LNT@SeNPs group, the PRP@SeNPs group had a smaller lesion area and a higher proportion of Nissl positive neurons (E). Figure 14 F).

[0042] In summary, these data indicate that PRP@SeNPs are superior to LNT@SeNPs in restoring motor function, improving conduction, restoring gait coordination, and protecting spinal cord tissue.

[0043] (3) PRP@SeNPs can enhance microtubule stability and axonal regeneration after spinal cord injury: To investigate cytoskeleton integrity and glial response during the chronic phase, the spinal cord was examined 35 days after injury.

[0044] Immunofluorescence staining of acetylated tubulin and tyrosine-substituted tubulin showed significant differences among the groups. Figure 15 A). In the spinal cord injury group, acetylated tubulin decreased while tyrosine-treated tubulin increased, consistent with the characteristics of microtubule instability and structural disorder. Both LNT@SeNPs and PRP@SeNPs improved microtubule structure, with PRP@SeNPs showing the most significant recovery effect. Quantitative analysis of regions of interest (ROIs) around the lesion proximal / distal to the injury center revealed that the acetylated / tyrosine-treated tubulin ratio in the PRP@SeNPs group was significantly higher than that in the LNT@SeNPs group and the spinal cord injury group. Figure 15 C); Distance binning analysis showed that PRP@SeNPs achieved microtubule stabilization over a larger area around the lesion ( Figure 15 D). These data indicate that PRP@SeNPs can induce a shift in tubulin encoding long-lived acetylated microtubules, a hallmark of axonal cytoskeleton stability. Next, astrocyte responsiveness and axonal continuity were assessed using co-staining with GFAP and NF200. Figure 15 B). The spinal cord injury group exhibited strong GFAP scarring and disruption of NF200 labeling at the lesion site. LNT@SeNPs partially alleviated these features, while PRP@SeNPs significantly reduced GFAP expression and restored the continuity of NF200-positive axons, including longer NF200-positive axonal extensions across the perilesional area. Skeletal-based quantitative indicators confirmed that, compared to the LNT@SeNPs group and the spinal cord injury group, the PRP@SeNPs group had a higher NF200 continuity index / total axonal length and a lower GFAP area ratio / mean intensity. Figure 15 E–F).

[0045] Overall, Figure 15 The results showed that the spinal cord treated with PRP@SeNPs had a higher acetylated / tyrosineized tubulin ratio, reduced GFAP expression, and restored NF200 continuity, all of which support axonal repair.

[0046] (4) PRP@SeNPs can activate GPX4, inhibit neuronal ferroptosis, and protect the myelin sheath after spinal cord injury: This study investigates whether different polysaccharide coronal delivery methods can achieve ferroptosis control and myelin protection in vivo.

[0047] NeuN / 4-HNE / GFAP three-color immunofluorescence showed extensive colocalization of 4-HNE and NeuN⁺ neurons in the spinal cord injury group (SCI), consistent with neuronal-centered lipid peroxidation characteristics. Both LNT@SeNPs and PRP@SeNPs reduced the 4-HNE load in neurons, with PRP@SeNPs showing the most significant reduction. Simultaneously, GFAP markers around the lesion were also reduced, indicating lower responsiveness of the glial environment. Figure 16 A).

[0048] Whole-spinal cord MBP / NF200 / DAPI staining further showed that, compared with LNT@SeNPs, PRP@SeNPs best maintained the continuity of myelinated axons, with a wider MBP coverage and a more continuous NF200 trajectory. Figure 16 B).

[0049] At the molecular level, Western blot analysis showed that PRP@SeNPs could restore GPX4 expression and inhibit ferroptosis-related proteins ACSL4 and TFR1. Among all groups, PRP@SeNPs had the most significant effect in shifting the proteome toward anti-ferroptosis. Figure 16 C, 11E).

[0050] Consistent with the structural examination results, Luxol LFB staining showed that the PRP@SeNPs group had the highest myelin retention rate. Figure 16 D, 11F).

[0051] In summary, these data indicate that although both LNT and PRP can act on the selenoprotein axis, the PRP canopy generates a more effective anti-ferroptosis response in tissues, reduces neuronal lipid peroxidation, and protects myelin and axonal structures. This protective effect, centered on GPX4, is consistent with the accelerated selenium uptake observed in PC12 cells.

[0052] (5) PRP@SeNPs can remodel the activated state of microglia and reduce neuroinflammation: Although LNT@SeNPs and PRP@SeNPs showed comparable effects on the selenium-GPX4 axis, their anti-inflammatory effects differed significantly. In BV2 cell experiments, cell proliferation / viability (as measured by CCK-8) was highest in the PRP@SeNPs group, followed by the LNT@SeNPs group, and lowest in the CS@SeNPs group. Figure 17 A). Under lipopolysaccharide (LPS) stimulation, the total superoxide dismutase (SOD) activity in the LPS group was inhibited, while both types of nanoparticles could restore their activity. PRP@SeNPs showed better results than CS@SeNPs, and were comparable to LNT@SeNPs. Figure 17B). The results of polarization marker detection also showed the same trend: Western blotting showed that the expression of Arg1 / IL-10 was most significantly increased in the PRP@SeNPs group, while the expression of iNOS / CD86 was most significantly decreased. Figure 17 C); Immunofluorescence further confirmed that after PRP@SeNPs treatment, the proportion of CD206⁺ cells was higher and the proportion of CD86⁺ cells was lower. Figure 17 D). This advantage may stem from two properties of the material: compared to other coated SeNPs, PRP@SeNPs have a more negative zeta potential and a smaller, more stable particle size, both of which are known to reduce opsonization / protein bridging and alleviate microglial activation in serum; furthermore, PRP itself has inherent anti-inflammatory activity, which can enhance the redox protective effect of selenium. In vivo evaluation mainly focused on PRP@SeNPs and LNT@SeNPs; CS@SeNPs were not included in subsequent studies due to their poor colloidal stability and in vitro antioxidant properties. At 7 days post-injury (7 dpi), CD86 / CD206 / DAPI staining showed a significant shift in microglia in the PRP@SeNPs group towards a repair phenotype—CD86 was decreased and CD206 was increased compared to the SCI and LNT@SeNPs groups. Figure 18 A). Western blot results at the same time point confirmed this pattern, namely, upregulation of CD206 / IL-10 and downregulation of CD86 / iNOS, with the PRP@SeNPs group showing the largest changes. Figure 18 (B) Mechanistically, these results are consistent with the following process: both LNT and PRP can activate the selenoprotein defense system, but PRP@SeNPs are more effective at maintaining antioxidant status and reducing lipid peroxidation load, thereby attenuating pro-inflammatory signaling and polarizing microglia towards the M2 type. This PRP-driven remodeling of the inflammatory microenvironment provides a reasonable explanation for the superior functional and histological recovery of PRP@SeNPs compared to LNT@SeNPs.

[0053] The above description discloses only preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention. Therefore, equivalent variations made in accordance with the claims of the present invention are still within the scope of the present invention.

Claims

1. A polysaccharide of Polygonatum multiflorum, characterized in that, Its chemical formula is shown in formula (Ⅰ): （Ⅰ）； m + n = 22 - 24, m ≠ 0, n ≠ 0.

2. The method for preparing Polygonatum multiflorum polysaccharide as described in claim 1, characterized in that, Includes the following steps:

1. Take the rhizome of Polygonatum multiflorum, dry it until the moisture content is ≤10%, pulverize it and pass it through a 20-40 mesh sieve to obtain Polygonatum multiflorum rhizome powder; 2. Mix the powdered rhizome of Polygonatum multiflorum with deionized water or buffer solution, and extract by stirring under conditions of 55–60 °C and pH 5.5–6.5 using inert gas headspace or low dissolved oxygen. Then, obtain the filtrate by hot filtration at 55–60 °C.

3. An anion-exchange cellulose column was used to elute and collect the neutral polysaccharide main peak with deionized water, followed by elution of acidic heteropolysaccharides with 0.1–0.5 M NaCl. The collected neutral polysaccharide main peak was then polished by Sephadex G-100 gel filtration to collect symmetrical single peaks. The aqueous eluent was desalted by ultrafiltration. IV. Freeze-drying to obtain the Polygonatum polysaccharide as described in claim 1.

3. The method for preparing Polygonatum multiflorum polysaccharide according to claim 2, characterized in that: The Polygonatum multiflorum root and stem powder in step two is obtained by pre-treating the Polygonatum multiflorum root and stem powder obtained in step one. The pre-treating includes the following steps: mixing the Polygonatum multiflorum root and stem powder obtained in step one with 60-80% ethanol at a mass-volume ratio of 1:8-1:12, stirring, filtering, taking the filter residue, and removing the residual ethanol from the filter residue.

4. The method for preparing Polygonatum multiflorum polysaccharide according to claim 2, characterized in that: In step two, the stirring extraction involves gentle stirring for 60–120 minutes, repeated twice, and the filtrates are combined.

5. The method for preparing Polygonatum multiflorum polysaccharide according to claim 2, characterized in that: In step four, the sample is pre-frozen at –40 °C for 6–12 h and then sublimated and dried for 24–36 h.

6. The application of the Polygonatum polysaccharide as described in claim 1 in the preparation of products that improve glucose and lipid metabolism or insulin resistance.

7. A selenium nanoparticle, characterized in that: It includes selenium nanoparticles and a coating layer modified on the outside of the selenium nanoparticles, wherein the coating layer is the Polygonatum polysaccharide as described in claim 1.

8. The method for preparing selenium nanoparticles as described in claim 7, characterized in that, Includes the following steps: (1) Disperse the Polygonatum polysaccharide as described in claim 1 in water to prepare an aqueous solution of Polygonatum polysaccharide; (2) Mix the selenium precursor solution with the polysaccharide aqueous solution of Polygonatum odoratum prepared in step (1) under magnetic stirring until homogeneous. Add the reducing agent solution dropwise while stirring and react. (3) Dialysis removes free small molecules to obtain purified selenium nanoparticles.

9. The method for preparing selenium nanoparticles according to claim 8, characterized in that: In step (3), dialysis is performed using a dialysis bag with a molecular weight cutoff of 6000–8000 kDa.

10. The use of the selenium nanoparticles as described in claim 7 in the preparation of a medicament for treating spinal cord injury.

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