Polyflavonoid polysaccharide of polygonatum cyrtonema and application thereof

Abstract

The present application relates to a kind of Polygonatum cyrtonema polysaccharide and its application.A new type of polysaccharide PRP is extracted and purified in Polygonatum cyrtonema, which is neutral inulin type polysaccharide.PRP has a node similar to sucrose α -D-glucose-(1↔2)- β -D-fructose is centered.C6 position of glucose has an additional β -fructose (2→6) substituent, while two chains are connected by (2→1)- β -fructose bond extends from the core, forming a Y-shaped moderately branched structure.The present application provides a selenium nanoparticle coated with Polygonatum cyrtonema polysaccharide, which has smaller initial particle size, more negative zeta potential, stable batch repeatability and excellent long-term stability compared to other polysaccharide stabilized selenium nanoparticles.

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):

[0008]

[0009] (I);

[0010] m + n = 22 - 24, m ≠ 0, n ≠ 0.

[0011] This invention further provides a method for preparing Polygonatum multiflorum polysaccharide as described above, comprising the following steps:

[0012] I. Take the rhizome of Polygonatum cyathophorum, dry it to moisture ≤10%, crush it and pass it through a 20-40 mesh sieve to obtain Polygonatum cyathophorum rhizome powder;

[0013] II. Mix the Polygonatum cyathophorum rhizome powder with deionized water or buffer, and under the conditions of 55-60 °C and pH 5.5-6.5, use inert gas headspace or low dissolved oxygen conditions to stir and extract, and then heat filter at 55-60 °C to obtain a filtrate;

[0014] III. Use an anion exchange cellulose column to elute the main peak of neutral polysaccharides collected with deionized water, and then elute the acidic heteropolysaccharides with 0.1-0.5 M NaCl. The main peak of neutral polysaccharides collected is polished again by Sephadex G-100 gel filtration, and the symmetric single peak is collected. The water phase eluent is desalted by ultrafiltration;

[0015] IV. Freeze-drying to obtain Polygonatum cyathophorum polysaccharide.

[0016] The Polygonatum cyathophorum rhizome powder in step II is obtained by pretreating the Polygonatum cyathophorum rhizome powder obtained in step I, and the pretreatment includes the following steps: mixing the Polygonatum cyathophorum rhizome powder obtained in step I with 60-80% ethanol at a mass-volume ratio of 1:8-1:12, stirring, filtering, taking the filter residue, and removing the residual alcohol from the filter residue.

[0017] In step II, the stirring extraction is gentle stirring for 60-120 min, repeated extraction 2 times, and the filtrates are combined.

[0018] In step IV, pre-freeze at -40 °C for 6-12 h, and sublimate dry for 24-36 h.

[0019] The application further provides the use of the Polygonatum cyathophorum polysaccharide as described above for preparing a product for improving glycolipid metabolism or insulin resistance.

[0020] The application further provides a selenium nanoparticle, which comprises a selenium nanoparticle and a coating layer modified on the outside of the selenium nanoparticle, and the coating layer is the Polygonatum cyathophorum polysaccharide as described above.

[0021] The application further provides a preparation method of the selenium nanoparticle as described above, comprising the following steps:

[0022] (1) Disperse the Polygonatum cyathophorum polysaccharide as described above in water to prepare a Polygonatum cyathophorum polysaccharide aqueous solution;

[0023] (2) Mix the selenium precursor solution with the Polygonatum cyathophorum polysaccharide aqueous solution prepared in step (1) uniformly under magnetic stirring, and add a reducing agent solution dropwise under stirring to react;

[0024] (3) dialysis to remove free small molecules to obtain purified selenium nanoparticles.

[0025] In step (3), dialysis is performed using a dialysis bag with a molecular weight cut-off of 6000-8000 kDa.

[0026] Use of the selenium nanoparticles as described above for the preparation of selenium supplements.

[0027] The beneficial effects of the present application are as follows:

[0028] 1. A novel polysaccharide PRP is obtained by extraction and purification from Polygonatum cyrtonema Hua, which 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 a node similar to sucrose α -D-glucose-(1↔2)- β -D-fructose as the center. The C6 position of glucose carries an extra β -fructose (2→6) substituent, and two chains are connected through (2→1)- β -fructose bond extending from the core (chain length m and n, m+n ≈ 23, and m, n are not 0), forming a Y-shaped moderate branching structure.

[0029] 2. The above-mentioned novel Polygonatum cyrtonema Hua polysaccharide PRP has antioxidant and anti-inflammatory activity. In vivo studies show that PRP can significantly reduce the weight loss, hyperglycemia, and liver and kidney damage of high-fat diet / streptozotocin (HFD / STZ)-induced type 2 diabetic mice.

[0030] 3. The present application provides selenium nanoparticles coated with polygonatum sibiricum polysaccharide (PRP@SeNPs). The PRP topological structure provides dense hydroxyl clusters that can be anchored at multiple sites of Se–OH / Se–O⁻ and construct a non-ionic high-hydrated space brush layer ("segment-loop-tail" crown layer) on the slip plane. This brush layer resists ion shielding, protein-mediated bridging and Ca²⁺-induced flocculation through steric hindrance / hydration repulsion, making PRP@SeNPs have smaller initial particle size, more negative zeta potential, stable batch repeatability and excellent long-term stability compared to other polysaccharide-stabilized selenium nanoparticles. Unlike chitosan or lentinan, which only have a physical stabilizing effect, in PRP@SeNPs, PRP not only provides stability, but also synergistically exerts pharmacological effects, such as ROS scavenging, inflammatory factor inhibition and immune balance promotion, thus achieving more significant efficacy in treating diseases such as spinal cord injury through "polysaccharide + selenium synergy". Moreover, the PRP coating makes the SeNPs surface negatively charged, with a zeta potential of about –36 mV, significantly improving the stability in the blood and tissue environment, avoiding rapid sedimentation and clearance, and enabling PRP@SeNPs nanoparticles to more effectively penetrate, distribute and reside in the spinal cord injury site, thereby increasing the local Se effective concentration. At the same time, the anti-inflammatory effect of PRP itself improves the local microenvironment, enabling SeNPs to better exert anti-ferroptosis and neuroprotective effects. In addition, the PRP coating layer can slow down the release rate of Se and enhance the stability of SeNPs in vivo, significantly widening the safety dose window of Se, enabling PRP@SeNPs to achieve the desired efficacy while requiring a lower dose than traditional SeNPs or inorganic Se supplements, thereby reducing the risk of liver and kidney toxicity and systemic side effects. BRIEF DESCRIPTION OF DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. Obviously, the drawings in the following description only constitute some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to these drawings without creative labor.

[0032] Figure 1 The infrared spectrum (FTIR) of polygonatum sibiricum polysaccharide PRP-W-1 prepared in Example 1;

[0033] Figure 2 Methylation assay of the hydrolysate of polygonatum sibiricum polysaccharide PRP-W-1 prepared in Example 1;

[0034] Figure 3Comparison of the liquid chromatogram of standard monosaccharides with the hydrolyzed polyphyllous polygonum multiflorum Thunb polysaccharide PRP-W-1 prepared in Example 1;

[0035] Figure 4 1H spectrum of the polyphyllous polygonum multiflorum Thunb polysaccharide PRP-W-1 prepared in Example 1;

[0036] Figure 5 13C spectrum of PRP-W-1;

[0037] Figure 6 HSQC spectrum of PRP-W-1;

[0038] Figure 7 HMBC spectrum of PRP-W-1;

[0039] Figure 8 COSY spectrum of PRP-W-1;

[0040] Figure 9 DEPT 135 spectrum of PRP-W-1;

[0041] Figure 10 A is the chemical structure of the polyphyllous polygonum multiflorum Thunb polysaccharide PRP-W-1, Figure 10 B is a simplified symbolic diagram of the structure of PRP-W-1;

[0042] Figure 11 A is a flowchart of the preparation of PRP@SeNPs in Example 2; Figure 11 B is a transmission electron microscope image of CS@SeNPs, LNT@SeNPs and PRP@SeNPs; Figure 11 C is the average particle size of CS@SeNPs, LNT@SeNPs and PRP@SeNPs; Figure 11 D is an infrared spectrum of PRP@SeNPs; Figure 11 E is the zeta potential of CS@SeNPs, LNT@SeNPs and PRP@SeNPs; Figure 11 F is the stability test results of CS@SeNPs, LNT@SeNPs and PRP@SeNPs;

[0043] Figure 12 A is the result of live / dead cell staining experiment; Figure 12 B is the CCK-8 detection result; Figure 12 C is the DCFH-DA imaging result; Figure 12 D is the flow cytometry experiment result; Figure 12 E is the HPLC-ICP-MS morphological analysis result; Figure 12F. The results of the influence of CS@SeNPs, LNT@SeNPs and PRP@SeNPs on the expression of GPX4 and TFR1; Figure 12 G. The results of the quantitative analysis of the influence of CS@SeNPs, LNT@SeNPs and PRP@SeNPs on the expression of GPX4 and TFR1;

[0044] Figure 13 A. The results of the influence of different treatments on the motor function of the hind limbs after spinal cord injury (SCI); Figure 13 A. The results of the influence of different treatments on the motor function of the hind limbs after spinal cord injury (SCI); Figure 13 B. The results of the footprint analysis of CS@SeNPs, LNT@SeNPs and PRP@SeNPs on SCI mice; Figure 13 C. The trajectory graph of mice in each experimental group based on DeepLabCut; Figure 13 D. The results of the quantitative analysis of the swing percentage and joint angle range (hip, knee, ankle) of mice in each experimental group;

[0045] Figure 14 A. The results of the influence of different treatments on the muscle electrophysiology, tissue morphology and neuronal function after spinal cord injury (SCI); Figure 14 A. The results of the influence of different treatments on the muscle electrophysiology, tissue morphology and neuronal function after spinal cord injury (SCI); Figure 14 B. The statistical results of the amplitude of the electrophysiological waveform; Figure 14 C. The radar chart of multi-index (hip joint angle amplitude, knee joint angle amplitude, sensory function, motor function, toe amplitude) integration; Figure 14 D. The results of HE staining; Figure 14 E. The results of Nissl staining; Figure 14 F. The results of the quantitative analysis of the injury area of mice in each experimental group;

[0046] Figure 15 A. The results of the influence of different treatments on the muscle electrophysiology, tissue morphology and neuronal function after spinal cord injury (SCI); Figure 15 A. The results of Tyr Tubulin / Ace Tubulin / DAPI staining; Figure 15 B. The results of GFAP / NF200 / DAPI staining; Figure 15 C. The quantitative analysis results of microtubule stability area; Figure 15 D. The fluorescence intensity ratio analysis of acetylated microtubules (AceTubulin) and tyrosinated microtubules (Tyr Tubulin); Figure 15 E. The quantitative results of GFAP fluorescence intensity; Figure 15 F. The quantitative results of NF200 fluorescence intensity;

[0047] Figure 16The effects of different treatments on neuron survival, myelin repair and ferroptosis after spinal cord injury (SCI) Figure 16 A is the NeuN / 4-HNE / GFAP three-color immunofluorescence result; Figure 16 B is the whole spinal cord MBP / NF200 / DAPI staining result; Figure 16 C, 11E are the Western blot results and quantitative analysis results; Figure 16 D, 11F are the Luxol Fast Blue (LFB) staining results and quantitative analysis results;

[0048] Figure 17 A is the BV2 cell proliferation experiment result; Figure 17 B is the total superoxide dismutase (SOD) activity analysis result; Figure 17 C is the detection result of polarization marker; Figure 17 D is the CD86 / CD206 / DAPI immunofluorescence experiment result;

[0049] Figure 18 The results of the regulation of different treatments on macrophage polarization after spinal cord injury (SCI) Figure 18 A is the CD86 / CD206 / DAPI immunofluorescence analysis result; Figure 18 B is the Western blot result of polarization-related protein. DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the present application clearer, the present application will be further described in detail below with reference to the drawings.

[0051] Example 1 Preparation and identification of polygonatum cyrtonema polysaccharide PRP

[0052] In this example, polygonatum cyrtonema polysaccharide PRP was extracted and purified from polygonatum cyrtonema rhizomes, and the specific process was as follows:

[0053] I. Crushing and sieving: Dry the polygonatum cyrtonema rhizome dry product at ≤40 °C to a moisture content of ≤10%, crush and sieve through a 20-40 mesh (preferably 30 mesh) sieve, and store in a sealed container to prevent moisture absorption;

[0054] II. Defatting / pre-treatment to remove small molecules: 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 recover the ethanol by suction filtration to remove pigments, oligosaccharides, free polyphenols and small molecules; remove residual alcohol (<0.5%) under ventilation or rotary evaporation, and prepare for use. This step is optional and is used to improve the clarity and color of the subsequent solution;

[0055] III. Extraction medium and conditions: The pretreated plant powder was mixed with deionized water or weak buffer (such as 10-20 mM phosphate, pH 6.0±0.2) at a solid-liquid ratio of 1:10-1:20 (w / v), and gently stirred at 55-60 °C for 60-120 min; the extraction can be repeated twice and the filtrate was combined; the pH was controlled at 5.5-6.5 throughout the process to avoid strong acid-induced fructan hydrolysis; the use of inert gas (nitrogen) headspace or low dissolved oxygen conditions can reduce Maillard reaction and browning;

[0056] IV. Coarse filtration and clarification: hot filtration (60 °C) to remove coarse residue, and if necessary, add 0.05-0.10% (w / v) diatomite to aid filtration, or use a 0.45 μm microfiltration membrane to clarify, to obtain a light transparent extract;

[0057] V. Purification: use anion exchange cellulose (DEAE) column (diameter: height ≈1:10) to elute the main peak of neutral polysaccharides (chicory-type polysaccharides are mostly neutral components, which can be eluted by water), and then elute the acidic heteropolysaccharides with 0.1-0.5 M NaCl; the main peak is polished by Sephadex G-100 gel filtration, and the symmetric single peak is collected. The eluent is an aqueous phase, and the salt is removed by ultrafiltration;

[0058] VI. Freeze-drying (research grade): pre-freeze at -40 °C for 6-12 h, sublimation dry for 24-36 h, and the finished product is loose, recorded as PRP-W-1.

[0059] The above process is completed under mild conditions of 55-60 °C, pH 5.5-6.5 and low oxygen environment, combined with fractional alcohol precipitation and membrane separation, which significantly reduces the chain breakage and depolymerization of fructan, stabilizes the molecular weight and distribution of the main peak, and keeps the chicory fingerprint clear in FT-IR; at the same time, the target component is obtained by one-pot fractionation, which takes into account the yield and retention of active structure.

[0060] Figure 1 The infrared spectrum (FTIR) of the prepared Polyphylla pratae polysaccharide PRP-W-1 can be seen at 3200-3600 cm -1 −OH peak, at 2800-3000 cm -1 −CH peak, at 1700 cm -1 −COO−R peak, at 1000-1200 cm -1 C−O−C peak.

[0061] Figure 2Total ion chromatogram of methylated sugar alcohol acetate of Polygonatum cyrtonema PRP-W-1, for determining the glycosidic linkage pattern of PRP-W-1, methylation analysis was performed. The main residue was 1,2-linked fructose (1,2-Fru), which accounted for more than 53%, identified as the main component of the backbone. Terminal glucose (t-Glc) was identified as the starting glucose residue of inulin structure, while 1,2,6-linked fructose (1,2,6-Fru) was part of the backbone forming branches, and 1,6-linked glucose (1,6-Glc) was found to be part of the side chain. 2-Fru residues were determined as terminal fructose units in the polysaccharide.

[0062] Figure 3 Comparison of liquid chromatogram of PRP-W-1 after hydrolysis with standard monosaccharides, wherein the standard monosaccharides include fucose (Fuc), arabinose (Ara), galactose (Gal), glucose (Glc), fructose (Fru), ribose (Rib), galacturonic acid (Gal-UA), guluronic acid (Gul-UA), glucuronic acid (Glc-UA), and mannuronic acid (Man-UA), it can be seen that PRP-W-1 after hydrolysis is mainly glucose (Glc) and fructose (Fru).

[0063] Figure 4 1H spectrum of PRP-W-1 prepared in Example 1; Figure 5 13C spectrum of PRP-W-1; Figure 6 HSQC spectrum of PRP-W-1; Figure 7 HMBC spectrum of PRP-W-1; Figure 8 COSY spectrum of PRP-W-1; Figure 9 DEPT 135 spectrum of PRP-W-1. Through Figures 4-9 The “composition + structure” comprehensive characterization of PRP-W-1 can be determined that PRP-W-1 is Figure 10 A shown in the chemical structure, Figure 10 B is a simplified symbolic diagram of the structure of PRP-W-1, and the blue circle represents α - glucose ( α -Glc), and the green pentagon represents β - fructose ( β -Fru), and the numbers (such as “1”, “2”, “6”) mark the connection position of the glycosidic bond, which can indicate that PRP has a sucrose-like node α -D-glucose-(1↔2)- β -D-fructose as the center. The C6 position of glucose has an additional β- fructose (2→6) substituents, with both chains extending away from the core (chain lengths m and n, m + n = 23, and m, n ≠ 0) to form a Y-shaped, moderately branched structure. β - fructose linkages extend away from the core (chain lengths m and n, m + n = 23, and m, n ≠ 0) to form a Y-shaped, moderately branched structure.

[0064] In vivo studies showed that PRP-W-1 treatment significantly alleviated the body weight loss, hyperglycemia, and liver and kidney injury in high-fat diet / streptozotocin (HFD / STZ)-induced type 2 diabetic mice. In addition, it was elucidated that PRP-W-1 exerts its regulatory effects through multiple signaling pathways, indicating that its mechanism of action is multifaceted. These findings highlight the potential of PRP-W-1 as a novel active polysaccharide in the treatment of diabetes.

[0065] Example 2 Preparation of selenium nanoparticles stabilized by polygonatum cyrtonema polysaccharide PRP-W-1 (PRP@SeNPs):

[0066] As shown in Figure 11 A, this embodiment uses sodium selenite (Na2SeO3) as a selenium precursor, vitamin C (Vc) as a reducing agent, and polygonatum cyrtonema polysaccharide PRP-W-1 prepared in Example 1 as a stabilizing agent to synthesize selenium nanoparticles stabilized by polygonatum cyrtonema polysaccharide (PRP@SeNPs) using a one-pot reduction method. The specific preparation steps are as follows:

[0067] (1) Prepare 5–20 mM Na2SeO3 aqueous solution, 20–50 mM Vc aqueous solution, and 1–10 mg / mL polygonatum cyrtonema polysaccharide PRP-W-1 aqueous solution;

[0068] (2) Mix 2 mL of the Na2SeO3 aqueous solution with 5 mL of the PRP-W-1 aqueous solution uniformly under magnetic stirring;

[0069] (3) Slowly add 1 mL of deionized water for dilution, and keep the reaction system uniform;

[0070] (4) Under stirring, add the Vc aqueous solution dropwise to reduce Se 4 ⁺ to elemental Se;

[0071] (5) Continue stirring for 24 h to form an orange transparent PRP@SeNPs solution at room temperature;

[0072] (6) Use a dialysis bag with a molecular weight cut-off of 6000–8000 kDa to dialyze and remove free small molecules to obtain purified PRP@SeNPs.

[0073] Comparative Example 1 Preparation of selenium nanoparticles stabilized by chitosan (CS@SeNPs):

[0074] Chitosan was used as stabilizer, and chitosan stabilized selenium nanoparticles (CS@SeNPs) were prepared under the same conditions as Example 2.

[0075] Preparation of lentinan stabilized selenium nanoparticles (LNT@SeNPs) in Comparative Example 2:

[0076] Lentinan was used as stabilizer, and chitosan stabilized selenium nanoparticles (CS@SeNPs) were prepared under the same conditions as Example 2.

[0077] Transmission electron microscopy showed that the particles of all coatings were uniformly dispersed, mainly in spherical shape, and PRP@SeNPs were smaller and more uniformly dispersed than CS and LNT modified nanoparticles (Fig. 1A). Figure 11 B). Dynamic light scattering results were consistent with the former: 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 the order remained consistent in repeated batches (Fig. 1B). Figure 11 C). Infrared spectroscopy confirmed the successful modification of the polysaccharide corona: 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 (Fig. 1C). 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 (Fig. 1D). Figure 11 E). Long-term stability tests (4 weeks) in PBS showed that the particle size of CS@SeNPs continued to increase and aggregate (about 400 nm by the 4th week), 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 (Fig. 1E). Figure 11 F). These results—smaller initial particle size, more negative zeta potential, stable batch reproducibility, and excellent long-term stability—indicate that PRP is an ideal coating material that can prepare compact, stable SeNPs.

[0078] Example 3. Application of PRP@SeNPs:

[0079] (1) PRP@SeNPs can promote the absorption of selenium by PC12 cells and inhibit the ferroptosis signaling pathway:

[0080] When selenium concentration was at 0-100 ng / mL (in the form of CS@SeNPs, LNT@SeNPs or PRP@SeNPs), there was no significant difference between the control group and the results of live / dead cell staining and CCK-8 detection; while when the concentration reached 200 ng / mL, all coated nanoparticles would reduce cell metabolic activity and increase cell death ( Figure 12 A-B). This "trace element window" supports the use of 100 ng / mL concentration in mechanism research to ensure that the differences between different formulations are due to different ways of delivering and processing selenium by cells.

[0081] Under ferroptosis stress (FINO2 treatment), DCFH-DA imaging and flow cytometry showed that the inhibitory effect of nanoparticle coating on reactive oxygen species (ROS) increased in a stepwise manner ( Figure 12 C-D). The proportion of ROS-positive cells was 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, which was consistent with the microscopic observation results, indicating that PRP@SeNPs had the most significant effect in reducing lipid peroxidation stress. Mechanistically, early control of ROS is important during ferroptosis, as it can block the positive feedback loop of lipid peroxide amplifying iron uptake / signaling.

[0082] HPLC-ICP-MS morphological analysis showed that compared with LNT@SeNPs or CS@SeNPs treated PC12 cells, SeCys2 formation was faster and reached a higher peak at 24 hours in PRP@SeNPs treated cells, which was consistent with the kinetic advantage in the selenium metabolism pathway ( Figure 12 E). Since SeCys2 is a direct source of selenium cysteine biosynthesis, this result indicates that PRP can accelerate the metabolic flux of selenium into selenoprotein synthesis. Consistently, all three formulations increased GPX4 expression at 24 hours (100 ng / mL), but transferrin receptor 1 (TFR1, a reflection of iron uptake signaling driven by ferroptosis) was only significantly reduced in the LNT@SeNPs and PRP@SeNPs treatment groups, with the largest reduction in the PRP@SeNPs treatment group ( Figure 12 F).

[0083] In summary, PRP@SeNPs not only inhibit FINO2-induced ROS production, but also accelerate selenium absorption (in the form of SeCys2), thereby meeting the synthesis needs of GPX4 in a timely manner and restoring iron uptake signaling to normal. This kinetic / mechanistic advantage at the neuronal level provides a reasonable basis for the excellent in vivo experimental results observed subsequently.

[0084] (2) PRP@SeNPs can promote locomotor functional recovery and protect spinal cord integrity in vivo:

[0085] To evaluate functional recovery and tissue protection after spinal cord injury (SCI), longitudinal behavioral / kinematic tracking, electrophysiological examination, and terminal histopathological examination were performed within 35 days.

[0086] For hindlimb kinematics, five anatomical landmarks (toe, ankle, knee, hip, and iliac crest) were labeled and tracked. As the first functional index, Basso Mouse Scale (BMS) was gradually improved in SeNPs-treated groups compared with the SCI group, and the improvement was most significant in the PRP@SeNPs group throughout the observation period ( Figure 13 A).

[0087] Footprint analysis confirmed these findings: the gait trajectory of SCI mice was irregular and asymmetric, while the gait pattern of PRP@SeNPs-treated mice was more continuous and coordinated ( Figure 13 B). Trajectory plots based on DeepLabCut further distinguished the differences between groups: the trajectory of injured mice was shortened and unstable, while PRP@SeNPs restored the trajectory close to the physiological state to some extent ( Figure 13 C). Quantitative analysis of swing percentage and joint angle range (hip, knee, ankle) showed that gait dynamics were significantly improved in the PRP@SeNPs group compared with the LNT@SeNPs and SCI groups ( Figure 13 D).

[0088] Next, the neuro-muscular electrophysiological index was evaluated as an independent functional correlation index. The motor / compound muscle action potential was significantly reduced and delayed in the SCI group, partially restored in the LNT@SeNPs group, and significantly improved in the PRP@SeNPs group, showing higher amplitude and shorter latency—indicating better axonal conduction and neuromuscular junction ( Figure 14 A–B). To integrate multi-parameter gait features, stride and joint indicators were summarized in a radar chart (hexagon), and the results showed that the PRP@SeNPs group had a wider range of motion and was closer to normal compared with the SCI and LNT@SeNPs groups ( Figure 14 C). Finally, terminal histological examination confirmed the functional results: HE staining showed extensive cavity formation and parenchymal loss in the SCI group, which was reduced in the LNT@SeNPs group, and most significantly in the PRP@SeNPs group ( Figure 14 D); Nissl staining showed a significant reduction in neurons after SCI, but the PRP@SeNPs-treated spinal cord preserved neuronal cell bodies ( Figure 14E). 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).

[0089] 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.

[0090] (3) PRP@SeNPs can enhance microtubule stability and axonal regeneration after spinal cord injury:

[0091] To investigate cytoskeleton integrity and glial response during the chronic phase, the spinal cord was examined 35 days after injury.

[0092] 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).

[0093] Overall, Figure 15PRP@SeNPs treatment showed higher acetylated / tyrosinated tubulin ratio, less GFAP expression, and restored NF200 continuity in the spinal cord, which all supported axonal repair.

[0094] (4) PRP@SeNPs can activate GPX4, inhibit neuronal ferroptosis, and protect myelin after spinal cord injury:

[0095] To investigate whether different polysaccharide crowns can deliver selenium to control ferroptosis and protect myelin in vivo.

[0096] NeuN / 4-HNE / GFAP triple immunofluorescence showed that 4-HNE and NeuN+ neurons were widely co-localized in the spinal cord injury (SCI) group, which was consistent with the characteristics of neuron-centered lipid peroxidation. Both LNT@SeNPs and PRP@SeNPs could reduce the 4-HNE load of neurons, with PRP@SeNPs showing the most significant effect; at the same time, the GFAP marker around the lesion also decreased accordingly, indicating a lower reactivity of the glial environment. Figure 16 A).

[0097] Whole spinal cord MBP / NF200 / DAPI staining further showed that PRP@SeNPs could best maintain the continuity of myelinated axons, with a wider MBP coverage range and more continuous NF200 tracks compared to LNT@SeNPs. Figure 16 B).

[0098] At the molecular level, Western blot analysis showed that PRP@SeNPs could restore GPX4 expression and inhibit ferroptosis-related proteins ACSL4 and TFR1, and PRP@SeNPs had the most significant effect on shifting the protein profile towards anti-ferroptosis in all groups. Figure 16 C, 11E).

[0099] Consistent with the structural detection results, Luxol fast blue (LFB) staining showed that the PRP@SeNPs group had the highest myelin preservation rate. Figure 16 D, 11F).

[0100] In summary, these data suggest that although both LNT and PRP can act on the selenium protein axis, the PRP crown can produce a more effective anti-ferroptosis response in tissues, reduce neuronal lipid peroxidation, and protect myelin and axon structure. This GPX4-centered protection is consistent with the observed accelerated selenium uptake in PC12 cells.

[0101] (5) PRP@SeNPs can remodel microglial activation and reduce neuroinflammation:

[0102] While LNT@SeNPs and PRP@SeNPs performed comparably in terms of selenium-GPX4 axis, their anti-inflammatory effects were significantly different. In BV2 cell experiments, cell proliferation / viability (detected by CCK-8) was highest in PRP@SeNPs group, followed by LNT@SeNPs group, and lowest in CS@SeNPs group (Fig. 2A). Under lipopolysaccharide (LPS) stimulation, total superoxide dismutase (SOD) activity was inhibited in LPS group, while both nanoparticles could restore its activity, with PRP@SeNPs outperforming CS@SeNPs and comparable to LNT@SeNPs (Fig. 2B). The detection results of polarization markers also showed the same trend: immunoblotting showed that Arg1 / IL-10 expression increased most significantly and iNOS / CD86 expression decreased most obviously in PRP@SeNPs group (Fig. 2C); immunofluorescence further confirmed that the proportion of CD206+ cells was higher and CD86+ cells were less after PRP@SeNPs treatment (Fig. 2D). This advantage might be attributed to two properties of this material: PRP@SeNPs had more negative zeta potential and smaller, more stable particle size compared to other coated SeNPs, both of which were known to reduce opsonization / protein bridging and attenuate microglial activation in serum; in addition, PRP itself had inherent anti-inflammatory activity that could enhance the redox protection of selenium. Figure 17 A). At the same time point, Western blot results confirmed this pattern, with CD206 / IL-10 upregulation and CD86 / iNOS downregulation, and the largest change in PRP@SeNPs group (Fig. 4B). Mechanistically, these results were consistent with the following processes: both LNT and PRP could activate the selenoprotein defense system, but PRP@SeNPs could more effectively maintain the antioxidant state, reduce lipid peroxidation load, and thus attenuate pro-inflammatory signals and polarize microglia to M2 type. This remodeling of the inflammatory microenvironment driven by PRP provided a reasonable explanation for the superior performance of PRP@SeNPs compared to LNT@SeNPs in terms of function and histological recovery. Figure 17 Figure 17 Figure 17 Figure 18 A). At the same time point, Western blot results confirmed this pattern, with CD206 / IL-10 upregulation and CD86 / iNOS downregulation, and the largest change in PRP@SeNPs group (Fig. 4B). Mechanistically, these results were consistent with the following processes: both LNT and PRP could activate the selenoprotein defense system, but PRP@SeNPs could more effectively maintain the antioxidant state, reduce lipid peroxidation load, and thus attenuate pro-inflammatory signals and polarize microglia to M2 type. This remodeling of the inflammatory microenvironment driven by PRP provided a reasonable explanation for the superior performance of PRP@SeNPs compared to LNT@SeNPs in terms of function and histological recovery. Figure 18

[0103] ​​​​The above merely provides the preferred embodiment of the application, and cannot allude the protection scope of the application, therefore any equivalent changes made according to the claims of the application shall be within the scope of the application.

Claims

1. 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 polysaccharide of Polygonatum odoratum with the chemical formula shown in formula (I); （Ⅰ）； m + n = 22 - 24, m ≠ 0, n ≠ 0; The preparation method of the polysaccharide from Polygonatum cyrtonema 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. The polysaccharide of Polygonatum odoratum was obtained by freeze-drying.

2. The selenium nanoparticles according to claim 1, 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.

3. The selenium nanoparticles according to claim 1, characterized in that: In step two, the stirring extraction involves gentle stirring for 60–120 minutes, repeated twice, and the filtrates are combined.

4. The selenium nanoparticles according to claim 1, 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.

5. The method for preparing selenium nanoparticles as described in claim 1, characterized in that, Includes the following steps: (1) Disperse the polysaccharide of Polygonatum multiflorum in water to prepare an aqueous solution of Polygonatum multiflorum 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.

6. The method for preparing selenium nanoparticles according to claim 5, characterized in that: In step (3), dialysis is performed using a dialysis bag with a molecular weight cutoff of 6000–8000 kDa.

7. The use of the selenium nanoparticles as described in claim 1 in the preparation of a drug for treating spinal cord injury.

Images