Polygonatum sibiricum polysaccharide nanoparticles and application thereof in preparation of sepsis prevention medicine

Abstract

The invention discloses a polygonatum sibiricum polysaccharide nano-particle, the average diameter of the polygonatum sibiricum polysaccharide nano-particle is in a range of 50-100 nm, and the Zeta potential is in a range of-(17-19mV). The invention further discloses a preparation method of the rhizoma polygonati polysaccharide nano-particles and application of the rhizoma polygonati polysaccharide nano-particles in preparation of drugs for preventing septicopyemia. The polygonatum polysaccharide nanoparticles disclosed by the invention can effectively relieve weight loss of a septicopyemia mouse model caused by escherichia coli infection, significantly improve the survival rate after escherichia coli infection, significantly promote Thp-1 cell proliferation and improve cell viability, show a positive regulation effect on Thp-1 cell growth, and can be used for preparing a medicine for treating septicopyemia. The application prospect of the polygonatum polysaccharide nano-particles in preparation of drugs for preventing septicopyemia and application of the polygonatum polysaccharide nano-particles as cell proliferation stimulating factors are shown.

Description

Technical Field

[0001] This invention relates to a nanomedicine delivery system based on natural polysaccharides, belonging to the field of pharmaceutical technology. Background Technology

[0002] Sepsis, a major global public health challenge, is characterized by a systemic, uncontrolled inflammatory response triggered by infection. The diagnostic criteria have been updated to Sepsis 3.0, emphasizing organ damage indicators such as elevated lactate and oliguria. Prevention and control of sepsis requires an integrated approach of "prevention-early diagnosis-standardized treatment." In recent years, the discovery of biomarkers such as bone morphogenetic protein 9 (BMP9) has provided new tools for early prognostic assessment, while integrated traditional Chinese and Western medicine therapies, such as Xuebijing injection, have been shown to reduce the 28-day mortality rate by 7.3%. High-risk groups include the elderly, immunocompromised individuals, and postoperative patients. Bacteria remain the primary cause, but the proportion of drug-resistant bacteria (such as MRSA) and fungal infections is rising, increasing the pressure on healthcare-associated infection control.

[0003] While antibiotic therapy is effective in controlling infection among existing preventative measures, overuse leads to an increase in drug-resistant bacteria (such as MRSA and ESBL-resistant bacteria) and has no direct effect on immunosuppression in the later stages of sepsis. For example, broad-spectrum antibiotics used in high-risk patients, such as vancomycin, are prone to causing drug resistance and intestinal flora imbalance. The use of immunomodulation requires individualized stratification, and while immunomodulators such as GM-CSF can enhance immune function, they are costly and may induce excessive inflammation.

[0004] The high penetration and controlled release advantages of nanodelivery systems enable precise prevention through targeted drug delivery, regulation of the immune microenvironment, and direct antibacterial action. Natural Chinese herbal polysaccharide nanoparticle delivery systems are novel drug delivery systems using natural Chinese herbal polysaccharides (such as astragalus polysaccharides and Ganoderma lucidum polysaccharides) as carrier materials. They offer multiple advantages: their natural origin ensures high biocompatibility, low toxicity, and easy metabolism, protecting drug stability and enhancing bioavailability; they achieve precise drug delivery through passive or active targeting; they utilize multilayer structures for sustained and controlled drug release; and the pharmacological activity of the polysaccharides themselves synergistically enhances the efficacy of the loaded drug, exerting multi-target effects. Furthermore, they improve the drug's water solubility and other physicochemical properties, and the preparation process is green and environmentally friendly.

[0005] Natural polysaccharides are highly safe, but their mechanisms of action are diverse. Their efficacy is limited by low delivery efficiency, requiring optimization based on specific structures (such as molecular weight and branching). For example, astragalus polysaccharides and Ganoderma lucidum polysaccharides inhibit inflammation through the TLR4 / NF-κB pathway, but their oral bioavailability is low. Existing nanocarriers have insufficient polysaccharide loading capacity and poor controlled-release performance. For instance, the Nano CO formulation developed by Central South University releases CO in response to ROS, inhibiting macrophage activation, clearing inflammatory mediators, and significantly improving the survival rate of septic mice (30%→80%). However, its absorption, distribution, metabolism, and excretion characteristics in vivo remain unclear, and its long-term safety needs further verification.

[0006] Polygonatum polysaccharides (chemical formula: (C6H)) 10 O5)(n) is a type of natural polysaccharide extracted from the plant Polygonatum sibiricum (Liliaceae family). Its molecular structure varies depending on the extraction method and plant variety. Studies have shown that Polygonatum sibiricum polysaccharides have a significant inhibitory effect on inflammatory factors such as monocyte chemoattractant protein 1 (MCP1) and NO induced by lipopolysaccharide (LPS) in RAW264.7 cells, and this effect is dose-dependent. Some researchers have found that Polygonatum sibiricum polysaccharides can increase the organ indices of the spleen and thymus in mice, enhance the proliferative response of spleen cells, promote the secretion of IFN-γ and IL-2 by lymphocytes, promote the secretion of IL-6 and TNF-α by macrophages, and enhance the phagocytic activity of peritoneal macrophages, thereby enhancing the body's immune function. Moreover, the macrophage activity of Polygonatum sibiricum polysaccharides after processing with alcohol is even stronger. In addition, the antioxidant effect of Polygonatum sibiricum polysaccharides can directly scavenge free radicals, inhibit bacterial biofilm formation, and reduce oxidative stress damage, thereby indirectly exerting an anti-inflammatory effect.

[0007] Given that current clinical treatments primarily rely on antibiotic therapy, this over-reliance leads to antibiotic abuse, fosters drug-resistant superbugs, and often fails to adequately improve patients' clinical symptoms. Therefore, the object of this invention is to provide a nanoformulation and its application in the preparation of medicaments for treating and / or preventing sepsis. Summary of the Invention

[0008] To achieve the above objectives, the present invention first provides Polygonatum polysaccharide nanoparticles, wherein the average diameter of the Polygonatum polysaccharide nanoparticles is in the range of 50-100 nm, and the Zeta potential is in the range of -(17-19 mV). In a specific embodiment of the present invention, the Zeta potential is -18 mV.

[0009] In a preferred embodiment, the monosaccharides comprising the Polygonatum polysaccharide include fucose (FUC), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), and galacturonic acid (Gal-UA).

[0010] In a more preferred embodiment, the molar ratio of monosaccharides is: fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid is (7.00-7.10): (2.80-2.90): (10.08-10.18): (2.80-2.90): (13.78-14.88): (20.37-20.47): (32.25-32.35): (5.86-5.96): (6.46-6.56).

[0011] In one specific embodiment of the present invention, the molar ratio of monosaccharides is: fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid is 7.05: 2.85: 10.13: 2.85: 14.83: 20.42: 32.30: 5.91: 6.51.

[0012] Secondly, this invention provides a method for preparing the aforementioned Polygonatum polysaccharide nanoparticles, the method comprising the following steps: (1) Grind the water extract of the Polygonatum odoratum slices into powder; (2) Spread the powder obtained in step (1) on a refractory material layer and bake it until a caramelized product is formed; (3) Cool the caramelized product obtained in step (2) to room temperature, remove the caramelized product from the refractory material layer and transfer it to a mortar pre-cooled at 4°C to lightly grind and obtain Polygonatum polysaccharide nanoparticles, and dry and store them.

[0013] In a preferred embodiment, the water extract of the Polygonatum odoratum slices in step (1) is carried out by water extraction and alcohol precipitation.

[0014] In a more preferred embodiment, in step (1), the obtained aqueous extract solution of Polygonatum odoratum slices is placed in a -80°C freezer for 1-3 hours. After the liquid is completely frozen, it is placed in a freeze dryer for 16-30 hours until it is completely freeze-dried. Then it is placed in a mortar and gently ground in a clockwise direction for 2-4 minutes until the powder is free of lumps. It is then sealed and dried for storage.

[0015] In another preferred embodiment, in step (1), the wine-processed Polygonatum sibiricum slices are Polygonatum sibiricum slices processed with wine.

[0016] In another preferred embodiment, in step (2), the powder obtained in step (1) is spread on the refractory material layer to form a thin film layer of 0.3-0.5 mm. The refractory material layer is placed at a distance of 2-3 cm vertically above the fire source and baked for 2-3 seconds. After an interval of 2 minutes, it is baked again. The baking step is repeated 4-6 times until a caramelized product is formed.

[0017] Furthermore, this invention provides the application of the aforementioned Polygonatum polysaccharide nanoparticles in the preparation of drugs for the treatment and / or prevention of sepsis.

[0018] The embodiments of the present invention show that, in the treatment of sepsis induced by Escherichia coli suspension in mice, the protective rate provided by Polygonatum polysaccharide nanoparticles is twice that of free Polygonatum polysaccharide, demonstrating excellent therapeutic effects and application prospects.

[0019] Finally, this invention provides the application of the aforementioned Polygonatum polysaccharide nanoparticles as a cell proliferation stimulating factor.

[0020] The embodiments of the present invention show that PSPs NPs have a significantly higher effect on stimulating the proliferation of THP-1 cells than free Polygonatum polysaccharide, demonstrating the application of PSPs NPs as cell proliferation stimulating factors, such as their application as components of cell culture media.

[0021] Given that THP-1 cells, as a human monocyte cell line, can mimic the responses and functional activation of innate immune cells through specific stimulating factors, they are commonly used in research on inflammation, infection, immune regulation, and drug screening. Since the PSPs NPs provided by this invention exhibit excellent ability to stimulate the growth and proliferation of THP-1 cells, they also show promise for applications similar to other THP-1 cell growth and proliferation stimulating factors, such as inducing monocyte differentiation into macrophages to achieve a more powerful pathogen clearance capacity of macrophages and antigen-presenting ability to initiate adaptive immune responses by presenting antigens to T cells via MHC class II molecules. Therefore, in a preferred embodiment, the cell proliferation stimulating factor is a stimulating factor that induces monocyte differentiation into macrophages.

[0022] The Polygonatum polysaccharide nanoparticles provided by this invention employ freeze-drying, pulverization, and thermo-induced nano-sizing, combining physical pulverization with thermal energy regulation. The pyrolysis or recombination during the baking stage overcomes the particle size limitations of purely mechanical methods while avoiding the complexity of chemical methods. Raw material utilization exceeds 95%, and the production cycle is shortened by 50% compared to chemical methods. This achieves zero chemical pollution, low energy consumption, and high activity retention, making it suitable for large-scale production of natural products. This process preserves the structural integrity and bioactivity of the polysaccharide, exhibiting low cytotoxicity, and provides an efficient, safe, and scalable universal platform for the precise delivery of natural polysaccharides in fields such as immune regulation and inflammation treatment.

[0023] After the above preparation process, transmission electron microscopy confirmed that the average diameter of the Polygonatum polysaccharide nanoparticles is in the range of 50-100 nm. The 50-100 nm Polygonatum polysaccharide nanoparticles are within the particle size range of macrophages that are efficiently taken up by macrophages (particle size <200 nm), which is conducive to being recognized and phagocytosed by macrophages, which are the main phagocytic cells of the immune system, through passive targeting.

[0024] Moreover, the Polygonatum polysaccharide nanoparticles provided by this invention can be fully taken up by macrophages, proving that through the preparation process of this invention, the surface of the Polygonatum polysaccharide nanoparticles exposes a sufficient number of mannose residues, which can bind to mannose receptors on the surface of macrophages, thereby forming a dual targeting mechanism of "passive size screening + active receptor recognition", which significantly improves the uptake efficiency of macrophages.

[0025] The improved uptake efficiency by macrophages ensures the application of the Polygonatum polysaccharide nanoparticles as a cell proliferation stimulating factor, especially as a stimulating factor for the differentiation of monocytes and macrophages, to participate in specific immune regulation processes and in the preparation of drugs for the prevention of sepsis. Attached Figure Description

[0026] Figure 1 Appearance of the ground Polygonatum polysaccharide powder; Figure 2 Dynamic light scattering (DLS) method was used to determine the average hydrodynamic diameter of free Polygonatum polysaccharide and Polygonatum polysaccharide nanoparticles; Figure 3 Dynamic light scattering (DLS) method was used to detect the zeta potential of free Polygonatum polysaccharide and Polygonatum polysaccharide nanoparticles; Figure 4 Microscopic morphology characterization of Polygonatum polysaccharide nanoparticles (top) and Free Polygonatum polysaccharide (bottom) by transmission electron microscopy (TEM); Figure 5 Detection of molecular weight, molecular size (and molecular weight distribution) of total polysaccharides from Polygonatum sibiricum; Figure 6 Component composition analysis of total polysaccharides from Polygonatum sibiricum; Figure 7 Experiment on the uptake of Free Polygonatum polysaccharides by Raw264.7 cells; Figure 8 . Raw264.7 cell uptake experiment of Polygonatum polysaccharide nanoparticles; Figure 9 Effects of free Polygonatum polysaccharide and Polygonatum polysaccharide nanoparticles on the survival rate of mice infected with Escherichia coli; Figure 10Effects of free Polygonatum polysaccharide and Polygonatum polysaccharide nanoparticles on body weight in mice infected with Escherichia coli; Figure 11 Effects of free Polygonatum polysaccharide and Polygonatum polysaccharide nanoparticles on the viability of Thp-1 cells. Detailed Implementation

[0027] The present invention will be further described below with reference to specific embodiments, and the advantages and features of the present invention will become clearer as a result of the description. However, these embodiments are merely exemplary and do not constitute any limitation on the scope of protection defined by the claims of the present invention.

[0028] Example 1. Preparation and characterization of PSPs and NPs (1) Extraction of Polygonatum polysaccharides: Polygonatum polysaccharides were extracted by water extraction and alcohol precipitation. The specific steps were as follows: Water decoction: Polygonatum slices (purchased from Chinese medicinal materials, batch number C12311286) were added to water (material-liquid ratio of about 1:10~1:7), and extracted by reflux for 2 hours to obtain an aqueous solution containing polysaccharides; Concentration and impurity removal: After rotary evaporation and concentration, centrifugation (3000 rpm, 10 minutes) was performed to remove insoluble impurities and collect the supernatant; Ethanol precipitation: 95% ethanol was added to the supernatant to adjust the ethanol concentration to about 80%, and after stirring, the mixture was allowed to stand at low temperature (4℃) overnight to precipitate polysaccharides; Separation and drying: The precipitate was collected by centrifugation and the residual solvent was removed by nitrogen blowing to obtain crude Polygonatum polysaccharides. The Polygonatum polysaccharides of this invention ( Polygonatum kingianum Coll. et Hemsl. is a perennial herb belonging to the genus *Polygonatum* in the family Liliaceae. It is mainly distributed in Yunnan, Sichuan, and Guizhou provinces of China, and also found in Myanmar and Vietnam. It is an important medicinal plant, with its rhizome used in medicine. It is similar to other members of the same genus, such as *Polygonatum sibiricum*. P. sibiricum Polygonatum multiflorum P. cyrtonema The medicinal ingredients differ slightly from each other.

[0029] (2) Freeze-drying and grinding of Polygonatum polysaccharide: The extracted crude Polygonatum polysaccharide aqueous solution was dispensed into 50mL centrifuge tubes and frozen at -80℃ for more than 1 hour. After the liquid was completely frozen, the cap was opened, a sealing film was placed on it, some small holes were poked in the film, and the tubes were freeze-dried in a freeze dryer for 24 hours. After complete freeze-drying, the tubes were placed in a mortar and ground gently in a clockwise direction in small batches until the powder was free of visible lumps. After grinding, the tubes were sealed immediately and stored in a desiccator. The degree of grinding was as follows: Figure 1 As shown. The crude polysaccharide of Polygonatum extracted in this invention, after complete freeze-drying, is a brownish-yellow or light brown powder with a reasonable uniformity, exhibiting a delicate texture and relatively consistent color. It is easily soluble in hot water and contains polysaccharides composed of monosaccharides such as glucose and mannose, as well as impurities such as proteins and pigments. It is hygroscopic and viscous.

[0030] (3) Nanoparticle synthesis: The ground Polygonatum polysaccharide powder (Free PSPs) is evenly spread on sterile tin foil to form a thin film layer with a thickness of about 0.3-0.5 mm and a spreading range of about 5 cm. Using sterile tweezers, the tin foil is symmetrically folded and fixed on a custom ceramic boat or high-temperature resistant support, ensuring that the material layer is 2-3 cm vertically away from the flame. After lighting the alcohol lamp, the support is held and swept horizontally across the outer flame area (400-600℃) at a uniform speed. The baking time for each batch is strictly controlled within 2-3 seconds. During the process, the color change of the material needs to be observed. When a uniform light brown caramelized layer appears on the surface and there are no black carbonized spots, it is immediately removed from the heat source. Repeat the operation 3-5 times. The entire baking time is controlled within 30 seconds to 1 minute. The sample that has undergone heat treatment needs to be cooled to room temperature in a clean workbench (about 5 minutes). Then, carefully peel off the caramelized product from the aluminum foil and transfer it to a pre-cooled (4°C) agate mortar and grind it for 30 seconds to obtain dried Polygonatum polysaccharide nanoparticles (PSPsNPs). Store them in a desiccator in a sealed container to prevent moisture absorption and aggregation.

[0031] 2. Characterization of Free PSPs and PSPs NPs Weigh the extracted Free PSPs and the prepared PSPs NPs, add ultrapure water, and prepare 2 mg / ml stock solutions respectively. Dilute 20 times before testing, drop the sample onto a copper grid and perform transmission electron microscopy (TEM) detection.

[0032] (1) Dynamic light scattering (DLS) method for detecting the hydrodynamic diameter and zeta potential of Free PSPs and PSPs NPs: Instruments and parameters used: Instrument: Malvern Zetasizer series; Measurement temperature: 25℃; Equilibrium time: 10s; Scattering angle: 173°; Measurement time: 10 seconds / measurement, repeated 5 times.

[0033] The hydrodynamic diameter of the obtained Free PSPs is around 450 nm; the hydrodynamic diameter of PSP NPs is below 200 nm, which is within the range of efficient macrophage uptake (see...). Figure 2 The Zeta potential results show that the potential of Free PSPs is around -15mV; the potential of PSPs NPs is around -18mV. The higher the absolute value of the potential, the more stable it is. In comparison, PSPs NPs have better stability (see...). Figure 3 ).

[0034] (2) Transmission electron microscopy (TEM) was used to further characterize the microstructure of Free PSPs and PSPs NPs. The TEM images showed that Free PSPs exhibited some aggregation, with an average nanoparticle diameter of 250-300 nm, and the particles were large (…). Figure 4 Below); PSPs and NPs are uniformly dispersed, without obvious agglomeration, exhibiting good monodispersity, and the particles are small, mostly in the range of 50-100 nm. Figure 4 superior).

[0035] 3. Determination of molecular weight, molecular size (and molecular weight distribution) of total polysaccharides from Polygonatum sibiricum. Instruments and parameters used: The chromatographic system employed was a gel chromatography-differential chromatography-multi-angle laser light scattering system. The liquid chromatography system was a U3000 (Thermo, USA), the differential detector was an Optilab T-rEX (Wyatt technology, CA, USA), and the laser light scattering detector was a DAWN HELEOS II (Wyatt technology, CA, USA). Specific column and elution conditions were as follows: Ohpak SB-805 HQ (300×8 mm) and Ohpak SB-803 HQ (300×8 mm) gel size exclusion columns were used in series. The column temperature was 45℃, the injection volume was 100 μL, the mobile phase was A (0.02% NaN3, 0.1M NaNO3), the flow rate was 0.6 mL / min, and the elution gradient was isocratic for 75 min.

[0036] By analyzing the total polysaccharides extracted from Polygonatum sibiricum using this invention, we found that the total polysaccharides mainly consist of two peaks. The main peak at retention time 22 min has a number-average molecular weight (Mn) of 117.93 kDa, a weight-average molecular weight (Mw) of 147.22 kDa, and a polydispersity (Mw / Mn) of 1.25. The main peak at retention time 33 min has a number-average molecular weight (Mn) of 78.06 kDa, a weight-average molecular weight (Mw) of 84.69 kDa, and a polydispersity (Mw / Mn) of 1.09. The spectra are shown below. Figure 5 The red line represents the multi-angle laser light scattering signal (LS, unit: V), and the intensity of the scattered light is proportional to the molecular size and molecular weight of the substance. The blue line represents the difference signal (RI, unit: RIU), and the response value depends on the change in the refractive index of the effluent after the column, and is related to the type, concentration, and molecular weight of the substance. The black line is the molecular weight obtained by fitting the two signals. The solvent peak of the mobile phase is around 37 min.

[0037] 4. Compositional Analysis of Total Polysaccharides from Polygonatum odoratum The total polysaccharides of Polygonatum odoratum were qualitatively and quantitatively analyzed using the TFA (trifluoroacetic acid) method.

[0038] The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), which used an electrochemical detector to analyze and detect monosaccharide components.

[0039] A Dionex™ CarboPac™ PA20 (150*3.0mm, 10μm) liquid chromatography column was used; the injection volume was 5 μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1 M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V). The chromatographic system used was the Thermo ICS 5000+ ion chromatography system (ICS 5000+, Thermo Fisher Scientific, USA), which used an electrochemical detector to analyze and detect monosaccharide components.

[0040] A Dionex™ CarboPac™ PA20 (150*3.0mm, 10μm) liquid chromatography column was used; the injection volume was 5 μl. Mobile phase A (H2O), mobile phase B (0.1M NaOH), mobile phase C (0.1 M NaOH, 0.2M NaAc), flow rate 0.5 ml / min; column temperature 30℃; elution gradient: 0 min A phase / B phase / C phase (95:5:0, V / V), 26 min A phase / B phase / C phase (85:5:10, V / V), 42 min A phase / B phase / C phase (85:5:10, V / V), 42.1 min A phase / B phase / C phase (60:0:40, V / V), 52 min A phase / B phase / C phase (60:40:0, V / V), 52.1 min A phase / B phase / C phase (95:5:0, V / V), 60 min A phase / B phase / C phase (95:5:0, V / V).

[0041] The composition of total polysaccharides in Polygonatum sibiricum included fucose (FUC), rhamnose (Rha), arabinose (Ara), galactose (Gal), glucose (Glc), xylose (Xyl), mannose (Man), and galacturonic acid (Gal-UA), with xylose, glucose, galactose, and arabinose being the main monosaccharides. The contents of each monosaccharide were as follows: xylose 45.75 mg / mg, glucose 34.70 mg / mg, galactose 25.20 mg / mg, arabinose 14.34 mg / mg, fucose 10.93 mg / mg, mannose 10.04 mg / mg, rhamnose 4.41 mg / mg, and galacturonic acid 11.93 mg / mg, totaling 157.30 mg / mg. The molar ratios of the monosaccharides are as follows: fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid 7.05: 2.85: 10.13: 2.85: 14.83: 20.42: 32.30: 5.91: 6.51 (see...) Figure 6 ).

[0042] Example 2. Experiment on the uptake capacity of Raw264.7 cells of Free PSPs and PSP NPs; 1. Experimental Procedure (1) Cell preparation: In a 12-well plate containing a coverslip, fill each well with 1 × 10⁶ cells. 5 RAW264.7 macrophages were seeded at a density of 100 cells and cultured in a 37°C, 5% CO2 incubator for 24 hours until the cells adhered to the incubator. (2) Fluorescent labeling of Polygonatum polysaccharides: Free PSPs and PSPs NPs stock solutions were prepared with a concentration of 1 mg / mL; Free PSPs and PSPs NPs were pre-labeled with Cy3-NHS: Cy3-NHS (concentration of 5 mg / mL) was added to centrifuge tubes at a mass ratio of 1:10000 with Free PSPs and PSPs NPs solutions, mixed by pipetting, wrapped with aluminum foil, and incubated overnight at 4°C in the dark. (3) Cy3-NHS-labeled Free PSPs and PSPs NPs were co-incubated with cells at concentrations of 2.5 μg / mL, 5 μg / mL, and 10 μg / mL for 12 and 24 hours, respectively; (4) After incubation, wash the cells three times with PBS and fix them with 4% paraformaldehyde at room temperature in the dark for 10 minutes. Discard the fixative, wash the cells twice with PBS for 5 minutes each time, and then stain the cell nuclei with DAPI (5ug / mL) for 15 minutes. (5) After staining, wash the cells three times with PBS; (6) Finally, the cells were observed using a laser confocal microscope (LSM880); (7) Imaging parameter settings: Cy3 channel: excitation wavelength 550 nm, emission wavelength 570 nm (detecting red fluorescence of Polygonatum polysaccharide); DAPI channel: excitation wavelength 358 nm, emission wavelength 461 nm (detecting blue fluorescence of cell nucleus); scanning mode: XYZ axis multi-layer scanning, resolution 1024×1024 pixels, magnification 40× or 63×.

[0043] 2. Results In the Free PSPs group, almost no obvious red fluorescence was observed in the Cy3 channel at both 12h and 24h, indicating that Raw264.7 cells had extremely weak uptake capacity for free Polygonatum polysaccharides. This may be because free polysaccharides have difficulty crossing the cell membrane or are easily metabolized and cleared (see [link to relevant documentation]). Figure 7 ).

[0044] PSPs NPs group: Time trend: The Cy3 red fluorescence in the 24h group was significantly stronger than that in the 12h group, indicating that the uptake of PSPs NPs by cells gradually increased with the extension of incubation time; Concentration trend: At the same time point (e.g., 24h), the red fluorescence in the 10 μg / mL group was stronger than that in the 2.5 μg / mL and 5 μg / mL groups, suggesting that the higher the concentration, the greater the amount of PSPs NPs uptake by cells (see...). Figure 8 ).

[0045] Raw264.7 cells uptake of Polygonatum polysaccharides depends on dosage form and time: PSPs NPs are significantly better than free PSPs, and the uptake increases with prolonged incubation time and higher concentration.

[0046] Mannose receptors are specifically highly expressed on macrophages (especially M2 macrophages). Polygonatum polysaccharides typically contain monosaccharides such as mannose, glucose, galactose, and arabinose. The results of this embodiment demonstrate that the Polygonatum polysaccharide nanoparticles provided by this invention have a sufficient number of mannose residues exposed on their surface during the preparation process, which can bind to mannose receptors on the surface of macrophages, thereby improving the targeting ability to macrophages and facilitating their full uptake by macrophages.

[0047] Example 3. Preventive effect of PSPs and NPs on sepsis 1. Experimental Procedure (1) Dissolving Free PSPs and PSPs NPs: Add Free PSPs and PSPs NPs powder to sterile saline at the required concentration to prepare a concentration of 10 mg / mL; (2) Female C57BL / 6 mice aged 6-8 weeks and weighing about 20g were selected and randomly divided into 3 groups (8 mice in each group) after 3 days of acclimatization: control group (C), sepsis group (M), free Polygonatum polysaccharide (Free PSPs) group, and Polygonatum polysaccharide nanoparticle (PSPs NPs) group; (3) The control group was not treated and was fed normally; the sepsis group was injected intraperitoneally with 100 μL of physiological saline every day; the Free PSPs group and the PSPs NPs group were injected intraperitoneally with Free PSPs solution and PSPs NPs solution at a dose of 500 μg / mouse for 5 days, and the weight of the mice was monitored daily during the period. (4) On day 6, except for the control group, the other groups were injected with 3.5×10 9 Sepsis was induced by CFU / mL E. coli suspension. The model was considered successful when all mice in group M died after 24 hours. After 48 hours, bronchoalveolar lavage fluid, blood, and internal organ samples such as heart, lung, liver, and kidney were collected from surviving mice in the Free PSPs group and PSPsNPs group. The mortality rate was recorded and the trend of weight change was analyzed simultaneously.

[0048] 2. Results (1) It was shown that all M group members died within 24 hours, 6 Free PSPs group members survived within 24 hours and 3 survived within 48 hours, with a survival rate of about 40%, and 6 PSPs NPs group members survived within both 24 hours and 48 hours, with a survival rate of 75%, which was 1 times higher than that of Free PSPs group.

[0049] (2) The survival rate curve (Probability of Survival) shows that PSPs and NPs can significantly improve the survival rate after E. coli infection. For example... Figure 9 As shown.

[0050] (3) The body weight curve shows that PSPs NPs can effectively alleviate weight loss caused by infection, comprehensively demonstrating that PSPs NPs have a protective effect against sepsis caused by Escherichia coli infection. Figure 10 As shown.

[0051] Example 4. Cell proliferation effect of PSPs and NPs 1. Experimental Procedure (1) Dissolution of Free PSPs and PSPs NPs: Add Free PSPs and PSPs NPs powder to RPMI 1640 complete medium to prepare concentrations of 3 μg / mL, 5 μg / mL, 10 μg / mL and 20 μg / mL respectively; (2) Thrp-1 cells (human monocytic leukemia cells) in good growth condition were seeded into 96-well plates, 2000 cells per well; (3) After the cells adhered for 12 hours, they were treated with a concentration gradient of Free PSPs and PSPs NPs for 72 hours; (4) The CCK8 assay was used to detect cell growth and to observe whether Free PSPs and PSPs NPs could promote the proliferation of Thrp-1 cells.

[0052] 2. Results Free PSPs group: As the concentration increased from 3 μg / mL to 20 μg / mL, the cell survival rate improved to a certain extent (from about 110% to 150%), but the overall improvement was smaller than that of the PSPs NPs group, and the difference between different concentrations within the group was not significant. This reflects the promoting effect of free Polygonatum polysaccharides on cell survival, but the effect is affected by the concentration and is weaker than that of nanoparticles.

[0053] PSPs NPs group: Cell survival rates were generally higher than those of the Free PSPs group at all concentrations. At the same concentrations (e.g., 3 μg / mL, 5 μg / mL, etc.), the survival rate of the PSPs NPs group was even higher, indicating that the promotion of cell survival was more significant after Polygonatum sibiricum polysaccharide was formulated into nanoparticles (PSPs NPs). Specifically, with the survival rate of the control group set as 100%, the survival rate of the PSPs NPs group at 3 μg / mL was 176%, significantly higher than the 117% of the Free PSPs group. At 5 μg / mL, the survival rate of the PSPs NPs group was 211%, much higher than the 123% of the Free PSPs group. Cell viability was highest at 10 μg / mL, with a survival rate of 222% in the PSPs NPs group, much higher than the 125% of the Free PSPs group. At 20 μg / mL, the survival rate of the PSPs NPs group was 193%, higher than the 152% of the Free PSPs group (e.g., 3 μg / mL, 5 μg / mL, etc.). Figure 11 (As shown).

Claims

1. A kind of Polygonatum polysaccharide nanoparticle, characterized in that, The average diameter of the Polygonatum polysaccharide nanoparticles is in the range of 50-100 nm, and the Zeta potential is in the range of -(17-19 mV).

2. The Polygonatum polysaccharide nanoparticles according to claim 1, characterized in that, The polysaccharides in Polygonatum include fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, and galacturonic acid.

3. The Polygonatum polysaccharide nanoparticles according to claim 2, characterized in that, The molar ratio of the monosaccharides is: fucose: rhamnose: arabinose: galactose: glucose: xylose: mannose: galacturonic acid is (7.00-7.10): (2.80-2.90): (10.08-10.18): (2.80-2.90): (13.78-14.88): (20.37-20.47): (32.25-32.35): (5.86-5.96): (6.46-6.56).

4. The method for preparing Polygonatum polysaccharide nanoparticles according to any one of claims 1-3, characterized in that, The method includes the following steps: (1) Grind the water extract of the Polygonatum odoratum slices into powder; (2) Spread the powder obtained in step (1) on a refractory material layer and bake it until a caramelized product is formed; (3) Cool the caramelized product obtained in step (2) to room temperature, remove the caramelized product from the refractory material layer and transfer it to a mortar pre-cooled at 4°C to lightly grind and obtain Polygonatum polysaccharide nanoparticles, and dry and store them.

5. The method for preparing Polygonatum polysaccharide nanoparticles according to claim 4, characterized in that, In step (1), the water extract of the Polygonatum odoratum slices is extracted by water extraction and alcohol precipitation.

6. The method for preparing Polygonatum polysaccharide nanoparticles according to claim 5, characterized in that, In step (1), the aqueous extract solution of the obtained Polygonatum odoratum slices is placed in a -80℃ freezer for 1-3 hours. After the liquid is completely frozen, it is placed in a freeze dryer for 16-30 hours until it is completely freeze-dried. Then it is placed in a mortar and gently ground in a clockwise direction for 2-4 minutes until the powder is free of lumps. Then it is sealed and dried for storage.

7. The method for preparing Polygonatum polysaccharide nanoparticles according to claim 4, characterized in that, In step (2), the powder obtained in step (1) is spread on the refractory material layer to form a thin film layer of 0.3-0.5 mm. The refractory material layer is placed 2-3 cm above the fire source and baked for 2-3 seconds. After a 2-minute interval, it is baked again. The baking steps are repeated 4-6 times until a caramelized product is formed.

8. The use of the Polygonatum polysaccharide nanoparticles according to claim 1 in the preparation of drugs for treating and / or preventing sepsis.

9. The application of the Polygonatum polysaccharide nanoparticles according to claim 1 as a cell proliferation stimulating factor.

10. The application according to claim 9, characterized in that, The cell proliferation stimulating factor is a stimulating factor that stimulates the differentiation of monocytes and macrophages.

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