A neutral homogeneous polysaccharide from Rehmannia glutinosa, its preparation method and application

Abstract

This invention discloses a neutral homogeneous polysaccharide from Rehmannia glutinosa, its preparation method, and its applications, belonging to the field of traditional Chinese medicine preparation technology. The polysaccharide is mainly composed of galactose, glucose, and fructose in a molar ratio of 2.72:1.17:1.11, with a weight-average molecular weight of 2152 Da. The neutral polysaccharide from Rehmannia glutinosa can exert a therapeutic effect on osteoporosis by increasing bone mineralization and bone density; it can also promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), showing significant application effects in the preparation of stem cell therapy drugs for bone defects. The neutral homogeneous polysaccharide from Rehmannia glutinosa shows great application potential in the preparation of drugs for treating osteoporosis and as a stem cell therapy (especially bone regeneration therapy based on bone marrow mesenchymal stem cells). This provides new candidate substances and research directions for developing efficient and safe treatment strategies for bone metabolic diseases derived from traditional Chinese medicine.

Description

Technical Field

[0001] This invention relates to a neutral homogeneous polysaccharide from Rehmannia glutinosa, its preparation method, and its application. It relates to the effects of a neutral homogeneous polysaccharide from Rehmannia glutinosa on osteoporosis and its role in promoting osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells, belonging to the field of traditional Chinese medicine preparation technology. Background Technology

[0002] Rehmannia glutinosa, a traditional Chinese medicine that can be used in health food, has the effects of nourishing vital energy and strengthening muscles and bones.

[0003] Bone-related diseases, such as osteoporosis, nonunion or delayed union of fractures, and large bone defects caused by trauma, infection, or tumor resection, have always been major challenges in orthopedic clinics. With the increasing aging of the global population, the incidence of osteoporotic fractures continues to rise, while high-energy injuries and other high-violence traumas are leading to a growing number of complex and refractory fractures and bone defects. Traditional treatments, including autologous / allogeneic bone grafting, internal fixation, and external fixation, while widely used and achieving some efficacy, each have limitations: autologous bone grafting has limited sources and can cause secondary damage to the donor site; allogeneic bone grafting may pose risks of immune rejection and disease transmission; internal fixation devices may have the risk of loosening or breakage; and for large bone defects, traditional methods often fail to achieve perfect structural and functional reconstruction. Therefore, exploring and innovating more efficient and safer bone repair strategies, such as tissue-engineered bone technology, bioactive materials, gene therapy, and 3D-printed personalized implants, has become a frontier and focus of common interest in orthopedics, biomaterials, and regenerative medicine, aiming to ultimately achieve perfect regeneration and functional restoration of bone tissue.

[0004] Bone marrow mesenchymal stem cells (BMSCs) have become the preferred seed cells for stem cell therapy of bone defects due to their strong osteogenic differentiation potential. However, traditional BMSCs suffer from problems such as low post-transplant survival rates, susceptibility to immune rejection, and suppressed osteogenic differentiation potential. Therefore, there is an urgent need to develop suitable carrier formulations to address these challenges.

[0005] Bone marrow mesenchymal stem cells (BMSCs) have become the most promising seed cells for stem cell therapy and research on bone defects due to their strong osteogenic differentiation potential, ease of acquisition, and relatively low immunogenicity. Their therapeutic mechanism primarily lies in the fact that, upon transplantation into the bone defect area, BMSCs can differentiate into osteoblasts under the induction of specific microenvironmental signals, directly participating in new bone formation. Furthermore, they exert a powerful paracrine effect by secreting various nutritional factors and cytokines, regulating the local immune microenvironment, promoting angiogenesis, and activating endogenous repair mechanisms, creating favorable conditions for bone regeneration. However, this therapy still faces many challenges in achieving clinical translation and application, such as: how to optimize cell survival and homing efficiency after transplantation; how to precisely control the direction and process of osteogenic differentiation in vivo; and how to develop more ideal biomaterials as scaffolds for their in vivo delivery and growth. Therefore, current research is focused on enhancing osteogenic capacity through gene editing, constructing cell-scaffold complexes using 3D printing to mimic natural bone structure, and exploring derivative therapies such as cell-free extracellular vesicles, with the ultimate goal of overcoming the regeneration challenge of refractory bone defects. Thus, there is an urgent need for an ideal biomaterial as a scaffold for the in vivo delivery and growth of bone marrow mesenchymal stem cells. Summary of the Invention

[0006] The purpose of this invention is to address the problems existing in the prior art by providing a neutral and homogeneous polysaccharide from Rehmannia glutinosa.

[0007] Another objective of this invention is to provide a method for preparing neutral homogeneous polysaccharides from Rehmannia glutinosa.

[0008] Another objective of this invention is to provide the application of Rehmannia glutinosa neutral homogeneous polysaccharide in the preparation of osteoporosis drugs and stem cell therapy.

[0009] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0010] A neutral homogeneous polysaccharide from Rehmannia glutinosa, wherein the polysaccharide is a homogeneous polysaccharide composed of galactose, glucose, and fructose in a molar ratio of 2.72:1.17:1.11, and the structural formula of the neutral polysaccharide from Rehmannia glutinosa is as follows:

[0011]

[0012] The ratio of a, b, and c is 3:1:2.

[0013] That is, the structural formula of Rehmannia glutinosa neutral polysaccharide is:

[0014]

[0015] In this context, Galp represents galactose, Fruf represents fructose, and Glcp represents glucose.

[0016]

[0017] According to some specific embodiments of the present invention, the weight-average molecular weight of the neutral homogeneous polysaccharide of Rehmannia glutinosa is 2152 Da.

[0018] According to some specific embodiments of the present invention, a method for preparing a neutral homogeneous polysaccharide from Rehmannia glutinosa includes the following steps:

[0019] (1) The Rehmannia glutinosa slices were mixed with purified water for extraction, and the crude polysaccharide of Rehmannia glutinosa was obtained by precipitating with ethanol after concentration.

[0020] (2) Dissolve the crude polysaccharide obtained in step (1) and adjust the pH using NaOH;

[0021] (3) Add the composite neutral polysaccharide purification reagent to the solution obtained in step (2) and stir continuously using a magnetic stirrer;

[0022] (4) Remove the gel-like precipitate from the solution obtained in step (3), and dialyze the supernatant to remove small molecules to obtain neutral refined polysaccharide of Rehmannia glutinosa.

[0023] (5) The neutral refined polysaccharide of Rehmannia glutinosa obtained in step (4) is separated by molecular size exclusion chromatography column to obtain the neutral homogeneous polysaccharide of Rehmannia glutinosa.

[0024] In step (3), the composite neutral polysaccharide purification reagent is an aqueous solution of polymethacrylamide propyltrimethylammonium chloride, hexadecyl pyridine bromide, and sodium dodecylbenzenesulfonate in a mass ratio of 2:1:1.

[0025] Specifically, a method for preparing a neutral homogeneous polysaccharide from Rehmannia glutinosa includes the following steps:

[0026] Step 1: Boil Rehmannia glutinosa slices with purified water at a mass-to-volume ratio of 1g:10mL at 100℃ for extraction. This extraction is repeated twice, with each extraction lasting 2 hours. The two extracts are combined and concentrated to a density of 1.10g / mL. 95% ethanol is added to precipitate the precipitate to a concentration of 80% ethanol. The precipitate is then dried under reduced pressure at 60℃ to obtain crude Rehmannia glutinosa polysaccharide.

[0027] Step 2: Prepare a 10 mg / mL aqueous solution of Rehmannia glutinosa crude polysaccharide and adjust the pH to 12 using a 0.1 M NaOH solution to obtain the Rehmannia glutinosa crude polysaccharide solution.

[0028] Step 3: Add 10 mg / mL of a complex neutral polysaccharide purification reagent to the crude polysaccharide solution obtained in Step 2, with a volume equivalent to 1:1 of the crude polysaccharide solution. The complex neutral polysaccharide purification reagent is composed of polymethacrylamide propyltrimethylammonium chloride, hexadecyl bromopyridine, and sodium dodecylbenzenesulfonate in a mass ratio of 2:1:1. Add the reagent while stirring at a stirring speed of 500 rpm for 3 hours.

[0029] Step 4: After stirring, use filter paper to remove the gel-like precipitate from the solution. Dialyze the supernatant with running water using a 1KD dialysis bag for 72 hours. After freeze-drying, obtain neutral refined polysaccharide of Rehmannia glutinosa.

[0030] Step 5: Separation of Rehmannia glutinosa neutral homogeneous polysaccharide using size exclusion chromatography column: 10 mg / mL Rehmannia glutinosa neutral purified polysaccharide was prepared using a preparative liquid chromatography system equipped with a Sephardex G100 dextran gel chromatography column. Purified water was used as the eluent, the flow rate was 0.8 mL / min, 10 mL was collected from each tube, and the eluents from tubes 12-17 were combined and freeze-dried to obtain Rehmannia glutinosa neutral homogeneous polysaccharide.

[0031] According to some specific embodiments of the present invention, the neutral homogeneous polysaccharide of Rehmannia glutinosa is used in the preparation of osteoporosis drugs.

[0032] According to some specific embodiments of the present invention, the application of the neutral homogeneous polysaccharide of Rehmannia glutinosa in stem cell therapy.

[0033] According to some specific embodiments of the present invention, the stem cells are bone marrow mesenchymal stem cells.

[0034] According to some specific embodiments of the present invention, the application of the neutral homogeneous polysaccharide of Rehmannia glutinosa in promoting the proliferation of bone marrow mesenchymal stem cells.

[0035] According to some specific embodiments of the present invention, the neutral homogeneous polysaccharide of Rehmannia glutinosa is used to promote osteogenic differentiation of bone marrow mesenchymal stem cells.

[0036] According to some specific embodiments of the present invention, the application of the neutral homogeneous polysaccharide of Rehmannia glutinosa in the preparation of biomaterials as in vivo delivery and growth scaffolds for stem cells.

[0037] A biomaterial for in vivo delivery and growth scaffolding of stem cells is prepared from the neutral homogeneous polysaccharide of Rehmannia glutinosa according to the present invention.

[0038] In summary, this invention provides a neutral homogeneous polysaccharide from Rehmannia glutinosa, its preparation method, and its applications, exhibiting unique activity in treating bone diseases and promoting stem cell differentiation. The method of this invention has the following advantages:

[0039] Neutral homogeneous polysaccharides from Rehmannia glutinosa show great promise in the preparation of drugs for treating osteoporosis and as stem cell therapy (especially bone regeneration therapy based on bone marrow mesenchymal stem cells). This provides new candidate substances and research directions for developing efficient and safe treatment strategies for bone metabolic diseases derived from traditional Chinese medicine.

[0040] This invention provides a neutral, homogeneous polysaccharide from Rehmannia glutinosa, its preparation method, and its applications. The polysaccharide is mainly composed of galactose, glucose, and fructose in a molar ratio of 2.72:1.17:1.11, and has a weight-average molecular weight of 2152. The neutral homogeneous polysaccharide structure of Rehmannia glutinosa consists of three repeating units: a, b, and c. The structure of part a is α-D-Galp-(1→6)-α-D-Galp-(1→), the structure of part b is →2)-β-D-Fruf-(1→2)-β-D-Fruf-(1→), and the structure of part c is α-D-Glcp-(1→6)-α-D-Galp-(1→). Rehmannia glutinosa neutral homogeneous polysaccharide can play a role in treating osteoporosis by increasing bone mineralization and bone density. It can also promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), showing significant application effects in the preparation of stem cell therapy drugs for bone defects. Rehmannia glutinosa neutral homogeneous polysaccharide provides a new candidate substance for the treatment of bone metabolic diseases and bone defect repair. Attached Figure Description

[0041] Figure 1 Elution curve of Rehmannia glutinosa neutral homogeneous polysaccharide dextran gel column;

[0042] Figure 2 Chromatogram of molecular weight determination of HPGPC-ELSD neutral homogeneous polysaccharide from Rehmannia glutinosa;

[0043] Figure 3 Chromatograms for the determination of monosaccharide composition; (A) Chromatograms of monosaccharide standards (1. fucose, 2. rhamnose, 3. arabinose, 4. galactose, 5. glucose, 6. xylose, 7. mannose, 8. fructose, 9. ribose, 10. galacturonic acid, 11. glucuronic acid, 12. guluronic acid, 13. mannuronic acid); (B) Chromatograms for the determination of monosaccharide composition of Rehmannia glutinosa homogeneous polysaccharide;

[0044] Figure 4 Infrared spectrum of neutral homogeneous polysaccharides from Rehmannia glutinosa;

[0045] Figure 5 Mass spectra of neutral homogeneous polysaccharide methylation determination in Rehmannia glutinosa; (A) Total ion chromatogram, including fragments 1, 2, 3, 4 and 5, (B) Mass spectrum of fragment 1, (C) Mass spectrum of fragment 2, (D) Mass spectrum of fragment 3, (E) Mass spectrum of fragment 4, (F) Mass spectrum of fragment 5.

[0046] Figure 6 Nuclear magnetic resonance chromatogram of neutral homogeneous polysaccharide from Rehmannia glutinosa; (A) 1 H NMR, (B) 13 C10 NMR, (C10) 1 H- 1 H COSY, (D) HSQC, (E) HMBC spectrum;

[0047] Figure 7 Photographs of zebrafish skulls stained with alizarin red pigment; the neutral polysaccharide of Rehmannia glutinosa prepared in Example 1 is designated as A, the neutral polysaccharide of Rehmannia glutinosa prepared in Comparative Example 1 is designated as B, and the neutral polysaccharide of Rehmannia glutinosa prepared in Comparative Example 2 is designated as C;

[0048] Figure 8 Quantitative results of alizarin red staining on zebrafish skulls; (A) Area of ​​alizarin red staining region in zebrafish skulls; (B) Cumulative optical density of alizarin red staining region in zebrafish skulls.

[0049] Figure 9 Effects of neutral homogeneous polysaccharides from Rehmannia glutinosa on osteogenic differentiation of bone marrow mesenchymal stem cells; (A) Alkaline phosphatase activity detection results, (B) Alizarin Red S staining quantitative analysis results;

[0050] Figure 10 Staining results of osteogenic differentiation of bone marrow mesenchymal stem cells; (A) alkaline phosphatase staining, (B) alizarin red S staining;

[0051] Figure 11 Histological and immunochemical analysis of bone defect repair; (A) HE staining, (B) Masson staining, (C) Immunochemical staining of type I collagen, (D) Immunochemical staining of osteocalcin. Detailed Implementation

[0052] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Example 1

[0053] The extraction and purification of neutral refined polysaccharides from Rehmannia glutinosa includes the following steps:

[0054] Rehmannia root slices were mixed with purified water at a mass-to-volume ratio of 1g:10mL and extracted twice. The mixture was boiled at 100℃ for 2 hours each time. The extracts were combined and concentrated to a density of 1.10g / mL. 95% ethanol was added to a concentration of 80% ethanol (the final ethanol concentration of the precipitate was 80%) to precipitate the product. The product was then dried under reduced pressure at 60℃ to obtain crude Rehmannia root polysaccharide.

[0055] The crude polysaccharide of Rehmannia glutinosa was prepared into a 10 mg / mL solution, and the pH was adjusted to 12 using 0.1 M NaOH solution. A 1:1 volume ratio of 10 mg / mL of a composite neutral polysaccharide purification reagent was added to the crude polysaccharide solution. The reagent consisted of polymethacrylamidopropyltrimethylammonium chloride, hexadecylpyridine bromide, and sodium dodecylbenzenesulfonate in a 2:1:1 mass concentration. The solution was added while stirring at a stirring speed of 500 rpm for 3 hours. After stirring, the gel-like precipitate in the solution was removed using filter paper. The supernatant was dialyzed with running water using a 1 KD dialysis bag for 72 hours. After freeze-drying, the neutral purified polysaccharide of Rehmannia glutinosa was obtained.

[0056] The extraction and purification of neutral homogeneous polysaccharides from Rehmannia glutinosa includes the following steps:

[0057] Separation of neutral homogeneous polysaccharides from Rehmannia glutinosa using size exclusion chromatography: 10 mg / mL neutral purified polysaccharides from Rehmannia glutinosa were prepared using a preparative liquid chromatography system equipped with a Sephardex G100 dextran gel chromatography column. Purified water was used as the eluent, the flow rate was 0.8 mL / min, and 10 mL was collected from each tube. The eluent signal was detected by a differential refractive index detector, and an elution curve was plotted (e.g., ...). Figure 1 Combine the eluents from tubes 12-17 and freeze-dry to obtain neutral homogeneous polysaccharide from Rehmannia glutinosa.

[0058] A neutral, homogeneous polysaccharide from Rehmannia glutinosa, composed of galactose, glucose, and fructose in a molar ratio of 2.72:1.17:1.11; its structural formula is as follows:

[0059]

[0060] Application of a neutral homogeneous polysaccharide from Rehmannia glutinosa in the preparation of osteoporosis drugs.

[0061] Application of a neutral homogeneous polysaccharide from Rehmannia glutinosa in the preparation of stem cell therapy drugs for bone defects.

[0062] Application of a neutral homogeneous polysaccharide from Rehmannia glutinosa in the preparation of biomaterials for in vivo delivery and growth scaffolds of stem cells.

[0063] Comparative Example 1

[0064] The preparation process of Rehmannia glutinosa neutral polysaccharide is the same as in Example 1, except for the formulation of the compound neutral polysaccharide purification reagent. The reagent in this comparative example is composed of polymethacrylamide propyltrimethylammonium chloride and hexadecyl bromopyridine at a mass concentration of 2:1.

[0065] Comparative Example 2

[0066] The preparation process of Rehmannia glutinosa neutral polysaccharide is the same as in Example 1, except for the formulation of the compound neutral polysaccharide purification reagent. The reagent in this comparative example is composed of polymethacrylamide propyltrimethylammonium chloride and sodium dodecylbenzenesulfonate at a mass concentration of 2:1.

[0067] Test Example 1

[0068] The structural characterization of the neutral homogeneous polysaccharide from Rehmannia glutinosa was obtained from Example 1, Comparative Example 1, and Comparative Example 2.

[0069] Molecular weight determination of neutral homogeneous polysaccharides from Rehmannia glutinosa: The homogeneity and molecular weight of Rehmannia glutinosa polysaccharides were evaluated using high-performance gel permeation chromatography combined with evaporative light scattering detector (HPGPC-ELSD). Rehmannia glutinosa polysaccharides were dissolved in purified water and injected into the HPGPC system (E2695, Waters Corporation, USA) for molecular weight determination. A molecular weight standard curve was plotted using dextran standards (50,000 Da, 25,000 Da, 12,000 Da, 5,000 Da, and 1,000 Da). The chromatographic conditions were as follows: Detector: Waters 2424 evaporative light scattering detector; Detection mode: 40 psi; Gain: 100; Nebulizer mode: Heating: 60℃; Drift tube temperature: 95℃; Column: TSK-GEL-GMPWXL (13μm, 7.8×300mm, Shimadzu, Japan); Column temperature: 30℃; Elution method: Isocratic elution; Injection volume: 10μL; Mobile phase: Double-distilled water; Flow rate: 0.8mL / min. A standard curve was plotted using the logarithm of the molecular weight (Mw) of the standard and its corresponding retention time (tR). The linear regression equation was: R 2 =0.9921. After software analysis, the weight-average molecular weight (Mw) of Rehmannia glutinosa polysaccharide was obtained, which was 2152 Da. Figure 2 ).

[0070] Determination of Monosaccharide Composition of Neutral Homogeneous Polysaccharide from Rehmannia glutinosa: Monosaccharide composition is the basis of polysaccharide structure and also affects polysaccharide activity. The monosaccharide content of neutral homogeneous polysaccharide from Rehmannia glutinosa was determined by the HPAEC-PAD method. 5 mg of Rehmannia glutinosa polysaccharide was dissolved in 2 M trifluoroacetic acid (TFA), heated at 60 °C for 1 hour, and then dried under nitrogen to eliminate TFA. Monosaccharide standards (containing fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, ribose, galacturonic acid, guluronic acid, glucuronic acid, and mannuronic acid) were used as analytical controls. Subsequently, the samples and controls were injected into an ICS5000 liquid chromatography system (ThermoFisher Scientific, USA) for analysis. The chromatographic conditions were as follows: Detector: electrochemical detector; Column: Dionex CarboPac TMPA20 column (10 μm, 150 × 3 mm, Thermo Fisher Scientific, USA); column temperature: 30 °C; flow rate: 0.5 mL / min; mobile phase: A, H₂O; B, 0.015 mol / L NaOH; C, 0.015 mol / L NaOH containing 0.1 mol / L NaOAc. Elution program: 0–26 min: 95% A, 5% B; 26–42 min: 85% A, 5% B; 42–52 min: 60% A, 40% C; 52–52.1 min: 60% A, 40% B; 52.1–60 min: 95% A, 5% B. Reference standard HPLC chromatogram as follows: Figure 3 As shown in Figure A, Rehmannia glutinosa polysaccharides are mainly composed of three monosaccharides: galactose, glucose, and fructose, with molar ratios of 2.72, 1.17, and 1.11, respectively (e.g., ...). Figure 3 B) indicates that Rehmannia polysaccharide is a neutral heteropolysaccharide.

[0071] Determination of the infrared spectrum of neutral homogeneous polysaccharide from Rehmannia glutinosa: Dried Rehmannia glutinosa polysaccharide was ground with KBr at a ratio of 1:100 and pressed into 1 mm thick slices. The spectra were determined using a Fourier transform infrared spectrophotometer (Bruker Tensor 27, Bruker, Germany) at 4000-400 cm⁻¹. -1 The polysaccharide spectra were obtained within the wavenumber range. The FT-IR spectra of Rehmannia glutinosa polysaccharides exhibited the characteristic absorption peaks typical of polysaccharides (e.g., ...). Figure 4 ). 1653 cm -1 and 3380 cm -1 The significant absorption peak at 1750 cm⁻¹ indicates the presence of hydroxyl groups, while the peak at 1750 cm⁻¹... -1 The absence of absorption peaks nearby indicates that Rehmannia glutinosa polysaccharide is a neutral polysaccharide. Furthermore, at 2929 cm⁻¹... -1 A weak peak was detected at 1026 cm⁻¹, indicating the presence of CH vibration. CO vibration was observed at 1026 cm⁻¹. -1 1072 cm -1 and 1145 cm -1 The absorption peaks at approximately 925 cm⁻¹ are correlated. -1 and 871 cm -1 The absorption peak at that point indicates the presence of β- and α-glycosidic bonds in the polysaccharide.

[0072] Determination of glycosidic bond linkages in neutral homogeneous polysaccharides from Rehmannia glutinosa: The linkage mode of sugar residues in Rehmannia glutinosa polysaccharides was determined by methylation analysis. Rehmannia glutinosa polysaccharides and sodium hydroxide were dissolved in anhydrous dimethyl sulfoxide under a nitrogen atmosphere. Iodomethane was slowly added to the solution under ice-water bath conditions with continuous stirring. The mixture was then shaken continuously at room temperature for 1 hour, and the reaction was terminated by adding pure water. The sample was hydrolyzed with trifluoroacetic acid and acetylated with acetic anhydride to obtain partially methylated diol esters (PMAAs). PMAAs were analyzed using gas chromatography-mass spectrometry (GC-MS, 6890A-5977B, Agilent Technologies, USA). Chromatographic conditions were as follows: Column: Agilent BPX70 (0.25 µm, 30 m × 0.25 mm, Australia); Carrier gas: High-purity helium (split ratio 10:1); Injection volume: 1 μL; Temperature program: Initial temperature 140℃ held for 2.0 min, then increased to 230℃ at a rate of 3℃ / min (heating time 3 min); Mass spectrometry range: 50-500 m / z. Qualitative identification was performed using the Complex Carbohydrate Research Center (CCRC) PMAA spectral database, and the relative molar ratios of each glycosidic bond were calculated using the PMAA peak area. Rehmannia glutinosa polysaccharide, after methylation with iodomethane, was analyzed by gas chromatography-mass spectrometry to elucidate its major glycosidic bond sites. Total ion chromatograms and fragment mass spectra are shown below. Figure 5 Table 1 shows the estimated molar ratios and linkages of sugar residues. Monosaccharide composition analysis results indicate that Rehmannia glutinosa polysaccharide contains Galp-(1→,→2)-Fruf-(1→,→6)-Glcp-(1→,→6)-Galp-(1→), with a molar ratio of 13.00:23.80:22.46:40.74.

[0073] Table 1. Glycosidic bond linkages of Rehmannia glutinosa polysaccharides

[0074]

[0075] Determination and Analysis of NMR Spectroscopy of Neutral Homogeneous Polysaccharide from Rehmannia glutinosa: Rehmannia glutinosa polysaccharide was dissolved in D2O and subjected to three freeze-thaw cycles to replace active hydrogen. Subsequently, one-dimensional and two-dimensional NMR spectra of the Rehmannia glutinosa polysaccharide were recorded using a 600 MHz NMR spectrometer (Bruker, Germany), including... 1 H NMR, 13 C NMR, 1 H- 1 H COSY, HSQC, and HMBC spectra. The scanning temperature was set to 25℃. Analysis of one-dimensional and two-dimensional NMR spectra (e.g., ...) was performed. Figure 6 This further elucidates the main chain structure of Rehmannia glutinosa polysaccharides. 1 In the 1H NMR spectrum, three coupling signal peaks were identified in the δ 4.3–5.4 ppm anodic carbon signal region; 13In the 10⁻⁶ C NMR spectrum, four coupled signal peaks were identified in the δ 90–110 ppm region. These results indicate that Rehmannia glutinosa polysaccharide contains four glycosyl residues, with corresponding anomeric carbon signals at δ 90.64, 96.58, 96.92, and 102.34 ppm. The δ 102.34 ppm signal has no corresponding hydrogen signal in the HSQC spectrum, indicating that it represents the terminal carbon signal of fructose. Subsequently, based on HMBC, COSY, and HSQC spectral data, chemical shifts with δ values ​​of 3.84, 3.99, 3.92, 3.66, and 3.55 / 3.60 ppm were identified as H1, H3, H4, H5, and H6 fructose residues, respectively; chemical shifts with δ values ​​of 60.01, 102.34, 74.94, 72.56, 79.88, and 61.00 ppm corresponded to C1, C2, C3, C4, C5, and C6 positions of fructose residues (labeled as glycosyl residue A). 1 H NMR, 13 Further ¹³C NMR and HSQC spectra confirmed that the anomeric signals of glycosyl residues B, C, and D in this sample were δ 5.19 / 90.64, 4.76 / 96.58, and 4.75 / 96.92 ppm, respectively. Based on the glycosidic bond linkage (methylation), anomeric signals, and literature synthesis data, it is speculated that residue A is →2)-β-D-Fruf-(1→, residue B is →6)-α-D-Glcp-(1→, residue C is →6)-α-D-Galp-(1→, and residue D is α-D-Galp-(1→).

[0076] The NMR signal assignment of sugar residue B is as follows: the anodic signal at δ values ​​of 5.39 / 91.64 ppm (H1 / C1) indicates that residue B may be an α-configuration glucose residue. Based on the COSY spectrum, H2 (3.34 ppm) was determined by the cross-peak at δ 5.19 / 3.34 ppm. Similarly, H3 (3.52 ppm), H4 (3.29 ppm), H5 (3.93 ppm), and H6 (3.44 / 3.80 ppm) of residue B were determined using the same method and can be attributed to the chemical shifts of hydrogen atoms on the sugar ring. Analysis of the CH cross-peaks in the HSQC spectrum identified the chemical shifts of C1-C6 as δ 90.64, 69.49, 71.27, 68.05, 69.82, and 64.44 ppm, respectively. The lower-field shifts of the C1 and C6 chemical shifts of residue B indicate that this residue undergoes substitution at the O-1 and O-6 positions. Based on the methylation analysis results and literature reports, residue A is inferred to be →6)-α-D-Glcp-(1→). The chemical shifts of the remaining residues can be assigned using a similar procedure, and the chemical shift results are shown in Table 2.

[0077] The linkage mode of Rehmannia glutinosa polysaccharides was determined by HMBC spectral analysis. According to the HMBC spectrum, H1 and C2 of residue A show a cross-peak at δ 3.44 / 102.34 ppm; H1 of residue B and C2 of residue A show a cross-peak at δ 5.19 / 102.34 ppm; H1 of residue A and C1 of residue C show a cross-peak at δ 3.54 / 96.58 ppm, H6 of residue C and C1 of residue C show a cross-peak at δ 3.59 / 96.58 ppm; H6 of residue B and C1 of residue B show a cross-peak at δ 3.80 / 90.64 ppm; H1 of residue B and C6 of residue B show a cross-peak at δ 5.19 / 64.44 ppm; H1 of residue D and C6 of residue B show a cross-peak at δ 4.85 / 64.44 ppm; H1 of residue D and C6 of residue C show a cross-peak at δ 5.75 / 65.04 ppm. A cross peak is formed at ppm.

[0078] Table 2 Chemical shifts of C and H in D2O for Rehmannia glutinosa polysaccharides

[0079]

[0080] Analysis revealed that the neutral and homogeneous polysaccharide structure of Rehmannia glutinosa consists of three repeating units: a, b, and c. The structure of part a is α-D-Galp-(1→6)-α-D-Galp-(1→), the structure of part b is →2)-β-D-Fruf-(1→2)-β-D-Fruf-(1→), and the structure of part c is α-D-Glcp-(1→6)-α-D-Galp-(1→). In Example 1, the polysaccharide structure with a ratio of a:b:c is 3:1:2; in Comparative Example 1, the polysaccharide structure with a ratio of a:b:c is 1:2:3; and in Comparative Example 2, the polysaccharide structure with a ratio of a:b:c is 1:3:2.

[0081] Test Example 2

[0082] Anti-osteoporosis activity of neutral polysaccharides from Rehmannia glutinosa;

[0083] The neutral polysaccharide of Rehmannia glutinosa prepared in Example 1 is designated as A, the neutral polysaccharide of Rehmannia glutinosa prepared in Comparative Example 1 is designated as B, and the neutral polysaccharide of Rehmannia glutinosa prepared in Comparative Example 2 is designated as C.

[0084] Culture and hatching of zebrafish: Take 5-6 pairs of wild-type AB lineage adult zebrafish and place them in a rearing tank. The culture temperature is 28.5℃ and the day-night cycle is controlled at 14h:10h. After they mate freely, collect the zebrafish fertilized eggs and place them in a culture dish. Add culture medium and place the dish in a constant temperature incubator at a temperature of 28.5±0.5℃.

[0085] Grouping and administration of zebrafish: After hatching, zebrafish juveniles 3 days after fertilization were placed in 6-well plates with 20 zebrafish per well, divided into 6 groups (blank group, model group, positive group, group A, group B, and group C), with 3 wells in each group. The blank group was cultured in blank culture medium, the model group was cultured in 0.1% DMSO culture medium containing 25 μM prednisolone, the positive group was cultured in 0.1% DMSO culture medium containing 25 μM prednisolone and 30 μg / mL etidronate disodium solution, and the administration group was cultured in 0.1% DMSO culture medium containing 25 μM prednisolone and 500 μg / mL different Rehmannia glutinosa neutral polysaccharides. The zebrafish were cultured until 7 days post-hatching (dpf), and the culture medium and drug solution were changed daily.

[0086] Alizarin Red Staining Analysis of Zebrafish Skeletal Mineralization: Zebrafish cultured to 7 days post-fertilization were anesthetized and killed in MS-222 solution. After removing the MS-222 solution, the zebrafish were fixed in 4% paraformaldehyde general-purpose tissue fixative for 0.5 h, then the fixative was removed. The zebrafish were washed three times with 50% ethanol, each time for no more than 3 min. Bleaching was performed for 2 h using a bleaching agent containing 1.5% H2O2 prepared with 0.5% KOH. After removing the bleaching agent, 0.01% alizarin red staining solution prepared with 0.5% KOH was added to stain the skull bones of juvenile zebrafish for 15 min. After removing the staining solution, the juveniles were cleared for 2 h using a 1:1 mixture of 1% KOH solution and glycerol. Finally, the zebrafish were stored in 100% glycerol. The ventral surface of the alizarin red-stained zebrafish skull was observed using a Zeiss stereomicroscope. Images were acquired using imaging software, and all images were taken with the same light intensity and exposure settings (e.g., ...). Figure 7 The area (AREA) and cumulative optical density (IOD) of the alizarin red stained region in zebrafish skulls were calculated using the professional image analysis software Image Pro pLus 6.0 to reflect bone mineralization and bone density, respectively. Compared with the control group, the area (AREA) and cumulative optical density (IOD) of the alizarin red stained region in zebrafish skulls were significantly reduced after prednisolone treatment (P<0.01), indicating that prednisolone successfully induced a zebrafish osteoporosis model. In terms of the area (AREA) and cumulative optical density (IOD) of the alizarin red stained region in zebrafish skulls, all three treatment groups showed significant differences compared with the model group (PD) (P<0.05), indicating that Rehmannia glutinosa polysaccharide has an anti-prednisolone-induced osteoporosis effect (e.g., Figure 8 Furthermore, the efficacy of polysaccharides in group A was significantly higher than that in groups B and C.

[0087] Test Example 3

[0088] Neutral polysaccharides from Rehmannia glutinosa promote osteogenic differentiation of BMSCs;

[0089] Alkaline phosphatase staining and activity assay: Bone marrow mesenchymal stem cells were stained with alkaline phosphatase at a concentration of 1×10⁻⁶. 5Cells were seeded at a density of approximately 80% in 12-well plates. Osteogenic induction was initiated when the cell density in each group reached approximately 80%. The culture medium was then replaced with osteogenic induction medium (OIM) containing 10 mM β-hydroxyproline (βGP), 100 nM deoxysarcosine (DXMS), and 50 μg / mL L-hydroxyproline (LA). 500 μg / mL Rehmannia glutinosa polysaccharide was added to groups B and C. Alkaline phosphatase staining was performed on days 7 and 14 of induction. Cells were first fixed with 4% paraformaldehyde general tissue fixative for 20 minutes. Then, the cells were incubated for 2 hours at room temperature in the dark using an alkaline phosphatase colorimetric kit. The reaction was terminated by washing once with distilled water. Images were then carefully observed and photographed using an inverted microscope. Cells were lysed using Western blotting and IP cell lysis buffer, and staining was performed using an ALP colorimetric kit, a BCA assay kit, and an ALP detection kit to measure alkaline phosphatase activity. ALP activity and total protein content were quantitatively analyzed, and ALP content was standardized. After osteogenic induction and Rehmannia glutinosa polysaccharide treatment of BMSCs for 7 and 14 days, ALP measurement results are as follows: Figure 10 As shown in Figure A, the quantitative results are as follows: Figure 9 A. Compared with groups B and C, group A showed a higher proportion of blue-purple positive staining areas.

[0090] Alizarin Red S staining: The effect of Rehmannia glutinosa polysaccharide on calcium deposition in BMSCs was assessed by alizarin red S staining, a slightly modified version of previous studies. Cells were seeded in 6-well plates (1×10⁶ cells / wells). 5 Cells were cultured at a density of 1000 mcg / mL and then divided into three groups (A, B, and C) for BMSC treatment. After 21 days of culture, cells were fixed with 4% polyformaldehyde general tissue fixative for 20 minutes, followed by 15 minutes of Alizarin Red S staining. After rinsing the cells with distilled water, images of calcium deposition in bone marrow mesenchymal stem cells were obtained. Finally, the cells were destained with 10% CPC, and the amount of calcium deposition was quantitatively analyzed by measuring the amount at 562 nm using a microplate reader. The results of Alizarin Red S staining and subsequent measurements are shown below. Figure 10 B, Quantitative results are as follows Figure 9 B. BMSCs in groups A, B, and C all showed more significant mineralization effects. Among them, group A was superior to groups B and C in both early osteogenic promotion and late mineralization promotion.

[0091] Test Example 4

[0092] In vivo pharmacodynamics of Rehmannia glutinosa neutral polysaccharide in repairing bone defects;

[0093] Animal grouping and administration: SPF-grade SD rats (8 weeks old, weighing 200-230 g) were placed in a constant temperature environment (23±2℃) with a 12-hour light-dark cycle and free access to food and water. After one week of acclimatization, the rats were randomly divided into three groups: Group A, Group B, and Group C. Group A was implanted with the polysaccharide obtained in Example 1, Group B with the polysaccharide obtained in Comparative Example 1, and Group C with the polysaccharide obtained in Comparative Example 2. Anesthesia was administered via intraperitoneal injection of 2% sodium pentobarbital (40 mg / kg body weight). A 2 mm × 2 mm × 2 mm defect was drilled on the lateral aspect of the distal left femur using an electric drill equipped with a φ2 mm drill bit. Residual bone fragments were removed with cold saline to prevent thermal necrosis. Each group was implanted with 500 μg / mL Rehmannia glutinosa polysaccharide and 1×10 5 Five BMSCs were implanted, and the gap was sealed with bone wax. Five weeks post-operation, the left femur was completely removed for subsequent experiments.

[0094] Histological and Immunohistochemical Analysis: To investigate the mechanisms of collagen deposition and bone regeneration in vivo, histological and immunohistochemical staining was performed. Bone samples were placed in a 37°C shaking incubator and decalcified using EDTA decalcification solution, which was changed weekly. After two months, the samples were dehydrated, paraffin-embedded, and sectioned to a thickness of 5 μm. After routine dewaxing to water, hematoxylin-eosin (HE) and Masson's trichrome (MT) staining were performed according to the manufacturer's instructions. After observation and imaging under a stereomicroscope, the sections were analyzed using ImageJ software at the same threshold range. The area of ​​newly mineralized regions in HE-stained tissue images was calculated using ImageJ software, and the volume fraction of collagen deposition was calculated based on the MT staining results. Finally, immunohistochemical (IHC) staining was used to assess the expression levels of type I collagen (ColⅠ) and osteocalcin (OCN), two proteins related to bone regeneration.

[0095] HE staining provides a more comprehensive analysis of the mechanisms of bone defect repair. First, such as Figure 11 As shown in Figure A, HE staining revealed that Group B exhibited a significant "cavity-like" structure and inflammatory infiltration, with loosely arranged surrounding tissue primarily composed of fibrous connective tissue, indicating that the defect had not yet been filled. In Group C, the defect area was extensively filled with new tissue, osteoblasts were densely packed, inflammatory infiltration and defect area were reduced, and tissue density was higher. Group A was almost completely filled with new tissue, with richer osteoid tissue, better tissue continuity, reduced inflammatory infiltration, and a morphology closer to normal bone tissue. This indicates that the Rehmannia glutinosa neutral polysaccharide group had a significant effect on new bone formation, and the efficacy of the polysaccharide in Group A was significantly higher than that in Groups B and C.

[0096] like Figure 11As shown in B, Masson staining further validated the above results. In the control group, the blue collagen fibers were sparsely scattered, mainly composed of disordered fibrous connective tissue, lacking the regular collagen arrangement characteristic of bone matrix; in groups B and C, the blue collagen fibers increased significantly, collagen deposition was obvious, and they began to show a directional arrangement trend similar to "bone-like trabeculae"; in group A, the blue area accounted for a higher proportion, the collagen arrangement was more compact and regular, and the polysaccharide efficacy in group A was significantly higher than that in groups B and C.

[0097] Immunohistochemical staining was used to assess protein expression in new bone formation and further analyze the bone defect repair effect. ColⅠ, as a non-specific marker of early osteogenic differentiation, reflects the initiation stage of matrix synthesis in bone repair; while OCN, as a late marker of osteogenic differentiation and mineralization, reflects the osteoblast maturation stage and active mineralization state. Combined detection comprehensively reflects the bone defect repair process from early matrix preparation to late mineralization maturation. Figure 11 C (Col I staining) and Figure 11 As shown by D (OCN staining), the staining intensity of group A was significantly higher than that of groups B and C, indicating that the efficacy of the polysaccharide in group A was significantly higher than that in groups B and C. This suggests that it has a more significant advantage in osteoblast maturation and mineralization, enabling the bone defect repair process to progress to a late stage characterized by high maturity and mineralization activity.

[0098] It should be understood that, in order to simplify this disclosure and aid in understanding one or more of the various aspects of the invention, features of the invention are sometimes grouped together in a single embodiment, figure, or description thereof in the above description of exemplary embodiments of the invention. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than expressly recited in each claim. Rather, as reflected in the claims, inventive aspects lie in fewer than all the features of the foregoingly disclosed embodiments. Therefore, the claims, following the detailed description, are hereby expressly incorporated into that detailed description, wherein each claim itself is a separate embodiment of the invention.

[0099] Although the invention has been described with reference to a limited number of embodiments, those skilled in the art will understand from the foregoing description that other embodiments are conceivable within the scope of the invention described herein. Furthermore, it should be noted that the language used in this specification has been chosen primarily for readability and instructional purposes, and not for the purpose of interpreting or limiting the subject matter of the invention. Therefore, many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the appended claims. The disclosure of the invention is illustrative and not restrictive, and the scope of the invention is defined by the appended claims.

[0100] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A neutral homogeneous polysaccharide from Rehmannia glutinosa, characterized in that, It is composed of galactose, glucose and fructose in a molar ratio of 2.72:1.17:1.11; Its preparation method includes the following steps: Step 1: Mix Rehmannia glutinosa slices with purified water for extraction, concentrate the extract, and then precipitate with ethanol to obtain crude Rehmannia glutinosa polysaccharide; Step 2: Dissolve the crude polysaccharide obtained in Step 1 and adjust the pH using NaOH; Step 3: Add the complex neutral polysaccharide purification reagent to the solution obtained in Step 2, and stir continuously using a magnetic stirrer; Step 4: Remove the gel-like precipitate from the solution obtained in Step 3, and dialyze the supernatant to remove small molecules, to obtain neutral purified polysaccharide of Rehmannia glutinosa. Step 5: Separate the neutral purified polysaccharide of Rehmannia glutinosa obtained in Step 4 by molecular size exclusion chromatography column to obtain neutral homogeneous polysaccharide of Rehmannia glutinosa. In step three, a 10 mg / mL composite neutral polysaccharide purification reagent, equivalent to a 1:1 volume of the crude polysaccharide solution obtained in step two, is added to the solution. The composite neutral polysaccharide purification reagent is composed of polymethacrylamide propyltrimethylammonium chloride, hexadecyl bromopyridine, and sodium dodecylbenzenesulfonate in a mass ratio of 2:1:

1. The reagent is added while stirring at a stirring speed of 500 rpm for 3 hours.

2. The method for preparing a neutral homogeneous polysaccharide from Rehmannia glutinosa according to claim 1, characterized in that, Includes the following steps: Step 1: Mix Rehmannia glutinosa slices with purified water for extraction, concentrate the extract, and then precipitate with ethanol to obtain crude Rehmannia glutinosa polysaccharide; Step 2: Dissolve the crude polysaccharide obtained in Step 1 and adjust the pH using NaOH; Step 3: Add the complex neutral polysaccharide purification reagent to the solution obtained in Step 2, and stir continuously using a magnetic stirrer; Step 4: Remove the gel-like precipitate from the solution obtained in Step 3, and dialyze the supernatant to remove small molecules, to obtain neutral purified polysaccharide of Rehmannia glutinosa. Step 5: Separate the neutral refined polysaccharide of Rehmannia glutinosa obtained in Step 4 using a size exclusion chromatography column to obtain a neutral homogeneous polysaccharide of Rehmannia glutinosa.

3. The preparation method according to claim 2, characterized in that, In step one, Rehmannia glutinosa slices were boiled and extracted with purified water at a mass-to-volume ratio of 1g:10mL at 100℃ for a total of 2 extractions, each extraction lasting 2 hours. The two extracts were combined and concentrated to a density of 1.10g / mL. 95% ethanol was added to precipitate the precipitate to a concentration of 80% ethanol. The precipitate was dried under reduced pressure at 60℃ to obtain crude Rehmannia glutinosa polysaccharide.

4. The preparation method according to claim 3, characterized in that, In step two, the crude polysaccharide of Rehmannia glutinosa is prepared into a 10 mg / mL aqueous solution, and the pH is adjusted to 12 using a 0.1 M NaOH solution to obtain the crude polysaccharide solution of Rehmannia glutinosa.

5. The preparation method according to claim 4, characterized in that, In step three, a 10 mg / mL composite neutral polysaccharide purification reagent, equivalent to a 1:1 volume of the crude polysaccharide solution obtained in step two, is added to the solution. The composite neutral polysaccharide purification reagent is composed of polymethacrylamide propyltrimethylammonium chloride, hexadecyl bromopyridine, and sodium dodecylbenzenesulfonate in a mass ratio of 2:1:

1. The reagent is added while stirring at a stirring speed of 500 rpm for 3 hours.

6. The preparation method according to claim 5, characterized in that, In step four, after stirring, the gel-like precipitate in the solution is removed using filter paper. The supernatant is dialyzed with running water using a 1KD dialysis bag for 72 hours and then freeze-dried to obtain neutral refined polysaccharide from Rehmannia glutinosa.

7. The preparation method according to claim 6, characterized in that, In step five, size exclusion chromatography was used to separate Rehmannia glutinosa neutral homogeneous polysaccharide: 10 mg / mL Rehmannia glutinosa neutral purified polysaccharide was prepared using a preparative liquid chromatography system equipped with a Sephardex G100 dextran gel chromatography column. Purified water was used as the eluent, the flow rate was 0.8 mL / min, 10 mL was collected from each tube, and the eluents from tubes 12-17 were combined and freeze-dried to obtain Rehmannia glutinosa neutral homogeneous polysaccharide.

8. The application of the neutral homogeneous polysaccharide of Rehmannia glutinosa according to claim 1 in the preparation of osteoporosis drugs.

9. The application of the neutral homogeneous polysaccharide of Rehmannia glutinosa according to claim 1 in the preparation of stem cell therapy drugs for bone defects, characterized in that, Stem cells include bone marrow mesenchymal stem cells.

10. The application of the neutral homogeneous polysaccharide of Rehmannia glutinosa according to claim 1 in the preparation of biomaterials as in vivo delivery and growth scaffolds for stem cells, characterized in that, Stem cells include bone marrow mesenchymal stem cells.

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