Modified monascus exopolysaccharide, and preparation method and application thereof

Abstract

The application provides a modified monascus exopolysaccharide and an application thereof, and the yield of the monascus exopolysaccharide is effectively improved by adding genistein in a fermentation medium, and the modified monascus exopolysaccharide with high biological activity is generated; the monascus exopolysaccharide provided by the application effectively improves the osteogenic differentiation effect of mesenchymal stem cells, and provides a new treatment drug and a treatment approach for the treatment of osteoporosis; the preparation method provided by the application is simple, green, environment-friendly, short in period, and low in cost and high in efficiency, and is favorable for realizing high-value utilization of monascus resources in China.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a modified Monascus purpureus extracellular polysaccharide, its preparation method, and its application. Background Technology

[0002] Monascus purpureus is a small, filamentous saprophytic fungus with a long history of medicinal and edible use, belonging to the Ascomycota family, and distributed in Fujian, Zhejiang, Taiwan, and Guangxi Zhuang Autonomous Region of China. Its fermentation metabolites have wide applications in the food and pharmaceutical industries. Monascus purpureus can secrete a series of beneficial secondary metabolites, including dimeritic acid, γ-aminobutyric acid (GABA), monacolin K, and red monacolin pigment, which possess anti-inflammatory, antifungal, hypoglycemic, antioxidant, and antitumor biological effects. Extracellular polysaccharides (EPS), another important metabolite, have attracted considerable attention in recent years due to their various biological functions, including antitumor, antibacterial, and antioxidant properties. However, the low yield of EPS severely limits the further application of Monascus purpureus resources. Therefore, liquid deep fermentation, which features short production cycles, easy quality control, and high levels of mechanization and automation, is conducive to large-scale industrial production and has become the main method for producing Monascus purpureus extracellular polysaccharides. With the development of biotechnology, the combined fermentation of medicinal and edible fungi with natural bioactive substances has become a very effective approach for developing medicinal and functional product resources.

[0003] With the increasing aging of my country's population, osteoporosis in the elderly and postmenopausal women has become a significant health problem for the entire society. Osteoporosis can lead to complications such as systemic bone pain, decreased height, and fractures of the vertebrae and hip. These complications not only severely affect the mobility and quality of life of the elderly but can also cause serious disability or even death. Osteoporosis has become the most common geriatric disease after hypertension, diabetes, and cardiovascular and cerebrovascular diseases, placing a huge burden on families and society. The pathophysiological mechanism of osteoporosis is complex. Its basic cause is the imbalance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption (i.e., bone remodeling), leading to bone loss, degeneration of bone microstructure, and increased risk of fracture. Multiple factors and signaling pathways are involved in the bone remodeling process.

[0004] Currently, clinical medications for treating osteoporosis are mainly divided into two categories: one is drugs that inhibit bone resorption, such as bisphosphonates, but long-term use of these drugs may be accompanied by complications such as osteonecrosis of the mandible; the other is drugs that promote bone formation, such as recombinant human parathyroid hormone, but these drugs are expensive and cannot be used for a long time.

[0005] Therefore, a new solution is needed to improve the above problems. Summary of the Invention

[0006] The present invention aims to provide a modified Monascus extracellular polysaccharide, its preparation method and application, to improve the problems of high cost and risk of complications from long-term use of existing drugs for treating osteoporosis.

[0007] In a first aspect, the present invention provides a modified Monascus purpureus extracellular polysaccharide G-EMP, wherein the main chain of the repeating structure of the extracellular polysaccharide G-EMP unit includes →4)-α-D-1, 4-Galp-(1→, →6)-α-D-2, 6-Glcp-(1→, →2)-β-D-1, 2-Manp-(1→;

[0008] The side chains in the extracellular polysaccharide G-EMP are formed by α-D-Manp-(1→ linked to →6)-α-D-2,6-Glcp-(1→) at the O-2 position.

[0009] Optionally, the extracellular polysaccharide G-EMP is a pyranose that exists in both α- and β-configurations.

[0010] Optionally, the extracellular polysaccharide G-EMP is composed of arabinose, xylose, mannose, galactose, and glucose.

[0011] Optionally, the extracellular polysaccharide G-EMP includes 12 linkage types, including t-Man(p), t-Glc(p), 3-Glc(p), 2-Man(p), 4-Man(p), 6-Man(p), 4-Gal(p), 4-Glc(p), 2,3-Man(p), 2,4-Man(p), 3,6-Glc(p) and 2,6-Glcp.

[0012] Secondly, the present invention provides the application of modified Monascus purpureus extracellular polysaccharide G-EMP in the treatment of osteoporosis, including pharmaceutical compositions and functional foods.

[0013] Optionally, the modified extracellular polysaccharide G-EMP in the application can induce osteogenic differentiation of mesenchymal stem cells.

[0014] Optionally, the activity of the modified extracellular polysaccharide G-EMP in inducing osteogenic differentiation of mesenchymal stem cells is related to the →4)-α-D-1,4-Galp-(1→, →2)-β-D-1,2-Manp-(1→ and →4)-α-D-1,4-Galp-(1→ glycosidic bonds in its structure.

[0015] Optionally, in this application, the extracellular polysaccharide G-EMP can enhance the activity of alkaline phosphatase in mesenchymal stem cells.

[0016] Optionally, in this application, the extracellular polysaccharide G-EMP can increase the calcium ion concentration in mesenchymal stem cells.

[0017] Thirdly, the present invention provides a method for preparing the extracellular polysaccharide G-EMP from Monascus purpureus, comprising the following steps:

[0018] S 1. Activate and culture Monascus purpureus to prepare seed liquid; the Monascus purpureus is purple Monascus purpureus 40269;

[0019] S2. The seed liquid is added to a fermentation culture medium to obtain a fermentation broth; the fermentation culture medium includes genistein.

[0020] S3. Centrifuge and concentrate the fermentation broth. After concentration, add anhydrous ethanol to obtain crude extracellular polysaccharide precipitate.

[0021] S4. Add protease solution to the precipitate for enzymatic hydrolysis. After enzymatic hydrolysis, remove the protein using the Sevage method to obtain crude extracellular polysaccharide.

[0022] S5. The crude extracellular polysaccharide is separated and purified to obtain Monascus purpureus extracellular polysaccharide G-EMP.

[0023] The beneficial effects of this invention include:

[0024] (1) In the method for modifying Monascus purpureus extracellular polysaccharide provided by the present invention, the addition of genistein to the fermentation culture medium can promote the synthesis of Monascus purpureus extracellular polysaccharide and improve the biological activity of Monascus purpureus extracellular polysaccharide.

[0025] (2) This invention provides the structure of modified Monascus purpureus extracellular polysaccharide. The structural information of the modified extracellular polysaccharide was comprehensively analyzed by means of methylation, nuclear magnetic resonance and other methods, which provides a reference for improving the bioactivity of Monascus purpureus polysaccharide and is conducive to realizing the high-value utilization of Monascus purpureus resources.

[0026] (3) The modified Monascus extracellular polysaccharide provided by the present invention exhibits high activity in the process of inducing osteogenic differentiation of mesenchymal tissue and can increase the concentration of alkaline phosphatase and calcium ions in cells. It has good application prospects in the preparation of drugs for treating osteoporosis and functional foods. Attached Figure Description

[0027] Figure 1 This is the elution diagram of pure G-EMP obtained on cellulose and Sepharose columns in this invention;

[0028] Figure 2 The effect of adding different flavonoids to the fermentation medium in this invention on the biomass and extracellular polysaccharide yield of Monascus purpureus;

[0029] Figure 3 The results of particle size and zeta potential detection for the extracellular polysaccharide G-EMP prepared in Example 2 of this invention;

[0030] Figure 4 The results of ultraviolet-visible spectrophotometry and infrared spectroscopy of the extracellular polysaccharide G-EMP prepared in Example 2 of this invention are shown below.

[0031] Figure 5 The X-ray diffraction pattern and thermogravimetric analysis results of the extracellular polysaccharide G-EMP prepared in Example 2 of this invention are shown.

[0032] Figure 6 This is a scanning electron microscope image of the extracellular polysaccharide G-EMP prepared in Example 2 of the present invention;

[0033] Figure 7 The nuclear magnetic resonance spectrum of the extracellular polysaccharide G-EMP prepared in Example 2 of this invention;

[0034] Figure 8 This is a schematic diagram of the structure of the extracellular polysaccharide G-EMP prepared in Example 2 of the present invention;

[0035] Figure 9 The nuclear magnetic resonance spectrum of the extracellular polysaccharide EMP prepared in Comparative Example 5 of this invention;

[0036] Figure 10 This is a schematic diagram of the structure of the extracellular polysaccharide EMP prepared in Comparative Example 5 of the present invention;

[0037] Figure 11 The effect of the extracellular polysaccharide G-EMP prepared in Example 1 of this invention on the osteogenic differentiation of hUC-MSCs cells. Detailed Implementation

[0038] The present invention will be further described in conjunction with the accompanying drawings and through the following embodiments.

[0039] In a first aspect, embodiments of the present invention provide a modified Monascus purpureus extracellular polysaccharide G-EMP, wherein the main chain of the repeating structure of the extracellular polysaccharide G-EMP unit includes →4)-α-D-1,4-Galp-(1→、→6)-α-D-2,6-Glcp-(1→、→2)-β-D-1,2-Manp-(1→;

[0040] The side chains in the extracellular polysaccharide G-EMP are formed by α-D-Manp-(1→ linked to →6)-α-D-2,6-Glcp-(1→) at the O-2 position.

[0041] In some embodiments, the extracellular polysaccharide G-EMP is a pyranose that exists in both α- and β-configurations.

[0042] In some embodiments, the extracellular polysaccharide G-EMP is composed of arabinose, xylose, mannose, galactose, and glucose.

[0043] In some embodiments, the extracellular polysaccharide G-EMP includes 12 linkage types, including t-Man(p), t-Glc(p), 3-Glc(p), 2-Man(p), 4-Man(p), 6-Man(p), 4-Gal(p), 4-Glc(p), 2,3-Man(p), 2,4-Man(p), 3,6-Glc(p), and 2,6-Glcp.

[0044] Secondly, embodiments of the present invention provide the application of Monascus purpureus extracellular polysaccharide G-EMP in the treatment of osteoporosis, including pharmaceutical compositions and functional foods.

[0045] In some embodiments, the extracellular polysaccharide G-EMP in the application can induce osteogenic differentiation of mesenchymal stem cells.

[0046] In some embodiments, the activity of the extracellular polysaccharide G-EMP in inducing osteogenic differentiation of mesenchymal stem cells is related to the →4)-α-D-1,4-Galp-(1→, →2)-pD-1,2-Manp-(1→ and →4)-α-D-1,4-Galp-(1→ glycosidic bonds in its structure.

[0047] In some embodiments, the extracellular polysaccharide can enhance the activity of alkaline phosphatase in mesenchymal stem cells.

[0048] In some embodiments, the extracellular polysaccharide can increase the calcium ion concentration in mesenchymal stem cells.

[0049] Thirdly, embodiments of the present invention provide a method for preparing the extracellular polysaccharide G-EMP from Monascus purpureus, comprising the following steps:

[0050] S1. Activate and culture Monascus purpureus to prepare seed liquid; the Monascus purpureus is purple Monascus purpureus 40269;

[0051] S2. The seed liquid is added to a fermentation culture medium to obtain a fermentation broth; the fermentation culture medium includes genistein.

[0052] S3. Centrifuge and concentrate the fermentation broth. After concentration, add anhydrous ethanol to obtain crude extracellular polysaccharide precipitate.

[0053] S4. Add protease solution to the precipitate for enzymatic hydrolysis. After enzymatic hydrolysis, remove the protein using the Sevage method to obtain crude extracellular polysaccharide.

[0054] S5. The crude extracellular polysaccharide is separated and purified to obtain Monascus purpureus extracellular polysaccharide G-EMP.

[0055] In some embodiments, the final concentrations of each component of the malt powder inorganic salt culture medium (MYF) are: 2-3% malt powder, 0.04-0.06% potassium dihydrogen phosphate, 0.8-1.0% magnesium sulfate heptahydrate, 0.08-0.10% dipotassium hydrogen phosphate, and 2-3% agar.

[0056] In some embodiments, the final concentrations of the components of the seed liquid culture medium are: 25-35 g / L glucose, 1.8-2.2 g / L potassium dihydrogen phosphate, 2.8-3.2 g / L sodium nitrate, 0.4-0.6 g / L potassium chloride, and 0.45-0.55 g / L magnesium sulfate heptahydrate.

[0057] In some embodiments, the final concentrations of each component in the fermentation medium are: 40-60 g / L sucrose, 3-4 g / L yeast extract, 0.8-1.0 g / L potassium dihydrogen phosphate, 1.5-2.1 g / L dipotassium hydrogen phosphate, 0.9-1.1 g / L magnesium sulfate heptahydrate, and 1.8-2.2 mL / L Tween 80.

[0058] In some embodiments, the osteogenic induction medium consists of: 190-195 mL of basal medium for inducing chondrogenic differentiation of mesenchymal stem cells, 190-200 μL of sodium pyruvate, 1.9-2 mL LITS, 1.9-2 mL mL LTGF-β3, 580-600 μL of ascorbic acid, 190-200 μL of proline, 18-20 μL of dexamethasone, and 1.9-2 mL of penicillin and streptomycin.

[0059] The purple red Monascus purpureus 40269 used in the embodiments of this invention was purchased from CICC Industrial Microbiology, and other reagents and kits were commercially available.

[0060] Example 1

[0061] Example 1 of this invention provides a method for preparing a fermentation medium that increases the yield of extracellular polysaccharides from Monascus purpureus, comprising the following steps:

[0062] S1. Activate and culture Monascus purpureus to prepare seed culture, including the following sub-steps:

[0063] S11. The low-temperature preserved purple red Monascus purpureus 40269 was inoculated into malt powder inorganic salt medium for activation and cultured at 28℃ for 12 days.

[0064] S12. After the culture is completed, add sterile water and stir with an inoculation loop to obtain a spore suspension;

[0065] S13. Inoculate 1.5 mL of spore suspension into a culture flask containing 100 mL of liquid seed culture medium and incubate at 30 °C for 2 days to obtain activated seed liquid.

[0066] S2. Add the activated seed culture to the fermentation medium to prepare the fermentation broth, including the following sub-steps:

[0067] S21. Add 100 mL of fermentation medium to different conical fermentation flasks, and add genistein powder with a purity of more than 95% to the fermentation flasks, with a final concentration of 3 g / L.

[0068] S22. Sonicate the fermentation flask at a frequency of 45-65KHz, with an ultrasonic power of 150W and an ultrasonic duration of 10-20min, so that the genistein is fully dissolved in the fermentation medium.

[0069] S23. After ultrasonication, the fermentation flask is autoclaved at 121℃ for 25 minutes to obtain a sterile fermentation medium.

[0070] S24. After sterilization, the seed liquid obtained in S13 is inoculated into a sterile fermentation medium containing genistein at an inoculation amount of 9% (the volume ratio of seed liquid to sterile fermentation medium). The medium is then cultured at 30°C and 180 rpm for 4 days to obtain a fermentation broth with a genistein concentration of 3 g / L.

[0071] Example 2

[0072] Embodiment 2 of the present invention provides a method for generating Monascus purpureus extracellular polysaccharides using the fermentation broth provided in Embodiment 1, comprising the following steps:

[0073] D1. Centrifuge and concentrate the fermentation broth. After concentration, add anhydrous ethanol to obtain crude extracellular polysaccharide precipitate, including the following sub-steps:

[0074] D11. After filtering the fermentation broth with a genistein concentration of 3 g / L obtained in step S24 through gauze, centrifuge at 10000×g for 15 min, and concentrate it to 1 / 5 of the original volume using a rotary evaporator to obtain a concentrated solution.

[0075] D12. Add anhydrous ethanol to the concentrate and precipitate for 24 hours to remove free pigments, small molecules, and residual genistein, thus obtaining crude extracellular polysaccharide precipitate.

[0076] D2. Add protease solution to the precipitate for enzymatic hydrolysis. After hydrolysis, remove proteins using the Sevage method to obtain crude extracellular polysaccharide, including the following sub-steps:

[0077] D21. Transfer the crude extracellular polysaccharide precipitate obtained in step S32 to ultrapure water containing papain, and enzymatically hydrolyze it at 55°C for 1 hour. The mass ratio of papain to crude extracellular polysaccharide precipitate is 2%. After enzymatic hydrolysis, inactivate papain at 90°C for 10 minutes to obtain the enzymatic hydrolysate.

[0078] D22. Mix 20 mL of enzymatic hydrolysate with 60 mL of Sevage reagent and perform deproteinization treatment: centrifuge at 10000×g for 15 min, collect the supernatant, and repeat centrifugation 3 times; dialyze the supernatant collected in the last centrifugation for 72 h, freeze dry, and obtain crude extracellular polysaccharide.

[0079] D3. Separating and purifying the crude extracellular polysaccharide to obtain Monascus purpureus extracellular polysaccharide G-EMP, including the following sub-steps:

[0080] D31. The crude extracellular polysaccharide obtained in step D22 was prepared into a 10 mg / mL solution and further purified using a DEAE-Cellulose 52 ion exchange column. Elution was optimized using 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl, respectively. Figure 1 As shown in A;

[0081] D32. Collect the eluent from step D31 in separate tubes and monitor it using the phenol-sulfuric acid method. When the presence or absence of sugar components is detected, stop collecting and freeze-dry the collected eluent.

[0082] D33. The elution group with the highest yield and total sugar content after freeze-drying and the highest concentration of 0.1 mol / L NaCl was fractionated into solutions, further purified using a Sepharose gel column, and eluted with purified water to obtain Monascus purpureus extracellular polysaccharide G-EMP. Figure 1 As shown in B in the diagram.

[0083] Comparative Example 1

[0084] Comparative Example 1 of the present invention provides a method for preparing a fermentation medium for Monascus purpureus extracellular polysaccharides. The difference between this method and the method for preparing the fermentation medium in Example 1 is that the fermentation medium added in step S21 is a flavonoid aglycone; other conditions and steps remain the same.

[0085] Comparative Example 2

[0086] Comparative Example 2 of the present invention provides a method for preparing a fermentation medium for Monascus purpureus extracellular polysaccharides. The difference between this method and the method for preparing the fermentation medium in Example 1 is that quercetin is added to the fermentation medium in step S21; other conditions and steps remain the same.

[0087] Comparative Example 3

[0088] Comparative Example 3 of the present invention provides a method for preparing a fermentation medium for Monascus purpureus extracellular polysaccharides. The difference between this method and the method for preparing the fermentation medium in Example 1 is that luteolin is added to the fermentation medium in step S21; other conditions and steps remain the same.

[0089] Comparative Example 4

[0090] Comparative Example 4 of the present invention provides a method for preparing a fermentation medium for Monascus purpureus extracellular polysaccharides. The difference between this method and the method for preparing the fermentation medium in Example 1 is that kaempferol is added to the fermentation medium in step S21; other conditions and steps remain the same.

[0091] Comparative Example 5

[0092] Comparative Example 5 of the present invention provides a method for preparing extracellular polysaccharide of Monascus purpureus, which differs from Example 1 in that genistein is not added to the fermentation medium in step S21, and EMP is prepared in step D33; other conditions and steps remain the same.

[0093] Property testing

[0094] 1. Comparison of mycelial and extracellular polysaccharide yields in the fermentation broths of Example 1 and Comparative Examples 1 to 4:

[0095] The fermentation broths from Example 1 and Comparative Examples 1 to 4 were used to detect the extracellular polysaccharide content and mycelial biomass; the results are as follows: Figure 2 As shown, the genistein fermentation broth prepared in Example 1 had the highest extracellular polysaccharide concentration, which was 1.57 g / L.

[0096] 2. Detection of the chemical composition of the Monascus purpureus extracellular polysaccharide G-EMP obtained in Example 2:

[0097] (1) Determination of neutral sugar content: phenol-sulfuric acid method;

[0098] (2) Determination of uronic acid content: carbazole method;

[0099] (3) Determination of protein content: Coomassie brilliant blue method;

[0100] (4) Determination of molecular weight: High performance liquid chromatography, including the following steps:

[0101] Standard solutions were prepared using T-series dextran with molecular weights of 10, 40, 50, 60, 70, 500, and 2000 kDa as standards.

[0102] Detection was performed by high-performance liquid chromatography (Agilent 1260) using an Ultrahydrogel column. TM -1000 (7.8mm×300mm). After the detection is completed, a linear regression equation is fitted, and the molecular weight is calculated based on the retention time of the sample peak.

[0103] (5) Determination of particle size and zeta potential: 1 mg / mg G-EMP solution was taken and measured using a potentiometer (ZS90, Malvern, UK) at 25℃; the results are as follows. Figure 3 As shown;

[0104] (6) Determination of monosaccharide composition: High performance anion exchange chromatography-pulse amperometric detection (HPAEC-PAD), including the following steps:

[0105] Add 5 mg of G-EMP to 0.5 mL of 12 mol / L H2SO4 and stir magnetically in an ice bath for 30 min.

[0106] After stirring, immediately add 2.5 mL of distilled water and react in an oil bath at 110 °C for 2.5 h;

[0107] After the reaction was completed, the sample was immediately cooled and diluted to a final concentration of 0.02 mg / mL. After filtration through a 0.22 μm aqueous membrane, the sample was injected into a sample vial for determination.

[0108] The measurement results are shown in Table 1:

[0109] Table 1. Detection results of chemical composition of Monascus purpureus extracellular polysaccharide G-EMP

[0110]

[0111] 3. Structural analysis of the Monascus purpureus extracellular polysaccharide G-EMP obtained in Example 2:

[0112] (1) Determination of nucleic acids and proteins:

[0113] The G-EMP prepared in Example 2 was formulated into a G-EMP solution with a concentration of 0.5 mg / mL. The UV-Vis absorption spectrum of G-EMP in the wavelength range of 200-800 nm was measured using a spectrophotometer (TU-1900, Pgenenal, Beijing, China). The results are as follows. Figure 4 As shown in A;

[0114] (2) Determination of functional groups:

[0115] 1 mg of G-EMP prepared in Example 2 was ground together with 100 mg of potassium bromide and pressed into transparent thin films; the films were analyzed using an FT-IR system (Nicolet 5700, Thermo Electron, USA), and the results are as follows. Figure 4 As shown in B;

[0116] (3) Determination of structure:

[0117] X-ray diffractometer (D8 ADVANCE, Bruker, Germany) was used at 25°C to determine whether the sample had a crystalline or amorphous structure. The parameters were set as follows: 2θ = 15-50°, counting time: 1 s per step, step size: 0.02°, Cu Ka radiation. The results are as follows. Figure 5 As shown in A;

[0118] (4) Thermal performance measurement:

[0119] 2 mg of G-EMP prepared in Example 2 was measured using a thermogravimetric analyzer (TGA-4000, PE Corporation, USA). The parameters were set as follows: heating in a N2 atmosphere at 30-600°C, with a flow rate of 20 mL / min. The results are as follows: Figure 5 As shown in B;

[0120] (5) Microstructure determination:

[0121] G-EMP was fixed to the sample stage with double-sided tape, and then gold was plated under vacuum ion current with a thickness of 10 nm.

[0122] The sample was magnified 1000 times, and the microstructure of G-EMP was observed using SEM (JEOLLtd, Japan). The results are as follows. Figure 6 As shown in A;

[0123] Using the same method, the microstructure of the EMP prepared in Comparative Example 5 was observed, and the results are as follows. Figure 6 As shown in B in the diagram.

[0124] (6) Determination of monosaccharide residue linkage:

[0125] Qualitative and quantitative analyses were performed on G-EMP in Example 2 and EMP in Comparative Example 5 using GC-MS, including the following steps:

[0126] Dissolve 2-3 mg of G-EMP sample in 500 μL of dimethyl sulfoxide, add 1 mg of sodium hydroxide, and incubate for 30 min.

[0127] After incubation, add 50 μL of iodomethane and react for 60 min.

[0128] After the reaction is complete, add 1 mL of water and 2 mL of dichloromethane, vortex for 30 seconds to mix, centrifuge for 10 minutes, discard the supernatant, and repeat the washing and centrifugation process 3 times.

[0129] After the final centrifugation, the lower layer of dichloromethane phase was removed and dried with nitrogen. 100 μL of 2 mol / L trifluoroacetic acid was added, and the mixture was hydrolyzed at 121 °C for 90 min.

[0130] After hydrolysis, the solution was evaporated to dryness at 30°C to remove residual trifluoroacetic acid; 50 μL of 2 mol / L ammonia solution and 50 μL of 1 mol / L sodium borodeuteride solution were added, mixed thoroughly, and reacted for 2.5 h.

[0131] After the reaction is complete, add 20 μL of acetic acid to terminate the reaction, blow dry with nitrogen, wash twice with 250 μL of methanol, and then blow dry with nitrogen.

[0132] After nitrogen blowing, add 250 μL of acetic anhydride, vortex for 30 seconds to mix, and then react at 100 °C for 2.5 h.

[0133] After the reaction is complete, add 1 mL of water and let stand for 10 min. Add 500 μL of dichloromethane, vortex for 30 s, mix well, centrifuge, discard the supernatant, and repeat the washing and centrifugation process 3 times.

[0134] After the final centrifugation, the lower layer of dichloromethane phase was removed and analyzed.

[0135] The results of G-EMP analysis are shown in Table 2; the results of EMP analysis are shown in Table 3.

[0136] Table 2 shows the results of monosaccharide residue linkage analysis in G-EMP.

[0137]

[0138] Table 3. Analysis results of monosaccharide residue linkage mode of EMP

[0139]

[0140] (7) Characterization of G-EMP and EMP by nuclear magnetic resonance (NMR):

[0141] The G-EMP obtained in Example 2 was dissolved in heavy water to prepare a G-EMP solution with a concentration of 40 mg / mL;

[0142] 500 μL was aspirated into an NMR tube and recorded in a 600 MHz NMR spectrometer (Brook, Germany) in one dimension. 1 H spectrum, 13 C-spectrum and two-dimensional COSY, HSQC, HMBC, and NOESY spectra;

[0143] The detection results of G-EMP are as follows Figure 7 As shown ( 1 The H NMR results are as follows Figure 7 As shown in A; 13 CNMR results are as follows Figure 7 As shown in B; 1 H- 1 H COSY spectrum as shown Figure 7 As shown in C; the NOESY spectrum is as follows. Figure 7 As shown in D; 1 H- 13 C-HSQC spectrum as shown Figure 7 As shown in E; the HMBC spectrum of G-EMP is shown in... Figure 7 (as shown in F in the diagram); the possible structure of G-EMP is as follows: Figure 8 As shown in the figure; the G-EMP characterization results are shown in Table 4;

[0144] EMP detection results are as follows Figure 9 As shown ( 1 The H NMR results are as follows Figure 9 As shown in A; 13 CNMR results are as follows Figure 9 As shown in B; 1 H- 1 H COSY spectrum as shown Figure 9 As shown in C; the NOESY spectrum is as follows. Figure 9 As shown in D; 1 H- 13 C-HSQC spectrum as shown Figure 9 As shown in E; the HMBC spectrum of EMP is shown in... Figure 9 (as shown in F); the possible structure of EMP is as follows Figure 10 As shown in the figure; the EMP characterization results are shown in Table 5;

[0145] Table 4 shows the carbon and hydrogen shifts of sugar residues in G-EMP.

[0146]

[0147] Table 5 shows the carbon and hydrogen shifts of sugar residues in EMP.

[0148]

[0149] Performance verification

[0150] This invention provides the application of Monascus purpureus extracellular polysaccharide G-EMP in inducing osteogenic differentiation of mesenchymal stem cells (MSCs), including four aspects: evaluation of G-EMP cytotoxicity to MSCs, detection of alkaline phosphatase (ALP) activity, detection of calcium ion content, and quantitative analysis of mineralized nodules.

[0151] 1. Evaluation of G-EMP's cytotoxicity against MSCs:

[0152] The potential cytotoxicity of G-EMP to MSCs was analyzed using the CCK-8 assay.

[0153] Mesenchymal stem cells (MSCs) derived from discarded human umbilical cords were added to basal medium (α-MEM) containing 10% fetal bovine serum and cultured at 37°C with 5% CO2. When the cells reached 80%-90% confluence, they were passaged.

[0154] 100 μL of MSCs were seeded into 96-well plates, with a cell density of 1 × 10⁶ cells per well. 4Cultured for 24 hours; wells without cell inoculation served as the control group;

[0155] After the culture was completed, the G-EMP prepared in Example 1 was added to the treatment group and diluted to different concentrations (25, 50, 100, 200 μg / mL) of G-EMP to incubate the MSCs.

[0156] In the control group, different concentrations of EMP prepared in Comparative Example 5 were added;

[0157] After incubation, the absorbance of cells at 450 nm at different concentrations of G-EMP was measured using a microplate reader (Thermo, USA); cell viability was calculated as follows:

[0158]

[0159] A 450 Treatment group: absorbance at 450 nm of cells, CCK-8 solution, and G-EMP solution;

[0160] A 450 Control group: absorbance at 450 nm of solutions containing cells, CCK-8 solution, and solutions without G-EMP;

[0161] A 450 Blank group: Absorbance at 450 nm of culture medium and CCK-8 solution containing no cells.

[0162] The results are as follows Figure 11 As shown in A; 0-200 μg / mL of G-EMP can maintain high cell viability, and 200 μg / mL of G-EMP was selected for subsequent detection.

[0163] 2. ALP activity assay:

[0164] MSCs were seeded in 12-well plates at a density of 2 × 10⁶ cells per well. 4 Once the cells have fused to 80-90%, switch to osteogenic induction medium containing G-EMP (190mL mesenchymal stem cell chondrogenic differentiation basal medium, 200μL sodium pyruvate, 2mL LITS, 2mL LTGF-β 3mL, 590μL ascorbic acid, 195μL proline, 19μL dexamethasone, 2mL penicillin and streptomycin).

[0165] Change the culture medium every 3 days and induce for 14 consecutive days. Discard the culture medium, wash the cells twice with 1×PBS, digest with trypsin and collect the cells, wash the cells with PBS, and add Western and IP lysis buffer (purchased from Shanghai Beyotime Biotechnology Co., Ltd.) to lyse the cells at a ratio of 75-150 μL of lysis buffer per well in a 12-well plate.

[0166] After lysis, the cells were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected. ALP activity in the cells was detected using an ALP detection kit purchased from Nanjing Jiancheng Biotechnology Co., Ltd., and the absorbance at 520 nm was measured. The results are as follows: Figure 11 As shown in B in the diagram.

[0167] 3. Calcium ion content detection:

[0168] MSCs were seeded in 12-well plates at a density of 2 × 10⁶ cells per well. 4 Once the cells have fused to 80%-90%, switch to osteogenic induction medium containing G-EMP.

[0169] Change the culture medium every 3 days and continue induction for 28 days. Discard the culture medium, wash the cells twice with 1×PBS, digest with trypsin and collect the cells, wash the cells with 1×PBS, and lyse the cells using Western lysis and IP lysis buffer.

[0170] After lysis, the cells were centrifuged at 12,000 × g for 10 min at 4 °C, and the supernatant was collected. The calcium ion content in the cells was detected using a calcium ion content detection kit, and the absorbance at 575 nm was measured. The results are as follows: Figure 11 As shown in C.

[0171] 4. Quantitative analysis of mineralized nodules:

[0172] MSCs were seeded in 12-well plates at a density of 2 × 10⁶ cells per well. 4 Once the cells have fused to 80%-90%, switch to osteogenic induction medium containing G-EMP.

[0173] Change the culture medium every 3 days and continue induction for 28 days; discard the culture medium, wash the cells twice with 1×PBS, add 4% paraformaldehyde to each well and fix for 10-15 min;

[0174] After fixation, wash three times with double-distilled water and stain with 0.2% Alizarin Red S solution for 30 min.

[0175] After staining, the staining solution was recovered, and the well plate was rinsed three times with double-distilled water. After drying, it was photographed and observed using an inverted phase-contrast microscope to calculate the number of mineralized nodules; the results are as follows. Figure 11 As shown in D in the diagram.

[0176] Results Analysis

[0177] See Figure 3 The Zavg potential is 386.05 ± 8.23 ​​nm. Figure 3 The A) and zeta potentials are -13.88 ± 0.46 mV. Figure 3 (B in the middle).

[0178] Referring to Table 1, the neutral sugar content was 8.41±0.26%, the uronic acid content was 0.56±0.05%, and the protein content was 1.03±0.11%. According to the obtained standard curve formula, logMw=-0.6438T+14.3496(R) 2 Calculated using (=0.996), the molecular weight of G-EMP is 56.4 kDa; and G-EMP is composed of 0.91±0.04% arabinose, 0.33±0.05% xylose, 37.26±0.39% mannose, 34.11±0.55% galactose and 27.39±0.89% glucose; among which mannose, galactose and glucose are the main monosaccharides in the composition of G-EMP, indicating that they mainly constitute the backbone of the red Monascus extracellular polysaccharide G-EMP prepared in this invention, which is closely related to its high biological activity.

[0179] See Figure 4 No obvious absorption peaks were detected at 260 nm and 280 nm for G-EMP, indicating that G-EMP... (0.1) It does not carry a large amount of nucleic acid or protein (<3%), such as Figure 4 As shown in A; the FT-IR spectrum of G-EMP at 4000 cm⁻¹ -1 -400cm -1 Similar polysaccharide absorption peaks were observed within the range, such as Figure 4 As shown in B; at 3481.63cm -1 The peak at 2911.89 cm⁻¹ is caused by the stretching vibration of the OH groups in the sugar molecule, while the peak at 2911.89 cm⁻¹ is caused by the stretching vibration of the OH groups in the sugar molecule. -1 The peak at 1745 cm⁻¹ is caused by the CH stretching vibration of the CH₂ and CH₃ groups; and, at 1745 cm⁻¹... -1 G-EMP (0.1) The absence of a significant peak indicates the absence of substantial aldehydes in G-EMP, consistent with the results in Table 1; furthermore, at 1631.09 cm⁻¹... -1 The nearby absorption peak belongs to the C=O stretching vibration, 1430.78 cm⁻¹. -1 The absorption peak at 1200–1000 cm⁻¹ represents the bending vibration of C=C; -1 The characteristic absorption peak nearby is due to the stretching vibration of pyranose; while 825.66 cm⁻¹ -1 and 917.63cm -1 The characteristic absorption peak at the location indicates the simultaneous presence of α- and β-configurations; therefore, the Monascus purpureus extracellular polysaccharide G-EMP provided by the present invention is a pyranose that simultaneously possesses α- and β-configurations.

[0180] See Figure 5G-EMP has a low overall crystallinity, and its X-ray diffraction (XRD) pattern contains the typical "bread-shaped" peaks of amorphous materials, such as... Figure 5 As shown in Figure A; a crystallization-related peak was observed at 2θ = 18.18, indicating that the Monascus purpureus extracellular polysaccharide G-EMP provided by this invention is a semi-crystalline polymer, such as... Figure 5 As shown in B in the diagram.

[0181] See Figure 6 The red Monascus extracellular polysaccharide G-EMP provided in Example 2 of this invention has a smooth surface with honeycomb-like pores. This structure may improve the water solubility of G-EMP, thereby enhancing its biological activity. In Comparative Example 5, the red Monascus extracellular polysaccharide EMP exhibits a relatively smooth surface, but a rough surface with many small pores.

[0182] See Table 2. 2,3,6-Tri-O-methylgalactitol may be 5-Gal(f) or 4-Gal(p). G-EMP identified 12 types of linkages, including t-Man(p) (13.41 mol%), t-Glc(p) (5.42 mol%), 3-Glc(p) (4.20 mol%), 2-Man(p) (17.61 mol%), 4-Man(p) (3.62 mol%), 6-Man(p) (6.18 mol%), and 4-Gal(p) (…). The content of 3,6-Glc (p) (31.87 mol%), 4-Glc (p) (3.68 mol%), 2,3-Man (p) (1.96 mol%), 2,4-Man (p) (1.48 mol%), 3,6-Glc (p) (1.13 mol%), and 2,6-Glcp (9.47 mol%) was relatively high, which is consistent with the results of monosaccharide analysis.

[0183] Referring to Table 3, ten types of linkages were identified in the Monascus purpureus extracellular polysaccharide EMP, which was rich in glycosyl sugar residues, including 2-Glc(p) (26.42 mol%), 6-Glcp (7.52 mol%), 4-Glcp (3.69 mol%), and 2,6-Glcp (8.02 mol%).

[0184] See Figure 7 And Table 4, Figure 7 The NMR results confirmed that 2,3,6-tri-O-methylgalactitol in Table 2 is 4-Gal(p) linked; Figure 7 As shown in A, G-EMP's 1The 1H NMR results showed four peaks in the anodic signal region (approximately 4.3–5.8 ppm), with values ​​of 5.16, 5.12, 5.07, and 4.95 ppm, indicating that G-EMP exists in both α- and β-configurations, which is consistent with the results of FT-IR analysis.

[0185] like Figure 7 As shown in B, G-EMP in 13 Four peaks were also identified in the anodic carbon region (95-110 ppm) of the C10 NMR spectrum, namely δ107.01, 107.25, 98.28 and 107.93 ppm; combined with two-dimensional... 1 H- 13 C HSQC spectrum ( Figure 7 The position results of the cross peaks in E) determined the assignment of the anodic region H-1 / C-1, namely δ5.16 / 107.01, δ5.12 / 107.25, δ5.07 / 98.28 and δ4.95 / 107.93ppm, and named sugar residues A, B, C and D, respectively.

[0186] By combining multiple two-dimensional NMR spectra, the carbon and hydrogen signal positions and structural correspondences of the four main residual sugars in the polysaccharide structure were sequentially assigned. The anomeric hydrogen of 5.16 ppm was determined to belong to the anomeric hydrogen H1 in the α-D-1,4-Gal residual sugar. This was further confirmed by identifying the correlation between adjacent hydrogen atoms. 1 H- 1 H COSY spectrum ( Figure 7 The signal associated with C) is 4.14 ppm, confirming the position of H2 at 4.14 ppm. Combined with the HSQC spectrum, the H2 / C2 signal is confirmed to be 4.14 / 76.19, thus determining the carbon spectral signal of the residual sugar at position 2 to be 76.19 ppm. Further analysis based on... 1 H- 1 Using HCl COSY information, a relevant signal of 4.14 / 3.59 was found, confirming that the 1H NMR position of H3 is 3.72 ppm. Combining this with HSQC, a value of 3.59 / 73.19 was found, determining the C3 NMR position to be 73.19 ppm. Following this method, the positions of H4 / C4, H5 / C5, and H6 / C6 were found to be 4.19 / 76.84, 3.63 / 66.54, and 3.61 / 60.11 ppm, respectively. Using the same method, the possible assignments of the four main residual sugars were determined, as shown in Table 2.

[0187] like Figure 7As shown in Figure D, in the NOESY spectrum of this polysaccharide, two correlation signals (5.16 / 4.02, 5.16 / .07) were found in the anodic region of the residual sugar α-D-1,4-Gal, confirming a long-range correlation between the anodic hydrogens of the residual sugar α-D-2,6-Glc (H6) and β-D-1,2-Man (H2). Similar correlation signals were also found in other major residual sugars, such as the correlation signals 5.12 / 4.02, 5.12 / 4.07 for α-Dt-Man, and the correlation signals 5.07 / 4.07 for α-D-2,6-Glc and 4.98 / 4.04 for β-D-1,2-Man.

[0188] like Figure 7 As shown in F, in the α-D-1,4-Gal remnants with higher content, anomeric hydrogen correlation signals were found in the HMBC spectrum: 5.17 / 75.55, 5.17 / 81.34, 5.18 / 83.21. Based on the above attribution, it was confirmed that the anomeric hydrogen of this remnant is remotely correlated with C2 in the same ring, C4 of the same residue, C6 of the α-D-2,6-Glc remnant, and C2 of β-D-1,2-Man. This further confirmed that the glycosidic bonds connecting C1→C4 of α-D-1,4-Gal and C1→C6 of the α-D-2,6-Glc remnant, and C1→C2 of the β-D-1,2-Man remnant are connected, which is consistent with the GC-MS results. Using the same method, the correlation signal 5.14 / 81.34 for the residual sugar α-Dt-Man was determined, confirming that the C1 position of this residual sugar is connected to the C4 position of α-D-1,4-Gal and the C2 position of the α-D-2,6-Glc residual sugar to form a glycosidic bond. The HMBC correlation signal 5.10 / 82.31 for the residual sugar α-D-2,6-Glc was found, confirming that H1 is correlated with the C6 position of the same residual sugar; the HMBC correlation signal 4.98 / 81.34 for the β-D-1,2-Man residual sugar was found, presumably connected to the C4 position of α-D-1,4-Gal and the C2 position of the α-D-2,6-Glc residual sugar to form a glycosidic bond.

[0189] See Figure 8 Based on the proportions in the monosaccharide composition results and the reference GC-MS results, it is inferred that the G-EMP main chain is mainly composed of →4)-α-D-1, 4-Galp-(1→, →6)-α-D-2, 6-Glcp-(1→ and →2)-β-D-1, 2-Manp-(1→, with α-D-Manp-(1→ linked at the O-2 positions of →6)-α-D-2, 6-Glcp-(1→, thus forming a side chain.

[0190] See Figure 9The NMR spectra of extracellular polysaccharides G-EMP and EMP are different. Therefore, the extracellular polysaccharide G-EMP prepared by adding genistein fermentation broth is a modified red Monascus extracellular polysaccharide. G-EMP and EMP have different compositions and structures.

[0191] See Figure 10 According to Table 5, EMP is formed by interconnecting →4)-β-D-Galp-(1→, →2)-β-D-Glcp-(1→) with a small amount of →6)-α-D-Glcp-(1→, →2,6)-α-D-Glcp-(1→, →4)-α-D-Manp-(1→) to form the main chain, and α-D-Manp-(1→) is connected to the O-2 position of →2,6)-α-D-Glcp-(1→ to form the side chain. It has a different main chain and side chain than G-EMP.

[0192] See Figure 11 ,like Figure 11 As shown in A, within the concentration range of 0-200 μg / mL, G-EMP had no effect on the proliferation of HUC-MSCs, and the cell viability in the G-EMP group was higher than that in the negative control group, which produced more EMP.

[0193] like Figure 11 As shown in B, ALP expression is relatively abundant in the early stage of osteogenic differentiation; the higher the ALP activity, the stronger the osteogenic differentiation ability of the cells and the more mature they are. With the increase of G-EMP concentration, ALP activity is greatly increased, and the ALP activity in the G-EMP group is higher than that in the EMP group. This indicates that G-EMP promotes early osteogenic differentiation of hUC-MSCs, and the addition of genistein fermentation improves the biological activity of Monascus purpureus extracellular polysaccharide.

[0194] like Figure 11 As shown in C, compared with the blank control group, the calcium ion content in the negative control group and the G-EMP group was significantly increased in a concentration-dependent manner; at the same time, the calcium ion content in the G-EMP group was higher than that in the negative control group, indicating that G-EMP can significantly promote osteogenic differentiation of hUC-MSCs.

[0195] During the late stages of osteogenic differentiation, calcium deposits form within cells, directly demonstrating successful osteogenic differentiation. Alizarin Red S staining solution is an anionic dye that can recognize calcium salt components in tissue cells, forming orange-red complexes with calcium ions, i.e., calcium nodules; the deeper the staining, the greater the amount of calcium salt deposited. Figure 11As shown in D, both the negative control group and the G-EMP group were conducive to the formation of calcified nodules. Compared with the blank control group, as the concentration of extracellular polysaccharide increased, the density of each concentration group of the negative control group and G-EMP increased accordingly. At the same time, the stained area increased significantly and the stained degree was also significantly enhanced. Moreover, as the concentration increased, the mineral nodules gradually became denser and the number also increased. Meanwhile, the effect of G-EMP was more obvious than that of the negative control group, proving that the extracellular polysaccharide G-EMP of Monascus purpureus provided by this invention has stronger activity than the extracellular polysaccharide produced without the addition of genistein fermentation broth.

[0196] While embodiments of the present invention have been described in detail above, it will be apparent to those skilled in the art that various modifications and variations can be made to these embodiments. However, it should be understood that such modifications and variations fall within the scope and spirit of the invention as set forth in the claims. Furthermore, the invention described herein may have other embodiments and can be implemented or carried out in various ways.

Claims

1. A modified Monascus purpureus extracellular polysaccharide G-EMP, characterized in that, The main chain of the repeating structure of the extracellular polysaccharide G-EMP unit includes →4)-α-D-1,4-Gal p -(1→、→6)-α-D-2,6-Glc p -(1→、→2)-β-D-1,2-Man p -(1→;The branched chains in the extracellular polysaccharide G-EMP are composed of α-D-Man p -(1→connected to→6)-α-D-2,6-Glc p -(1→ is formed at the O-2 position; the preparation method of the modified extracellular polysaccharide G-EMP includes the following steps: S1. Activate and culture Monascus purpureus to obtain seed culture; the Monascus purpureus is Monascus purpureus var. purple. Monascus purpureus 40269; S2. The seed liquid is added to a fermentation culture medium to obtain a fermentation broth; the fermentation culture medium includes genistein. S3. Centrifuge and concentrate the fermentation broth. After concentration, add anhydrous ethanol to obtain crude extracellular polysaccharide precipitate. S4. Add protease solution to the precipitate for enzymatic hydrolysis. After enzymatic hydrolysis, remove the protein using the Sevage method to obtain crude extracellular polysaccharide. S5. The crude extracellular polysaccharide is separated and purified to obtain Monascus purpureus extracellular polysaccharide G-EMP; the specific steps of separation and purification are as follows: D1. The crude extracellular polysaccharide was prepared into a 10 mg / mL solution and further purified using a DEAE-Cellulose 52 ion exchange column. Elution was optimized using 0, 0.1, 0.2, 0.3, 0.4, and 0.5 mol / L NaCl, respectively. D2. Collect the eluent from step D1 in separate tubes and monitor it using the phenol-sulfuric acid method. When the presence or absence of sugar components is detected, stop collecting and freeze-dry the collected eluent. D3. The elution group with the highest yield and total sugar content after freeze-drying and the highest 0.1 mol / L NaCl was prepared into a solution, further purified using a Sepharose gel column, and eluted with purified water to obtain the Monascus purpureus extracellular polysaccharide G-EMP.

2. The Monascus purpureus extracellular polysaccharide G-EMP according to claim 1, characterized in that, The extracellular polysaccharide G-EMP is a pyranose that exists in both α- and β-configurations.

3. The Monascus purpureus extracellular polysaccharide G-EMP according to claim 1, characterized in that, The extracellular polysaccharide G-EMP is composed of arabinose, xylose, mannose, galactose, and glucose.

4. The Monascus purpureus extracellular polysaccharide G-EMP according to claim 1, characterized in that, The extracellular polysaccharide G-EMP includes 12 linkage types, including t-Man(p), t-Glc(p), 3-Glc(p), 2-Man(p), 4-Man(p), 6-Man(p), 4-Gal(p), 4-Glc(p), 2,3-Man(p), 2,4-Man(p), 3,6-Glc(p), and 2,6-Glcp.

5. The use of the Monascus purpureus extracellular polysaccharide G-EMP as described in any one of claims 1-4 in the preparation of a medicament for treating osteoporosis.

6. The application according to claim 5, characterized in that, In this application, the extracellular polysaccharide G-EMP can induce osteogenic differentiation of mesenchymal stem cells.

7. The application according to claim 6, characterized in that, In this application, the extracellular polysaccharide G-EMP can enhance the activity of alkaline phosphatase in mesenchymal stem cells.

8. The application according to claim 6, characterized in that, In this application, the extracellular polysaccharide G-EMP can increase the calcium ion concentration in mesenchymal stem cells.

Images