Konjac glucomannan, application of derivative of konjac glucomannan, glucomannan octoate, nano-particles of glucomannan octoate, coating of glucomannan octoate, preparation method of glucomannan

Abstract

The invention discloses application of konjac glucomannan and derivatives thereof, glucomannan octoate, nano-particles of glucomannan octoate, a coating of glucomannan octoate, a preparation method of glucomannan octoate and application of glucomannan octoate, and belongs to the technical field of medicine and material chemistry. Konjac glucomannan and derivatives thereof can be used as an extracellular matrix filler, and glucomannan octoate obtained by modifying the extracellular matrix filler through an esterification reaction, and nanoparticles or a glucomannan octoate coating further obtained from the glucomannan octoate can be used for preparing drugs or materials for regulating nucleus pulposus cell functions. Particularly, the compound can be used for preparing medicines or materials for relieving or treating intervertebral disc degeneration. The glucomannan octoate has the characteristics of no toxicity, easiness in obtaining, high histocompatibility and the like, and can be prepared into different dosage forms according to requirements. The invention provides a new thought for improving the intervertebral disc degeneration microenvironment, and also provides a new application of a polysaccharide derivative-based biological material.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority of Chinese patent application No. 202311098026.X filed with the China Patent Office on August 29, 2023, and entitled “Application of konjac glucomannan and its derivatives, glucomannan caprylate and its nanoparticles, coating and its preparation method and application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The invention relates to the technical field of medicine and material chemistry, and in particular to applications of konjac glucomannan and derivatives thereof, glucomannan caprylate and nanoparticles thereof, a coating and a preparation method and application thereof. Background Art

[0004] Lumbar disc degeneration is one of the most important causes of low back pain. Epidemiological surveys show that about 80% of adults have a history of lower back pain more than once. As a motor system disease closely related to age and exercise, with the rapid development of the aging population and the popularization of national sports, more and more individuals will face the troubles of disc degeneration and corresponding nerve compression symptoms. The typical characteristics of disc degeneration are the progressive degeneration of disc tissue, especially nucleus pulposus tissue. At present, drugs and physical therapy can only improve the symptoms of some patients, but cannot effectively intervene in the process of tissue degeneration. Most patients can only choose surgical treatment as the final treatment. However, the instability of local mechanical structure caused by postoperative tissue loss, the continuous degeneration of the remaining degenerated nucleus pulposus tissue, and the postoperative microenvironment disorder are important reasons for aggravating tissue degeneration and causing re-protrusion and re-surgery. How to effectively regulate the postoperative degenerative microenvironment, prevent recurrence, and promote the repair of nucleus pulposus tissue is the current mainstream accelerated rehabilitation strategy.

[0005] Pathological studies have found that when the intervertebral disc degenerates, the extracellular matrix (ECM) of the nucleus pulposus tissue will accelerate degradation, the number of nucleus pulposus cells will decrease, and the phenotype will begin to be lost. Therefore, in clinical practice, typical changes such as weakened nucleus pulposus MR T2 signal and reduced intervertebral disc height can be observed. Basic research further reveals that as degeneration progresses, matrix decomposition-related enzymes (such as MMP2, HYAL2, etc.) in the nucleus pulposus tissue gradually become highly expressed, while the content of ECM components such as collagen and hyaluronic acid will gradually decrease. In view of the changing characteristics of ECM, researchers have proposed the use of injectable and filling materials to fill the defects after intervertebral disc surgery in order to maintain height, improve the degenerative microenvironment, and improve treatment efficiency, but there are currently no reports of effective related materials.

[0006] In view of this, the present invention is proposed. Summary of the invention

[0007] The purpose of the present invention is to provide an application of konjac glucomannan and its derivatives, glucomannan caprylate and its nanoparticles, coating and its preparation method and application to solve the above technical problems.

[0008] This application can be implemented as follows:

[0009] In a first aspect, the present application provides a use of konjac glucomannan and its derivatives as extracellular matrix fillers.

[0010] In a second aspect, the present application provides a glucomannan caprylate, which is obtained by modifying konjac glucomannan through an esterification reaction.

[0011] In a third aspect, the present application provides a method for synthesizing glucomannan caprylate as described in the aforementioned embodiment, comprising the following steps: modifying konjac glucomannan with a caprylyl group.

[0012] In an optional embodiment, konjac glucomannan is reacted with trifluoroacetic anhydride and caprylic acid, the reaction solution obtained by the reaction is subjected to solid-liquid separation, the liquid phase is collected for a first alcohol precipitation, the first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the second precipitate obtained is collected.

[0013] In an optional embodiment, konjac glucomannan is added to the first reaction solution obtained by reacting trifluoroacetic anhydride with octanoic acid, and the reaction is continued to obtain a second reaction solution; the second reaction solution is separated into solid and liquid, the liquid phase is collected for a first alcohol precipitation, the first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove the product insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the obtained second precipitate is collected;

[0014] In an alternative embodiment, the reaction of trifluoroacetic anhydride and octanoic acid is carried out at 50-55° C. for 15-25 min.

[0015] In an optional embodiment, the reaction of konjac glucomannan with the first reaction solution is carried out at 50-55° C. for 3-4 hours.

[0016] In an optional embodiment, the usage ratio of konjac glucomannan, trifluoroacetic anhydride and caprylic acid is 500 mg:(8-12) mL:(12-14) mL.

[0017] In a fourth aspect, the present application provides a nanoparticle obtained by nano-processing the glucomannan caprylate according to the aforementioned embodiment.

[0018] In a fifth aspect, the present application provides a method for preparing nanoparticles as described in the aforementioned embodiment, comprising the following steps: subjecting glucomannan caprylate to nano-processing.

[0019] In an optional embodiment, glucomannan caprylate is subjected to ultrasonic nano-processing in the presence of polyvinyl alcohol, and the organic solvent is removed and the product is dried.

[0020] In a sixth aspect, the present application provides a glucomannan caprylate coating, which is obtained by coating the glucomannan caprylate of the aforementioned embodiment.

[0021] In a seventh aspect, the present application provides a use of glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment in the preparation of a drug or material for regulating the function of nucleus pulposus cells.

[0022] In an optional embodiment, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare drugs or materials for regulating the adhesion and physiological functions of nucleus pulposus cells.

[0023] In an alternative embodiment, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare a drug or material for protecting the phenotype of nucleus pulposus cells by intervening in the interactions between cells-matrix and cells-cells.

[0024] In an alternative embodiment, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare a drug or material that highly expresses a nucleus pulposus phenotype.

[0025] In an optional embodiment, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare a drug or material enriched in milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype includes Collagen2.

[0026] In an eighth aspect, the present application provides a use of glucomannan caprylate according to the aforementioned embodiment, or the nanoparticles according to the aforementioned embodiment, or the glucomannan caprylate coating according to the aforementioned embodiment in the preparation of a drug or material for alleviating or treating intervertebral disc degeneration.

[0027] In an optional embodiment, glucomannan caprylate or nanoparticles or glucomannan caprylate coating is used to prepare a drug for delaying water loss in nucleus pulposus tissue, or for preparing a drug for reducing the reduction of intervertebral disc height.

[0028] In a ninth aspect, the present application provides a drug, the active ingredient of which includes the glucomannan caprylate of the aforementioned embodiment or the nanoparticles of the aforementioned embodiment or the glucomannan caprylate coating of the aforementioned embodiment.

[0029] In a tenth aspect, the present invention provides a repair material, wherein the active ingredient of the repair material includes the glucomannan caprylate of the aforementioned embodiment or includes the nanoparticles of the aforementioned embodiment or the glucomannan caprylate coating of the aforementioned embodiment.

[0030] The beneficial effects of this application include:

[0031] The konjac glucomannan and its derivatives provided in the present application can be used as extracellular matrix fillers, and the glucomannan caprylate obtained by modification of the esterification reaction, as well as the nanoparticles or glucomannan caprylate coating further obtained from the glucomannan caprylate, can be used to prepare drugs or materials for regulating the function of nucleus pulposus cells, especially for preparing drugs or materials for relieving or treating intervertebral disc degeneration. The above-mentioned glucomannan caprylate has the characteristics of being non-toxic, easy to obtain, and having high tissue compatibility, and can be prepared into different dosage forms according to needs. The present application provides a new idea for improving the microenvironment of intervertebral disc degeneration, and also provides a new use of biomaterials based on polysaccharide derivatives. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0033] Figure 1 This is the mass spectrum of the enzymatic hydrolysis product of hyaluronic acid in Example 1;

[0034] Figure 2 The mass spectrum of the enzymatic hydrolysis product of KGM in Example 1;

[0035] Figure 3 This is a comparison diagram of infrared spectra of KGM and GMOC in Example 2;

[0036] Figure 4 This is a comparison of the nuclear magnetic resonance images of KGM and GMOC in Example 2;

[0037] Figure 5 This is a diagram showing the cell morphology changes after GMOC and nucleus pulposus cells were co-cultured in Example 3;

[0038] Figure 6 This is the result diagram of GMOC regulating cell phenotype in Example 3;

[0039] Figure 7 This is a diagram showing the enrichment of Mfge8 protein by GMOC in Example 3;

[0040] Figure 8 This is the result of the nuclear magnetic resonance imaging in Example 4;

[0041] Fig. 9 This is the X-ray imaging result diagram in Example 4. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0043] The following is a detailed description of the application of konjac glucomannan and its derivatives, glucomannan caprylate and its nanoparticles, coatings and their preparation methods and applications provided in the present application.

[0044] The present application proposes the use of konjac glucomannan (KGM) and its derivatives as extracellular matrix fillers.

[0045] The rich and varied molecular configurations endow polysaccharides with great potential in tissue repair. Konjac glucomannan, as a water-soluble high molecular weight polysaccharide, is a polymer of glucose and mannan connected by β-1,4-glycosidic bonds in konjac tubers. In addition, it can exert specific biological effects by modifying the polysaccharide molecules and giving them additional functional groups. For example, multiple octanoyl groups can be modified on konjac glucomannan through esterification reactions to regulate cell functions.

[0046] At the same time, konjac glucomannan has excellent anti-enzymatic degradation properties and can remain stably in the local area for a long time to replenish the ECM lost due to degeneration. Ultimately, it achieves the therapeutic goal of delaying disc degeneration by improving the microenvironment of disc degeneration tissue.

[0047] The present application prepares the filling material by selecting konjac glucomannan (KGM) and its derivatives composed of non-nucleus pulposus ECM, which can effectively resist degradation by common matrix-degrading enzymes in situ and achieve long-term retention of the filling material in the defect site.

[0048] In addition, the present application also provides a glucomannan caprylate, which is obtained by modifying konjac glucomannan through an esterification reaction.

[0049] Accordingly, the synthesis method of the above-mentioned glucomannan caprylate may include the following steps: modifying konjac glucomannan with caprylyl groups.

[0050] In some embodiments, konjac glucomannan is reacted with trifluoroacetic anhydride (TFAA) and caprylic acid, the reaction solution obtained by the reaction is subjected to solid-liquid separation, the liquid phase is collected for a first alcohol precipitation, the first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove the product insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, the second precipitate obtained is collected, and the second precipitate can be used as glucomannan caprylate.

[0051] The reaction equation involved in the above process is as follows:

[0052]

[0053] The trifluoroacetic anhydride is mainly used as a catalyst in the synthesis reaction process to efficiently produce the target product. The use of octanoic acid can ensure the simplicity, stability and feasibility of the reaction and can avoid the generation of by-products.

[0054] It should be noted that the product obtained by the first alcohol precipitation is glucomannan caprylate containing more impurities, and a relatively pure product can be obtained by purifying the product again.

[0055] In some specific embodiments, konjac glucomannan can be added to the first reaction solution obtained by the reaction of trifluoroacetic anhydride and caprylic acid to continue the reaction to obtain a second reaction solution; the second reaction solution is separated into solid and liquid, the liquid phase is collected for a first alcohol precipitation, the first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove the product insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation to collect the obtained second precipitate.

[0056] For reference, the reaction of trifluoroacetic anhydride and octanoic acid can be carried out at 50-55°C (such as 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, etc.) for 15-25 min (such as 15 min, 18 min, 20 min, 22 min or 25 min, etc.). In some embodiments, the reaction of trifluoroacetic anhydride and octanoic acid is carried out at 50°C for 20 min.

[0057] The reaction of konjac glucomannan with the first reaction solution can be carried out at 50-55° C. (such as 50° C., 51° C., 52° C., 53° C., 54° C. or 55° C.) for 3-4 h (such as 3 h, 3.2 h, 3.5 h, 3.8 h or 4 h, etc.). In some embodiments, the reaction of konjac glucomannan with the first reaction solution is carried out at 50° C. for 3.5 h.

[0058] The amount ratio of konjac glucomannan, trifluoroacetic anhydride and caprylic acid can be 500 mg: (8-12) mL: (12-14) mL, such as 500 mg: 8 mL: 12 mL, 500 mg: 8 mL: 13 mL, 500 mg: 8 mL: 14 mL, 500 mg: 10 mL: 12 mL, 500 mg: 10 mL: 13 mL, 500 mg: 10 mL: 14 mL, 500 mg: 12 mL: 12 mL, 500 mg: 12 mL: 13 mL, 500 mg: 12 mL: 14 mL, etc. In some embodiments, the amount ratio of konjac glucomannan, trifluoroacetic anhydride and caprylic acid is 500 mg: 10 mL: 13 mL.

[0059] The alcohol used in the above-mentioned alcohol precipitation processes can be ethanol, for example but not limited thereto, and its amount can be 3-5 times the volume of the solution to be treated.

[0060] Furthermore, the collected second precipitate can be vacuum dried.

[0061] It should be noted that in other embodiments, the above reaction conditions can be appropriately adjusted as needed. In addition, the second precipitate can be further dissolved in chloroform, and the liquid phase dissolved in chloroform can be subjected to a third alcohol precipitation. In other words, the number of alcohol precipitations can be specifically adjusted as needed.

[0062] Based on the above-mentioned glucomannan caprylate, the present application also provides a nanoparticle, which is obtained by nano-processing the above-mentioned glucomannan caprylate.

[0063] For reference, the method for preparing the nanoparticles may include the following steps: subjecting glucomannan caprylate to nano-processing.

[0064] In some embodiments, glucomannan caprylate may be subjected to ultrasonic nano-processing in the presence of polyvinyl alcohol (PVA), and the nanoparticles may be collected, and then the organic solvent may be further removed and dried.

[0065] Specifically, glucomannan caprylate can be first dissolved in chloroform to obtain a glucomannan caprylate solution, and then the glucomannan caprylate solution is added dropwise to a polyvinyl alcohol solution with a concentration of 4-6% at a volume ratio of 1:8-12, and ultrasonically crushed at 15-20 Hz for 2-4 minutes. Excess PVA is added and stirred overnight, and the organic solvent is removed by rotary evaporation, and finally, the nanoparticles are obtained by freeze drying.

[0066] The above-mentioned PVA can utilize its surfactant properties to promote the nanoparticles generated by ultrasound to be more evenly dispersed in the solution, maintain stability, and avoid the fusion or rupture of the nanoparticles.

[0067] It should be noted that the methods and conditions for nano-processing are not limited to those listed above, and similar processing can also be performed by referring to relevant existing technologies.

[0068] Based on the above-mentioned glucomannan caprylate, the present application also provides a glucomannan caprylate coating, which is obtained by coating the above-mentioned glucomannan caprylate.

[0069] It should be noted that conventional methods can be used for coating substrates and coating methods, and no further limitations are given herein.

[0070] In addition, the above-mentioned glucomannan caprylate can also be prepared into different dosage forms according to needs, for example, capsules, soft capsules, powders, granules, tablets, pills, honey pastes, oral liquids, wine preparations or injections, etc.

[0071] Furthermore, the present application also provides the use of the above-mentioned glucomannan caprylate or nanoparticles containing glucomannan caprylate or glucomannan caprylate coating in the preparation of drugs or materials for regulating the function of nucleus pulposus cells.

[0072] In some embodiments, the above-mentioned glucomannan caprylate or nanoparticles or glucomannan caprylate coating can be used to prepare drugs or materials for regulating the adhesion and physiological functions of nucleus pulposus cells.

[0073] In some embodiments, the above-mentioned glucomannan caprylate or nanoparticles or glucomannan caprylate coating can be used to prepare drugs or materials for protecting the phenotype of nucleus pulposus cells by intervening in the interactions between cells-matrix and cells-cells.

[0074] In some embodiments, the above-mentioned glucomannan caprylate or nanoparticles or glucomannan caprylate coating can be used to prepare drugs or materials that highly express nucleus pulposus phenotype.

[0075] In some embodiments, the above-mentioned glucomannan caprylate or nanoparticles or glucomannan caprylate coating can be used to prepare drugs or materials enriched in milk fat globule epidermal growth factor 8 (Mfge8 protein) secreted by nucleus pulposus cells; wherein the nucleus pulposus phenotype can exemplarily but not limitatively include Collagen2, etc.

[0076] In addition, the present application also provides the use of the above-mentioned glucomannan caprylate or nanoparticles containing glucomannan caprylate or glucomannan caprylate coating in the preparation of drugs or materials for relieving or treating intervertebral disc degeneration.

[0077] In some embodiments, the above-mentioned glucomannan octanoate or nanoparticles or glucomannan octanoate coating can be used to prepare a drug for delaying water loss in nucleus pulposus tissue. In other embodiments, the above-mentioned glucomannan octanoate or nanoparticles or glucomannan octanoate coating can be used to prepare a drug for reducing the reduction of intervertebral disc height.

[0078] Correspondingly, the present application also provides a drug, the active ingredient of which includes the above-mentioned glucomannan caprylate or the above-mentioned nanoparticles or the above-mentioned glucomannan caprylate coating.

[0079] Similarly, the present application also provides a repair material, the active ingredients of which include the above-mentioned glucomannan caprylate or the above-mentioned nanoparticles or the above-mentioned glucomannan caprylate coating.

[0080] It should be noted that in the prior art, supplementing the intervertebral disc with exogenous collagen or hyaluronic acid can increase tissue water content and improve tissue microenvironment in the short term. However, the matrix decomposing enzymes expressed in large quantities will further decompose the supplemented exogenous ECM, which may lead to repair failure.

[0081] In the cell-cell and cell-ECM interactions, Mfge8 is secreted by cells as a "bridge protein" and expressed in the cell membrane and ECM, participating in mediating cell recognition and adhesion behavior, thereby further regulating physiological functions. The present application adds an octanoyl group to konjac glucomannan through an esterification reaction, and the modified product exhibits an extremely high Mfge8 protein enrichment ability. By preparing glucomannan caprylate coatings and nanoparticles, they are contacted with nucleus pulposus cells in vitro and in vivo, respectively, and the changes in nucleus pulposus cell function are mediated by enriching Mfge8 protein. At the same time, glucomannan caprylate material has excellent resistance to enzyme degradation properties and can remain stably in the local area for a long time to supplement the ECM lost due to degeneration. Ultimately, the therapeutic goal of delaying disc degeneration is achieved by improving the microenvironment of disc degeneration tissue.

[0082] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0083] Example 1

[0084] Verify the anti-enzymatic performance of KGM

[0085] 50 mg of KGM and hyaluronic acid (molecular weight 180K) were added to 10 mL of buffer solution (PBS saline solution, pH = 5.3), stirred and fully dissolved, and then 2 mg of hyaluronidase was added, and the reaction was fully reacted at 37 ° C for 6 hours, and then the temperature was raised to 90 ° C to inactivate the enzyme for 5 minutes to end the reaction. After cooling, the sample was filtered through a 0.22 μm filter to remove insoluble particles. Mass spectrometry was used to detect whether there were oligosaccharide products after enzymatic hydrolysis in the reaction solution to verify the anti-enzymatic ability of the substrate.

[0086] The mass spectrometry results are as follows Figure 1 and Figure 2 As shown, Figure 1 The hyaluronic acid in the sample was used as the control group. After enzymatic hydrolysis, a large number of oligosaccharide peaks appeared, indicating that it was enzymatically hydrolyzed. Figure 2 The absence of significant oligosaccharide peaks in KGM indicated that it had good resistance to enzymatic hydrolysis.

[0087] Example 2

[0088] Synthesis of GMOC by esterification reaction

[0089] Weigh 13.2 mL of octanoic acid and slowly add it dropwise to 10 mL of TFAA, react in an oil bath at 50 ° C for 20 min to obtain the first reaction solution. Add 500 mg of KGM to the first reaction solution and react for 3.5 h to obtain the second reaction solution. Centrifuge the second reaction solution, take the supernatant and dropwise add it to a four-fold volume of ethanol solution for the first alcohol precipitation, centrifuge and collect the first precipitate, further dissolve it in chloroform, remove the insoluble product, and then precipitate the liquid phase dissolved in chloroform for the second alcohol precipitation, collect the second precipitate, that is, obtain the modified reaction product GMOC, and vacuum dry it for use.

[0090] 1 mg of the obtained GMOC was characterized by infrared spectroscopy and nuclear magnetic resonance, and the modification was successful. Figure 3 and Figure 4 As shown, it is calculated that by controlling the reaction time, a GMOC product with a substitution rate of about 1.77 can be stably obtained.

[0091] Example 3

[0092] Verify the role of GMOC in regulating nucleus pulposus cell function

[0093] The GMOC prepared in Example 2 was coated on the surface of the slide and co-cultured with the nucleus pulposus cells. The expression of the nucleus pulposus cell phenotype and the changes in cell morphology were observed by cell immunofluorescence staining. The results are as follows: Figure 5 As shown in the figure, it can be seen that the nucleus pulposus cells show significant colony growth on GMOC and can highly express nucleus pulposus phenotypes such as collagen2. At the same time, GMOC can also upregulate the high expression of genes such as collagen2 in nucleus pulposus cells (such as Figure 6 ).

[0094] Further proteomic analysis suggested that the GMOC coating could significantly adsorb the Mfge8 protein secreted by nucleus pulposus cells and in subsequent validation experiments (such as Figure 7 ) was confirmed.

[0095] Among them, proteomics detection refers to the use of 4D-label free technology to detect the types of nucleus pulposus cell membrane proteins specifically adsorbed by GMOC. The steps are: after co-culturing the nucleus pulposus cell membrane with GMOC for 24 hours, PBS is repeatedly washed three times, and the proteins adsorbed on the surface of GMOC are digested into peptides using pancreatic enzymes, and then desalted and tested on the machine.

[0096] Figure 7The corresponding experiment is as follows: Nucleus pulposus cells were planted on the surface of Glass and GMOC materials and cultured for 24 hours. Cells adhering to the surface were removed using EDTA. PBS was washed three times, and Glass and GMOC materials were incubated with mfge8 primary antibody at 4 degrees overnight, and washed three times with PBS. HRP-labeled secondary antibody was added, incubated at room temperature for 1 hour, and washed three times with PBS. ECL chemiluminescent solution was added, and the material was exposed to light under the instrument to observe the adsorption of mfge8 protein on the surface of the material.

[0097] Figure 6 and Figure 7 In the figure, Control represents a conventionally used cell culture plate; Glass represents a cell culture plate, i.e., a glass slide; GMOC represents the glucomannan caprylate coating provided in the present application, which is coated on the surface of the cell culture plate.

[0098] Example 4

[0099] Animal model verifies the therapeutic effect of GMOC in alleviating intervertebral disc degeneration

[0100] 8-week-old SD female rats weighing 250 g were selected. After anesthesia, a 21G needle was used to puncture the intervertebral space halfway to establish an intervertebral disc degeneration model (IDD). A microsyringe was used to inject 10 μL of a 5% GMOC nanoparticle PBS solution into the defect site in situ as the treatment group, where the GMOC was prepared by Example 2. The segments injected with the same amount of PBS were used as the control group. 3.0t nuclear magnetic resonance and X-ray were used to detect the water content of the nucleus pulposus tissue and the height of the intervertebral space after 3 and 7 days of treatment, respectively, to evaluate the therapeutic effect of GMOC. The results are shown in Figure 2. Figure 8 and Fig. 9 shown.

[0101] Among them, control is the healthy control group, that is, the control group without any treatment; IDD group is the degeneration group, that is, only puncture is performed without any treatment; PBS group is injected with PBS solution after puncture; GMOC group is injected with GMOC solution after puncture.

[0102] Depend on Figure 8 and Fig. 9 It can be seen that compared with the IDD group, GMOC treatment can significantly delay the water loss of nucleus pulposus tissue and reduce the degree of reduction in intervertebral disc height.

[0103] In summary, the sugar-like biomaterial provided by the present application is obtained by modifying konjac glucomannan (KGM) through esterification reaction to obtain glucomannan octanoate (GMOC), which can regulate the adhesion and physiological function of nucleus pulposus cells by enriching milk fat globule epidermal growth factor 8 (Mfge8), and further protect the phenotype of nucleus pulposus cells by intervening in cell-matrix and cell-cell interactions, thereby improving the microenvironment of intervertebral disc degeneration. This sugar derivative material (GMOC) has the characteristics of being non-toxic, easy to obtain, and having high tissue compatibility, and can be prepared into different dosage forms according to needs. On this basis, the present application provides a new idea for improving the microenvironment of intervertebral disc degeneration, as well as a new use of biomaterials based on the above-mentioned polysaccharide derivatives.

[0104] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of konjac glucomannan and its derivatives as extracellular matrix fillers.

2. A glucomannan caprylate, characterized in that The glucomannan caprylate is obtained by modifying konjac glucomannan through an esterification reaction.

3. A method for synthesizing glucomannan caprylate as claimed in claim 2, characterized in that: The following steps are involved: The konjac glucomannan is modified with an octanoyl group; Preferably, konjac glucomannan is reacted with trifluoroacetic anhydride and octanoic acid, the reaction solution obtained by the reaction is subjected to solid-liquid separation, the liquid phase is collected for a first alcohol precipitation, a first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove products insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the obtained second precipitate is collected; More preferably, konjac glucomannan is added to the first reaction solution obtained by reacting trifluoroacetic anhydride with octanoic acid, and the reaction is continued to obtain a second reaction solution; the second reaction solution is separated into solid and liquid, the liquid phase is collected for a first alcohol precipitation, the first precipitate obtained by the first alcohol precipitation is dissolved in chloroform to remove the product insoluble in chloroform, and then the liquid phase dissolved in chloroform is subjected to a second alcohol precipitation, and the obtained second precipitate is collected; Preferably, the reaction of the trifluoroacetic anhydride and the octanoic acid is carried out at 50-55° C. for 15-25 min; Preferably, the reaction of the konjac glucomannan with the first reaction solution is carried out at 50-55° C. for 3-4 hours; Preferably, the usage ratio of the konjac glucomannan, the trifluoroacetic anhydride and the caprylic acid is 500 mg:(8-12) mL:(12-14) mL.

4. A nanoparticle, characterized in that: The nanoparticles are obtained by subjecting the glucomannan caprylate described in claim 2 to nano-processing.

5. A method for preparing nanoparticles according to claim 4, characterized in that: The following steps are involved: The glucomannan caprylate is subjected to nano-processing; Preferably, the glucomannan caprylate is subjected to ultrasonic nano-processing in the presence of polyvinyl alcohol, the organic solvent is removed, and the product is dried.

6. A glucomannan caprylate coating, characterized in that: The glucomannan caprylate coating is obtained by coating the glucomannan caprylate according to claim 2.

7. Use of the glucomannan caprylate according to claim 2, the nanoparticles according to claim 4, or the glucomannan caprylate coating according to claim 6 in the preparation of a drug or material for regulating the function of nucleus pulposus cells; Preferably, the glucomannan caprylate or the nanoparticles or the glucomannan caprylate coating is used to prepare a drug or material for regulating the adhesion and physiological function of nucleus pulposus cells; Preferably, the glucomannan caprylate or the nanoparticle or the glucomannan caprylate coating is used to prepare a drug or material for protecting the phenotype of nucleus pulposus cells by intervening in the interaction between cells-matrix and cells-cells; Preferably, the glucomannan caprylate or the nanoparticles or the glucomannan caprylate coating is used to prepare a drug or material that highly expresses a nucleus pulposus phenotype; Preferably, the glucomannan caprylate or the nanoparticles or the glucomannan caprylate coating is used to prepare a drug or material enriched with milk fat globule epidermal growth factor 8 secreted by nucleus pulposus cells; wherein, The nucleus pulposus phenotype includes Collagen2.

8. Use of the glucomannan caprylate according to claim 2, the nanoparticles according to claim 4, or the glucomannan caprylate coating according to claim 6 in the preparation of a drug or material for alleviating or treating intervertebral disc degeneration; Preferably, the glucomannan caprylate or the nanoparticles or the glucomannan caprylate coating is used to prepare a drug for delaying water loss in nucleus pulposus tissue, or for preparing a drug for alleviating the reduction in intervertebral disc height.

9. A drug, characterized in that The active ingredient of the drug includes the glucomannan caprylate according to claim 2 or the nanoparticles according to claim 4 or the glucomannan caprylate coating according to claim 6.

10. A repair material, characterized in that: The active ingredient of the repair material includes the glucomannan octanoate according to claim 2 or the nanoparticles according to claim 4 or the glucomannan octanoate coating according to claim 6.

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