Composition as well as preparation method and application thereof

Abstract

The invention belongs to the field of microbial medicines, and more specifically relates to a composition, and a preparation method and application thereof. According to the invention, mannuronic acid disaccharide, guluronic acid disaccharide and aceran A are combined according to a certain proportion, so that the composition with the capabilities of inhibiting A beta aggregation and reducing ROS (reactive oxygen species) generated by catalysis of an A beta-Cu complex is obtained, and a new potential method is provided for research and treatment of nerve injury and / or degeneration.

Description

Technical Field

[0001] The present invention belongs to the field of microbial medicine, and more specifically, relates to a composition comprising mannuronic acid disaccharide, guluronic acid disaccharide and acerol A, and a preparation method and use thereof. Background Art

[0002] Alzheimer's disease (AD) is the neurodegenerative disease with the highest incidence. With the aging of the population, the incidence of AD has shown a clear upward trend. The characteristic pathological changes of AD include the deposition of β-amyloid protein (Aβ) in the brain, forming amyloid aggregates. The most common Aβ short peptide sequence contains 42 amino acid residues. The N-terminus of the Aβ(1-42) short peptide is hydrophilic, and the C-terminus is hydrophobic. The aggregation process of Aβ includes the folding and orderly arrangement of Aβ short peptides to form a β-folded structure (β-sheet), exposing the hydrophilic structure to the outside of the aggregate. Aβ aggregates are amphiphilic and can therefore bind to amphiphilic compounds. The hydrophobic structure of the compound can also be inserted into the middle of the β-sheet, destroying the ordered arrangement structure inside the aggregate, reducing the aggregation degree of Aβ, and providing conditions for reducing Aβ toxicity and clearing Aβ in the body.

[0003] The hydrophilic end of Aβ is the metal ion complexing end. Since the hydrophilic N-terminus of Aβ aggregates is exposed to the outside of the aggregates and participates in metal ion complexing, the complexing ability of Aβ aggregates with metal ions is stronger than that of Aβ monomers. Studies have shown that the complexes formed by Aβ and variable-valent metals (such as Aβ-Cu, Aβ-Fe) have a strong ability to catalyze the formation of reactive oxygen species (ROS), among which Fe(III) is easy to form precipitation and is not easy to exist stably in the body fluid environment. Therefore, the oxidative damage caused by Aβ-Cu complexes has become an important cause of AD neurooxidative damage. Considering the strong complexing ability of Aβ aggregates and divalent copper ions, the aggregation of Aβ will further accelerate the oxidative damage of nerve cells. Based on the above analysis, the development of drugs that can inhibit Aβ aggregation and reduce the ability of Aβ-Cu to catalyze the generation of ROS is crucial for the treatment of AD.

[0004] Guluronate disaccharide has a low relative molecular mass and can bind to divalent copper ions, seize divalent copper ions in Aβ-Cu complexes, and reduce the damage of Aβ-Cu catalyzing the production of ROS. There are a large number of reducing compounds in the human body that can convert divalent copper ions into monovalent copper ions. Monovalent copper ions still have a strong complexing ability with Aβ, and are converted into divalent copper ions under the action of reactive oxygen in the body, continuing to accelerate the production of ROS. Guluronate disaccharide can bind to monovalent copper ions, seize monovalent copper ions in Aβ-Cu, and reduce the catalytic activity of Aβ-Cu complexes. Since guluronate disaccharide has a stronger ability to capture monovalent copper ions from Aβ-Cu complexes than divalent copper ions, the presence of reducing substances is of great significance for reducing the catalytic activity of Aβ-Cu. However, guluronate disaccharide can promote the aggregation of free Aβ and cannot be directly applied in vivo.

[0005] Mannosuronate disaccharide can effectively inhibit Aβ aggregation and has a certain reducibility. Mixing guluronate disaccharide with mannuronate disaccharide is very beneficial for removing Aβ aggregates and reducing Aβ-Cu catalytic toxicity. However, the effects of mixing guluronate disaccharide and mannuronate disaccharide for inhibiting Aβ aggregation and depolymerizing Aβ are not significant. This may be due to the contradictory effects of the two compounds on Aβ aggregation: guluronate disaccharide promotes Aβ aggregation, and mannuronate disaccharide inhibits Aβ aggregation. Therefore, it is impossible to alleviate the toxicity of Aβ aggregation by simply mixing the two compounds. At the same time, the reducibility of sugars is limited, and the efficiency of reducing divalent copper ions to monovalent copper ions is not high, so it is impossible to significantly reduce and eliminate the ROS generated by Aβ-Cu catalytic activity.

[0006] Acer A is a natural small molecule polyphenol compound with strong reducing properties. It contains both hydrophilic and hydrophobic groups, which can effectively embed into Aβ aggregates to achieve the disaggregation of Aβ aggregates. However, Acer A has poor water solubility and is easy to aggregate in aqueous solution, so a compound is needed as its stabilizer in practical applications. The amphiphilicity of mannuronic acid disaccharide and guluronic acid disaccharide has a good effect in reducing the aggregation of Acer A.

[0007] Therefore, there is an urgent need in the art for a composition comprising three natural small molecule compounds, namely, mannuronate disaccharide, guluronate disaccharide and acerol A, which can reduce Aβ aggregation and reduce ROS damage, providing a new method for the treatment of AD. However, how to reasonably configure the three compounds to retain their advantages while overcoming their shortcomings has become the research focus of the present invention. Summary of the invention

[0008] As mentioned above, in existing studies, the simple mixture of guluronic acid disaccharide and mannuronic acid disaccharide has no significant effect on inhibiting Aβ aggregation and depolymerization in practical applications, and cannot significantly reduce ROS generated by the catalytic action of Aβ-Cu complex. Therefore, there is an urgent need in the art to provide a new composition that can both inhibit Aβ aggregation and reduce the ability of Aβ-Cu complex to generate ROS, so as to be applied to the research and treatment of neural damage and / or degeneration, and provide a new potential method for the treatment of Alzheimer's disease.

[0009] In view of this, in a first aspect, the present invention provides a composition comprising guluronate disaccharide, mannuronate disaccharide and acerol A.

[0010] In a second aspect, the present invention provides a method for preparing the composition of the first aspect of the present invention, comprising the following steps: 1) dissolving and mixing guluronate disaccharide, mannuronate disaccharide and acerol A in a non-protonic polar solvent, performing a first ultrasonic treatment, and then performing a first freeze-drying to obtain a first freeze-dried powder; 2) dissolving the first lyophilized powder in a buffer solution having a pH value of 7.2-8.0, performing a second ultrasonic treatment, and then performing a second freeze-drying to obtain a second lyophilized powder.

[0011] In a third aspect, the present invention provides use of the composition of the first aspect of the present invention in inhibiting aggregation of β-amyloid protein.

[0012] In a fourth aspect, the present invention provides use of the composition of the first aspect of the present invention in disaggregating β-amyloid protein aggregates.

[0013] In a fifth aspect, the present invention provides use of the composition of the first aspect of the present invention in reducing reactive oxygen species generated by catalysis of copper ions and / or β-amyloid protein-copper complexes.

[0014] In a sixth aspect, the present invention provides use of the composition of the first aspect of the present invention in reducing the toxicity of β-amyloid protein.

[0015] In a seventh aspect, the present invention provides use of the composition of the first aspect of the present invention in the preparation of a medicament for treating and / or preventing nerve damage and / or degeneration.

[0016] In an eighth aspect, the present invention provides use of the composition of the first aspect of the present invention in the preparation of a medicament for treating and / or preventing a neurodegenerative disease.

[0017] The beneficial effects of the present invention include one or more of the following: 1) The composition of the present invention has good antioxidant properties, inhibits the production of ROS catalyzed by copper ions and / or Aβ-Cu complexes, reduces oxidative stress, and thus reduces the damage of ROS to nerve cells; 2) The composition of the present invention can inhibit Aβ aggregation and depolymerize Aβ polymers, thereby alleviating the toxic damage of Aβ aggregates to nerve cells; 3) The composition of the present invention can effectively dissolve mannuronate disaccharide, guluronate disaccharide and acerol A, thereby giving full play to the functions of the three natural small molecule compound components, while achieving synergistic enhancement among the components. The composition is made from natural materials, is safe and non-toxic, and is easy to produce and store. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other implementation plans can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The L929 cytotoxicity of the compositions is shown.

[0020] Figure 2 The results of ThT assays showing that the compositions inhibit A[beta](1-42) aggregation.

[0021] Figure 3 The effect of the composition on the cytotoxicity of Aβ(1-42) SH-SY5Y cells by inhibiting its aggregate formation is shown.

[0022] Figure 4 The results of ThT assays showing that the compositions disaggregate A[beta](1-42) aggregates.

[0023] Figure 5 The effect of the composition on SH-SY5Y cell cytotoxicity by disaggregating Aβ(1-42) aggregates is shown.

[0024] Figure 6 The composition is shown to inhibit the generation of ROS catalyzed by free divalent copper ions.

[0025] Figure 7 The effect of the composition on SH-SY5Y cell cytotoxicity by inhibiting the production of ROS catalyzed by free divalent copper ions is shown.

[0026] Figure 8 The composition is shown to inhibit the generation of ROS catalyzed by the Aβ-Cu complex.

[0027] Fig. 9The effect of the composition on the cytotoxicity of SH-SY5Y cells by inhibiting the production of ROS catalyzed by Aβ-Cu complex is shown. DETAILED DESCRIPTION

[0028] The present invention will be described clearly and completely below in conjunction with the embodiments and drawings of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments that can be obtained by those of ordinary skill in the art based on the embodiments of the present invention belong to the scope of protection of the present invention.

[0029] Unless otherwise specified, all terms used herein should be understood as the common meanings known in the art. Other more specific definitions of certain terms used in this application are described below, and are intended to be uniformly applied throughout the specification and invention patent scope, unless the definitions explicitly listed provide a broader definition.

[0030] Throughout the specification and the scope of the invention patent application, the expressions "including", "comprising" and their variations are open terms and are not intended to exclude other technical features, additives, components or steps. In addition, the word "including" covers the case of "consisting of..." By reading the specification, other objects, advantages and features of the present invention will become apparent to those skilled in the art, or can be learned by practicing the present invention. In addition, the present invention covers all possible combinations of the specific and particular embodiments described herein.

[0031] As mentioned above, in existing studies, although guluronate disaccharide has a certain reducing property and mannuronate disaccharide can effectively inhibit Aβ aggregation, the effects of both when used alone or simply mixed are not ideal and have limitations.

[0032] Therefore, the object of the present invention is to provide a novel composition that can inhibit Aβ aggregation and reduce the ability of Aβ-Cu complex to generate ROS, so as to be applied to the research and treatment of nerve damage and / or degeneration.

[0033] Guluronate disaccharide is a linear compound connected by α-1,4-glycosidic bonds obtained by hydrolysis of brown algae oligosaccharides. Related studies have shown that 2-12 sugar units are generally required to bind to divalent copper ions; and in the presence of monovalent copper ions and divalent copper ions, guluronate disaccharide can selectively complex with monovalent copper ions. In addition, guluronate disaccharide can capture divalent copper ions and monovalent copper ions from the complex of β-amyloid protein and copper ions, and will not capture copper ions with normal physiological effects, thereby inhibiting the aggregation toxicity of β-amyloid protein and reducing the catalytic oxidation of the complex of β-amyloid protein and divalent copper ions. It has the potential to prepare drugs for preventing and treating copper ion-induced reactive oxygen damage and β-amyloid protein aggregation toxicity, but guluronate disaccharide can promote the aggregation of free Aβ and cannot be directly used in vivo.

[0034] The inventor first thought of combining galactoside A with guluronic acid disaccharide to overcome the aggregation of Aβ. Galactoside A has strong reducing properties and can effectively intercalate into Aβ aggregates to achieve the disaggregation of Aβ aggregates. However, galactoside A has poor water solubility, is unstable in aqueous solution systems and is easy to aggregate. Therefore, in practical applications, a compound is needed as a stabilizer to stabilize galactoside A and promote its dissolution. On this basis, the inventor thought of adding mannuronic acid disaccharide as a stabilizer for galactoside A. Mannuronic acid disaccharide itself has reducing properties and can effectively inhibit Aβ aggregation, which is beneficial to reducing Aβ aggregates and ROS toxicity. Finally, the composition of the present invention was obtained.

[0035] In the composition of the present invention, guluronate disaccharide combines with monovalent copper ions to reduce free copper ions and simultaneously capture monovalent copper ions in Aβ-Cu to reduce the generation of ROS. Mannonate disaccharide and acerol A inhibit Aβ aggregation on the one hand, and assist in ROS clearance on the other hand through their own reducing properties, converting divalent copper ions into monovalent copper ions, thereby improving the complexing ability of guluronate disaccharide for copper ions and further reducing the oxidative damage of ROS. On this basis, the three natural small molecule compound components can give full play to their respective roles and produce a synergistic effect to achieve better results.

[0036] In the present application, the term "synergistic effect" means that if a composition comprising multiple components is administered, it provides a measurable physiological effect, such as a therapeutic effect, if the actual effect produced by the composition comprising the multiple components is greater than the effect predicted by the sum of the actual physiological effects of the individual components. Specifically, when each component individually provides some measurable effect, and the measurable effect provided by the composition comprising the multiple components is greater than the effect provided by the sum of each component, a synergistic effect is provided. More specifically, when one component individually does not provide a measurable effect, another or two components individually provide some measurable effect, and the measurable effect provided by the composition comprising the multiple components is greater than the effect provided by one or two of the components, a synergistic effect is provided. More specifically, when each component individually does not provide any measurable effect, but the composition comprising the multiple components can provide a measurable effect, a synergistic effect is provided.

[0037] Therefore, in a first aspect, the present invention provides a composition comprising guluronate disaccharide, mannuronate disaccharide and acerol A.

[0038] Guluronate disaccharide and mannuronate disaccharide have a low molecular weight, contain more hydrophilic hydroxyl groups and carboxyl groups outside the molecular skeleton, and have good solubility in water. At the same time, the molecule also has a hydrophobic skeleton of an alkane, which is conducive to its combination with hydrophobic small molecule drugs. The preliminary research results of the present invention show that the ratio of the total amount of guluronate disaccharide and mannuronate disaccharide mixed with acerol A directly affects the solubility of acerol A. The inventors have found that when the ratio of guluronic acid disaccharide: mannuronic acid disaccharide: acerol A is 1: (4-10): (3-7), the solubility of acerol A in water can be improved, and the higher the ratio of the total amount of guluronic acid disaccharide and mannuronic acid disaccharide to acerol A, the more conducive it is to the dissolution of acerol A. However, considering the cost of the sample and the experimental effect, the inventors found that the optimal ratio of guluronic acid disaccharide, mannuronic acid disaccharide and acerol A is 1:9:5. The basis for the optimal ratio is that after the aqueous solution is left for 2 hours, no insoluble acerol A is precipitated visible to the naked eye.

[0039] Therefore, in some embodiments, the molar ratio of guluronic acid disaccharide, mannuronic acid disaccharide and acerol A is 1: (4-10): (3-7); in some preferred embodiments, the molar ratio of guluronic acid disaccharide, mannuronic acid disaccharide and acerol A is 1:9:5, and the obtained composition can be uniformly dissolved in water to form a stable aqueous solution.

[0040] In a second aspect, the present invention provides a method for preparing the composition of the first aspect of the present invention, comprising the following steps: 1) dissolving and mixing guluronate disaccharide, mannuronate disaccharide and acerol A in a non-protonic polar solvent, performing a first ultrasonic treatment, and then performing a first freeze-drying to obtain a first freeze-dried powder; 2) dissolving the first lyophilized powder in a buffer solution having a pH value of 7.2-8.0, performing a second ultrasonic treatment, and then performing a second freeze-drying to obtain a second lyophilized powder.

[0041] When preparing the composition of the present invention, the inventor first dissolves and mixes guluronic acid disaccharide, mannuronic acid disaccharide and galactin A in a non-protonic polar solvent. In this step, the three components can be dissolved in the non-protonic polar solvent first and then the solutions of the three components are mixed, or the three components can be mixed to form a mixture and then dissolved in the non-protonic polar solvent. Since the three components, especially galactin A, are difficult to dissolve, first dissolving the three components in the non-protonic polar solvent and then mixing the solutions of the three components can usually obtain a better dissolution effect and a higher dissolution rate. In some embodiments, guluronic acid disaccharide, mannuronic acid disaccharide and galactin A are dissolved in the non-protonic polar solvent respectively and then mixed together.

[0042] The term "aprotic solvent" refers to a type of solvent in which the proton self-transfer reaction of the solvent is extremely weak or has no self-transfer tendency. It can be further divided into aprotic non-polar solvents and aprotic polar solvents. The molecules of aprotic polar solvents are polar, so they will have an effect on the solute molecules and produce a solvation effect. Examples of aprotic polar solvents include: dimethyl sulfoxide (DMSO), N,N-dimethylformamide, acetone, etc.

[0043] In some embodiments, the aprotic polar solvent is DMSO.

[0044] In the method of the present application, the solution after ultrasonic dispersion is freeze-dried to obtain a freeze-dried powder. Those skilled in the art should understand that any freeze-drying that does not affect the properties or functional activity of the composition of the present invention can be adopted. The inventors found that liquid nitrogen freeze-drying has the characteristics of short freezing time and high efficiency compared to other freeze-drying methods, so it can achieve rapid freezing of samples, maintain the uniform dispersion state of guluronic acid disaccharide, mannuronic acid disaccharide and acerol A, and the obtained freeze-dried sample is loose and porous, which is conducive to subsequent dissolution.

[0045] In some embodiments, the first freeze drying and the second freeze drying are liquid nitrogen freeze drying.

[0046] In some embodiments, the buffer solution is a phosphate buffer solution.

[0047] In addition, the two ultrasonic treatments in the method of the present application are both intended to increase the uniform distribution of guluronic acid disaccharide, mannuronic acid disaccharide and acerol A in different solvents (non-protonic polar solvents or buffer solutions), promote the dispersion of acerol A in guluronic acid disaccharide and mannuronic acid disaccharide, and provide favorable conditions for the solubilization of acerol A in the aqueous phase system.

[0048] In some embodiments, the time of the first ultrasonic treatment is 15 to 120 minutes, and the time of the second ultrasonic treatment is 10 to 100 minutes; in some preferred embodiments, the time of the first ultrasonic treatment and the second ultrasonic treatment is 30 minutes respectively.

[0049] Subsequently, the inventors verified the effects of the composition of the present invention on Aβ aggregation at the molecular and cellular levels, including the inhibition of Aβ aggregation and the disaggregation of Aβ aggregates. The experimental results showed that the composition of the present invention can significantly inhibit the aggregation of Aβ(1-42) and disaggregate Aβ(1-42) aggregates to reduce its aggregation degree, thereby reducing its toxicity.

[0050] Therefore, in a third aspect, the present invention provides use of the composition of the first aspect of the present invention in inhibiting the aggregation of β-amyloid protein (Aβ).

[0051] In a fourth aspect, the present invention provides use of the composition of the first aspect of the present invention in disaggregating Aβ aggregates.

[0052] Furthermore, the inventors verified the effect of the composition of the present invention on reactive oxygen species (ROS) generated by copper ions and / or Aβ-Cu at the molecular and cellular levels. The experimental results showed that the composition of the present invention can significantly inhibit the generation of ROS catalyzed by both free divalent copper ions and Aβ-Cu complexes, thereby reducing their toxicity.

[0053] Therefore, in a fifth aspect, the present invention provides use of the composition of the first aspect of the present invention in reducing active oxygen generated by copper ions and / or Aβ-Cu complexes.

[0054] It can be seen from the above experimental results that the composition of the present invention can reduce the toxicity of Aβ to cells by reducing Aβ aggregation and reducing ROS generated by copper ions and / or Aβ-Cu complexes.

[0055] Therefore, in a sixth aspect, the present invention provides use of the composition of the first aspect of the present invention in reducing the toxicity of Aβ.

[0056] In a seventh aspect, the present invention provides use of the composition of the first aspect of the present invention in the preparation of a medicament for treating and / or preventing nerve damage and / or degeneration.

[0057] The term "neural injury" refers to the damage to the integrity and function of neural structures due to trauma or certain pathological causes, including but not limited to cerebrovascular disease, genetic diseases, tumors, inflammation, autoimmune reactions or diseases, neurodegenerative diseases, pulmonary encephalopathy, hepatic encephalopathy, etc. The term "neurodegeneration" refers to the loss of neuronal number, structure and function, which can be caused by a variety of reasons such as oxidative stress, apoptosis, inflammatory response and protein aggregation.

[0058] As used herein, "prevention" refers to preventive interventions performed on normal or susceptible individuals to prevent the individual from developing a disease or condition. "Treatment" refers to therapeutic interventions performed on diseased individuals to prevent the disease or condition from developing, or to alleviate or cure the disease or condition in the diseased individual relative to before the therapeutic intervention.

[0059] In an eighth aspect, the present invention provides use of the composition of the first aspect of the present invention in the preparation of a medicament for treating and / or preventing a neurodegenerative disease.

[0060] Neurodegenerative diseases are caused by the loss of neurons and / or their myelin sheaths, which worsen over time and cause functional impairment. They can be divided into acute neurodegenerative diseases and chronic neurodegenerative diseases. The former mainly includes cerebral ischemia (CI), brain injury (BI), and epilepsy; the latter includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), different types of spinocerebellar ataxia (SCA), Pick's disease, etc.

[0061] In some embodiments, the neurodegenerative disease is Alzheimer's disease.

[0062] Example 1: Preparation of a composition of mannuronate disaccharide, guluronate disaccharide and acerol A and an Aβ(1-42) solution

[0063] Guluronate disaccharide, mannuronate disaccharide and truncatin A were dissolved in DMSO respectively, and then the solutions of guluronate disaccharide, mannuronate disaccharide and truncatin A were mixed and prepared into a mixed solution with a molar ratio of 1:9:5, wherein the concentrations of guluronate disaccharide, mannuronate disaccharide and truncatin A in the mixed solution were 1 mM, 9 mM and 5 mM, respectively. The mixed solution was ultrasonically treated for half an hour, then frozen with liquid nitrogen, and dried with a freeze dryer to form a loose and porous freeze-dried powder. A phosphate buffer with a pH value of 7.2-8.0 was prepared, and the freeze-dried powder was dissolved in the buffer solution, ultrasonically treated for half an hour, divided into portions, and then frozen with liquid nitrogen and dried with a freeze dryer to form a loose and porous dry powder. Ultrapure water was added to the sub-filling bottle to prepare a solution, and the concentration of guluronate in the solution was 500 μM for subsequent experiments.

[0064] Aβ(1-42) was dissolved in HFIP solution at 1 mg / mL, sonicated for half an hour, and dried after freezing in liquid nitrogen. The dried Aβ(1-42) was dissolved in 20 mM NaOH solution to prepare a 5 mM solution, and then the solution was diluted to 100 μM with PBS (10 mM) buffer.

[0065] Example 2: Cytotoxicity of a Composition of Mannosuronate Disaccharide, Guluronate Disaccharide and Acer striata A

[0066] The composition solution prepared in Example 1 was diluted so that the concentrations of guluronate disaccharide, mannuronate disaccharide and acerol A were 1 mM, 9 mM and 5 mM, respectively. The solution was diluted 100 times with cell culture medium and co-cultured with L929 cells for 24 h. The cytotoxicity was determined by MTT method. The experimental results showed that the addition of the composition did not affect the survival rate of L929 cells ( Figure 1 ), indicating that the composition of mannuronic acid disaccharide, guluronic acid disaccharide and tea maple A has no obvious cytotoxicity. In addition, the experimental results also show that the composition has the ability to promote the proliferation of L929, which may be due to the fact that guluronic acid, mannuronic acid and tea maple A molecules all have reducing groups, which are beneficial to the removal of oxidative active free radicals generated by cell metabolism, and thus are beneficial to the proliferation of L929 cells.

[0067] Example 3: Combination of mannuronate disaccharide, guluronate disaccharide and acerol A reduces the toxicity of Aβ(1-42) by inhibiting its aggregation

[0068] After the Aβ(1-42) prepared in Example 1 was mixed with the solution of the composition and incubated in a 37°C water bath, the aggregation of Aβ(1-42) was measured over time by thioflavin T (ThT) fluorescence method. The concentrations of Aβ(1-42), guluronic acid disaccharide, mannuronic acid disaccharide and acerol A in the mixed solution during the incubation process were 20, 10, 90 and 50 μM, respectively. The solution was diluted 2 times with PBS during the test, and the ThT concentration used was 20 μM. The control group used a 20 μM Aβ(1-42 solution and was incubated and tested under the same conditions.

[0069] The mixture of Aβ(1-42) and the combination treated in the same way (treatment group) and the single Aβ(1-42) solution (control group) were incubated in a 37°C water bath for 24 h, and then mixed with SH-SY5Y cells for 24 h, and the cell survival rate was determined by the MTT method.

[0070] The experimental results showed that the combination of mannuronate disaccharide, guluronate disaccharide and Acer truncatum A can significantly inhibit the aggregation of Aβ(1-42) and reduce its aggregation degree ( Figure 2 ), and significantly increased cell survival rate after mixed culture with SH-SY5Y cells ( Figure 3 ), indicating that the composition can reduce the toxicity of Aβ(1-42) by inhibiting its aggregation.

[0071] Example 4: Combination of mannuronosyl disaccharide, guluronosyl disaccharide and acerol A reduces the toxicity of Aβ(1-42) by disaggregating Aβ(1-42) aggregates

[0072] The Aβ(1-42) solution prepared in Example 1 was first incubated in a 37°C water bath for 24 hours, and then a composition of guluronate disaccharide, mannuronate disaccharide and acerol A was added and incubated under the same conditions. The aggregation of Aβ(1-42) was measured over time by thioflavin T (ThT) fluorescence method. Among them, the concentrations of Aβ(1-42), guluronate disaccharide, mannuronate disaccharide and acerol A in the mixed solution during the incubation process were 20, 10, 90 and 50 μM, respectively. The mixture was diluted 2 times with PBS during the test, and the ThT concentration used was 20 μM. The control group used a 20 μM Aβ(1-42) solution, which was incubated and detected under the same conditions.

[0073] The mixture of Aβ(1-42) and the combination treated in the same way (treatment group) and the single Aβ(1-42) solution (control group) were incubated in a 37°C water bath for 24 h, and then mixed with SH-SY5Y cells for 24 h, and the cell survival rate was determined by the MTT method.

[0074] The experimental results showed that the combination of mannuronate disaccharide, guluronate disaccharide and Acer truncatula A can depolymerize Aβ(1-42) aggregates and reduce their aggregation degree ( Figure 4 ), and significantly increased cell survival rate after mixed culture with SH-SY5Y cells ( Figure 5 ), indicating that the composition can reduce the toxicity of Aβ(1-42) aggregates by achieving disaggregation.

[0075] Example 5: Combination of mannuronate disaccharide, guluronate disaccharide and acerol A inhibits the production of ROS toxicity catalyzed by free copper ions

[0076] H2O2 was selected as the reactant. In the presence of ascorbic acid (AA), the amount of ·OH produced by free copper ions was detected by the coumarin-3-carboxylic acid (3-CCA) method. The concentrations of H2O2, AA, copper ions, 3-CCA, guluronic acid disaccharide, mannuronic acid disaccharide and acerol A were 100, 120, 10, 200, 20, 180 and 100 μM, respectively, and the reaction time was 15 minutes. The control group used samples without adding guluronic acid disaccharide, mannuronic acid disaccharide and acerol A under the same conditions.

[0077] The mixture of H2O2, AA, copper ions, guluronosyl disaccharide, mannuronosyl disaccharide and acerol A in the previous step was cultured with SH-SY5Y cells for 24 h, and the cell survival rate was determined by MTT method to determine the protective effects of guluronosyl disaccharide, mannuronosyl disaccharide and acerol A on nerve cell maintenance damage. In the control group, H2O2, AA and copper ions were cultured with SH-SY5Y cells for 24 h.

[0078] The experimental results showed that the combination of mannuronate disaccharide, guluronate disaccharide and Acer striata A can significantly inhibit the production of ROS catalyzed by free divalent copper ions ( Figure 6 ), and significantly increased cell survival rate after mixed culture with SH-SY5Y cells ( Figure 7 ), indicating that the composition can reduce the toxicity of ROS by inhibiting the catalysis of free divalent copper ions, thereby playing a protective role against nerve cell maintenance damage.

[0079] Example 6: Combination of mannuronate disaccharide, guluronate disaccharide and Acer striata A inhibits the production of ROS toxicity catalyzed by Aβ-Cu complex

[0080] The Aβ(1-42) obtained in Example 1 was mixed with the composition, and the amount of ·OH produced by free copper ions was detected by 3-CCA method in the presence of H2O2 and AA. The concentrations of H2O2, AA, Aβ(1-42), copper ions, 3-CCA, guluronic acid disaccharide, mannuronic acid disaccharide and acerol A used were 100, 120, 20, 20, 200, 20, 180 and 100 μM, respectively, and the reaction time was 15 minutes.

[0081] The mixture of H2O2, AA, Aβ(1-42), copper ions, guluronate disaccharide, mannuronate disaccharide and acerol A in the previous step was cultured with SH-SY5Y cells for 24 h, and the cell survival rate was determined by MTT method to determine the protective effects of guluronate disaccharide, mannuronate disaccharide and acerol A on nerve cell maintenance damage. In the control group, H2O2, AA, Aβ(1-42) and copper ions were cultured with SH-SY5Y cells for 24 h. The control group used samples without adding guluronate disaccharide, mannuronate disaccharide and acerol A under the same conditions. ( Fig. 9 ) The experimental results showed that the combination of mannuronate disaccharide, guluronate disaccharide and Acer striata A can significantly inhibit the production of ROS catalyzed by Aβ-Cu complex. Figure 8 ), and significantly increased cell survival rate after mixed culture with SH-SY5Y cells ( Fig. 9 ), indicating that the composition can reduce the toxicity of Aβ-Cu complex by inhibiting the production of ROS, thereby protecting the nerve cells from damage.

Claims

1. A composition comprising guluronate disaccharide, mannuronate disaccharide and acerol A. 2 . The composition according to claim 1 , wherein the molar ratio of guluronic acid disaccharide, mannuronic acid disaccharide and acerol A is 1:9:

5.

3. A method for preparing the composition according to claim 1 or 2, comprising the following steps: 1) dissolving and mixing guluronate disaccharide, mannuronate disaccharide and acerol A in a non-protonic polar solvent, performing a first ultrasonic treatment, and then performing a first freeze-drying to obtain a first freeze-dried powder; 2) dissolving the first lyophilized powder in a buffer solution having a pH value of 7.2-8.0, performing a second ultrasonic treatment, and then performing a second freeze-drying to obtain a second lyophilized powder. The method according to claim 3 , wherein the first freeze drying and the second freeze drying are liquid nitrogen freeze drying.

5. The method according to claim 3 or 4, wherein the aprotic polar solvent is DMSO.

6. Use of the composition according to claim 1 or 2 in inhibiting the aggregation of β-amyloid protein.

7. Use of the composition according to claim 1 or 2 in disaggregating β-amyloid protein aggregates.

8. Use of the composition according to claim 1 or 2 in reducing active oxygen generated by copper ions and / or β-amyloid protein-copper complexes.

9. Use of the composition according to claim 1 or 2 in reducing the toxicity of β-amyloid protein.

10. Use of the composition according to claim 1 or 2 in the preparation of a medicament for treating and / or preventing nerve damage and / or nerve degeneration.

11. Use of the composition according to claim 1 or 2 in the preparation of a medicament for treating and / or preventing a neurodegenerative disease.

12. The use according to claim 11, wherein the neurodegenerative disease is Alzheimer's disease.

Images