Application of sea cucumber chondroitin sulfate in preparation of medicine for preventing or treating Alzheimer disease

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of sea cucumber chondroitin sulfate in preparation of a medicine for preventing or treating Alzheimer's disease. According to the invention, chondroitin sulfate is extracted from sea cucumber by adopting a specific preparation method, and research finds that the sea cucumber chondroitin sulfate can effectively reduce deposition of Abeta protein in an Alzheimer disease (AD) model mouse body and remarkably improve the learning and memory ability of the mouse, and shows the potential of the sea cucumber chondroitin sulfate in the aspects of neuroprotection and cognitive function recovery. Therefore, the sea cucumber chondroitin sulfate is expected to provide a safe and effective treatment choice for the patients with the Alzheimer disease, and a new breakthrough is brought to the current treatment means.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of sea cucumber chondroitin sulfate in the preparation of a drug for preventing or treating Alzheimer's disease. Background Art

[0002] Alzheimer's disease (AD) is an age-related neurodegenerative disease and a major dementia disease, mainly manifested as progressive memory decline and cognitive dysfunction. The etiology of AD is complex and involves the dysfunction of multiple systems such as the nervous, immune, and blood circulation systems. Although studies have revealed multiple factors closely related to the occurrence and development of AD, such as the dysfunction of the central cholinergic system, the tangling and precipitation of β-amyloid protein (Aβ), the hyperphosphorylation of Tau protein, continuous immune response and inflammation, and the disorder of the central metabolic system, etc., the drug research and development targeting these pathological mechanisms has been slow and the success rate is extremely low.

[0003] Currently, the US FDA has approved 6 therapeutic drugs: rivastigmine, galantamine, donepezil, memantine, the combination of memantine and donepezil, and tacrine. These drugs only temporarily increase the central neurotransmitters in the brain to improve symptoms, cannot reduce the pathological changes of AD, nor can they reverse or slow down the disease process. Therefore, in-depth study of the pathogenesis of AD, discovery of new markers, and development of safe and effective drugs are of great significance for the prevention and treatment of AD. Summary of the Invention

[0004] The purpose of the present invention is to overcome the deficiencies in the prior art and provide the application of sea cucumber chondroitin sulfate in the preparation of a drug for preventing or treating Alzheimer's disease.

[0005] To achieve the above purpose, the technical solutions adopted by the present invention include:

[0006] In the first aspect, the present invention provides the application of sea cucumber chondroitin sulfate in the preparation of a drug for preventing or treating neurodegenerative diseases.

[0007] Preferably, the neurodegenerative disease includes Alzheimer's disease.

[0008] Through experimental exploration, it was found that chondroitin sulfate from sea cucumbers (FCS) can effectively reduce the deposition of Aβ protein in the bodies of Alzheimer's disease (AD) model mice and significantly improve the learning and memory abilities of the mice. Compared with chondroitin sulfate (CS) from shark cartilage, the chondroitin sulfate from sea cucumbers described in the present invention shows better effects in reducing Aβ protein deposition and improving learning and memory. This finding indicates that chondroitin sulfate from sea cucumbers has stronger biological activity and can more effectively intervene in the pathogenesis of Alzheimer's disease. Therefore, chondroitin sulfate from sea cucumbers is expected to become a new potential drug for the treatment of Alzheimer's disease, bringing new treatment options for Alzheimer's disease patients.

[0009] Preferably, the effective dose of the chondroitin sulfate from sea cucumbers is 100 - 200 mg / kg.

[0010] Through experimental exploration, it was found that in the effect of chondroitin sulfate from sea cucumbers (FCS) on improving the pathological characteristics of AD, the reduction in the number of plaque areas in the hippocampus and motor cortex of the model mice is dose-dependent with FCS. When the dose is 100 - 200 mg / kg, the number of plaque areas in both the hippocampus and motor cortex shows significant reduction, indicating its significant role in improving the pathological characteristics of AD. When the FCS dose is lower than the above preferred dose range, there is a tendency for the number of plaque areas to decrease, but there is no statistical significance compared with the blank group. Therefore, the effective dose of FCS is of great significance for improving or treating AD. A reasonable dose range can not only maximize its efficacy but also provide important reference for clinical application.

[0011] Preferably, the preparation method of the chondroitin sulfate from sea cucumbers includes the following steps:

[0012] S1. Cut the body wall of dried sea cucumbers into pieces and homogenize them, then perform degreasing treatment with a chloroform / methanol mixture to obtain sea cucumber residue;

[0013] S2. Mix the degreased sea cucumber residue with a papain solution containing EDTA and cysteine for enzymatic hydrolysis. After enzymatic hydrolysis, centrifuge and collect the supernatant;

[0014] S3. Add cetylpyridinium chloride solution to the supernatant, stir until precipitation is complete, redissolve the precipitate with a NaCl / ethanol mixture, then add ethanol, let it stand, centrifuge, and collect the polysaccharide precipitate;

[0015] S4. Dissolve the polysaccharide precipitate collected in step S3 in water for dialysis, and then lyophilize to obtain crude polysaccharide;

[0016] S5. Perform anion exchange chromatography on the crude polysaccharide obtained in step S4, collect the eluate, and lyophilize to obtain the chondroitin sulfate from sea cucumbers.

[0017] Preferably, it includes at least one of the following (1)-(6):

[0018] (1) In step S1, the volume ratio of chloroform to methanol in the chloroform / methanol mixture is (3-5):1;

[0019] (2) In step S2, the concentration of EDTA is 4-5 mM, and the concentration of cysteine is 4-5 mM;

[0020] (3) In step S2, the specific conditions for enzymatic hydrolysis are: enzymatic hydrolysis at 55-65 °C for 8-12 h;

[0021] (4) In step S3, the volume ratio of NaCl to ethanol in the NaCl / ethanol mixture is 10:(1-2), and the concentration of NaCl is 1-3 M;

[0022] (5) In step S3, the specific conditions for standing are: standing at 2-6 °C for 10-14 h;

[0023] (6) In step S4, the cut-off molecular weight of the dialysis bag used for dialysis is 3-4 kDa.

[0024] Studies have found that different preparation methods will lead to differences in the biological activities of sea cucumber chondroitin sulfate. Through experimental verification, the sea cucumber chondroitin sulfate extracted by the specific preparation method described in the present invention can effectively reduce the deposition of Aβ protein in the body of AD model mice in a dose-dependent manner, thereby showing significant biological activities in the treatment of Alzheimer's disease and enhancing its potential in the treatment of Alzheimer's disease. Therefore, optimizing the preparation process is crucial for the efficacy of sea cucumber chondroitin sulfate in clinical applications.

[0025] In a second aspect, the present invention provides a method for preparing sea cucumber chondroitin sulfate, comprising the following steps:

[0026] S1. Cut the dried sea cucumber body wall into pieces and homogenize it, and then perform defatting treatment with a chloroform / methanol mixture to obtain sea cucumber residues;

[0027] S2. Mix the defatted sea cucumber residues with a papain solution containing EDTA and cysteine for enzymatic hydrolysis, centrifuge after enzymatic hydrolysis, and collect the supernatant;

[0028] S3. Add a cetylpyridinium chloride solution to the supernatant, stir until precipitation is complete, redissolve the precipitate with an NaCl / ethanol mixture, then add ethanol, let stand, centrifuge, and collect the polysaccharide precipitate;

[0029] S4. Dissolve the polysaccharide precipitate collected in step S3 in water for dialysis, and lyophilize to obtain crude polysaccharide;

[0030] S5. Subject the crude polysaccharide obtained in step S4 to anion exchange chromatography, collect the eluate, and lyophilize it to obtain the sea cucumber chondroitin sulfate.

[0031] Preferably, it includes at least one of the following (1)-(6):

[0032] (1) In step S1, the volume ratio of chloroform to methanol in the chloroform / methanol mixture is (3-5):1;

[0033] (2) In step S2, the concentration of EDTA is 4-5 mM, and the concentration of cysteine is 4-5 mM;

[0034] (3) In step S2, the specific conditions for enzymatic hydrolysis are: enzymatic hydrolysis at 55-65 °C for 8-12 h;

[0035] (4) In step S3, the volume ratio of NaCl to ethanol in the NaCl / ethanol mixture is 10:(1-2), and the concentration of NaCl is 1-3 M;

[0036] (5) In step S3, the specific conditions for standing are: standing at 2-6 °C for 10-14 h;

[0037] (6) In step S4, the cut-off molecular weight of the dialysis bag used for dialysis is 3-4 kDa.

[0038] In the third aspect, the present invention provides a drug for preventing or treating neurodegenerative diseases, and the drug includes sea cucumber chondroitin sulfate.

[0039] Preferably, the sea cucumber chondroitin sulfate is prepared by the described preparation method.

[0040] Preferably, the drug further includes a pharmaceutically acceptable carrier or excipient.

[0041] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0042] The present invention extracts chondroitin sulfate from sea cucumbers by a specific preparation method. It is found that this sea cucumber chondroitin sulfate can effectively reduce the deposition of Aβ protein in the bodies of Alzheimer's disease (AD) model mice, and significantly improve the learning and memory abilities of mice, showing its potential in neuroprotection and cognitive function recovery. Therefore, sea cucumber chondroitin sulfate is expected to provide a safe and effective treatment option for Alzheimer's disease patients and bring new breakthroughs to current treatment methods. Description of the Drawings

[0043] Figure 1 It is the infrared spectrogram of sea cucumber chondroitin sulfate (FCS) and its low molecular weight fragments;

[0044] Figure 2 This is the effect diagram of sea cucumber chondroitin sulfate (FCS) improving the pathological characteristics in Example 3. Among them, Figure 2 A is the representative diagram for detecting the change in Aβ expression by fluorescence microscopy, scale bar = 500 μm; Figure 2 B - C are the statistical charts for detecting the change in Aβ expression by immunofluorescence before and after drug administration; **P < 0.01***P < 0.001;

[0045] Figure 3 This is the effect diagram of sea cucumber chondroitin sulfate (FCS) improving the pathological characteristics in Example 4. Among them, Figure 3 A - B are the representative diagram and quantitative analysis result diagram of Aβ immunofluorescence labeling (n = 6); scale bar = 500 μm; Figure 3 C is the statistical chart of the alternation percentage in the Y - maze test (n = 10); Figure 3 D is the statistical chart of the ratio of the total number of entries in the maze test (n = 10); **P < 0.01***P < 0.001. Detailed implementation manners

[0046] To make the objectives, technical solutions and effects of the present invention clearer and more definite, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0047] Unless otherwise specified, the reagents used in the embodiments are all conventional reagents in the art and can be obtained through commercial channels. The experimental operations not specifically described in the embodiments are all conventional operations in the art or can be understood or known by those skilled in the art according to the existing technology or common general knowledge they have mastered.

[0048] The sea cucumber chondroitin sulfate (FCS) described in the present invention is also called fucosylated chondroitin sulfate from sea cucumber (FCS - Pg). Therefore, FCS and FCS - Pg involved in the embodiments are the same substance.

[0049] Example 1

[0050] This embodiment provides a preparation method of sea cucumber chondroitin sulfate, including the following steps:

[0051] 1. Pretreatment of raw materials

[0052] (1) Selection of raw materials: Select the body wall of dried sea cucumbers (Pearsonothuria graeffei), requiring no mildew and few impurities, and the storage conditions meet the food raw material standards;

[0053] (2) Surface cleaning: Rinse the surface of dried sea cucumbers with pure water to remove the attached impurities and avoid mechanical damage;

[0054] (3) Defatting treatment: The cleaned sea cucumbers are shredded and homogenized, then soaked in a chloroform / methanol mixture (4:1, v / v) to remove fat-soluble impurities. The operation is repeated until the solvent is clear, and sea cucumber residues are obtained.

[0055] 2. Polysaccharide extraction and separation

[0056] (1) Enzymatic extraction: The defatted sea cucumber residues are mixed with a papain solution containing 5 mM EDTA and 5 mM cysteine, and enzymatically hydrolyzed in a constant temperature water bath at 60 °C for 10 hours. After enzymatic hydrolysis, centrifugation is carried out (4000 rpm, 20 minutes), and the supernatant is collected for standby.

[0057] (2) Polysaccharide precipitation: Cetylpyridinium chloride solution is slowly added to the supernatant and stirred until precipitation is complete; the precipitate is redissolved with 2M NaCl:ethanol (100:15, v / v), 95% ethanol is added to a final concentration of 80%, and it is left to stand at 4 °C for 12 hours, and the polysaccharide precipitate is collected by centrifugation.

[0058] 3. Purification and refinement

[0059] (1) Dialysis for impurity removal: The precipitate is dissolved in ultrapure water, filled into a dialysis bag (molecular weight cut-off 3.5 kDa), and dialyzed against running water for 24 hours, and then freeze-dried to obtain crude polysaccharide.

[0060] (2) Ion exchange chromatography: Use a DEAE-cellulose anion exchange column and elute with a 0-1.5M NaCl gradient to collect the elution peak containing polysaccharide.

[0061] (3) After combining the target components, it is freeze-dried to obtain high-purity sea cucumber chondroitin sulfate (FCS).

[0062] Example 2

[0063] In this example, the degradation products of the sea cucumber chondroitin sulfate (FCS) prepared in Example 1 were characterized and analyzed. The specific method is as follows:

[0064] 1. Irradiation degradation

[0065] (1) Solution preparation: The purified FCS is formulated into aqueous solutions with different concentrations (1%-10% w / w).

[0066] (2) 60Co irradiation: Irradiation treatment is carried out at a dose rate of 10 kGy / h at room temperature, and the total dose range is 10-100 kGy.

[0067] (3) Post-treatment: After irradiation, centrifugation is carried out (12000 rpm, 10 minutes), and the supernatant is taken and freeze-dried to obtain different molecular weight fragments.

[0068] 2. Quality control and analysis

[0069] (1) Molecular weight determination: Using a TSK-G3000 PWXL gel chromatography column (mobile phase: 0.1 M NaNO, flow rate 0.8 mL / min), the average molecular weight of degraded FCS was calculated through the standard dextran molecular weight curve.

[0070] (2) Composition analysis:

[0071] Monosaccharide composition: The sample was acid hydrolyzed (2 M TFA, 120 °C, 4 h), derivatized with PMP, and analyzed by HPLC (C18 column, detection wavelength 250 nm).

[0072] Sulfate content: Ion chromatography (Dionex ICS-3000 system) was used, with a mobile phase of 4.5 mM NaCO and a flow rate of 1.2 mL / min.

[0073] Structural characterization: Infrared spectroscopy (IR) was used to analyze the characteristic absorption peaks of glycosidic bonds.

[0074] 3. Results of molecular characterization analysis

[0075] (1) The monosaccharide composition of FCS and its low molecular weight fragments was analyzed by HPLC, derivatized with PMP, and the sulfate content was determined by ion chromatography. The effect of irradiation dose on the chemical composition of FCS fragments is shown in Table 1. Native FCS contains glucuronic acid (GlcA), galactosamine (GalNAc), fucose (Fuc), and sulfate, with a molar ratio of 1.3:1.0:1.7:3.3 (Table 1). In addition, the effect of FCS solution concentration on the chemical composition of FCS fragments after irradiation was also investigated in this study (detailed in Table 1).

[0076] Table 1 Composition analysis of native FCS and its low molecular weight fragments prepared by 60Co irradiation

[0077]

[0078] Note: There are significant differences (p < 0.01) between the means with different superscripts (a - e) in the columns. There are significant differences (p < 0.01) between the means with different superscripts (a, f - i) in the columns.

[0079] * The molar ratios of sugars and sulfates were compared by defining the GalNAc content as 1 mole.

[0080] **DfCS-1, DfCS-2, DfCS-3, and DfCS-4 were prepared by 60Co radiation degradation of FCS-Pg solution (1%) at doses of 10, 20, 50, and 100 kGy; while DfCS-1′, DfCS-2′, DfCS-3′, and DfCS-4′ were prepared by 60Co radiation degradation of FCS-Pg solution at a degradation dose of 50 kGy with solution concentrations of 1%, 2%, 5%, and 10% respectively.

[0081] (2) Infrared spectroscopy analysis

[0082] The IR spectra of native FCS and its low molecular weight fragments (DfCS-1, DfCS-2, DfCS-3, DfCS-4, and DfCS-1′, DfCS-2′, DfCS-3′, DfCS-4′) are as Figure 1 (A - B) shown. Both native and low molecular weight FCS fragments showed similar spectral bands. (1) In the 4000 - 1800 cm -1 region, characteristic O - H and C - H stretching vibrations were observed at 3471 and 2960 cm -1 respectively. (2) The 1800 - 400 cm -1 region contains characteristic bands of glycosaminoglycans, including amide I band (1646 cm -1 ), amide II vibration (1545 cm -1 ), and C - N vibration of N - acetyl group (1423 cm -1 ). (3) The band at 1372 cm -1 was assigned to the symmetric deformation of CH3. (4) Three characteristic signals assigned to sulfate groups, including S = O asymmetric stretching vibration (1240 cm -1 ), symmetric C - O - S stretching vibration (850 cm -1 ), and S - O stretching vibration (589 cm -1 ). In addition, the absorption bands in the IR spectra of native FCS and its fragments were similar to those of fCS of other types of sea cucumbers reported in the literature.

[0083] Example 3

[0084] This example explored the effect of sea cucumber chondroitin sulfate (FCS) prepared in Example 1 on improving the pathological characteristics of AD, and the specific method is as follows:

[0085] Dissolve FCS in a solution prepared with physiological saline and intragastrically administer different doses of FCS (50 mg / kg, 100 mg / kg, 200 mg / mg) to 6-month-old 5xFAD mice for a total of 28 days. Then sacrifice the mice, remove the whole brain, and perform frozen sectioning to obtain the drug administration group. Intragastrically administer physiological saline in the same method as the solvent group. Then use thioflavin S staining to detect and analyze the levels of Aβ in the hippocampus and motor cortex of male 6-month-old mice in the drug administration group and the solvent group.

[0086] Figure 2 The results showed that the reduction in the plaque area and number in the hippocampus and motor cortex of the model mice was dose-dependent with FCS. Compared with the mice in the solvent group, the plaque area and number in the hippocampus and motor cortex of the mice in the 100 mg and 200 mg drug administration groups were significantly reduced. Compared with the mice in the solvent group, there was a tendency of reduction in the 50 mg group, but it was not statistically significant.

[0087] Example 4

[0088] To further study the comparative effects of the FCS prepared in Example 1 and CS (a chondroitin sulfate derived from shark cartilage) in AD, this example used immunofluorescence staining to observe the deposition of Aβ plaques in the brains of 5xFAD mice after 28 days of FCS treatment. The result analysis showed that compared with the mice in the solvent group and the CS group, the plaque area and number in the hippocampus and cortex of the FCS group were significantly reduced ( Figure 3 A - B), and the difference was statistically significant (both P < 0.001). In addition, the Y-maze behavioral experiment was used to detect the alternation behavior of four groups of mice, namely WT-vehicle, 5xFAD-vehicle, 5xFAD-CS, and 5xFAD-FCS, for five consecutive days. The result analysis showed that compared with the two groups of mice, 5xFAD-vehicle and 5xFAD-CS, the correct rate of 5xFAD-FCS was significantly improved ( Figure 3 C - D), and the difference was statistically significant (both P < 0.01). There was no significant difference in the number of times the four groups of mice entered the arms (both P > 0.05).

[0089] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of sea cucumber chondroitin sulfate in the preparation of drugs for preventing or treating neurodegenerative diseases.

2. The use according to claim 1, characterized in that The neurodegenerative diseases include Alzheimer's disease.

3. The use according to claim 1, characterized in that The effective dosage of the sea cucumber chondroitin sulfate is 100-200 mg / kg.

4. The use according to claim 1, characterized in that The preparation method of sea cucumber chondroitin sulfate comprises the following steps: S1, cutting the dried sea cucumber body wall into pieces and homogenizing them, and then defatting them with a chloroform / methanol mixture to obtain sea cucumber residues; S2, mixing the defatted sea cucumber residue with a papain solution containing EDTA and cysteine ​​for enzymolysis, centrifuging after enzymolysis, and collecting the supernatant; S3, adding cetylpyridinium chloride solution to the supernatant, stirring until the precipitation is complete, using NaCl / ethanol mixed solution to redissolve the precipitate, then adding ethanol, standing, centrifuging, and collecting the polysaccharide precipitate; S4, dissolving the polysaccharide precipitate collected in step S3 in water, dialyzing, and freeze-drying to obtain crude polysaccharide; S5. Perform anion exchange chromatography on the crude polysaccharide obtained in step S4, collect the eluate, and freeze-dry to obtain the sea cucumber chondroitin sulfate.

5. The use according to claim 4, characterized in that Include at least one of the following (1)-(6): (1) In step S1, the volume ratio of chloroform to methanol in the chloroform / methanol mixture is (3-5):1; (2) In step S2, the concentration of EDTA is 4-5 mM, and the concentration of cysteine ​​is 4-5 mM; (3) In step S2, the specific conditions of the enzymatic hydrolysis are: enzymatic hydrolysis at 55-65°C for 8-12 hours; (4) In step S3, the volume ratio of NaCl to ethanol in the NaCl / ethanol mixture is 10:(1-2), and the concentration of NaCl is 1-3M; (5) In step S3, the specific conditions of the standing are: standing at 2-6°C for 10-14 hours; (6) In step S4, the dialysis is performed using a dialysis bag having a molecular weight cut-off of 3-4 kDa.

6. A method for preparing sea cucumber chondroitin sulfate, characterized in that: The following steps are involved: S1, cutting the dried sea cucumber body wall into pieces and homogenizing them, and then defatting them with a chloroform / methanol mixture to obtain sea cucumber residues; S2, mixing the defatted sea cucumber residue with a papain solution containing EDTA and cysteine ​​for enzymolysis, centrifuging after enzymolysis, and collecting the supernatant; S3, adding cetylpyridinium chloride solution to the supernatant, stirring until the precipitation is complete, using NaCl / ethanol mixed solution to redissolve the precipitate, then adding ethanol, standing, centrifuging, and collecting the polysaccharide precipitate; S4, dissolving the polysaccharide precipitate collected in step S3 in water, dialyzing, and freeze-drying to obtain crude polysaccharide; S5. Perform anion exchange chromatography on the crude polysaccharide obtained in step S4, collect the eluate, and freeze-dry to obtain the sea cucumber chondroitin sulfate.

7. The preparation method according to claim 6, characterized in that: Include at least one of the following (1)-(6): (1) In step S1, the volume ratio of chloroform to methanol in the chloroform / methanol mixture is (3-5):1; (2) In step S2, the concentration of EDTA is 4-5 mM, and the concentration of cysteine ​​is 4-5 mM; (3) In step S2, the specific conditions of the enzymatic hydrolysis are: enzymatic hydrolysis at 55-65°C for 8-12 hours; (4) In step S3, the volume ratio of NaCl to ethanol in the NaCl / ethanol mixture is 10:(1-2), and the concentration of NaCl is 1-3M; (5) In step S3, the specific conditions of the standing are: standing at 2-6°C for 10-14 hours; (6) In step S4, the dialysis is performed using a dialysis bag having a molecular weight cut-off of 3-4 kDa.

8. A drug for preventing or treating neurodegenerative diseases, characterized in that: The medicine comprises sea cucumber chondroitin sulfate.

9. The drug according to claim 8, characterized in that The sea cucumber chondroitin sulfate is prepared by the preparation method according to claim 6 or 7.

10. The drug according to claim 8, characterized in that The medicine also includes a pharmaceutically acceptable carrier or excipient.

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