Biological treatment medicine for cognitive impairment

Abstract

The invention relates to a biological treatment medicine for cognitive impairment, and belongs to the technical field of neuromedicine. The active ingredient of the medicine is litchi chinensis bacterium oligosaccharide extracted and prepared from litchi chinensis bacteria, and the preparation method comprises the following steps: (1) cleaning fresh litchi chinensis bacterium entities, freeze-drying and crushing to obtain litchi chinensis bacterium powder; (2) carrying out multi-enzyme synergistic enzymolysis on the polysaccharide; (3) adjusting the pH value, and then carrying out microwave-assisted acidolysis treatment; (4) separating and purifying by using an ultrafiltration membrane and a nanofiltration membrane to remove small molecular impurities; and (5) freeze-drying to obtain high-purity litchi chinensis bacteria oligosaccharide powder. Experimental results show that the litchi chinensis bacterium oligosaccharide prepared by the invention has a remarkable protection effect on nerve cell injury induced by an A beta1-42 oligomer, and can effectively improve the learning ability and the memory ability of an AD (Alzheimer's disease) model mouse, so that the overall cognitive function of the AD model mouse is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of neurology, and particularly relates to a biotherapeutic drug for cognitive impairment. Background Art

[0002] Cognitive dysfunction refers to the decline in an individual's abilities in aspects such as attention, memory, language, executive function, and social cognition. Such dysfunctions have a significant impact on the patient's daily life, social skills, and mental health, resulting in a significant decline in the quality of life. Although cognitive dysfunction can be caused by various factors, including brain injury, mental illness, and neurodegenerative diseases, Alzheimer's disease (AD) is one of the main causes of cognitive dysfunction.

[0003] The pathological mechanism of cognitive dysfunction is complex and involves multiple neurobiological processes. Common pathological features in the brains of AD patients include the deposition of β-amyloid plaques and the formation of neurofibrillary tangles, which lead to neuron death, synaptic loss, and neural network reorganization, thereby affecting cognitive function. In addition, neuroinflammation, oxidative stress, and neurotransmitter imbalance are also important influencing factors for cognitive dysfunction.

[0004] Currently, the clinical treatment for cognitive dysfunction mainly focuses on drugs that improve symptoms, such as cholinesterase inhibitors and NMDA receptor antagonists. These drugs can relieve symptoms to a certain extent, but have limited effects on disease progression, and patients may face adverse reactions during the treatment. Since existing treatment methods cannot significantly improve cognitive dysfunction and have relatively large side effects, there is an urgent need to develop new biotherapeutic drugs. Summary of the Invention

[0005] The purpose of the present invention is to provide a biotherapeutic drug for cognitive impairment, thereby reducing side effects while treating cognitive dysfunction caused by AD.

[0006] To achieve the above purpose, the present invention provides the following technical solutions:

[0007] First, the present invention provides a biotherapeutic drug for cognitive dysfunction, and the active ingredient of the drug is the oligopolysaccharide extracted from Termitomyces albuminosus.

[0008] Preferably, the oligopolysaccharide of Termitomyces albuminosus is prepared by the following preparation method:

[0009] (1) Wash, freeze-dry, and pulverize the fresh Termitomyces albuminosus fruiting body to obtain Termitomyces albuminosus powder;

[0010] (2) Mix the Termitomyces albuminosus powder with a buffer solution, and add cellulase and xylanase for enzymatic hydrolysis;

[0011] (3) After adjusting the pH, add β-glucanase and pectinase and continue enzymatic hydrolysis;

[0012] (4) After centrifuging and concentrating the enzymatic hydrolysate, add anhydrous ethanol to precipitate crude polysaccharides;

[0013] (5) Dissolve the crude polysaccharides in an acidic solution and perform microwave-assisted acid hydrolysis treatment;

[0014] (6) Separate and purify the acid hydrolysate through ultrafiltration membrane and nanofiltration membrane to obtain a concentrated solution of litchi mushroom oligosaccharides;

[0015] (7) Lyophilize to obtain litchi mushroom oligosaccharides.

[0016] Preferably, the specific steps of step (2) are as follows: Mix the litchi mushroom powder with a citric acid-disodium hydrogen phosphate buffer solution at pH 5.0 - 5.5 according to a material-liquid ratio of 1:15 to 1:25, add 1% - 1.5% cellulase and 1% - 1.5% xylanase, and perform enzymatic hydrolysis in a water bath at 45°C to 50°C for 2 hours;

[0017] The specific steps of step (3) are as follows: Adjust the pH to 4.5 - 5.0, add 0.5% - 1.0% β-glucanase and 0.5% - 1.0% pectinase, and continue enzymatic hydrolysis for 1.5 hours;

[0018] The specific steps of step (4) are as follows: After inactivating the enzyme, centrifuge the enzymatic hydrolysate to obtain the supernatant, concentrate the supernatant to 1 / 5 to 1 / 10 of the original volume with a rotary evaporator, add anhydrous ethanol to a final concentration of 70% - 80%, stand at 4°C for 12 hours, and centrifuge to collect the crude polysaccharides;

[0019] The specific steps of step (5) are as follows: Dissolve the crude polysaccharides in a 1% citric acid solution according to a material-liquid ratio of 1:20 to 1:30, adjust the pH to 3.5, place it in a microwave reactor, perform microwave-assisted acid hydrolysis at 60°C with a power of 350W to 400W for 15 minutes, cool to room temperature and then centrifuge to obtain the supernatant;

[0020] The specific steps of step (6) are as follows: Pass the acid hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, collect the permeate, and concentrate the permeate through a nanofiltration membrane with a molecular weight cut-off of 150 Da to obtain a concentrated solution of litchi mushroom oligosaccharides;

[0021] The specific steps of step (7) are as follows: Lyophilize the concentrated solution of litchi mushroom oligosaccharides at -50°C to obtain litchi mushroom oligosaccharide powder.

[0022] Preferably, the cognitive impairment is AD cognitive impairment caused by Aβ1-42 oligomers;

[0023] The drug carrier of the biotherapeutic drug is physiological saline;

[0024] In the biotherapeutic agent, the concentration of the litchi mushroom oligosaccharide is 50 - 200 μg / mL.

[0025] Preferably, the AD cognitive dysfunction includes learning ability disorder and memory ability disorder.

[0026] Secondly, the present invention provides a method for preparing oligosaccharides with the ability to treat AD cognitive dysfunction, and the method includes the following steps:

[0027] (1) Take fresh litchi mushroom fruiting bodies, rinse with deionized water to remove impurities, drain, freeze-dry, pulverize and sieve to obtain litchi mushroom powder;

[0028] (2) Mix the litchi mushroom powder with a citric acid - disodium hydrogen phosphate buffer solution at pH 5.0 - 5.5 at a solid - liquid ratio of 1:15 to 1:25, add 1.0% - 1.5% cellulase and 1.0% - 1.5% xylanase, and enzymatically hydrolyze in a water bath at 45°C to 50°C for 2 hours;

[0029] (3) Adjust the pH to 4.5 - 5.0, add 0.5% - 1.0% β - glucanase and 0.1% - 1.0% pectinase, and continue enzymatic hydrolysis for 1.5 hours;

[0030] (4) After inactivating the enzymes, centrifuge the enzymatic hydrolysate to obtain the supernatant, concentrate the supernatant to 1 / 5 to 1 / 10 of the original volume with a rotary evaporator, add absolute ethanol to a final concentration of 70% - 80%, stand at 4°C for 12 hours, and centrifuge to collect the crude polysaccharide;

[0031] (5) Dissolve the crude polysaccharide in a 1% citric acid solution at a solid - liquid ratio of 1:20 to 1:30, adjust the pH to 3.5, place it in a microwave reactor, and carry out microwave - assisted acid hydrolysis at 60°C with a power of 350W to 400W for 15 minutes. After cooling to room temperature, centrifuge to obtain the supernatant;

[0032] (6) Pass the acid hydrolysate through an ultrafiltration membrane with a molecular weight cut - off of 3 kDa, collect the permeate, and concentrate the permeate through a nanofiltration membrane with a molecular weight cut - off of 150 Da to obtain a concentrated litchi mushroom oligosaccharide solution;

[0033] (7) Freeze - dry the concentrated litchi mushroom oligosaccharide solution at - 50°C to obtain litchi mushroom oligosaccharide powder.

[0034] Preferably, in step (2), the pH is 5.0, the solid - liquid ratio is 1:25, the addition amount of cellulase is 1.5%, the addition amount of xylanase is 1.0%, and the temperature of the water bath is 50°C;

[0035] In step (3), the pH is 4.5, the addition amount of β - glucanase is 1.0%, and the addition amount of pectinase is 0.5%;

[0036] In step (4), the supernatant was concentrated to 1 / 10 of the original volume by a rotary evaporator, and absolute ethanol was added to a final concentration of 80%;

[0037] In step (5), the material-liquid ratio was 1:20 and the power was 400 W.

[0038] In addition, the present invention provides an application of the Termitomyces albuminosus oligosaccharide in the preparation of a biotherapeutic drug for treating AD cognitive dysfunction, and the Termitomyces albuminosus oligosaccharide is prepared by the method described in claim 6.

[0039] Preferably, the AD cognitive dysfunction is AD cognitive dysfunction caused by Aβ1-42 oligomers;

[0040] The AD cognitive dysfunction includes learning ability disorder and memory ability disorder;

[0041] In the drug, the concentration of the Termitomyces albuminosus oligosaccharide is 50-200 μg / mL.

[0042] Finally, the present invention provides an application of the Termitomyces albuminosus oligosaccharide in the preparation of a biotherapeutic drug for treating nerve cell damage caused by Aβ1-42 oligomers, and the concentration of the Termitomyces albuminosus oligosaccharide is 50-200 μg / mL.

[0043] The beneficial effects of the present invention are as follows:

[0044] Experimental results show that the Termitomyces albuminosus oligosaccharide prepared by the steps of enzymatic hydrolysis treatment, microwave-assisted acid hydrolysis, separation and purification, etc. in the present invention has a significant protective effect on nerve cell damage induced by Aβ1-42 oligomers. While Termitomyces albuminosus polysaccharide could not significantly improve cell survival rate under the same conditions. This comparison further highlights the unique advantages of Termitomyces albuminosus oligosaccharide.

[0045] Secondly, Termitomyces albuminosus oligosaccharide a also shows a certain protective effect on nerve cell damage induced by H2O2, but its effect is far less significant than the protective effect on damage induced by Aβ1-42 oligomers ( Figure 2 ). This indicates that the mechanism of action of Termitomyces albuminosus oligosaccharide may be specific and mainly targets Aβ1-42-related neurotoxic damage.

[0046] In addition, the results of animal experiments show that the Termitomyces albuminosus oligosaccharide prepared by the special preparation method in the present invention can effectively improve the learning disorder and memory disorder of AD mice, thereby improving the cognitive dysfunction of AD mice, thus first revealing the potential value of Termitomyces albuminosus oligosaccharide in the treatment of cognitive dysfunction and filling the blank in the research field of Termitomyces albuminosus oligosaccharide in neurodegenerative diseases.

[0047] Finally, the present invention uses natural lychee mushroom as a raw material, and prepares lychee mushroom oligosaccharides through mild enzymatic hydrolysis, acid hydrolysis and purification processes, ensuring the safety and biocompatibility of the drug. Description of the Drawings

[0048] Figure 1 The protective effects of lychee mushroom oligosaccharides a, b and lychee mushroom polysaccharides a, b at different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL) on Aβ1-42 oligomer-induced nerve cell damage;

[0049] Figure 2 The protective effects of lychee mushroom oligosaccharide a at different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL) on H2O2-induced nerve cell damage;

[0050] Figure 3 The differences in the number of platform crossings and the residence time in the target quadrant of each group of mice in the exploration experiment. Detailed Embodiments

[0051] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the protection scope of the present invention.

[0052] Termitomyces albuminosus polysaccharide is an active polysaccharide extracted from Termitomyces albuminosus (scientific name: Termitomyces albuminosus). Existing research has found that Termitomyces albuminosus polysaccharide has various functions such as immune regulation and anti-tumor. However, there is less research on lychee mushroom, which is also in the order Agaricales, and the functions of lychee mushroom polysaccharide and oligosaccharide are completely unknown. Therefore, the present invention explores the functions of lychee mushroom polysaccharide and oligosaccharide in cognitive dysfunction, and attempts to obtain active polysaccharides or oligosaccharides through special preparation methods.

[0053] Example 1

[0054] Preparation method of lychee mushroom oligosaccharide 1:

[0055] (1) Take fresh lychee mushroom entities, rinse with deionized water to remove impurities, drain and freeze-dry (-50 °C, 48 h), pulverize and pass through a 60-mesh sieve to obtain lychee mushroom powder;

[0056] (2) Mix the lychee mushroom powder with a citric acid-disodium hydrogen phosphate buffer solution at pH 5.0 according to a solid-liquid ratio of 1:25 (g / mL), add 1.5% cellulase and 1.0% xylanase, and enzymatically hydrolyze in a water bath at 50 °C for 2 h;

[0057] (3) Adjust the pH to 4.5, add 1.0% β-glucanase and 0.5% pectinase, and continue enzymatic hydrolysis for 1.5 h;

[0058] (4) After inactivating the enzyme (95 °C, 10 minutes), centrifuge the enzymatic hydrolysate (8000 rpm, 10 min) to obtain the supernatant. Concentrate the supernatant to 1 / 10 of the original volume using a rotary evaporator, add absolute ethanol to a final concentration of 80%, let it stand at 4 °C for 12 h, and centrifuge (5000 rpm, 10 min) to collect the crude polysaccharide;

[0059] (5) Dissolve the crude polysaccharide in a 1% citric acid solution at a solid-liquid ratio of 1:20 (g / mL), and adjust the pH to 3.5;

[0060] (6) Place it in a microwave reactor, perform microwave-assisted acid hydrolysis at 60 °C for 15 min (power 400 W), cool to room temperature, and centrifuge (8000 rpm, 5 min) to obtain the supernatant;

[0061] (7) Pass the acid hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, and collect the permeate;

[0062] (8) Concentrate the permeate through a nanofiltration membrane (molecular weight cut-off 150 Da) to remove small molecule impurities (monosaccharides, salts) to obtain the concentrated solution of Termitomyces albuminosus oligosaccharides;

[0063] (9) Freeze-dry the concentrated solution of Termitomyces albuminosus oligosaccharides (-50 °C, 48 h) to obtain the powder of Termitomyces albuminosus oligosaccharides.

[0064] Example 2

[0065] The preparation method of Termitomyces albuminosus oligosaccharide 2 is as follows:

[0066] (1) Take fresh Termitomyces albuminosus fruiting bodies, rinse with deionized water to remove impurities, drain, and then freeze-dry (-50 °C, 48 h), pulverize and pass through a 60-mesh sieve to obtain Termitomyces albuminosus powder;

[0067] (2) Mix the Termitomyces albuminosus powder with a citric acid-disodium hydrogen phosphate buffer solution at pH 5.5 at a solid-liquid ratio of 1:15 (g / mL), add 1.0% cellulase and 1.5% xylanase, and perform enzymatic hydrolysis in a water bath at 45 °C for 2 h;

[0068] (3) Adjust the pH to 5, add 0.5% β-glucanase and 1.0% pectinase, and continue enzymatic hydrolysis for 1.5 h;

[0069] (4) After inactivating the enzyme (95 °C, 10 minutes), centrifuge the enzymatic hydrolysate (8000 rpm, 10 min) to obtain the supernatant. Concentrate the supernatant to 1 / 5 of the original volume using a rotary evaporator, add absolute ethanol to a final concentration of 70%, let it stand at 4 °C for 12 h, and centrifuge (5000 rpm, 10 min) to collect the crude polysaccharide;

[0070] (5) Dissolve the crude polysaccharide in 1% citric acid solution at a solid-liquid ratio of 1:30 (g / mL), and adjust the pH to 3.5.

[0071] (6) Place it in a microwave reactor, carry out microwave-assisted acid hydrolysis at 60 °C for 15 min (power 350 W), then cool to room temperature, and centrifuge (8000 rpm, 5 min) to obtain the supernatant.

[0072] (7) Pass the acid hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, and collect the permeate.

[0073] (8) Concentrate the permeate through a nanofiltration membrane (molecular weight cut-off 150 Da) to remove small molecule impurities (monosaccharides, salts) to obtain the concentrated solution of Litchi mushroom oligosaccharides.

[0074] (9) Freeze-dry the concentrated solution of Litchi mushroom oligosaccharides (-50 °C, 48 h) to obtain the Litchi mushroom oligosaccharide powder.

[0075] Example 3

[0076] The preparation method of Litchi mushroom polysaccharide 1 is as follows:

[0077] (1) Take fresh Litchi mushroom fruiting bodies, rinse with deionized water to remove impurities, drain, and then freeze-dry (-50 °C, 48 h), and crush through a 60-mesh sieve to obtain Litchi mushroom powder.

[0078] (2) Mix the Litchi mushroom powder with deionized water at a solid-liquid ratio of 1:20 (g / mL), place it in a water bath at 60 °C for extraction for 3 h, and stir every 30 min during this period.

[0079] (3) After the extraction is completed, cool to room temperature, centrifuge (8000 rpm, 10 min), and take the supernatant.

[0080] (4) Add absolute ethanol to the supernatant to a final concentration of 80%, and let it stand at 4 °C for 12 h to promote polysaccharide precipitation.

[0081] (5) Dissolve the crude polysaccharide in deionized water (1% concentration), add Sevag reagent (chloroform: n-butanol = 4:1, v / v), mix well, and then centrifuge (5000 rpm, 10 min) to take the upper aqueous phase, and repeat the operation until no obvious protein precipitation occurs.

[0082] (6) Pass the crude polysaccharide solution through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, and collect the retentate.

[0083] (7) Freeze-dry the retentate (-50 °C, 48 h) to obtain the Litchi mushroom polysaccharide powder.

[0084] Example 4

[0085] The preparation method of Litchi mushroom polysaccharide 2 is as follows:

[0086] (1) Take fresh Hericium coronarium fruiting bodies, rinse them with deionized water to remove impurities, drain them, and then freeze-dry (-50 °C, 48 h), pulverize and sieve through a 60-mesh sieve to obtain Hericium coronarium powder;

[0087] (2) Mix the Hericium coronarium powder with deionized water at a solid-liquid ratio of 1:15 (g / mL), place it in a water bath at 60 °C for extraction for 3 h, and stir every 30 min during this period;

[0088] (3) After the extraction, cool it to room temperature, centrifuge (8000 rpm, 10 min), and take the supernatant;

[0089] (4) Add absolute ethanol to the supernatant to a final concentration of 80%, and let it stand at 4 °C for 12 h to promote polysaccharide precipitation;

[0090] (5) Dissolve the crude polysaccharide in deionized water (3% concentration), add Sevag reagent (chloroform:n-butanol = 4:1, v / v), mix well, centrifuge (5000 rpm, 10 min), take the upper aqueous phase, and repeat the operation until no obvious protein precipitation occurs;

[0091] (6) Pass the crude polysaccharide solution through an ultrafiltration membrane with a molecular weight cut-off of 10 kDa, and collect the retentate;

[0092] (7) Freeze-dry the retentate (-50 °C, 48 h) to obtain Hericium coronarium polysaccharide powder.

[0093] Example 5

[0094] Detect the effects of Hericium coronarium oligosaccharides and Hericium coronarium polysaccharides on nerve damage caused by Aβ1-42 oligomers

[0095] (1) Cell culture

[0096] Digest the SH-SY5Y cells in the logarithmic growth phase with trypsin to prepare a single-cell suspension (density of 1×10 5 cells / mL).

[0097] Inoculate into a 96-well plate, add 100 μL of cell suspension to each well, place the 96-well plate in an incubator at 37 °C and 5% CO2 for culture, and perform subsequent experiments when the cell density reaches 80%-90%.

[0098] (2) Preparation of Aβ1-42 oligomers

[0099] Dissolve Aβ1-42 in sterile PBS to prepare a 1 mM stock solution, aliquot and store at -80 °C.

[0100] Dilute the Aβ1-42 stock solution to 20 μM and 40 μM before the experiment, and incubate at 37 °C for 24 h to form oligomers.

[0101] (3) Preparation of litchi mushroom oligosaccharide and polysaccharide solutions

[0102] Weigh litchi mushroom oligosaccharide a, litchi mushroom oligosaccharide b, litchi mushroom polysaccharide a and litchi mushroom polysaccharide b respectively, dissolve each of them with DMEM and dilute them to concentrations of 100 μg / mL, 200 μg / mL, and 400 μg / mL respectively.

[0103] After filtering and sterilizing the solutions, mix the solutions with 40 μM Aβ1-42 solution in a volume ratio of 1:1 before the experiment to obtain solutions with final concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL.

[0104] (4) Experimental grouping

[0105] Control group: SH-SY5Y cells cultured normally (only DMEM medium added), treated for 24 h.

[0106] Model group: Add 20 μM Aβ1-42 oligomer solution, treated for 24 h.

[0107] Litchi mushroom oligosaccharide / polysaccharide treatment group:

[0108] Add litchi mushroom oligosaccharide a, b or litchi mushroom polysaccharide a, b at different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL), and co-treat with 20 μM Aβ1-42 oligomer for 24 h.

[0109] Set 5 replicate wells for each treatment.

[0110] (5) Cell viability detection

[0111] After the incubation is over, add 10 μL of MTT solution (5 mg / mL) to each well and continue to incubate for 4 h.

[0112] Aspirate the supernatant, add 100 μL of DMSO to each well, and shake for 10 min to fully dissolve the crystals.

[0113] Use an enzyme-linked immunosorbent assay (ELISA) reader to measure the absorbance value (OD value) at a wavelength of 570 nm and calculate the cell survival rate.

[0114] The detection results are as Figure 1 shown. It can be clearly observed from the figure that when treating cells with litchi mushroom polysaccharide a and litchi mushroom polysaccharide b, neither of these two substances can significantly improve the decrease in cell survival rate caused by Aβ1-42 oligomers. This result indicates that litchi mushroom polysaccharide a and litchi mushroom polysaccharide b obtained according to the preparation method of the present invention lack effectiveness in alleviating Aβ1-42 oligomer-induced nerve cell damage.

[0115] However, for litchi mushroom oligosaccharide a and litchi mushroom oligosaccharide b, the experimental results showed completely different trends. At a concentration of 50 μg / mL, these two oligosaccharides showed obvious protective effects and could effectively reduce the decrease in cell viability caused by Aβ1-42 oligomers. As the concentration increased, the protective effect was further enhanced and reached the best effect at a concentration of 200 μg / mL. Specific data showed that when the concentration of litchi mushroom oligosaccharide a was 200 μg / mL, the cell viability increased to 87.52%; at the same concentration, the cell viability of litchi mushroom oligosaccharide b was 77.50%. Thus, compared with litchi mushroom oligosaccharide b, litchi mushroom oligosaccharide a showed better effects in alleviating Aβ1-42 oligomer-induced nerve cell damage, and the difference was very significant, showing unexpected technical effects.

[0116] Example 6

[0117] Since the effects of litchi mushroom oligosaccharide a and litchi mushroom oligosaccharide b were similar and litchi mushroom oligosaccharide a had better effects, only litchi mushroom oligosaccharide a was studied in the subsequent experiments of the present invention. To further explore the effect of litchi mushroom oligosaccharide a, the alleviating effect of litchi mushroom oligosaccharide a on H2O2-induced nerve cell damage was detected in this example.

[0118] (1) Cell culture

[0119] The SH-SY5Y cells in the logarithmic growth phase were digested with trypsin to prepare a single-cell suspension (density: 1×10 5 cells / mL).

[0120] They were inoculated into a 96-well plate, 100 μL of cell suspension was added to each well, and the 96-well plate was placed in an incubator at 37 °C and 5% CO2 for culture. Subsequent experiments were carried out when the cell density reached 80%-90%.

[0121] (2) Preparation of H2O2 solution

[0122] H2O2 was prepared into solutions with concentrations of 200 μM and 400 μM.

[0123] (3) Preparation of litchi mushroom oligosaccharide a solution

[0124] Litchi mushroom oligosaccharide a was weighed separately and dissolved and diluted with DMEM to the following concentrations: 100 μg / mL, 200 μg / mL, 400 μg / mL.

[0125] After filtering and sterilizing the solution, the solution was mixed with 400 μM H2O2 solution in a ratio of 1:1 before the experiment to obtain solutions with final concentrations of 50 μg / mL, 100 μg / mL, and 200 μg / mL.

[0126] (4) Experimental grouping

[0127] Control group: SH-SY5Y cells cultured normally (only DMEM medium was added), and treated for 24 h.

[0128] Model group: 200 μM H2O2 solution was added and treated for 24 h.

[0129] Treatment group with litchi mushroom oligosaccharide / polysaccharide:

[0130] Litchi mushroom oligosaccharides a and b at different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL) were added and co-treated with 200 μM H2O2 for 24 h.

[0131] Five replicate wells were set for each treatment.

[0132] (5) Detection of cell viability

[0133] After incubation, 10 μL of MTT solution (5 mg / mL) was added to each well and incubation was continued for 4 h.

[0134] The supernatant was aspirated, and 100 μL of DMSO was added to each well and shaken for 10 min to fully dissolve the crystals.

[0135] The absorbance value (OD value) was measured at a wavelength of 570 nm using a microplate reader, and the cell survival rate was calculated.

[0136] From Figure 2 the results, it can be seen that although litchi mushroom oligosaccharide a also has a certain alleviating effect on H2O2-induced nerve cell damage, the effect is much worse than that on Aβ1-42 oligomer-induced nerve cell damage. This result indicates that the mechanism of action of litchi mushroom oligosaccharide a may have a certain specificity, and there are significant differences in its ability to intervene in different types of nerve damage.

[0137] Example 7

[0138] In this example, the therapeutic effect of litchi mushroom oligosaccharide a on cognitive impairment in AD mice was detected. Experimental grouping:

[0139] Experimental group: 10 6-month-old male APP / PS1 double transgenic AD mice were injected with litchi mushroom oligosaccharide a (prepared with normal saline) at a dose of 2 mg / kg per day for 3 months;

[0140] Model group: 10 6-month-old male APP / PS1 double transgenic AD mice were injected with an equal amount of normal saline for 3 months;

[0141] Control group: 10 6-month-old wild-type mice were injected with an equal amount of normal saline for 3 months.

[0142] Experimental procedure:

[0143] After the experimental treatment, the mice were subjected to the Morris water maze test, including a 1-day adaptation period, a 5-day learning test for finding the hidden platform, and an exploration test 24 hours after the learning test.

[0144] During the Morris water maze test, the room temperature was 26 ± 2 °C and the water temperature was 22 ± 1 °C.

[0145] (1) Adaptation experiment

[0146] Without using the platform, the mice in each group were sequentially placed into the water maze for the adaptation experiment.

[0147] (2) Learning experiment

[0148] The mice in each group were placed into the water from any quadrant (repeated once for each of the 4 quadrants), and allowed to find the hidden platform underwater within a limited time (60 s). The time taken for the mice to find the hidden platform was the latency.

[0149] (3) Exploration experiment

[0150] 24 hours after the learning experiment ended, the platform was removed, and the mice in each group were subjected to the exploration experiment to detect the number of platform crossings and the residence time in the target quadrant.

[0151] During the experiment, no obvious toxic side effects or adverse reactions were observed, and all the mice survived.

[0152] Table 1 Differences in the latency of the Morris water maze test for mice in each group

[0153]

[0154]

[0155] According to the experimental results in Table 1, after 5 days of learning and training, the mice in the experimental group showed a significantly shortened latency compared with the model group. This result indicates that after treating the Alzheimer's disease (AD) model mice with the Hericium erinaceus oligosaccharides prepared by the present invention, their learning ability can be effectively improved. This improvement may be related to the regulatory effect of Hericium erinaceus oligosaccharides on the nervous system function, further verifying its potential value in the treatment of cognitive impairment.

[0156] In addition, from Figure 2 and Figure 3In the data analysis, it can be observed that in the exploratory experiment, the performance of the experimental group of mice was significantly better than that of the model group. Specifically, the number of platform crossings of the experimental group of mice increased significantly, and at the same time, the residence time in the target quadrant also increased significantly. The improvement of these behavioral indicators indicates that after treatment with Termitomyces albuminosus oligosaccharide, the memory ability of AD mice has been significantly improved.

[0157] Based on the above results, after the Termitomyces albuminosus oligosaccharide prepared by the present invention is dissolved in physiological saline to prepare a drug, it can significantly improve the learning ability and memory ability of AD model mice, thereby effectively improving their overall cognitive function. This finding provides an important experimental basis for the development of new drugs for the treatment of cognitive impairment and demonstrates the broad application prospects of Termitomyces albuminosus oligosaccharide in the field of neurodegenerative disease treatment.

Claims

1. A biological therapeutic drug for cognitive dysfunction, characterized in that, The active ingredient of the drug is litchi mushroom oligosaccharide extracted from litchi mushroom.

2. The biotherapeutic drug according to claim 1, characterized in that, The litchi mushroom oligosaccharide is prepared by the following preparation method: (1) Wash the fresh litchi mushroom fruiting body, freeze-dry and then crush it to obtain litchi mushroom powder; (2) Mix the litchi mushroom powder with a buffer solution, and add cellulase and xylanase for enzymatic hydrolysis; (3) After adjusting the pH, add β-glucanase and pectinase and continue enzymatic hydrolysis; (4) The enzymatic hydrolysate is centrifuged and concentrated, and then anhydrous ethanol is added to precipitate crude polysaccharide; (5) The crude polysaccharide is dissolved in an acidic solution and subjected to microwave-assisted acidolysis treatment; (6) The acidolysis solution is separated and purified by an ultrafiltration membrane and a nanofiltration membrane to obtain a litchi mushroom oligosaccharide concentrate; (7) Freeze-dry to obtain litchi mushroom oligosaccharide.

3. The biotherapeutic drug according to claim 2, characterized in that, The specific steps of step (2) are as follows: Mix the litchi mushroom powder with a citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.0 - 5.5 at a material-liquid ratio of 1:15 to 1:25, add 1% - 1.5% cellulase and 1% - 1.5% xylanase, and carry out enzymatic hydrolysis in a water bath at 45°C to 50°C for 2 hours; The specific steps of step (3) are as follows: Adjust the pH to 4.5 - 5.0, add 0.5% - 1.0% β-glucanase and 0.5% - 1.0% pectinase, and continue enzymatic hydrolysis for 1.5 hours; The specific steps of step (4) are as follows: After inactivating the enzyme, the enzymatic hydrolysate is centrifuged to take the supernatant, the supernatant is concentrated to 1 / 5 to 1 / 10 of the original volume with a rotary evaporator, add anhydrous ethanol to a final concentration of 70% - 80%, stand at 4°C for 12 hours, and centrifuge to collect the crude polysaccharide; The specific steps of step (5) are as follows: Dissolve the crude polysaccharide in a 1% citric acid solution at a material-liquid ratio of 1:20 to 1:30, adjust the pH to 3.5, place it in a microwave reactor, and carry out microwave-assisted acidolysis at 60°C with a power of 350W to 400W for 15 minutes. After cooling to room temperature, centrifuge to take the supernatant; The specific steps of step (6) are as follows: The acidolysis solution passes through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, the permeate is collected, and the permeate is concentrated by a nanofiltration membrane with a molecular weight cut-off of 150 Da to obtain a litchi mushroom oligosaccharide concentrate; The specific steps of step (7) are as follows: Freeze-dry the litchi mushroom oligosaccharide concentrate at -50°C to obtain litchi mushroom oligosaccharide powder.

4. The biotherapeutic drug according to claim 3, wherein, The cognitive impairment is AD cognitive impairment caused by Aβ1-42 oligomers; The drug carrier of the biotherapeutic drug is physiological saline; In the biotherapeutic drug, the concentration of the litchi mushroom oligosaccharide is 50 - 200 μg / mL.

5. The biotherapeutic agent according to claim 4, wherein The AD cognitive impairment includes learning ability impairment and memory ability impairment.

6. A method for preparing oligosaccharides with therapeutic effects on AD cognitive dysfunction, characterized in that, The method includes the following steps: (1) Take fresh litchi mushroom fruiting bodies, rinse them with deionized water to remove impurities, drain them, freeze-dry, crush them and then sieve them to obtain litchi mushroom powder; (2) Mix the litchi mushroom powder with a citric acid-disodium hydrogen phosphate buffer solution with a pH of 5.0 - 5.5 at a material-liquid ratio of 1:15 to 1:25, add 1.0% - 1.5% cellulase and 1.0% - 1.5% xylanase, and carry out enzymatic hydrolysis in a water bath at 45°C to 50°C for 2 hours; (3) Adjust the pH to 4.5 - 5.0, add 0.5% - 1.0% β-glucanase and 0.1% - 1.0% pectinase, and continue enzymatic hydrolysis for 1.5 hours; (4) After inactivating the enzyme, centrifuge the enzymatic hydrolysate to obtain the supernatant. Concentrate the supernatant to 1 / 5 to 1 / 10 of the original volume using a rotary evaporator, add absolute ethanol to a final concentration of 70% - 80%, and let it stand at 4°C for 12 hours. Then centrifuge to collect the crude polysaccharide; (5) Dissolve the crude polysaccharide in 1% citric acid solution at a solid-liquid ratio of 1:20 to 1:30, adjust the pH to 3.5, place it in a microwave reactor, and carry out microwave-assisted acid hydrolysis at 60°C with a power of 350W to 400W for 15 minutes. After cooling to room temperature, centrifuge to obtain the supernatant; (6) Pass the acid hydrolysate through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, collect the permeate, and concentrate the permeate through a nanofiltration membrane with a molecular weight cut-off of 150 Da to obtain the concentrated solution of Litchi mushroom oligosaccharides; (7) Freeze-dry the concentrated solution of Litchi mushroom oligosaccharides at -50°C to obtain the powder of Litchi mushroom oligosaccharides.

7. The method according to claim 6, characterized in that, In the step (2), the pH is 5.0, the solid-liquid ratio is 1:25, the addition amount of cellulase is 1.5%, the addition amount of xylanase is 1.0%, and the temperature of the water bath is 50°C; In the step (3), the pH is 4.5, the addition amount of β-glucanase is 1.0%, and the addition amount of pectinase is 0.5%; In the step (4), the supernatant is concentrated to 1 / 10 of the original volume using a rotary evaporator, and absolute ethanol is added to a final concentration of 80%; In the step (5), the solid-liquid ratio is 1:20, and the power is 400W.

8. Use of litchi mushroom oligosaccharide in the preparation of a biotherapeutic drug for treating AD cognitive dysfunction, characterized in that, The Litchi mushroom oligosaccharides are prepared by the method described in claim 6.

9. The application according to claim 8, wherein The AD cognitive impairment is the AD cognitive impairment caused by Aβ1 - 42 oligomers; The AD cognitive impairment includes learning ability impairment and memory ability impairment; In the drug, the concentration of the Litchi mushroom oligosaccharides is 50 - 200 μg / mL.

10. Use of litchi mushroom oligosaccharide in the preparation of a biotherapeutic drug for treating nerve cell damage caused by Aβ1-42 oligomers, characterized in that, The concentration of the Litchi mushroom oligosaccharides is 50 - 200 μg / mL.

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