A biological therapeutic drug for cognitive impairment

Abstract

The present invention relates to a biological therapeutic drug for cognitive dysfunction, and belongs to the field of neuromedicine technology. The active ingredient of the drug is litchi oligosaccharide extracted and prepared from litchi mushroom, and its preparation method comprises the following steps: (1) fresh litchi mushroom entity is cleaned, freeze-dried and then crushed to obtain litchi mushroom powder; (2) polysaccharide is hydrolyzed by multi-enzyme collaboration; (3) microwave-assisted acid hydrolysis is performed after pH adjustment; (4) ultrafiltration membrane and nanofiltration membrane are used for separation and purification to remove small molecule impurities; (5) freeze-drying is used to obtain high-purity litchi oligosaccharide powder. Experimental results show that the litchi oligosaccharide prepared by the present invention has a significant protective effect on nerve cell damage induced by Aβ1‑42 oligomers, and can effectively improve the learning ability and memory ability of Alzheimer's disease (AD) model mice, thereby improving their overall cognitive function.

Description

Technical Field

[0001] The present invention belongs to the technical field of neuromedicine, and in particular relates to a biological therapeutic drug for cognitive impairment. Background Art

[0002] Cognitive impairment refers to a decline in an individual's ability to focus, remember, speak, perform executive functions, and social cognition. This impairment significantly impacts patients' daily lives, social skills, and mental health, leading to a significant decline in their quality of life. While cognitive impairment can be caused by a variety of factors, including brain injury, mental illness, and neurodegenerative diseases, Alzheimer's disease (AD) is a leading cause of cognitive impairment.

[0003] The pathological mechanisms of cognitive impairment are complex, involving 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. These pathological features lead to neuronal death, synaptic loss, and neural network reorganization, thereby affecting cognitive function. In addition, neuroinflammation, oxidative stress, and neurotransmitter imbalance are also important factors affecting cognitive impairment.

[0004] Currently, clinical treatments for cognitive impairment primarily focus on symptom-modifying medications, such as cholinesterase inhibitors and NMDA receptor antagonists. While these medications can alleviate symptoms to a certain extent, their impact on disease progression is limited, and patients may experience adverse reactions during treatment. Because existing treatments fail to significantly improve cognitive impairment and are associated with significant side effects, the development of novel biotherapeutic drugs is urgently needed. Summary of the Invention

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

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

[0007] First, the present invention provides a biological therapeutic drug for cognitive dysfunction, wherein the active ingredient of the drug is litchi oligosaccharide extracted from litchi mushroom.

[0008] Preferably, the litchi oligosaccharide is prepared by the following preparation method:

[0009] (1) washing fresh lychee mushrooms, freeze-drying and then crushing to obtain lychee mushroom powder;

[0010] (2) mixing the litchi mushroom powder with a buffer solution, and adding cellulase and xylanase for enzymatic hydrolysis;

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

[0012] (4) the enzymatic hydrolysate is centrifuged and concentrated, and then anhydrous ethanol is added to precipitate the crude polysaccharide;

[0013] (5) The crude polysaccharide was dissolved in an acidic solution and subjected to microwave-assisted acid hydrolysis;

[0014] (6) separating and purifying the acid hydrolyzate through an ultrafiltration membrane and a nanofiltration membrane to obtain a litchi oligosaccharide concentrate;

[0015] (7) Lychee oligosaccharides were obtained by freeze drying.

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

[0017] The specific steps of step (3) are: adjusting the pH to 4.5-5.0, adding 0.5%-1.0% β-glucanase and 0.5%-1.0% pectinase, and continuing enzymolysis for 1.5 hours;

[0018] The specific steps of step (4) are as follows: after the enzyme is inactivated, the enzymatic hydrolyzate is centrifuged to obtain the supernatant, the supernatant is concentrated to 1 / 5 to 1 / 10 of the original volume using a rotary evaporator, anhydrous ethanol is added to a final concentration of 70%-80%, and the mixture is allowed to stand at 4°C for 12 hours, and the crude polysaccharide is collected by centrifugation;

[0019] The specific steps of step (5) are as follows: dissolving the crude polysaccharide in a 1% citric acid solution at a material-liquid ratio of 1:20 to 1:30, adjusting the pH to 3.5, placing the crude polysaccharide in a microwave reactor, performing microwave-assisted acid hydrolysis at 60° C. and a power of 350 W to 400 W for 15 minutes, cooling the crude polysaccharide to room temperature, and then centrifuging the resulting solution to obtain the supernatant;

[0020] The specific steps of step (6) are: the acid hydrolyzate is passed through an ultrafiltration membrane with a molecular weight cut-off of 3 kDa, the permeate is collected, and the permeate is concentrated through a nanofiltration membrane with a molecular weight cut-off of 150 Da to obtain a litchi oligosaccharide concentrate;

[0021] The specific steps of step (7) are: freeze-drying the litchi oligosaccharide concentrate at -50°C to obtain litchi oligosaccharide powder.

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

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

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

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

[0026] Secondly, the present invention provides a method for preparing oligosaccharides for treating AD cognitive dysfunction, the method comprising the following steps:

[0027] (1) fresh lychee mushrooms were washed with deionized water to remove impurities, drained, freeze-dried, crushed, and sieved to obtain lychee mushroom powder;

[0028] (2) mixing litchi mushroom powder with citric acid-sodium hydrogen phosphate buffer at a pH of 5.0-5.5 at a material-liquid ratio of 1:15 to 1:25, adding 1.0%-1.5% cellulase and 1.0%-1.5% xylanase, and enzymolyzing 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 the enzyme is inactivated, the enzymatic hydrolyzate is centrifuged to obtain the supernatant, which is concentrated to 1 / 5 to 1 / 10 of the original volume using a rotary evaporator, and anhydrous ethanol is added to a final concentration of 70%-80%. The mixture is allowed to stand at 4°C for 12 hours, and the crude polysaccharide is collected by centrifugation;

[0031] (5) The crude polysaccharide was dissolved in 1% citric acid solution at a solid-liquid ratio of 1:20 to 1:30, the pH was adjusted to 3.5, and the solution was placed in a microwave reactor for microwave-assisted acid hydrolysis at 60°C and a power of 350W to 400W for 15 minutes. The solution was cooled to room temperature and centrifuged to obtain the supernatant.

[0032] (6) the acid hydrolyzate was passed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, the permeate was collected, and the permeate was concentrated through a nanofiltration membrane with a molecular weight cutoff of 150 Da to obtain a litchi oligosaccharide concentrate;

[0033] (7) The litchi oligosaccharide concentrate was freeze-dried at -50°C to obtain litchi oligosaccharide powder.

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

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

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

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

[0038] In addition, the present invention provides a use of litchi oligosaccharide in the preparation of a biotherapeutic drug for treating AD cognitive dysfunction, wherein the litchi oligosaccharide is prepared by the method according to claim 6.

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

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

[0041] In the medicine, the concentration of the litchi oligosaccharide is 50-200 μg / mL.

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

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

[0044] Experimental results showed that the litchi oligosaccharide, prepared through enzymatic hydrolysis, microwave-assisted acid hydrolysis, and separation and purification, significantly protected against neuronal damage induced by Aβ1-42 oligomers. However, litchi polysaccharide failed to significantly improve cell survival under the same conditions. This comparison further highlights the unique advantages of litchi oligosaccharide.

[0045] Secondly, litchi oligosaccharide a also showed a certain protective effect on H2O2-induced nerve cell damage, but its effect was far less significant than that on Aβ1-42 oligomer-induced damage ( Figure 2 This suggests that the mechanism of action of litchi oligosaccharides may be specific, mainly targeting Aβ1-42-related neurotoxic damage.

[0046] In addition, the results of animal experiments showed that the litchi oligosaccharides prepared by the special preparation method of the present invention can effectively improve the learning and memory disorders of AD mice, thereby improving the cognitive dysfunction of AD mice. This revealed for the first time the potential value of litchi oligosaccharides in the treatment of cognitive dysfunction and filled the gap in the research field of litchi oligosaccharides in neurodegenerative diseases.

[0047] Finally, the present invention uses natural litchi mushroom as raw material and prepares litchi mushroom oligosaccharides through mild enzymatic hydrolysis, acid hydrolysis and purification processes, thereby ensuring the safety and biocompatibility of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0050] Figure 3 The differences in the number of platform crossings and the time spent in the target quadrant among the mice in each group during the exploration experiment. DETAILED DESCRIPTION

[0051] The following is a clear and complete description of the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without making any creative efforts shall fall within the scope of protection of the present invention.

[0052] Termitomyces albuminosus polysaccharide is an active polysaccharide extracted from the Termitomyces albuminosus mushroom. Existing research has found that Termitomyces albuminosus polysaccharide exhibits various effects, including immunomodulatory and anti-tumor effects. However, research on the litchi mushroom, also a member of the Agaricales order, is less extensive, and the functions of its polysaccharides and oligosaccharides are completely unknown. Therefore, the present invention investigates the effects of litchi polysaccharides and oligosaccharides on cognitive impairment, attempting to obtain active polysaccharides or oligosaccharides through a unique preparation method.

[0053] Example 1

[0054] Preparation method of litchi oligosaccharide 1:

[0055] (1) Fresh lychee mushrooms were washed with deionized water to remove impurities, drained, freeze-dried (-50°C, 48 h), and crushed through a 60-mesh sieve to obtain lychee mushroom powder;

[0056] (2) Litchi mushroom powder was mixed with pH 5.0 citric acid-sodium hydrogen phosphate buffer at a solid-liquid ratio of 1:25 (g / mL), 1.5% cellulase and 1.0% xylanase were added, and enzymatic hydrolysis was carried out in a 50°C water bath 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 inactivation of the enzyme (95°C, 10 min), the enzymatic hydrolyzate was centrifuged (8000 rpm, 10 min) and the supernatant was collected. The supernatant was concentrated to 1 / 10 of the original volume using a rotary evaporator, and anhydrous ethanol was added to a final concentration of 80%. The mixture was allowed to stand at 4°C for 12 h and centrifuged (5000 rpm, 10 min) to collect the crude polysaccharide.

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

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

[0061] (7) The acid hydrolyzate was passed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the permeate was collected;

[0062] (8) The permeate was concentrated through a nanofiltration membrane (molecular weight cut-off 150 Da) to remove small molecule impurities (monosaccharides, salts) to obtain a litchi oligosaccharide concentrate;

[0063] (9) The litchi oligosaccharide concentrate was freeze-dried (-50°C, 48h) to obtain litchi oligosaccharide powder.

[0064] Example 2

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

[0066] (1) Fresh lychee mushrooms were washed with deionized water to remove impurities, drained, freeze-dried (-50°C, 48 h), and crushed through a 60-mesh sieve to obtain lychee mushroom powder;

[0067] (2) Litchi mushroom powder was mixed with citric acid-sodium hydrogen phosphate buffer (pH 5.5) at a solid-liquid ratio of 1:15 (g / mL), 1.0% cellulase and 1.5% xylanase were added, and enzymatic hydrolysis was carried out in a 45°C water bath 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 inactivation of the enzyme (95°C, 10 min), the enzymatic hydrolyzate was centrifuged (8000 rpm, 10 min) and the supernatant was collected. The supernatant was concentrated to 1 / 5 of the original volume using a rotary evaporator, and anhydrous ethanol was added to a final concentration of 70%. The mixture was allowed to stand at 4°C for 12 h and centrifuged (5000 rpm, 10 min) to collect the crude polysaccharide.

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

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

[0072] (7) The acid hydrolyzate was passed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the permeate was collected;

[0073] (8) The permeate was concentrated through a nanofiltration membrane (molecular weight cut-off 150 Da) to remove small molecule impurities (monosaccharides, salts) to obtain a litchi oligosaccharide concentrate;

[0074] (9) The litchi oligosaccharide concentrate was freeze-dried (-50°C, 48h) to obtain litchi oligosaccharide powder.

[0075] Example 3

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

[0077] (1) Fresh lychee mushrooms were washed with deionized water to remove impurities, drained, freeze-dried (-50°C, 48 h), and crushed through a 60-mesh sieve to obtain lychee mushroom powder;

[0078] (2) Litchi mushroom powder was mixed with deionized water at a solid-liquid ratio of 1:20 (g / mL) and extracted in a 60°C water bath for 3 h, stirring every 30 min.

[0079] (3) After extraction, cool to room temperature, centrifuge (8000 rpm, 10 min), and collect the supernatant;

[0080] (4) Anhydrous ethanol was added to the supernatant to a final concentration of 80% and allowed to 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 thoroughly, and centrifuge (5000 rpm, 10 min). Take the upper aqueous phase and repeat the operation until no obvious protein precipitation occurs.

[0082] (6) The crude polysaccharide solution was passed through an ultrafiltration membrane with a molecular weight cut-off of 5 kDa, and the retentate was collected;

[0083] (7) The retentate was freeze-dried (-50°C, 48 h) to obtain litchi polysaccharide powder.

[0084] Example 4

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

[0086] (1) Fresh lychee mushrooms were washed with deionized water to remove impurities, drained, freeze-dried (-50°C, 48 h), and crushed through a 60-mesh sieve to obtain lychee mushroom powder;

[0087] (2) Litchi mushroom powder was mixed with deionized water at a solid-liquid ratio of 1:15 (g / mL) and placed in a 60°C water bath for extraction for 3 h, stirring every 30 min.

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

[0089] (4) Anhydrous ethanol was added to the supernatant to a final concentration of 80% and allowed to 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 thoroughly, and centrifuge (5000 rpm, 10 min). Take the upper aqueous phase and repeat the operation until no obvious protein precipitation occurs.

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

[0092] (7) The retentate was freeze-dried (-50°C, 48 h) to obtain litchi polysaccharide powder.

[0093] Example 5

[0094] Detection of the effects of litchi oligosaccharides and litchi polysaccharides on neuronal damage caused by Aβ1-42 oligomers

[0095] (1) Cell culture

[0096] SH-SY5Y cells in the logarithmic growth phase were digested with trypsin and prepared into a single-cell suspension (density of 1×10 5 cells / mL).

[0097] The cells were seeded into a 96-well plate, 100 μL of cell suspension was added to each well, and the 96-well plate was placed in a 37°C, 5% CO2 incubator for culture. Subsequent experiments were performed when the cell density reached 80%-90%.

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

[0099] Aβ1-42 was dissolved in sterile PBS to prepare a 1 mM stock solution, which was aliquoted and stored at -80°C.

[0100] Before the experiment, Aβ1-42 stock solution was diluted to 20 μM and 40 μM and incubated at 37°C for 24 h to form oligomers.

[0101] (3) Preparation of Litchi Oligosaccharide and Polysaccharide Solutions

[0102] Litchi oligosaccharide a, litchi oligosaccharide b, litchi polysaccharide a and litchi polysaccharide b were weighed respectively, dissolved in DMEM and diluted to concentrations of 100 μg / mL, 200 μg / mL and 400 μg / mL respectively.

[0103] After the solution was sterilized by filtration, it was mixed with 40 μM Aβ1-42 solution at 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 groups

[0105] Control group: Normally cultured SH-SY5Y cells (only DMEM medium was added) were treated for 24 hours.

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

[0107] Litchi oligosaccharide / polysaccharide treatment group:

[0108] Different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL) of litchi oligosaccharides a, b or litchi polysaccharides a, b were added and co-treated with 20 μM Aβ1-42 oligomers for 24 h.

[0109] Five replicate wells were set up for each treatment.

[0110] (5) Cell viability assay

[0111] After the incubation, 10 μL of MTT solution (5 mg / mL) was added to each well and the cells were incubated for another 4 h.

[0112] The supernatant was removed by aspiration, 100 μL DMSO was added to each well, and the mixture was shaken for 10 min to fully dissolve the crystals.

[0113] The absorbance (OD value) was measured at a wavelength of 570 nm using a microplate reader to calculate the cell survival rate.

[0114] Test results such as Figure 1 As shown in the figure, it can be clearly observed that when cells were treated with litchi polysaccharide a and litchi polysaccharide b, neither substance significantly improved the decrease in cell survival caused by Aβ1-42 oligomers. This result indicates that litchi polysaccharide a and litchi polysaccharide b obtained according to the preparation method of the present invention are not effective in alleviating neuronal cell damage induced by Aβ1-42 oligomers.

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

[0116] Example 6

[0117] Since the effects of litchi oligosaccharide a and litchi oligosaccharide b are similar and litchi oligosaccharide a is more effective, the subsequent experiments of the present invention only study litchi oligosaccharide a. To further explore the effect of litchi oligosaccharide a, this example tests the effect of litchi oligosaccharide a on the alleviation of H2O2-induced nerve cell damage.

[0118] (1) Cell culture

[0119] SH-SY5Y cells in the logarithmic growth phase were digested with trypsin and prepared into a single-cell suspension (density of 1×10 5 cells / mL).

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

[0121] (2) Preparation of H2O2 solution

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

[0123] (3) Preparation of Litchi Oligosaccharide A Solution

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

[0125] After the solution was sterilized by filtration, it was mixed with 400 μM H2O2 solution at 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 groups

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

[0128] Model group: Add 200 μM H2O2 solution and treat for 24 h.

[0129] Litchi oligosaccharide / polysaccharide treatment group:

[0130] Different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL) of litchi oligosaccharides a and b were added and treated with 200 μM H2O2 for 24 h.

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

[0132] (5) Cell viability assay

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

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

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

[0136] from Figure 2 The results show that although litchi oligosaccharide a has a certain effect on alleviating H2O2-induced nerve cell damage, the effect is far inferior to the alleviating effect on nerve cell damage caused by Aβ1-42 oligomers. This result suggests that the mechanism of action of litchi oligosaccharide a may have certain specificity, and its ability to intervene in different types of nerve damage varies significantly.

[0137] Example 7

[0138] This example tests the therapeutic effect of litchi oligosaccharide a on cognitive dysfunction in AD mice and divides the experimental groups into the following groups:

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

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

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

[0142] Experimental process:

[0143] After the experimental treatment, the mice were subjected to a water maze experiment, which included a 1-day adaptation period, a 5-day learning experiment to find the hidden platform, and an exploration experiment 24 hours after the learning experiment.

[0144] During the water maze experiment, the room temperature was 26±2℃ and the water temperature was 22±1℃.

[0145] (1) Adaptation experiment

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

[0147] (2) Learning Experiment

[0148] Each group of mice was placed into the water from any quadrant (repeat once for each of the four quadrants) and asked to find a hidden platform under the water within a limited time (60 seconds). The time it took for the mice to find the hidden platform was the latency period.

[0149] (3) Exploratory experiments

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

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

[0152] Table 1 Differences in water maze latency among mice in each group

[0153]

[0154]

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

[0156] In addition, from Figure 2 and Figure 3Data analysis revealed that the experimental group significantly outperformed the model group in the exploration experiment. Specifically, the experimental group significantly increased their platform crossings and spent significantly longer in the target quadrant. These behavioral improvements suggest that treatment with litchi oligosaccharides significantly enhanced the memory abilities of AD mice.

[0157] Based on the above results, the prepared lychee oligosaccharide, when dissolved in physiological saline and prepared as a drug, can significantly improve the learning and memory abilities of AD model mice, thereby effectively improving their overall cognitive function. This discovery provides important experimental evidence for the development of new drugs for the treatment of cognitive impairment and demonstrates the broad application prospects of lychee oligosaccharides in the treatment of neurodegenerative diseases.

Claims

1. A drug for treating cognitive dysfunction in Alzheimer's disease, characterized in that: The active ingredient of the drug is litchi oligosaccharide extracted from litchi mushroom; The litchi oligosaccharide is prepared by the following preparation method: (1) Take fresh lychee mushrooms, rinse with deionized water to remove impurities, drain and freeze-dry, crush and sieve to obtain lychee mushroom powder; (2) Mix the litchi mushroom powder with a pH 5.0-5.5 citric acid-sodium hydrogen phosphate buffer at a material-liquid ratio of 1:15 to 1:25, add 1.0%-1.5% cellulase and 1.0%-1.5% xylanase, and perform enzymolysis 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 the enzyme is inactivated, the enzymatic hydrolysate is centrifuged to obtain the supernatant, which is concentrated to 1 / 5 to 1 / 10 of the original volume using a rotary evaporator. Anhydrous ethanol is added to a final concentration of 70%-80%, and the mixture is allowed to stand at 4°C for 12 hours. The crude polysaccharide is then collected by centrifugation. (5) The crude polysaccharide was dissolved in 1% citric acid solution at a solid-liquid ratio of 1:20 to 1:30, the pH was adjusted to 3.5, and the solution was placed in a microwave reactor for microwave-assisted acid hydrolysis at 60°C and a power of 350W to 400W for 15 minutes. The solution was cooled to room temperature and centrifuged to obtain the supernatant. (6) The acid hydrolyzate was passed through an ultrafiltration membrane with a molecular weight cutoff of 3 kDa, and the permeate was collected. The permeate was concentrated through a nanofiltration membrane with a molecular weight cutoff of 150 Da to obtain a concentrated solution of litchi oligosaccharides; (7) The litchi oligosaccharide concentrate was freeze-dried at -50°C to obtain litchi oligosaccharide powder.

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

3. The drug according to claim 2, characterized in that The Alzheimer's disease cognitive dysfunction is Alzheimer's disease cognitive dysfunction caused by Aβ1-42 oligomers; The drug carrier of the drug is normal saline; In the medicine, the concentration of the litchi oligosaccharide is 50-200 μg / mL.

4. The drug according to claim 3, characterized in that The cognitive dysfunction of Alzheimer's disease includes learning impairment and memory impairment.

5. A use of litchi oligosaccharide in the preparation of a drug for treating cognitive dysfunction in Alzheimer's disease, characterized in that: The litchi oligosaccharide is prepared by the preparation method described in claim 1.

6. The use according to claim 5, characterized in that The Alzheimer's disease cognitive dysfunction is Alzheimer's disease cognitive dysfunction caused by Aβ1-42 oligomers; The cognitive dysfunction of Alzheimer's disease includes learning impairment and memory impairment; In the medicine, the concentration of the litchi oligosaccharide is 50-200 μg / mL.

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