Armillariella mycelium polysaccharide with effect of improving cognitive impairment as well as preparation method and application of armillariella mycelium polysaccharide

Abstract

The invention discloses armillariella mycelium polysaccharide ATMP capable of improving cognitive impairment and a preparation method and application of the armillariella mycelium polysaccharide ATMP. The polysaccharide is composed of two components, and the molecular weights of the two components are 2.30 * 10 < 4 > Da and 4.56 * 10 < 5 > Da respectively; the monosaccharide composition comprises mannose (Man), glucose (Glc) and galactose (Gal), and the molar ratio of the mannose (Man) to the glucose (Glc) to the galactose (Gal) is 1: 16.64: 1.96. Animal experiment results show that the armillariella mycelium polysaccharide ATMP can improve cognitive impairment induced by lipopolysaccharide (LPS) by improving the spatial memory ability of mice, repairing the disorder state and deletion injury of neurons in a hippocampal region and relieving neuroinflammatory response, so that the obvious neuroprotective effect is achieved. As a natural brain-benefiting active component, the armillariella tabescens mycelium polysaccharide ATMP has a wide application prospect in the related fields of brain health.

Description

Technical Field

[0001] This invention belongs to the field of bioactive polysaccharide application technology, specifically relating to a polysaccharide of *Bacillus glomeratus* mycelium with the effect of improving cognitive impairment, its preparation method and application. Background Technology

[0002] Cognitive impairment is a group of neurological diseases characterized by declining learning and memory abilities and progressive cognitive function. Its incidence is increasing annually with the accelerating aging of the population, becoming a major challenge threatening global public health. Research indicates that neuroinflammation is one of the core pathological mechanisms driving the development of cognitive impairment. Neuroinflammation is essentially a defense strategy formed by the body in response to the invasion of foreign pathogens. However, if the body is in a state of neuroinflammation for a long time, microglia will be overactivated, becoming a key "trigger" for the onset of neurological diseases. Therefore, intervening in neuroinflammation is considered a key breakthrough in improving cognitive impairment. Currently, drugs developed for neuroinflammation mainly include chemically synthesized drugs (non-steroidal anti-inflammatory drugs, glucocorticoids, etc.), biological agents (recombinant anti-inflammatory protein, targeted antibodies), and traditional natural products. While chemically synthesized drugs can rapidly inhibit the release of inflammatory factors, they suffer from poor targeting and significant side effects. Biological agents, although highly targeted, have high production costs, short half-lives in vivo, and are prone to causing immunogenic reactions. Natural active products have attracted much attention due to their high efficacy and low toxicity.

[0003] Bright fungus, scientific name Armillaria mellea ( Armillariella tabescens (Scop. : Fr.) Sing *Armillaria*, belonging to the phylum Basidiomycota, class Agaricomycetes, family Tricholomataceae, and genus *Armillaria*, has been reported to contain various active ingredients, including leucinogen A, leucinogen B, and leucinogen polysaccharides. Leucinogen A, as the main active ingredient, is commonly used clinically to treat cholecystitis, hepatitis, and leukopenia caused by radiotherapy and chemotherapy. Our laboratory's previous patent application (CN115010822B) disclosed a *Armillaria* mycelial polysaccharide for the prevention and treatment of oral ulcers, demonstrating the significant anti-inflammatory potential of *Armillaria* mycelial polysaccharides. Currently, it remains unclear whether *Armillaria* mycelial polysaccharides can improve cognitive impairment by alleviating neuroinflammation. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to propose a new use of *Bacillus lucida* mycelial polysaccharide in improving cognitive impairment.

[0005] The present invention solves the above-mentioned technical problems through the following technical means:

[0006] The first aspect of this invention discloses the use of *Aureobasidium brevicornu* mycelial polysaccharide (ATMP) in the preparation of a medicament for improving cognitive impairment, wherein the *Aureobasidium brevicornu* mycelial polysaccharide contains two components with molecular weights of 2.30 × 10⁻⁶ and 2.30 × 10⁻⁶ respectively. 4 Da, 4.56 × 105 Da; The monosaccharide composition and molar ratio of the polysaccharide of the *Bacillus globosum* mycelium are mannose (Man): glucose (Glc): galactose (Gal) = 1:16.64:1.96.

[0007] Preferably, the improvement of cognitive impairment refers to enhancing cognitive and spatial memory abilities, restoring hippocampal neuronal disorder and loss, increasing the number of Nissl bodies, and reducing the levels of brain inflammatory factors TNF-α, IL-6, and IL-1β.

[0008] Preferably, the concentration of the *Bacillus lucida* mycelial polysaccharide administered is 400 mg / mL.

[0009] Preferably, the administration method of the *Bacillus glomeratus* mycelial polysaccharide is by gavage.

[0010] Preferably, the drug further includes a pharmaceutically acceptable carrier.

[0011] "Pharmaceutical acceptable" means non-toxic materials that do not reduce the active ingredient. Such pharmaceutically acceptable excipients and carriers are well known in the art (see Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Company (1990) and hand book of Pharmaceutical Excipients, 3rd edition, edited by A. Kibbe, Pharmaceutical Press (2000)).

[0012] Preferably, the pharmaceutically acceptable carrier is selected from one or more of diluents, binders, wetting agents, disintegrants, absorption enhancers, surfactants, and lubricants.

[0013] Preferably, the drug is formulated into a pharmaceutically acceptable dosage form.

[0014] Preferably, the dosage form includes tablets, capsules, granules, pills, syrups, powders, granules, suppositories, drops, emulsions, injections, oral liquids, aerosols, or suspensions.

[0015] A second aspect of the present invention provides a method for preparing the above-mentioned *Aureobasidium brevicornu* mycelial polysaccharide (ATMP), comprising the following steps: (1) Grind the mycelium of Glossy fungus into powder and extract it with hot water at 80-95°C at a material-to-liquid ratio of 1g:(15-25)mL. (2) Concentrate the extract, centrifuge, and precipitate with 75-85% (v / v) ethanol; then remove the alcohol. (3) The product obtained in (2) was subjected to Sevag method to remove protein and rotary evaporation to remove residual organic reagents; (4) The product obtained in (3) was dialyzed with running water using a dialysis bag, and the dialyzed solution was rotary evaporated and freeze-dried to obtain the mycelial polysaccharide ATMP of Glossy Bacteria.

[0016] Preferably, in (2), the concentration of the extract refers to concentrating it to 1 / 10 to 1 / 20 of the original volume of the extract.

[0017] Preferably, in (4), the molecular weight cutoff of the dialysis bag is 3.5~4.0×10⁻⁶. 3 Da.

[0018] A third aspect of the present invention provides the ATMP mycelial polysaccharide prepared by the above preparation method.

[0019] This invention evaluates the neuroprotective effect of *Gymnocytogenes* mycelial polysaccharide through mouse behavioral experiments, pathological sections, and the levels of inflammatory factors in mouse brain tissue.

[0020] The neuroprotective function described in this invention refers to enhancing the cognitive ability of mice, restoring hippocampal neuronal disorder and the number of Nissl bodies, and reducing the levels of brain inflammatory factors TNF-α, IL-6, and IL-1β.

[0021] The mycelial polysaccharide of *Gynostemma pentaphyllum* described in this invention can significantly restore the memory and spatial cognition abilities of mice. Behavioral results show that after administration of ATMP, the mice in this group recovered to 90.40%~94.74% of the level of the control group.

[0022] The mycelial polysaccharide of *Gymnospermum* described in this invention can significantly increase the number of Nissl bodies in the hippocampus, with the number of Nissl bodies in the CA1 and CA3 regions increasing by 152% and 126% respectively compared to the model group.

[0023] The mycelial polysaccharide of *Gynostemma pentaphyllum* described in this invention has a significant inhibitory effect on inflammatory factors in mouse brain tissue, reducing TNF-α, IL-6, and IL-1β by 18.6%, 26.6%, and 27.6%, respectively.

[0024] This invention analyzes the neuroprotective potential of *Gymnospermum globosum* mycelial polysaccharide using an animal model with intraperitoneal injection of lipopolysaccharide (LPS).

[0025] The beneficial effects of this invention are as follows: (1) This invention discovers a neuroprotective polysaccharide ATMP from *Gymnosus lucida* mycelium. The neuroprotective activity of the polysaccharide was evaluated through animal experiments based on its basic physicochemical properties. This invention obtains a mycelial polysaccharide ATMP from *Gymnosus lucida* mycelium using hot water extraction. Activity experiments show that it has significant anti-neuroinflammatory and neuroprotective functions, providing a valuable reference for the industrial application of *Gymnosus lucida* mycelial polysaccharides.

[0026] (2) Animal experiments showed that ATMP mycelial polysaccharide can alleviate neuroinflammation, protect the nervous system, and improve memory function in mice. When the ATMP concentration reached 400 mg / mL, the relevant indicators of the model mice could be restored to the level of the blank group. At the behavioral level, the number of times mice crossed the platform, the proportion of the distance traveled, and the time spent on the platform were detected by the water maze test, which confirmed that ATMP can improve cognitive impairment by alleviating neuroinflammation, protecting the nervous system, and improving cognitive impairment.

[0027] (3) HE staining results showed that ATMP could significantly reduce the loss of neurons in the hippocampus caused by neuroinflammation and restore the disordered arrangement of neurons. In NISSL staining, the number of Nissl bodies in the model group was greatly reduced, and after ATMP administration, it recovered to more than 90% of the level in the blank group. Biochemical index measurements showed that the inflammatory factors TNF-α, IL-6, and IL-1β in the brain were reduced by 18.6%, 26.6%, and 27.6%, respectively, after intervention with 400 mg / mL ATMP. These results demonstrate the ability of ATMP mycelial polysaccharide to resist neuroinflammation and improve cognitive impairment. Attached Figure Description

[0028] Figure 1 The following are chromatograms showing the composition and molecular weight of ATMP monosaccharides in Example 2 of the present invention (A: High-performance liquid chromatography (HPLC) chromatogram of molecular weight of *Bacillus thuringiensis* polysaccharide, B: HPLC chromatogram of monosaccharide composition of *Bacillus thuringiensis* polysaccharide, C: HPLC chromatogram of monosaccharide standard).

[0029] Figure 2 The animal experimental modeling scheme and weight change diagram in Embodiment 3 of the present invention are shown in Figure 3 (A: Animal experimental design diagram, B: Weight change diagram).

[0030] Figure 3 The data graphs for the water maze experiment in the animal experiment of Embodiment 3 of the present invention are as follows: (A: escape distance, B: escape time, C: trajectory, D: number of platform crossings, E: time spent in the target quadrant, F: ratio of target quadrant distance to total distance).

[0031] Figure 4 This is a scanned image of an HE-stained section from Example 3 of the present invention.

[0032] Figure 5 The image shows the scanning and quantification results of Nissl stained sections in Example 3 of the present invention (A: Nissl stained section scan, B: number of CA1 Nissl bodies, C: number of CA3 Nissl bodies).

[0033] Figure 6The image shows the results of brain inflammatory factor measurement in Example 3 of the present invention (A: TNF-α, B: IL-1β, C: IL-6). Detailed Implementation

[0034] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0035] Unless otherwise specified, the test materials and reagents used in the following examples are commercially available or prepared by known methods.

[0036] Unless otherwise specified, all techniques or conditions described in the embodiments can be performed in accordance with the techniques or conditions described in the literature in this field or in the product manual. Unless otherwise specified, the quantitative experiments in the following embodiments are all repeated three times or more, and the results are averaged.

[0037] Example 1: Preparation of polysaccharides from *Bacillus lucida* mycelium (1) Place the mycelium of Gynostemma pentaphyllum (refer to Example 1 in CN115010822B for strain and inoculation method) in an oven at 65°C to dry, grind into powder, pass through an 80-mesh sieve, and set aside for use; (2) Weigh the powdered *Bleucanthus glomeratus* mycelium and place it in a beaker. Prepare 80% ethanol in advance. Add the mycelium to the ethanol at a ratio of 1:5 (material to liquid), stir well, cover with plastic wrap, and place in a water bath at 60 ℃ for 2 h, stirring once every 0.5 h. Then filter using 8 layers of degreased cotton gauze, collect the filter residue, and dry at 65 ℃.

[0038] (3) Take out the decolorized and dried *Gynostemma pentaphyllum* mycelium powder, weigh 10 g, add pure water at a ratio of 1 g: 20 mL, incubate in a 90 ℃ water bath for 2 h, stirring once every 0.5 h, filter with 8 layers of gauze, collect the filtrate, and continue to add pure water to the filter residue in the water bath, repeating 3 times. Combine the extracts.

[0039] (4) The extract was rotary evaporated and concentrated under reduced pressure at 75 °C to 1 / 20 of the original volume. Four times the volume of anhydrous ethanol was added to achieve a final ethanol concentration of 80% (v / v). The polysaccharides were precipitated at 4 °C for 12 h. The precipitate was collected by centrifugation at 3000 rpm for 10 min in a refrigerated centrifuge.

[0040] (5) The obtained precipitate was deproteinized by the Sevag method and the residual organic reagents were removed by rotary evaporation.

[0041] (6) Using a dialysis bag with a molecular weight of 3500 Da, dialyze with running water for 2 days, and then concentrate and freeze-dry the dialysate to obtain the ATMP polysaccharide.

[0042] (7) The sugar content of ATMP was 83.56% and the protein content was 1.3% as determined by the phenol-sulfuric acid method and the Coomassie brilliant blue method.

[0043] Example 2: Determination of the physicochemical properties of polysaccharides from *Bacillus lucida* mycelium The molecular weight of the *Gynostemma pentaphyllum* polysaccharide obtained in Example 1 was determined using high-performance liquid chromatography (HPLC). An appropriate mass of *Gynostemma pentaphyllum* polysaccharide ATMP was weighed and prepared into a 5 mg / mL polysaccharide solution. This solution was then filtered through a 0.22 μm aqueous filter membrane and added to a vial, which was placed in the HPLC sample chamber. The nitrogen valve was turned on, the HPLC program was adjusted, air bubbles were removed, and the column was equilibrated. Injection was then initiated, with an injection volume of 20 μL and a flow rate of 0.600 mL / min. The elution time was detected, and the molecular weight of *Gynostemma pentaphyllum* polysaccharide ATMP was calculated based on the elution time of the dextran.

[0044] The monosaccharide composition of the *Glechotomyces* mycelial polysaccharides obtained in Example 1 was analyzed using an acid hydrolysis-pre-column 1-phenyl-3-methyl-5-pyrazolone (PMP) derivatization method, followed by high-performance liquid chromatography (HPLC). Specifically, 8-10 mg of sample was accurately weighed and dissolved in 5 mL of trifluoroacetic acid. Nitrogen gas was then purged, the test tube was sealed, and the tube was placed in an oil bath at 110 °C or 120 °C for 6 h. The solution was repeatedly evaporated to dryness, methanol was added to remove acidity until neutral, then pure water was added and evaporated to remove methanol. The solution was then evaporated to dryness again, and 1 mL of pure water was added to dissolve the sample.

[0045] For the derivatization of monosaccharide standards, PMP was accurately weighed and dissolved in 1 mL of pure water at a concentration of 3-4 mg. 1 mL of the previously obtained sample and 1 mL of the monosaccharide mixture were then aspirated into separate 10 mL centrifuge tubes. 1 mL of PMP and 1 mL of sodium hydroxide (NaOH) were added, and the mixture was incubated in a 70 °C water bath for 2 h, protected from light. Afterward, 1 mL of hydrochloric acid (HCl) was added to neutralize the solution. An equal volume of chloroform (CHCl3) was added for extraction 2-3 times, collecting the supernatant until no yellow color appeared in the lower layer. The extract was then filtered through a 0.22 μm aqueous filter membrane for later use. This completes the sample preparation process.

[0046] Prepare the four mobile phases required by vacuum filtration, adjust all programs, and then start sample injection. Compare the peak elution time of the sample and the mixed standard, and calculate the molar ratio of each monosaccharide based on the peak area.

[0047] The molecular weight and monosaccharide composition of ATMP were determined according to the above experimental protocol. For example... Figure 1 As shown in Figure A, ATMP mainly consists of 2.30 × 10 4Da and 4.56×10 5 Da is composed of two components. For example Figure 1 As shown in Figures B and C, the results indicate that ATMP is mainly composed of mannose, glucose, and galactose, with a molar ratio of Man: Glc: Gal = 1: 16.64: 1.96.

[0048] Example 3: Establishment of an animal model and drug administration Four- to five-week-old Kunming mice were purchased from the Experimental Animal Center of Anhui Medical University. The animals were divided into four groups: a control group, a model group, a Ganlutner (GV-971) group, and an ATMP group, with 15 mice in each group. After one week of acclimatization feeding, ATMP was administered via gavage. During this period, mouse weight was measured every four days, and food and water intake were recorded. Data showed that compared with the model group, the ATMP group mice had a significantly increased body weight, and ATMP administration had no effect on food and water intake. After five weeks of continuous gavage, mice were intraperitoneally injected with LPS at a dose of 1 mg / kg / day. During intraperitoneal injection, mice exhibited increased eye discharge, piloerection, lethargy, decreased food and water intake, and significant weight loss. Seven days after continuous intraperitoneal LPS injection, behavioral tests were immediately performed on the mice. The mice were then euthanized, and brain tissue was collected for staining and inflammatory factor determination.

[0049] Results analysis: such as Figure 2 As shown, intraperitoneal injection of LPS resulted in a significant decrease in mouse body weight. Figure 3 As shown in Figures A and B, the results indicate that in the orientation and heading experiment, the escape distance and escape time of the mice gradually decreased with the increase of training days. The cognitive abilities of the mice were assessed through a spatial exploration experiment. Figure 3 As shown in Figure C, the number of times mice crossed the platform in the model group was significantly lower than that in the control group. After gavage administration of ATMP, the number of times mice crossed the platform increased, and the effect was comparable to that in the GV-971 group, indicating that ATMP can restore LPS-induced cognitive impairment. Figure 3 As shown in D, E, and F, the results further indicate that the mycelial polysaccharide ATMP of *Lithocarpus lucida* can alleviate neuroinflammation and protect the nervous system. Figure 4 The arrangement of neurons in the DG, CA1, and CA3 regions of the hippocampus was shown. As shown in the figure, the hippocampal neurons in the control group were arranged neatly and roundedly; while in the model group, the hippocampal neurons were disordered, with many neurons shrunken and some missing, indicating severe brain damage in the model group mice. Administration of *Ligustrum lucidum* mycelial polysaccharide ATMP significantly improved the disordered arrangement of hippocampal neurons. Figure 5 As shown in Figure A, Nissl staining further revealed the brain inflammation status in different groups, and ImageJ was used to quantify Nissl bodies in different regions of the mouse hippocampus. The results are as follows: Figure 5As shown in Figures B and C, the number of Nissl bodies in the model group mice was significantly reduced, while administration of ATMP significantly restored the number of Nissl bodies. These results indicate that ATMP can restore neuronal disorder in the hippocampus, increase the number of Nissl bodies, and thus alleviate LPS-induced cognitive impairment. Figure 6 The study demonstrated the levels of inflammatory factors in the brains of mice in different groups. Specifically, the levels of inflammatory factors in the model group mice were significantly higher than those in the control group, while gavage administration of *Gymnosus leuciscus* mycelial polysaccharide ATMP significantly reduced the levels of inflammatory factors to those in the control group, and there was no significant difference in the improvement effect compared to GV-971. This result more directly proves that *Gymnosus leuciscus* mycelial polysaccharide ATMP can significantly improve cognitive impairment caused by neuroinflammation and exert a neuroprotective function.

[0050] Example 4: The difference between this embodiment and Embodiment 1 is that: In step (3), the material-to-liquid ratio is 1g:15mL, and the extraction is carried out using hot water at 80℃. In step (4), the extract is concentrated to 1 / 10 of its original volume and then precipitated with 85% ethanol. In step (6), the molecular weight cutoff of the dialysis bag used is 3.8 × 10⁻⁶. 3 Da. The rest is the same as in Example 1.

[0051] Example 5: The difference between this embodiment and Embodiment 1 is that: In step (3), the material-to-liquid ratio is 1g:25mL, and the extraction is carried out using hot water at 95℃. In step (4), the extract is concentrated to 1 / 15 of its original volume and then precipitated with 75% ethanol. In step (6), the molecular weight cutoff of the dialysis bag used is 4.0 × 10⁻⁶. 3 Da. The rest is the same as in Example 1.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. The application of a *Gynostemma pentaphyllum* mycelial polysaccharide in the preparation of a drug for improving cognitive impairment, characterized in that: The *Gymnocypris glomeratus* mycelial polysaccharide contains two components with molecular weights of 2.30 × 10⁻⁶ and 2.30 × 10⁻⁶ respectively. 4 Da, 4.56 × 10 5 Da; The monosaccharide composition and molar ratio of the polysaccharide in the mycelium of the Brillianthus lenticulum are mannose: glucose: galactose = 1: 16.64: 1.

96.

2. The application according to claim 1, characterized in that, The improvement of cognitive impairment refers to enhancing cognitive and spatial memory abilities, restoring disordered and missing hippocampal neurons, increasing the number of Nissl bodies, and reducing the levels of brain inflammatory factors TNF-α, IL-6, and IL-1β.

3. The application according to claim 1, characterized in that, The concentration of the *Gymnocypris lucida* mycelial polysaccharide administered is 400 mg / mL; the administration method of the *Gymnocypris lucida* mycelial polysaccharide is by gavage.

4. The application according to claim 1, characterized in that, The drug also includes a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable carrier is selected from one or more of the following: diluents, binders, wetting agents, disintegrants, absorption enhancers, surfactants, and lubricants.

6. The application according to claim 1, characterized in that, The drug is formulated into a pharmaceutically acceptable dosage form.

7. A method for preparing polysaccharides from *Bacillus lucida* mycelium, characterized in that, Includes the following steps: (1) Grind the mycelium of Glossy fungus into powder and extract it with hot water at 80-95°C at a material-to-liquid ratio of 1g:(15-25)mL. (2) Concentrate the extract, centrifuge, and precipitate with 75-85% ethanol; then remove the alcohol. (3) The product obtained in (2) was subjected to Sevag method to remove protein and rotary evaporation to remove residual organic reagents; (4) The product obtained in (3) was dialyzed with running water using a dialysis bag, and the dialyzed solution was rotary evaporated and freeze-dried to obtain the mycelial polysaccharide ATMP of Glossy Bacteria.

8. The preparation method according to claim 7, characterized in that, In (2), the concentration of the extract refers to concentrating it to 1 / 10 to 1 / 20 of the original volume of the extract.

9. The preparation method according to claim 7, characterized in that, In (4), the molecular weight cutoff of the dialysis bag is 3.5~4.0×10⁻⁶. 3 Da.

10. The mycelial polysaccharide of *Gymnocypris lucida* prepared by the preparation method according to any one of claims 7 to 9.

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