阅读说明：本技术 灵孢多糖在制备治疗中枢神经系统损伤药物中的应用及其制备方法 (Application of lingspore polysaccharide in preparing medicine for treating central nervous system injury and preparation method thereof ) 是由 杨育平 王翰斌 杨立志 于 2021-09-01 设计创作，主要内容包括：本发明涉及医药技术领域,具体涉及灵孢多糖在制备治疗中枢神经系统损伤药物中的应用,同时公开了灵孢多糖的制备方法。具体制备方法如下：以赤芝孢子为原料,经脱脂、水提、去蛋白、醇沉、干燥后得到灵孢多糖。通过细胞增殖实验检测发现,灵孢多糖可促进神经元细胞增殖,减轻甲基苯丙胺引起的神经元损伤；能改善甲基苯丙胺引起的小鼠神经行为障碍以及学习记忆障碍,减少神经元丢失,并增加神经生长因子(BDNF)的表达。因此灵孢多糖对神经元损伤具有很好的保护作用,可用于中枢神经系统损伤的治疗。这种灵孢多糖在制备治疗中枢神经系统损伤药物中的应用,拓宽了灵孢多糖的应用领域,同时为中枢神经系统损伤的治疗提供了一种新的治疗方案。(The invention relates to the technical field of medicines, in particular to application of lingpo polysaccharide in preparation of a medicine for treating central nervous system injury, and discloses a preparation method of lingpo polysaccharide. The preparation method comprises the following steps: ganoderma lucidum spores are used as raw materials, and the ganoderma lucidum polysaccharides are obtained by degreasing, water extraction, protein removal, alcohol precipitation and drying. Cell proliferation experiment detection shows that the lingosporin polysaccharide can promote the proliferation of neuron cells and reduce the neuron damage caused by methamphetamine; can improve mouse neurobehavioral disturbance and learning and memory disturbance caused by methamphetamine, reduce neuron loss, and increase nerve growth factor (BDNF) expression. Therefore, the lingosporin polysaccharide has good protection effect on neuron damage and can be used for treating central nervous system damage. The application of the lingpo polysaccharide in preparing the medicine for treating the central nervous system injury widens the application field of the lingpo polysaccharide, and provides a new treatment scheme for treating the central nervous system injury.)

1. Use of Ganoderma spore polysaccharide in preparing medicine for treating central nervous system injury is provided.

2. Use of the lingosporin polysaccharide according to claim 1 for the manufacture of a medicament for the treatment of central nervous system injury, wherein the central nervous system injury is brain tissue damage caused by drugs or psychoactive substances.

3. Use of the lingosporin polysaccharide according to claim 1 for the manufacture of a medicament for the treatment of central nervous system injury, wherein the drug or psychoactive substance is methamphetamine.

4. The use of the lingosporin polysaccharide according to claim 1 in the preparation of a medicament for treating central nervous system injury, wherein the lingosporin polysaccharide has a molecular weight of 5KD or less, 5KD to 100KD or 100KD to 300 KD.

5. The use of the lingosporin polysaccharide according to claim 4 in the preparation of a medicament for treating central nervous system injury, wherein the lingosporin polysaccharide has a molecular weight of 5-100 KD.

6. The use of the lingosporin polysaccharide according to claim 5 for the preparation of a medicament for the treatment of central nervous system injury, wherein the lingosporin polysaccharide is used in a dose of 22.5 μ g/kg to 90.0 μ g/kg for the preparation of a medicament for the treatment of SD rats.

7. The use of the lingosporin polysaccharide according to claim 6 in the preparation of a medicament for treating central nervous system injury, wherein the lingosporin polysaccharide is used in a dosage converted to that used in SD rats when used in the preparation of a medicament for human administration; the scaling method is according to a general scaling method in the art; including but not limited to the blackbell method, the dolley method, the Fibonacci method, the NOAEL method.

8. The use of the lingosporin polysaccharide according to claim 1 in the preparation of a medicament for the treatment of central nervous system injury, wherein the medicament is in the form of an injection, a granule, a tablet or a capsule.

9. The preparation method of the ganoderma spore polysaccharide is characterized by comprising the following steps of:

(1) breaking cell wall of Ganoderma spore, defatting with nonpolar organic solvent, and oven drying;

(2) adding 30-40 times of purified water into the dried ganoderma lucidum spore wall-broken powder by weight, heating to 80-90 ℃, decocting for 2-3h, centrifuging, filtering, and concentrating the supernatant into a fluid extract; adding 80-120U/ml protease into the fluid extract, stirring for 1-2 h at normal temperature, inactivating enzyme, centrifuging, filtering, and taking supernatant;

(3) concentrating the supernatant into a fluid extract, adding medicinal ethanol to enable the ethanol to account for 80-90% of the total volume, precipitating with ethanol, and drying the precipitate to obtain crude polysaccharide;

(4) dissolving the crude polysaccharide in water, filtering with tangential flow ultrafiltration membrane with molecular weight cut-off of 5KD, 100KD and 300KD, respectively collecting three components with molecular weight of less than or equal to 5KD, 5 KD-100 KD and 100 KD-300 KD, and vacuum drying to obtain the final product.

10. The method of claim 9, wherein the ganoderma lucidum spores are ganoderma lucidum spores.

Technical Field

The invention relates to the technical field of medicines, in particular to application of lingpo polysaccharide in preparing a medicine for treating central nervous system injury, and also relates to a preparation method of lingpo polysaccharide extract.

Background

Nerve cells, the most important class of cells of the nervous system, also called neurons, are the basic units that make up the structure and function of the nervous system, and their primary functions are to receive stimuli and transmit information. Nerve cells are composed of cell bodies and cell processes, and can transmit to an excited endocrine cell. Nerve cells are indispensable constituent cells of the human body. Wherein the sensory neurons can receive in vivo and in vitro stimulation and conduct nerve impulses to the central nerves. Motor neurons pass through the cell bodies of the neurons to the nerve endings, causing muscle contraction and glandular secretion. The connecting neuron receives nerve impulses transmitted from other neurons, and plays a role in connection. Pathological changes in a variety of central nervous system disorders in humans include neuronal damage, loss, necrosis and apoptosis. Protecting neurons by reducing neuronal damage and loss, inhibiting neuronal apoptosis, is an important direction in the treatment of diseases with damaged central nervous system.

In recent years, neuronal damage caused by drugs has attracted attention from researchers. Long-term intake of "synthetic drugs" such as Methamphetamine (MA) and the like can lead to the misunderstanding of thinking association, poor logic, paranoia or delusions of abusers, and severe anxiety and depressed mood, and the abusers are easy to take violent behaviors such as suicide or killers and the like under the control of the hallucination and the depressed mood. Drug abuse can induce various ultrastructural pathological changes of mouse multi-part neuron retraction, water sample degeneration, apoptosis, necrosis and the like, and induce changes of water sample degeneration, degenerative degeneration, myelin sheath layer separation, fracture and the like of brain neuron axons, dendrites and synapses in different degrees. The above studies all prove that abuse of drugs can cause pathological damage to neurons of the central nervous system, thereby affecting the functions of the central nervous system. Therefore, the search for substances having protective effect on neuronal damage has been a hot point of research.

Polysaccharides are a class of macromolecular substances commonly existing in organisms and widely participate in the regulation of various physiological activities of cells. Researches show that the polysaccharide has pharmacological effects of resisting tumor, oxidation and inflammation and regulating immunity, and literature reports that the polysaccharide has a protective effect on central nerve injury, for example, the yam polysaccharide has a protective effect on anoxic/reoxygenation nerve cells and can inhibit apoptosis, the morinda officinalis polysaccharide has a protective effect on a neuron cell injury model caused by beta amyloid protein, and senile dementia can be prevented and treated by activating brain energy metabolism and improving cholinergic system injury. A large number of studies indicate that polysaccharides can protect central nerve damage in multiple ways and layers.

Ganoderma spore is the germ cell of Ganoderma, it contains abundant protein, polysaccharide, amino acids, glycopeptide, vitamins, carotene, sterols, triterpenes, alkaloids, fatty acids etc. many active ingredient, wherein the polysaccharide is one of the main effective constituents, modern research shows that Ganoderma spore polysaccharide has biological activity such as anti-tumor, antioxidation, immunoregulation, but research and application in the neuroprotection field pay attention to inadequately. Therefore, the further development of the research of the lingpo polysaccharide on the aspect of protecting the nervous system is beneficial to widening the application range of the lingpo polysaccharide, can provide a new active substance for repairing the damage of the nervous system, and can promote the application of the lingpo polysaccharide in the medical field.

Disclosure of Invention

In view of the above situation, the present invention provides the use of lingpo polysaccharide in preparing medicine for treating central nervous system injury, and the present invention also provides the preparation process of lingpo polysaccharide.

The applicant takes methamphetamine-induced brain injury as an example, studies the protective effect of the prodigiosin on central nervous system injury, and the results are as follows:

(1) through cell proliferation experiments, tests show that the lingosporin polysaccharide can promote cell proliferation and reduce neuron damage caused by methamphetamine.

(2) The neuroethology score shows that the methamphetamine group mice have obvious neuroethology disorder, the learning and memory ability of the Morris water maze detection model group mice is damaged, the cortical tissue structure of the histology observation model group mice is abnormal, the expression of nerve growth factor (BDNF) is reduced, the neuroethology disorder and the learning and memory disorder caused by methamphetamine can be improved after the treatment of the polyporus frondosus polysaccharide, the neuron loss is reduced, and the expression of the BDNF is increased.

The applicant also finds that the effect of the ganoderma lucidum polysaccharide on treating the central nervous system injury has a certain relation with the preparation method and the molecular weight of the polysaccharide, and the ganoderma lucidum polysaccharide prepared by the following method has the strongest activity, and the method comprises the following steps:

(1) breaking cell wall of Ganoderma spore, defatting with nonpolar organic solvent, and oven drying;

(2) adding 30-40 times of purified water into the dried ganoderma lucidum spore wall-broken powder by weight, heating to 80-90 ℃, decocting for 2-3h, centrifuging, filtering, and concentrating the supernatant into fluid extract. Adding 80-120U/ml protease into the fluid extract, stirring for 1-2 h at normal temperature, inactivating enzyme, centrifuging, filtering, and taking supernatant.

(3) Concentrating the supernatant into a fluid extract, adding medicinal ethanol to enable the ethanol to account for 80-90% of the total volume, precipitating with ethanol, and drying the precipitate to obtain crude polysaccharide;

(4) dissolving the crude polysaccharide in water, filtering with tangential flow ultrafiltration membrane with molecular weight cut-off of 5KD, 100KD and 300KD, respectively collecting three components with molecular weight of less than or equal to 5KD, 5 KD-100 KD and 100 KD-300 KD, and vacuum drying to obtain the final product.

Preferably, the ganoderma lucidum spores are ganoderma lucidum spores.

Preferably, the nonpolar organic solvent in the step 1) is tetrachloromethane or petroleum ether.

Preferably, in step 2), 100U/ml protease is added to the flow soak.

Preferably, in step 3, ethanol is made to account for 85% of the total volume.

Experiments prove that the polysaccharide MAP2 component (namely the molecular weight range is 5 KD-100 KD) has the strongest activity in obtaining three polysaccharide extract components.

The invention has definite curative effect, and is a natural product, so the invention has less side effect, safety and reliability; the preparation method has high yield and high content of active ingredients. The lingosporin polysaccharide (5 KD-100 KD) disclosed by the invention has a good protection effect on neuron damage, and can be used for treating central nervous system damage. The application of the lingpo polysaccharide in preparing the medicine for treating the central nervous system injury widens the application field of the lingpo polysaccharide, and provides a new treatment scheme for treating the central nervous system injury.

Drawings

FIG. 1 is a graph showing the effect of the MAP1 fraction on the proliferation of PC-12 cells in example 1;

FIG. 2 is a graph showing the effect of the MAP2 fraction on PC-12 cell proliferation in example 1;

FIG. 3 is a graph showing the effect of the MAP3 fraction on PC-12 cell proliferation in example 1;

FIG. 4 is a graph of the effect of P.nebrodensis polysaccharide on hippocampal BDNF expression in rats infected with methamphetamine.

Detailed Description

The present invention will be described in more detail with reference to the accompanying drawings and examples, which are provided for illustration only and are not intended to limit the scope of the present invention.

Example 1

The preparation method of the ganoderma spore polysaccharide comprises the following steps:

(1) breaking cell wall of Ganoderma spore, defatting with 1.5 times of carbon tetrachloride, filtering, collecting filter cake, and vacuum drying;

(2) adding 40 times of purified water into the dried Ganoderma spore wall-broken powder, heating to 85 deg.C, decocting for 3 hr, centrifuging, filtering, and concentrating the supernatant to obtain fluid extract. Adding 100U/ml protease into the fluid extract, stirring at room temperature for 1h, heating to inactivate enzyme, cooling to room temperature, centrifuging, filtering, and collecting supernatant.

(3) Concentrating the supernatant into fluid extract, adding medicinal ethanol to make ethanol account for 85% of the total volume, precipitating with ethanol for 12 hr, filtering, collecting filter cake, and drying the precipitate to obtain crude polysaccharide;

(4) dissolving the crude polysaccharide in water, filtering with tangential flow ultrafiltration membrane with molecular weight cut-off of 5KD, 100KD and 300KD, respectively collecting three components with molecular weight of less than or equal to 5KD, 5 KD-100 KD and 100 KD-300 KD, and vacuum drying to obtain the final product.

Through calculation: the polysaccharide yield of the three components (the proportion of each component in the total sugar content of the medicinal materials) is as follows: the yields of MAP1, MAP2, and MAP3 were 20.5%, 38.6%, and 9.9%, respectively.

Example 2

The preparation method of the ganoderma spore polysaccharide comprises the following steps:

(1) breaking the wall of ganoderma lucidum spore, degreasing with 2 times of petroleum ether, filtering and collecting a filter cake, and then drying in vacuum;

(2) adding 30 times of purified water into the dried Ganoderma spore wall-broken powder, heating to 80 deg.C, decocting for 2 hr, centrifuging, filtering, and concentrating the supernatant to obtain fluid extract. Adding 90U/ml protease into the fluid extract, stirring at normal temperature for 1h, heating to inactivate enzyme, cooling to normal temperature, centrifuging, filtering, and collecting supernatant.

(3) Concentrating the supernatant into fluid extract, adding medicinal ethanol to make ethanol account for 90% of the total volume, precipitating with ethanol for 12 hr, filtering, collecting filter cake, and drying the precipitate to obtain crude polysaccharide;

(4) dissolving the crude polysaccharide in water, filtering with tangential flow ultrafiltration membrane with molecular weight cut-off of 5KD, 100KD and 300KD, respectively collecting three components with molecular weight of less than or equal to 5KD, 5 KD-100 KD and 100 KD-300 KD, and vacuum drying to obtain the final product.

Through calculation: the polysaccharide yield of the three components (the proportion of each component in the total sugar content of the medicinal materials) is as follows: the yields of MAP1, MAP2, and MAP3 were 16.2%, 31.5%, and 5.2%, respectively.

Example 3

The preparation method of the ganoderma spore polysaccharide comprises the following steps:

(1) breaking the wall of ganoderma lucidum spore, degreasing with 2 times of petroleum ether, filtering and collecting a filter cake, and then drying in vacuum;

(2) adding 40 times of purified water into the dried Ganoderma spore wall-broken powder, heating to 90 deg.C, decocting for 2 hr, centrifuging, filtering, and concentrating the supernatant to obtain fluid extract. Adding 80U/ml protease into the fluid extract, stirring at room temperature for 1h, heating to inactivate enzyme, cooling to room temperature, centrifuging, filtering, and collecting supernatant.

(3) Concentrating the supernatant into fluid extract, adding medicinal ethanol to make ethanol account for 80% of the total volume, precipitating with ethanol for 12 hr, filtering, collecting filter cake, and drying the precipitate to obtain crude polysaccharide;

(4) dissolving the crude polysaccharide in water, filtering with tangential flow ultrafiltration membrane with molecular weight cut-off of 5KD, 100KD and 300KD, respectively collecting three components with molecular weight of less than or equal to 5KD, 5 KD-100 KD and 100 KD-300 KD, and vacuum drying to obtain the final product.

Through calculation: the polysaccharide yield of the three components (the proportion of each component in the total sugar content of the medicinal materials) is as follows: the yields of MAP1, MAP2, and MAP3 were 17.8%, 35.5%, and 6.6%, respectively.

Test examples

The function of the polysaccharide of the lingeri spore is verified by cell and animal experiments, the polysaccharide extract obtained in example 1 is the test object, and the polysaccharide extracts obtained in examples 1 and 2 can be verified by the same method and achieve similar effects.

Test example 1

Effect of Lysimachia polysaccharide on the proliferation of PC-12 cells

The experimental method comprises the following steps:

rat chromaffin neuroblastoma PC-12 cells were purchased from the cell resource center of Shanghai Life sciences research institute of Chinese academy of sciences, and cultured at 37 ℃ and 5% CO2 saturated humidity with complete medium (RPMI-1640 medium containing 10% FBS and 100U streptomycin solution).

Taking PC-12 cells of logarithmic growth phase, digesting with 0.25% trypsin, beating with complete culture medium, preparing into single cell suspension, adjusting cell concentration to 1 × 10⁵One cell/ml of the cells were inoculated into a 96-well plate at 100. mu.l/well, and cultured in a 5% CO2 incubator at 37 ℃ for 24 hours. Divided into 10 groups according to experimental requirements, treated with 0, 1.56, 3.12, 6.25, 12.5, 25, 50, 100, 200 and 400 μ g/ml of the lingerie polysaccharide MAP1 of example 1, setting 6 multiple wells per concentration, and culturing at 37 ℃ for 24h while setting blank wells. After the treatment, the supernatant was aspirated and washed 3 times with sterile PBS, 90. mu.l of complete medium was added to each well, 10. mu.l of sterile MTT (final concentration 5mg/ml) solution was added, and the culture was continued in the incubator for 4 hours. After the culture is finished, carefully absorbing and removing the supernatant, adding 150 mu l of dimethyl sulfoxide (DMSO) into each hole, oscillating the mixture for 10min by using an oscillator to completely dissolve the purple crystals, detecting an absorbance value (A) by using a 570nm wavelength of an microplate reader, and calculating the survival rate T/C (%) of each group of cells according to the following formula: T/C (%) ═ (sample a-blank a)/(control a-blank a) × 100%.

The experimental procedures for MAP2 and MAP3 in example 1 were the same as for MAP 1.

The experimental results are as follows: as shown in figures 1, 2 and 3, the lingosporin polysaccharides with three molecular weights have no cytotoxicity in the range of 1.56-400 mu g/ml and can promote the proliferation of PC-12 cells, wherein the MAP2 has the best effect, and the survival rate of cells in a MAP2 treatment group is up to 171.1 +/-5.3%.

Test example 2

Protective effect of ganoderma lucidum polysaccharide on PC-12 cell injury caused by Methamphetamine (MA)

The experimental method comprises the following steps:

taking PC-12 cells of logarithmic growth phase, digesting with 0.25% trypsin, beating with complete culture medium, preparing into single cell suspension, adjusting cell concentration to 1 × 10⁵One cell/ml of the cells were inoculated into a 96-well plate at 100. mu.l/well, and cultured in a 5% CO2 incubator at 37 ℃ for 24 hours. The test is divided into 7 groups according to the experimental requirements: normal control group, 3mmol/lMA lesion group, 25. mu.g/ml MAP1+ MA treated group, 50. mu.g/ml MAP1+ MA treated group, 100. mu.g/ml MAP1+ MA treated group, 200. mu.g/ml MAP1+ MA treated group, and 400. mu.g/ml MAP1+ MA treated group. Then adding the test substances with different concentrations according to the above groups, setting 6 multiple wells for each concentration, continuing culturing at 37 ℃, and simultaneously setting blank wells. After the treatment, the supernatant was aspirated and washed 3 times with sterile PBS, 90. mu.l of complete medium was added to each well, 10. mu.l of sterile MTT (final concentration 5mg/ml) solution was added, and the culture was continued in an incubator for 4 hours. After the culture is finished, carefully absorbing and removing the supernatant, adding 150 mu l of dimethyl sulfoxide (DMSO) into each hole, oscillating for 10min by using an oscillator to completely dissolve the purple crystals, detecting an absorbance value (A) by using a 570nm wavelength of an enzyme-labeling instrument, and calculating the survival rate of each group of cells.

The experimental procedures for MAP2 and MAP3 in example 1 were the same as for MAP 1.

As a result: as shown in tables 1-3, MAP1, MAP2, and MAP3 all reduced neuronal damage caused by MA, with MAP2 being most effective.

TABLE 1 protective Effect of MAP1 on MA-damaged PC-12 cells

TABLE 2 protective Effect of MAP2 on MA-damaged PC-12 cells

Note: p <0.05 in experimental group 4 compared to the induction group and P <0.01 in experimental group 5 compared to the induction group.

TABLE 3 protective Effect of MAP3 on MA-damaged PC-12 cells

Note: compared with the induction group, P of the experimental group 4 and the group 5 is less than 0.05

Test example 3

Protective effect of ganoderma lucidum polysaccharide on brain injury of methamphetamine-infected rat

The experimental method comprises the following steps:

(1) animals were grouped into rats and treated

SD male rats (weight 180-220 g) are raised in a constant temperature air-conditioning SPF-level animal room. Light and dark are alternately circulated for 12 hours, and free drinking and eating are kept. Rats were randomly divided into 6 groups of rats, 10 rats per group, normal control group, MA lesion group, positive control group, MAP2 low dose group: MA + MAP 222.5. mu.g/kg, MAP2 dose group: MA + MAP 245.0. mu.g/kg, MAP2 high dose group: MA + MAP 290.0. mu.g/kg.

In the first week, normal control group rats were injected with normal saline intraperitoneally, the remaining rats were injected with 1mg/kgMA intraperitoneally, normal control group rats and MA group rats were gavaged with normal saline, MAP2 low dose group: MA + MAP 222.5. mu.g/kg, MAP2 dose group: MA + MAP 245.0. mu.g/kg, MAP2 high dose group: MA + MAP290.0 μ g/kg, and the positive control group rats were intragastrically injected with commercial Lysimachia foenum-graecum polysaccharide at a dose of 90.0 μ g/kg, and were intraperitoneally injected and intragastrically administered once a day for one week.

Starting from the second week, normal control rats and MA rats were gavaged with saline, MAP2 low dose group: MA + MAP 222.5. mu.g/kg, MAP2 dose group: MA + MAP 245.0. mu.g/kg, MAP2 high dose group: MA + MAP290.0 μ g/kg, and positive control group rat gavage 90.0 μ g/kg lingerie polysaccharide, once a day for three weeks.

(2) Rat spatial learning and memory capacity detection

After the experiment is finished, the learning and memory ability of the rat is detected by using the Morris water maze. The basic method is as follows: the Morris water maze is a stainless steel cylindrical water pool, the diameter is 120cm, the height is 36cm, the wall of the water pool marks 4 water entry points of east, west, south and north, and the boundary points of 4 quadrants and the middle points of the first quadrant, the third quadrant and the fourth quadrant are marked on the boundary of the water maze barrel according to water maze software. A platform of approximately 6cm diameter was fixed at the very center of the second quadrant. Water was added until the platform was submerged 1cm below the horizontal surface. The swimming track of each rat is collected and input into a tracking system through a camera arranged at the top of the water maze, and the position of the rat on the water surface, the escape latency (the time required by searching for a target), the movement track, the swimming speed and other movement related information are recorded in real time.

(3) Rat hippocampal BDNF expression level detection

After the experiment, 4 rats in each group were decapitated and brains were collected, and the hippocampus was rapidly separated on ice. Hippocampus tissues were homogenized with 0.4ml ice-cold homogenization buffer

(Tris-HMA50mmol/L, pH7.4, NaMA150mmol/L, 0.5% Triton X-100, ethical 1mmol/L, phenylmethylisulfonyfluoride 1mol/L, aprotinin 5mg/L), homogenized in ice bath at 14,000g and 4 ℃ for 30 min. The supernatant was collected and used as total protein. Polyacrylamide gel electrophoresis is adopted, the concentration of the separation gel is 8 percent, the pH value is 8.8, the concentration of the concentration gel is 5 percent, and the pH value is 6.8. After electrophoresis, proteins were transferred from polyacrylamide gel to nitrocellulose membrane, which was placed in TBS buffer containing 5% skim milk powder overnight at 4 ℃ and incubated for 2h at room temperature with BDNF monoclonal rabbit anti-mouse antibody (1: 500). After washing the membrane with TBS, incubating the membrane with a goat anti-rabbit secondary antibody labeled with horseradish peroxidase for 1h at room temperature, after washing the membrane with TBS, mixing a chemiluminescence agent and an enhancer according to a ratio of 1:1, dropwise adding the mixture onto a nitrocellulose membrane, and capturing an image through a chemiluminescence imaging system.

(4) Morphological examination

The rats in each group were deeply anesthetized, their brains (ice-cold physiological saline and 4% paraformaldehyde) were perfused through the heart, fixed overnight at 4 ℃ with 4% paraformaldehyde, removed, fixed externally at 4 ℃ with 4% paraformaldehyde, dehydrated, embedded in paraffin, coronal sections 3mm and 4.5mm from bregma were selected at equal intervals, the slice thickness was 5 μm, and they were observed under a light microscope after HE staining.

As a result:

(1) influence of ganoderma lucidum polysaccharide on spatial learning and memory ability of methamphetamine-infected rats

After the time for searching the platform by the rats in the normal control group is trained for 4 days, the time for finding the platform by the rats is obviously shortened, the time for searching the platform by the rats in the MA group is obviously longer than that of the rats in the normal group, the time for searching the platform by the rats in each dosage group of MAP2 on the 5 th day is obviously shortened, and the target property is also obviously enhanced. The normal control group rat latency is 12.25 + -3.3 s, the MA group rat latency is 32.8 + -3.22 s, the MAP2 low, middle and high group rats latency is 31.23 + -3.62 s, 22.11 + -3.45 s, 15.23 + -3.22 s, and the positive control group rat latency is 27.05 + -3.89 s. The incubation period of MAP2 rats in the medium and high dose groups was significantly shortened compared with MA rats (P <0.05), but the rats in the low dose MAP2 group and the rats in the positive control group were shortened compared with MAP rats, but there was no statistical difference (P > 0.05).

(2) Influence of ganoderma lucidum polysaccharide on expression change of hippocampal BDNF protein of rat infected with methamphetamine

The immunoblotting result is shown in fig. 4, which shows that the expression level of rat hippocampal tissue BDNF protein is changed, the expression level of MA group rat BDNF protein is lower than that of normal control group rat, the expression levels of positive control group rat, MAP2 low, medium and high dose group rat BDNF protein are all up-regulated, and the expression level of MAP2 high dose group rat is significantly increased compared with that of MA group rat (P < 0.05).

(3) Influence of Lysimachia polysaccharide on morphological changes of cortex of methamphetamine-infected rat

HE staining can be used to examine pathological changes in methamphetamine that lead to brain tissue damage. Morphological changes in the cortical tissue of the rats in the MA group were evident: loss of nerve cells, deepening of nuclear staining, gliosis, nuclear condensation, and the like. After MAP2(22.5 mu g/kg, 45.0 mu g/kg and 90.0 mu g/kg) treatment, the mice can obviously relieve the morphological change, which indicates that the prodigiosin has protective effect on the brain tissue damage caused by methamphetamine.

Test example 4

Clinical trial

According to the results of test example 3, the doses were classified into a MAP2 low dose group (3.0mg/d), a MAP2 medium dose group (4.5mg/d), and a MAP2 high dose group (6.0mg/d) in terms of human dose.

The test of the protective effect of the prodigiosin on the brain injury of a person infected with methamphetamine is carried out on 200-position persons infected with methamphetamine at a certain drug rehabilitation place. 200 persons were randomly divided into 5 groups, 40 persons in each group were respectively a positive control group (olanzapine oral 20mg/d), a negative control group (saline injection), a MAP2 low dose group (3.0mg/d), a MAP2 medium dose group (4.5mg/d), and a MAP2 high dose group (6.0mg/d), and the scores thereof (average score in each group) were compared by performing measurements on the simplified mental state assessment Scale (MMSE) and the Montreal cognitive assessment Scale (MoCA) before and after the test (3 months later), respectively. MMSE decision criterion: the highest score is 30 points, the score is normal when being 27-30 points, the score is less than 27 points, the cognitive dysfunction is judged, and the severity grading method is that mild MMSE is more than or equal to 21 points; moderate, MMSE10-20 points; the severity is less than or equal to 9 minutes in MMSE;

MoCA scale judgment standard: the MoCA scale is 30 points full, not less than 26 points normal, 18-26 Mild Cognitive Impairment (MCI), 10-17 moderate and less than 10 severe. The specific scores are shown in Table 4.

TABLE 4 MMSE and MoCA scale

	MMSE (before treatment)	MMSE (after treatment)	MoCA (before treatment)	MoCA (after treatment)
					MAP2 Low dose group	21.5	27.5	17.6	28.2
MAP2 Medium dose groups	21.7	28.1	17.7	28.4
					MAP2 high dose group	21.4	28.6	17.3	28.8
Positive control group	21.5	27.1	17.6	27.6
					Negative control group	21.5	22.5	17.5	23.1

According to the results in table 4, the lingosporin polysaccharide disclosed by the invention has a relatively obvious effect on improving the brain injury of a methamphetamine-infected person under an effective dose, has an effect superior to that of olanzapine serving as a positive control, and is worthy of further research on various dosage forms and pharmaceutical compositions of the lingosporin polysaccharide.

The above are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and all the equivalent changes and modifications made by the claims and the summary of the invention should be covered by the protection scope of the present patent application.