CN113308495B

CN113308495B - Gastrodia elata fermentation product polar component for assisting in improving memory and application thereof

Info

Publication number: CN113308495B
Application number: CN202110531153.9A
Authority: CN
Inventors: 梁建东; 田维毅; 向丽; 杨长福; 曹峰; 刘明; 代永东; 陈万浩; 任秀秀; 冯丽娴; 罗成浩; 郭兴
Original assignee: Guizhou University of Traditional Chinese Medicine
Current assignee: Guizhou University of Traditional Chinese Medicine
Priority date: 2021-05-16
Filing date: 2021-05-16
Publication date: 2023-05-05
Anticipated expiration: 2041-05-16
Also published as: CN113308495A

Abstract

The invention discloses a gastrodia elata fermentation product polar component capable of remarkably improving memory and application thereof. According to the invention, through researches, the n-butanol component in the fermentation product alcohol extract of the ganoderma lucidum cordyceps militaris and gastrodia elata has the effect of remarkably improving the memory, can be applied to the preparation of medicines for treating Alzheimer's disease or functional woolen foods and health-care products for improving the memory, provides a basis for further researching and developing traditional Chinese medicine anti-Alzheimer's disease medicines with remarkable curative effect and ideal memory improving effect, and has good application prospect.

Description

Gastrodia elata fermentation product polar component for assisting in improving memory and application thereof

Technical Field

The invention relates to a gastrodia elata fermentation product polar component for assisting in improving memory and application thereof, and belongs to the technical field of medicines.

Background

Cognitive disorders, which are predominantly memory decline, are a common neurological disorder, including Alzheimer's Disease (AD), the most common type of Alzheimer's disease, parkinson's Disease (PD), multiple Sclerosis (MS), etc., and AD is the most severe manifestation thereof. The number of people suffering from senile dementia is 4680 ten thousand worldwide, and in the elderly people over 60 years old, about 5-7% of people are affected by dementia, and after 65 years old, the incidence rate is doubled by about 5 years. Therefore, the research of effective medicaments for treating cognition disorder patients has great social and economic significance. Learning memory impairment is a typical symptom of dementia, and loss of memory severely affects the quality of life of dementia patients. To date, there are only two classes of drugs approved for the treatment of cognitive disorders, one being cholinesterase inhibitors, such as tacrine, donepezil, rivastigmine, galantamine; second, N-methyl D-aspartate antagonists such as memantine. Although these drugs can improve or alleviate cognitive impairment and mental symptoms to some extent, the drugs with efficacy are very few in the two types of drugs, and the two types of drugs have large side effects and are expensive.

Disclosure of Invention

The invention aims to solve the technical problem of providing a gastrodia elata fermentation product polar component for assisting in improving memory and application thereof, and provides a new choice for Alzheimer disease and memory disorder medicaments, memory-improving functional foods and health-care products.

In order to solve the technical problems, the invention adopts the following technical scheme:

the preparation method of the gastrodia elata fermentation product polar component for assisting in improving memory comprises the following steps of:

(1) Preparing a seed liquid culture medium: cleaning 20g of potato, peeling, cutting into small pieces, placing into a pot, adding 100mL of water, boiling for 10-15 minutes, filtering, adding 2g of glucose into the filtrate, heating and stirring uniformly, cooling, naturally pH, sterilizing at 121 ℃ for 30 minutes for later use;

(2) Activating strains: respectively transferring Cordyceps militaris strain B1528 and Ganoderma strain B1.4 onto PDA plate, and culturing at 25deg.C in dark for 7 d; the ganoderma lucidum strain B1.4 is preserved in China center for type culture collection, and the preservation number is as follows: CCTCCNO: m2019790, cordyceps militaris strain B1528 is deposited in China center for type culture Collection, accession number: CCTCCNO: m2019789;

(3) Seed liquid preparation: under the aseptic condition, taking activated ganoderma lucidum strain B1.4 and cordyceps militaris strain B1528 with diameters of 5mm from the flat plate in the step (2) by using a puncher, inoculating into a culture medium filled with 100mL of seed liquid, and shake culturing for 7d under the dark condition in a shaking incubator with the speed of 200r/min and the temperature of 25 ℃ for later use;

(4) The preparation of the alcohol extract of the fermentation product of the ganoderma lucidum, the cordyceps militaris and the gastrodia elata comprises the following steps: adding sucrose as carbon source 18.2g/L, peptone as nitrogen source 30.0g/L, potassium dihydrogen phosphate as inorganic salt 0.9g/L, cordyceps militaris-Ganoderma lucidum inoculation ratio 1:2, rhizoma Gastrodiae addition 30.0g/L, culturing at 25deg.C, shaking table 200r/min rotation speed, and dark culturing for 15d to obtain fermentation product; centrifuging the obtained fermentation product at 1000r/min to obtain fermentation liquor and solid matters; freeze drying the solid, pulverizing, adding 10 times of 95% ethanol, leaching at room temperature for three times for 24 hr, mixing the extractive solutions, concentrating under reduced pressure until no alcohol smell exists, to obtain ethanol extract of fermentation product of Ganoderma Cordyceps militaris and rhizoma Gastrodiae;

(5) Preparing n-butanol polar part of fermentation product of Ganoderma lucidum, cordyceps militaris and rhizoma Gastrodiae: taking the lucid ganoderma cordyceps militaris gastrodia elata fermentation product alcohol extract in the step (4), adding warm water, uniformly mixing to obtain a suspension, transferring the suspension into a separating funnel, adding petroleum ether according to the ratio of 1:1, slightly shaking the separating funnel, fully and uniformly mixing the petroleum ether and the fermentation product alcohol extract, and then placing the mixture on a funnel stand to be layered; after layering, the upper layer of liquid similar to transparent oil is the petroleum ether extraction part, the lower layer is the part left after petroleum ether extraction, and the upper layer part and the lower layer part are respectively collected into a beaker; transferring the lower layer part left by extraction into a separating funnel, respectively adding ethyl acetate and n-butanol with the same proportion according to the method, sequentially extracting each solvent for 6 times and 0.5h each time, and finally extracting the rest water part; concentrating the solution obtained by n-butanol extraction under reduced pressure until no smell is present, to obtain extract of n-butanol polar part of fermentation product of Ganoderma Cordyceps militaris and rhizoma Gastrodiae.

As a preferable scheme, the lucid ganoderma cordyceps militaris rhizoma gastrodiae fermentation product alcohol extract prepared in the step (4) of claim 1 can be used for preparing medicines for treating Alzheimer disease and dysmnesia or preparing functional foods and health-care products for improving memory.

As a further preferable scheme, the n-butanol polar part of the fermentation product of the ganoderma lucidum, the cordyceps militaris and the gastrodia elata, which are prepared in the step (5) in the claim 1, can be used for preparing medicines for treating Alzheimer disease and dysmnesia or functional foods and health care products for improving memory.

In the application, the n-butanol polar part of the fermentation product of the ganoderma lucidum cordyceps militaris and gastrodia elata can obviously reduce the content of IL-1 beta, IL-6, TNF-alpha and NO in serum and sea horse of a rat with dysmnesia, improve the content of IL-10 in serum and sea horse, and promote the expression level of BDNFmRNA of sea horse; can also improve damage of rat hippocampal neurons caused by lipopolysaccharide.

The application can take the ethanol extract of the fermentation product of the ganoderma lucidum, the cordyceps militaris and the gastrodia elata, and prepare the ethanol extract into pharmaceutically acceptable dosage forms including granules, capsules, tablets, injections, oral liquid and dripping pills.

The application can also take extractum of n-butanol polar parts of the fermentation product of ganoderma lucidum cordyceps militaris and gastrodia elata, and prepare acceptable dosage forms including granules, capsules, tablets, injections, oral liquid and dripping pills.

The beneficial effects are that: in the previous subject group research, the applicant finds that the alcohol extract of the fermentation product of the ganoderma lucidum cordyceps militaris and the gastrodia elata can obviously improve the effect of the lipopolysaccharide on learning and memory impairment of rats, but the effective parts of the alcohol extract are not clear. Therefore, the patent has been developed, and researches show that the petroleum ether component, the ethyl acetate component and the n-butanol component in the ethanol extract of the fermentation product of the ganoderma lucidum cordyceps militaris and the gastrodia elata have the effect of assisting in improving the memory, and the most effective component is the n-butanol component.

Compared with the prior art, the invention further discovers and confirms that the n-butanol polar part of the fermentation product of the ganoderma lucidum cordyceps militaris and gastrodia elata can assist in improving memory on the basis of the earlier research results of ganoderma lucidum cordyceps militaris and gastrodia elata fermentation products (prepared by taking ganoderma lucidum and cordyceps militaris as fermentation strains and taking gastrodia elata as drug property matrixes through two-way liquid co-culture fermentation), can be applied to preparing medicines for treating Alzheimer disease or functional foods and health care products for assisting in improving memory, and provides a basis for further developing traditional Chinese medicine anti-Alzheimer disease medicines with obvious curative effect and ideal memory improving effect or functional foods and health care products for improving memory.

Drawings

FIG. 1 is a schedule of drug treatment and assessment of learning and memory in rats;

FIG. 2 is a graph showing the change in body weight of rats in each experimental group;

FIG. 4 is a graph showing comparison of the results of the dark avoidance experiments for rats of each experimental group;

FIG. 5 is a graph of the morphological changes of neurons in the CA1 region of the hippocampus of rats in the experimental group (400X), with arrows indicating neuronal degeneration or necrosis;

FIG. 6 is a graph showing the effect of effective active sites on rat body weight;

FIG. 7 is a graph showing the effect of each experimental group on the escape latency of rats;

FIG. 8 is a graph showing comparison of the results of the dark avoidance experiments for rats of each experimental group;

FIG. 9 is a graph of the morphological changes of neurons in the CA1 region of the hippocampus of rats (400X) for each experimental group, with arrows indicating neuronal degeneration or necrosis;

FIG. 10 is a graph showing comparison of IL-1β content in serum and sea horse of rats of each experimental group;

FIG. 11 is a graph showing comparison of IL-6 content in serum and sea horse of rats in each experimental group;

FIG. 12 is a graph showing comparison of TNF- α levels in serum and sea horses from each experimental group;

FIG. 13 is a graph showing comparison of NO content in serum and hippocampus of rats of each experimental group;

FIG. 14 is a graph showing comparison of IL-10 content in serum and sea horse of rats of each experimental group

FIG. 15 is a graph showing comparison of IL-1. Beta. And TNF-. Alpha.mRNA expression levels in the hippocampus of rats of each experimental group;

FIG. 16 is a graph showing comparison of IL-10 and BDNFmRNA expression levels in the hippocampus of rats of each experimental group.

Detailed Description

A first part: screening of active ingredients

1 Experimental materials

1.1 fermentation strains: ganoderma GanodermaLucidum B1.4 (preserved in China center for type culture Collection, 10.9, with a preservation number of CCTCC NO: M2019790 and Cordyceps militaris CordycephamiltarisB 1528 (preserved in China center for type culture collection, 10.9, with a preservation number of CCTCC NO: M2019789)

1.2 experimental animals: healthy SPF-grade SD rats weighing 180-220g and 8 weeks old were selected and provided by Changsha laboratory animal center, laboratory animal license number: SCXK (Hunan) 2019-0014, the temperature in the feeding room is kept at 20-25 ℃, solid particles are fed on time every day to maintain feed and sterile water (high-pressure sterilization), and the feeding environment is kept quiet.

1.3 experimental drugs and reagents: gastrodia elata, petroleum ether, ethyl acetate, n-butanol, 95% ethanol, lipopolysaccharide, piracetam tablets, 4% paraformaldehyde fixing solution, hematoxylin-eosin staining kit, absolute ethanol, xylene and the like.

1.4 laboratory apparatus: electronic balance, constant temperature culture table, superclean bench, autoclave, freeze dryer, rotary evaporator, circulating water vacuum pump, morris water maze system, dark shuttle tester, tissue embedding machine, tissue slicing machine, and tissue spreading and baking machine. A microscope camera CCD camera system, etc.

2 Experimental methods

2.1 preparation of Medium

2.1.1 conventional PDA Medium

2.1.2 seed liquid culture medium: 20g of potato, 2g of glucose, 100mL of water, natural pH value, and sterilization at 121 ℃ for 30min for later use.

2.2 activating the strain: cordyceps militaris strain B1528 and Ganoderma strain B1.4 are respectively transferred onto PDA plate, and cultured in dark at 25deg.C for 7d for use.

2.3 seed liquid preparation: under the aseptic condition, activated strains B1528 and B1.4 with the diameters of 5mm are taken out from the flat plate of the item "2.2" by a puncher, inoculated into 100mL of seed liquid culture medium and shake-cultured for 7d in a shaking table under the dark condition at 25 ℃ for later use.

2.4 preparation of the ethanol extract of the gastrodia elata product by fermenting the ganoderma lucidum and the cordyceps militaris: adding sucrose as carbon source 18.2g/L, peptone as nitrogen source 30.0g/L, potassium dihydrogen phosphate as inorganic salt 0.9g/L, inoculating Cordyceps militaris and Ganoderma lucidum at a ratio of 1:2 (inoculating amount 30 mL), adding rhizoma Gastrodiae at 30.0g/L, culturing at 25deg.C, shaking table 200r/min, and culturing in darkness for 15d to obtain fermentation product. Centrifuging (1000 r/min) the obtained fermentation product to obtain fermentation liquor and solid matter, freeze drying the solid matter, pulverizing into powder, adding 10 times of 95% ethanol, leaching at room temperature for three times (each leaching time is 24 h), mixing the extractive solutions, concentrating under reduced pressure until no alcohol smell exists, and obtaining the alcoholic extract of the fermentation gastrodia elata product of the ganoderma lucidum cordyceps militaris.

2.5 preparation of different polarity portions of Gastrodia elata product by fermentation of Ganoderma lucidum and Cordyceps militaris

Taking the ethanol extract of the fermentation product of the item "2.4", adding warm water with equal amount, uniformly mixing to obtain suspension, transferring into a separating funnel, adding petroleum ether according to the ratio of 1:1, gently shaking the separating funnel, fully and uniformly mixing the petroleum ether and the ethanol extract of the fermentation product, placing on a funnel stand, and allowing the mixture to stand and delaminate. After layering, the upper layer of liquid similar to transparent oil is the petroleum ether extraction part, the lower layer is the part left after petroleum ether extraction, and the upper layer part and the lower layer part are respectively collected into a beaker. Transferring the lower layer part left after extraction into a separating funnel, adding ethyl acetate and n-butanol with the same proportion respectively according to the method, sequentially extracting (each solvent is extracted for 6 times and 0.5h each time), and finally extracting the rest water part. Mixing the solution obtained by petroleum ether, ethyl acetate and n-butanol extraction and the water extract, concentrating under reduced pressure until odorless, respectively obtaining extracts with different polarities, and calculating extraction rate. Extraction rate (%) =weight of extract after extraction/weight of alcoholic extract of fermentation product×100%.

2.6 grouping and administration of animals

SPF-class male SD rats, after 3 days of adaptive feeding, were eliminated by Morris water maze test as either oversensitive (stations found within 10 s) or overstardy (stations not found within 120 s), and the screened qualified rats were randomly divided into blank group, model group, piracetam tablet group, ethanol extract group, petroleum ether position group, ethyl acetate position group, n-butanol position group, and water position group. The rats of each group, except the blank group and model group, were administered and injected with an equal volume of physiological saline, and were interfered with the corresponding drugs according to a dosing regimen, once daily for 28 consecutive days, as shown in table 1.

2.7 method of Forming mold

In the whole experimental process, medicines at different polarity parts of the gastrodia elata product fermented by lucid ganoderma cordyceps militaris are continuously interfered for 28 days, from the 15 th day of interference, rats in a blank group are injected with an equal volume of physiological saline, and the other rats in each group are injected with 250 mug/kg of lipopolysaccharide in the abdominal cavity for one week. From day 22, the rats of each experimental group were subjected to a behavioural experiment (Morris water maze, dark avoidance experiment) for one week, and the learning and memory abilities of the rats were examined. Serum, brain and hippocampal tissue were obtained 2h after the behavioral test. FIG. 1 depicts the time of administration and the time of cognitive behavioral studies with respect to lipopolysaccharide and different polarity sites.

2.8Morris Water maze experiment

Gently placing the rat into water from the opposite quadrant of the platform towards the pool wall and recording the time it takes the rat to find the platform within 120s as escape latency; if the rat does not find the platform within 120s, stop timing and guide the rat to find the platform with stick and stay for 10s to achieve the learning and memory process, at which time the escape latency is 120s.

2.9 dark avoidance experiments

Rats entered the darkroom due to darkness habit, learning and memory ability was evaluated by entering the darkroom latency, and the time of first suffering from electric shock (darkness avoidance latency) was recorded for each group of rats within 300s, and if rats did not enter the darkroom within 300s, darkness avoidance latency was recorded as 300s, and the number of times of suffering from electric shock within 300s (darkness error count).

2.10 analysis of hippocampal tissue morphology

The rats of each group were subjected to behavioural examination for 2 hours and then were subjected to head breakage and brain extraction. Then 4% paraformaldehyde solution is used for fixing brain tissue to prevent autolysis or decomposition after cell death, so that the cells can keep original form and structure. The specific operation method is as follows:

(1) Repairing organization: the fixed brain tissue was removed from the paraformaldehyde, the brain tissue was repaired, the rat hippocampal region was excised along the coronal plane and placed into a labeled embedding cassette, which was flushed under tap water overnight to flush out excess paraformaldehyde.

(2) Dehydrating and transparentizing: gradually removing water in the washed brain tissue by using gradient alcohol, and then, using xylene for transparency, so that paraffin can smoothly permeate into the tissue. (3) wax dipping: and (3) sequentially immersing the transparent brain tissue blocks in paraffin I, paraffin II and paraffin III at 60 ℃.

(4) Embedding

1, the embedding machine is started in advance, the temperature is set at 60-65 ℃ until paraffin is completely melted for standby.

2, placing the brain tissue which is completely immersed in the wax into a metal embedding frame, embedding the brain tissue by using an embedding machine, placing the brain tissue in the middle of the embedding frame, avoiding the generation of bubbles, covering the embedding frame, slowly (1) horizontally moving the brain tissue onto an ice table for cooling, taking down the embedded tissue after the paraffin is completely solidified, marking, and slicing.

(5) Slicing: the embedded tissue is placed on a slicing machine according to the requirement, the slicing machine is firstly adjusted to a trimming mode, and when the tissue is completely displayed, the brain tissue is cut to be 5 mu m thick.

(6) Spreading: placing the cut tissue slices in a water bath kettle at 40 ℃ for spreading, taking out the slices by using a glass slide after spreading, attaching the slices to proper positions of the glass slide, and baking the slices overnight in an oven at 37 ℃.

(7) Specific HE dyeing procedure

1 slice dewaxing: paraffin sections were sequentially placed in xylene I, II, III for 20min each, absolute ethanol I, II for 5min each, ethanol 95%, 90%, 80%, 70% and distilled water for 5min each.

2 hematoxylin staining: hematoxylin dye solution is used for dyeing for 2min.

3 differentiation: hydrochloric acid alcohol is differentiated for 1s, and tap water is used for pickling to turn blue.

4 eosin staining: eosin dye liquor is used for dyeing for 3min, and washing and soaking are carried out for 30s.

5, dehydrating, transparentizing and sealing: absolute ethyl alcohol I, II, n-butanol, xylene I, II are respectively used for 5min, neutral gum is used for sealing the tablet, and shooting is carried out after the neutral gum is air-dried.

(8) Shooting: and shooting HE stained slice images by using a microscope, observing the whole tissue of each slice under a 100-time mirror, selecting a rat hippocampal CA1 region to acquire 400-time images, and further observing pathological structural changes of the rat hippocampal CA1 region.

2.11 statistical analysis: SPSS25 performs statistical analysis on experimental data, the statistical analysis is represented by mean value +/-standard deviation, the data accords with normal distribution, single factor analysis of variance (oneway-ANOVA) is adopted under the condition of variance alignment, LSD method is adopted in pairwise comparison between groups, non-normal distribution data is considered to have significant significance by non-parametric inspection method according to P <0.05, and P <0.01 is considered to have significant statistical significance.

3 results of experiments

3.1 Experimental results of different polarity portions of Gastrodia elata product fermented with Ganoderma lucidum Cordyceps militaris

After the cultivation is finished, the obtained fermentation product is subjected to centrifugal solid-liquid separation to obtain 15380g of solid, the solid is subjected to freeze drying to obtain 1691.9g, and then the obtained solid is leached by 95% ethanol, and the obtained solid is concentrated under reduced pressure to obtain 338.9g of ethanol extract with the extraction rate of 20.03%. And 40g, 9.4g, 47.2g and 141g of extractum with different polarities are obtained after extracting the ethanol extract of the fermentation gastrodia elata product of the lucid ganoderma cordyceps militaris by petroleum ether, ethyl acetate and n-butyl alcohol and the rest is water, and the extraction rates are 12.16%, 2.86%, 13.93% and 41.61% in sequence.

3.2 general physical State of rats in each experimental group

During the experiment, the spirit and activity status of rats in each experimental group were observed. The blank group has good mental state, normal diet and drinking water, free movement and glossiness of skin and hair. After lipopolysaccharide injection, rats in the model group showed typical disease behavior signs such as listlessness, reduced activity, reduced appetite, prone position, extrados, etc. The administration group rats have better mental condition than the model group, but have the phenomena of reduced activity, inappetence, drowsiness, prone position and the like, and generally recover to a normal state after 2-3 hours.

3.3 influence of different polarity portions of Gastrodia elata product fermented with Ganoderma lucidum and Cordyceps militaris on rat weight

The influence of different polarity parts of the fermented gastrodia elata product of the ganoderma lucidum cordyceps militaris on the weight of rats is shown in figure 2. The results show that the experimental rats undergo two stages of administration and molding, and the body weight of the rats tends to rise in each stage. After the modeling is finished, the weight of the rats in the model group is slowly increased, the weight of the rats in the model group is obviously reduced compared with that of the rats in the blank group, the weight of the rats in the n-butanol part group is obviously increased compared with that of the rats in the model group, and the weight of the rats in the water part group is also relatively increased. There was no significant change in body weight of rats before dosing and before molding.

3.4 influence of different polarity portions of Gastrodia elata Blume product fermented by Ganoderma lucidum and Cordyceps militaris on rat water maze experiment

Morris water maze is a worldwide accepted test method for evaluating learning and memory of experimental animals. As can be seen from fig. 3, the escape latency of rats in the model group was significantly prolonged compared to that in the blank group, suggesting that intraperitoneal injection of lipopolysaccharide could damage the learning and memory of rats. On days 3-4, the escape latency of the ethanol extract group, the ethyl acetate group and the water part group is obviously prolonged compared with that of the blank group, and the rest administration groups have no obvious difference. Compared with the model group, on days 2-7 of the test, each administration group can shorten the escape latency of rats, and particularly the piracetam tablet group and the n-butanol fraction group are more remarkable. Along with the extension of learning time, the escape latency of the ethanol extract group is obviously shortened in the 5 th to 7 th days compared with that of the model group, the escape latency of the petroleum ether part group is obviously shortened in the 4 th and 5 th days, and the escape latency is obviously shortened in the 3 th, 6 th and 7 th days. The escape latency of the water-based group was reduced at day 5 compared to the model group, and was significantly reduced at days 6-7.

Compared with the piracetam tablet group, the ethanol extract group has opposite escape latency at 3 and 4 days. Petroleum ether fraction escape latency was longer at

days

4,5 than that of piracetam tablet. The escape latency of the ethyl acetate group was also significantly prolonged at days 3-5 of the test, with no significant differences in the remaining groups. Compared with the ethanol extract group, the n-butanol fraction group was significantly shortened in the escape latency of rats at

days

3,4 and 6 of the test, and was shortened at day 7, and there was no significant difference between the rest of the time and each group. Compared with the n-butanol part group, the petroleum ether part group escape latency is obviously prolonged on the 6 th day of the test, the ethyl acetate part group escape latency is obviously prolonged on the 5 th day, the 3 rd and the 6 th days, the water part group escape latency is obviously prolonged on the 3 rd day, the 6 th day is longer, and the rest groups are not different. 3.5 influence of different polarity portions of Gastrodia elata Blume product fermented by Ganoderma lucidum and Cordyceps militaris on dark-avoiding experiment of rats

The influence of different polarity parts of the ganoderma lucidum cordyceps militaris fermented gastrodia elata product on the learning and memory of rats is tested by dark-avoiding experiments, and the experimental results are shown in figure 4. The data show that compared with a blank group, the dark-avoiding latency of rats in a model group is obviously reduced, and the number of dark-avoiding errors is obviously increased, so that lipopolysaccharide can induce learning and memory disorders of the rats, and the success of modeling is prompted. Compared with the rats in the model group, the piracetam tablet group, the ethyl acetate part group and the n-butanol part group have a trend of obviously increasing dark latency, and the dark latency of the ethanol extract group, the petroleum ether part group and the water part group is also increased. The medicine with different polarity positions can effectively improve the learning and memory disorder of rats caused by lipopolysaccharide, and the n-butanol effect is optimal.

Each dosing group reduced the number of dark errors in rats compared to the model group. Compared with the piracetam tablet group, the dark-avoiding latency of the rats in the ethanol extract group and the petroleum ether part group is obviously reduced, and the number of dark-avoiding errors of the rats in the ethanol extract group is Yu Bila. Compared with the ethanol extract group, the dark-avoiding latency of the rats in the ethyl acetate part group is prolonged, the dark-avoiding latency of the rats in the n-butanol part group is obviously increased (P < 0.01), and the dark-avoiding error frequency is obviously reduced (P < 0.01).

The result of the dark avoidance experiment shows that the effect of the n-butanol part group on improving learning and memory disorder is most obvious.

3.6 influence of different polarity portions of Gastrodia elata Blume product fermented by Ganoderma lucidum Cordyceps militaris on the nerve cell structure of CA1 region of rat Hippocampus

As shown in FIG. 5, the neuronal cells in the CA1 region of the hippocampus of rats in the blank group were orderly arranged, round and complete in structure, and the nuclei were clear without obvious swelling necrosis. Compared with the blank group, the nerve cells in the CA1 region of the hippocampus of the rat in the model group are extremely disordered, loose, irregular in size, obviously reduced in quantity, blurred in cell structure, different in morphology, deeper in coloring, and have pathological phenomena such as shrinkage, apoptosis, abnormal arrangement and the like.

Compared with the model group, the arrangement of the nerve cells in the CA1 region of the hippocampus of the rat in each of the other administration groups is relatively tidy and compact, the structure is relatively normal and clear, the deformity and shrinkage phenomenon of the nerve cells in the CA1 region of the hippocampus of the rat in the n-butanol part group are obviously improved, the arrangement of the nerve cells in the hippocampus is more compact, the structure is clearer, and the structure of the nerve cells is more complete. The result shows that the ganoderma lucidum cordyceps militaris fermented gastrodia elata product extracted by different polar solvents can improve damage of rat hippocampal neurons caused by lipopolysaccharide, and has the most obvious effect on n-butanol group.

Action 1 experimental material for improving and enhancing learning and memory disorder of rats by using second part of n-butanol polar component

1.1 experimental animals and drugs: the extract of the n-butanol part obtained by the experimental animal of the first part "1.2" and the experimental animal of the second part "2.5".

1.2 Experimental reagents

TABLE 2.1 major reagents

1.3 laboratory apparatus

TABLE 2.2 Main laboratory instruments

2 Experimental methods

2.1 grouping and administration of animals: SPF-class male SD rats, after 3 days of adaptive feeding, were eliminated from Morris water maze, and were either oversensitive (platform found within 10 s) or overstardy (platform not found within 120 s), and the screened qualified rats were randomly divided into 8 groups of blank, model, piracetam tablet, n-butanol high, medium, and low doses. The dosage (0.12 g/kg) of the n-butanol fraction in the first part is taken as the medium dosage, and the high dosage (0.24 g/kg) and the low dosage (0.06 g/kg) are set on the basis of the medium dosage, and the dosage is the same as that of the first part.

2.2, molding method: as in the first section "2.7".

2.3Morris water maze experiment: as in the first section "2.8".

2.4 dark avoidance experiments: as in the first section "2.9".

2.5 analysis of hippocampal tissue morphology: as in the first section "2.10".

2.6ELISA method for detecting the content of IL-1 beta, IL-6, TNF-alpha, NO and IL-10 in rat serum and sea horse

2.6.1 extraction and preservation of rat serum samples

The rats were anesthetized by intraperitoneal injection with anesthetic based on their body weight. After anesthesia, the rats were fixed on an operating table, the abdomen was fully exposed, and after sterilization with alcohol, blood was taken from the abdominal aorta. After blood taking is completed, standing for 1-2 h at room temperature, centrifuging for 15min at the temperature of 4 ℃ and the speed of 3000r/min, transferring the supernatant into an EP tube of 1.5mL, numbering, and freezing in a refrigerator at the temperature of-80 ℃ for later use.

2.6.2 collection and preservation of rat hippocampal tissue specimens

After blood taking is completed, the rat is immediately subjected to head breaking and brain taking, bilateral hippocampal tissues are separated on ice, and the two-sided hippocampal tissues are placed in a sterilized 1.5mLEP tube, immediately placed in liquid nitrogen for quick freezing, and frozen in a refrigerator at-80 ℃ for later use. When preparing the tissue homogenate, the tissue mass (g): pre-chilled saline was added at a ratio of saline volume (mL) =1:10, homogenized, and the supernatant was transferred to an EP tube of 1.5mL, numbered, frozen in a refrigerator at-80 ℃ for later use by centrifugation at 3000r/min for 15 min.

Method for measuring IL-1 beta, IL-6, TNF-alpha, NO and IL-10 in 2.6.3 rat serum and sea horse

Taking out the preserved serum and Hippocampus sample from the refrigerator at-80deg.C, thawing, taking out ELISA kit in the refrigerator at 4deg.C, standing at room temperature for 60min, and designing standard hole, blank hole and sample hole to be tested on the ELISA plate. All manipulations were performed strictly according to ELISA kit instructions.

2.7qRT-PCR method for detecting relative expression levels of IL-1 beta, TNF-alpha, IL-10 and BDNFmRNA in rat hippocampus

2.7.1 sample preparation

The hippocampal tissue preserved at-80℃was taken out, 50mg was placed in a 1.5mL sterile EP tube, 1mL of the lysate was added, and homogenized by a tissue grinder to prepare a homogenate.

2.7.2 extraction of Total RNA

The procedure was as per the kit instructions using conventional extraction methods.

2.7.3 Total RNA purity test

Dissolving the RNA solution in DEPC water, testing ultraviolet absorbance by using a nucleic acid protein detector, measuring the A260/A280 ratio, and judging the purity of the RNA. The RNA concentration was calculated according to the formula RNA=A260×2 (μg/. Mu.L) and adjusted to 0.5 μg/. Mu.L.

2.7.4 reverse transcription cDNA

1 removal of genomic DNA residues from RNA: mixing, and centrifuging briefly at 37deg.C for 5min.

TABLE 2.3 reverse transcription reaction System

2 the first strand cDNA synthesis step was performed by directly adding the following components necessary for the reverse transcription reaction to the reaction tube.

TABLE 2.4 reverse transcription reaction System

3 gently mixing, instantaneous centrifuging, and incubating at 42℃for 30min.

Heating at 485 deg.C for 5min to deactivate StartScript RTMix.

5, placing the cDNA obtained after the reaction is finished in a refrigerator at the temperature of minus 20 ℃ for standby.

2.7.5 primer design and synthesis

The Primer to be tested was designed by Primer software and synthesized by Shanghai Biotechnology Co. The primer sequences required for the reaction are shown in the following table:

TABLE 2.5 mRNA primer sequences of genes to be tested

2.7.6qRT-PCR amplification

1, constructing a PCR amplification system, wherein the PCR amplification system is specifically shown in the table:

TABLE 2.6PCR amplification reaction dosage

2 after the preparation of the reaction system, the amplification reaction is carried out on a real-time fluorescent quantitative PCR instrument, and the reaction is carried out in the following sequence.

TABLE 2.7PCR amplification reaction procedure

2.7.7 fluorescent quantitative PCR reading Cq value

After the reaction is finished, the PCR instrument automatically calculates the Cq value of the gene. The Cq value is the number of cycles required for the fluorescent signal of the sample in each reaction tube to reach the set threshold. By 2 ^-ΔΔCq The results were analyzed by the method and the calculation formula is as follows: ΔΔcq= [ (sample Cq value-sample internal reference Cq value) - (control sample Cq value-control sample internal reference Cq value)]Finally get 2 ^-ΔΔCq The relative expression level of the target gene.

2.8 statistical analysis: as in the first section "2.11".

3 results

3.1 general State of physical signs in rats of each Experimental group

Before molding, all rats in the experimental group were free to move and eat and drink water normally. After modeling, the activity of rats in the model group was significantly reduced, the response was retarded, and the water intake and food intake were also reduced. The high, medium and low dose intervention groups of n-butanol and the intervention group of piracetam tablet have reduced activity, poor mental condition, anorexia, drowsiness, prone position and the like in the first day of intraperitoneal injection, but return to normal in the next day, and then the response activity of each administration group becomes active gradually.

3.2 Effect of effective active sites on rat body weight

The average change in the effective active site (n-butanol) over the body weight of the rats is shown in FIG. 6. After the modeling is finished, the weight of rats in each experimental group is in a trend of increasing, but the weight of rats in the model group is lower in trend of increasing, compared with the blank group, the weight of rats is obviously reduced, and after the drug intervention, the weights of rats in the piracetam tablet group, the n-butanol high, medium and low dose groups are obviously increased compared with the model group.

3.3 Effect of effective active sites on rat Water maze experiments

In the water maze test, as can be seen from fig. 7, the escape latency of each group of rats gradually decreased with the increase of learning time. The escape latency of each group of rats was not significantly different on day 1 of the water maze test. Compared with the blank group, the escape latency of rats in the model group is obviously prolonged from the 2 nd day of the test, which indicates that the intraperitoneal injection of lipopolysaccharide can successfully induce learning and memory disorder of the rats. On days 2-3, the escape latency of the n-butanol high dose group was longer than that of the blank group, the escape latency of the n-butanol medium dose group was significantly prolonged on

days

2, 3, and 6, the escape latency of the n-butanol low dose group was prolonged on day 7 than that of the blank group, and the escape latency of the n-butanol low dose group was significantly prolonged on days 2-7 of the test.

Compared with the model group, the piracetam tablet group, the n-butanol high-dose group and the medium-dose group have the tendency of shortening escape latency on days 2-7 of the water maze test, and particularly the piracetam tablet group and the n-butanol high-dose group are most obvious, which indicates that the n-butanol part can effectively improve the learning and memory of rats. Whereas the n-butanol low dose group had a slightly shortened escape latency only on the last day of the test.

Compared with the piracetam tablet group, the escape latency of the rats in the high-dose n-butanol group is longer on the 3 rd day, the escape latency of the rats in the low-dose n-butanol group is prolonged on the 2 nd day, the escape latency of the rats in the low-dose n-butanol group is prolonged remarkably on the 3 rd day, and the escape latency of the rats in the low-dose n-butanol group is prolonged remarkably on the 2 nd-7 th day, so that the other groups have no obvious change.

The escape latency of rats in the n-butanol low dose group was significantly prolonged at days 4-6 compared to the n-butanol high dose group, and prolonged at the last day of the experiment.

The results show that the high and medium dosage groups of n-butanol can obviously improve the learning and memory impairment of rats caused by lipopolysaccharide.

3.4 Effect of effective active sites on rat dark avoidance experiments

The dark-avoiding experiment has higher sensibility to the memory process of rats. The experiment was carried out on rats for 7 days to avoid darkness, and the result is shown in fig. 8. Compared with a blank group, the dark-avoiding latency period of rats in the model group is obviously shortened, the number of dark-avoiding errors is obviously increased, and the lipopolysaccharide is suggested to induce the occurrence of neuroinflammation and damage the learning and memory of the rats. The dark-avoiding latency of the rats in the n-butanol low-dose group is shortened compared with that in the blank group, the dark-avoiding error times of the rats in the n-butanol low-dose group are obviously increased compared with that in the blank group, and the dark-avoiding error times of the rats in the n-butanol low-dose group are increased compared with that in the blank group. Compared with the model group, the dark-avoiding latency of the piracetam tablet group, the n-butanol high-dose group and the n-butanol medium-dose group is obviously increased, the dark-avoiding latency of the n-butanol low-dose group is increased, and the dark-avoiding error times of the piracetam tablet group, the n-butanol high-dose group, the n-butanol medium-dose group and the low-dose group are obviously reduced compared with the model group. The number of dark errors was slightly increased in rats in the low dose group compared to the high dose group of n-butanol.

3.5 Effect of effective active site on rat hippocampal CA1 region neural cell Structure

As shown in FIG. 9, neuronal cells in the CA1 region of the hippocampus of rats in the blank group were orderly arranged, round and complete in structure, and the nuclei were clear without obvious swelling necrosis. Compared with the blank group, the model group rat hippocampal CA1 region nerve cells are extremely disordered, loose, irregular in size, blurred in cell structure, various in morphology, abnormal in arrangement and other pathological phenomena. No significant changes were seen in the n-butanol low dose group of rats compared to the hippocampal neurons and model group of rats. The arrangement of neurons in the CA1 region of the rat hippocampus of the piracetam tablet intervention group, the normal butanol high and medium dose intervention group is tidy, the cell nucleus is clear, and the malformation phenomenon is obviously improved. The results show that the high and medium dose intervention groups of n-butanol can relieve the lipopolysaccharide-induced damage of rat hippocampal neurons.

3.6 Effect of effective active site on IL-1 beta, IL-6, TNF-alpha, NO, IL-10 content in rat serum and sea horse

3.6.1 Effect of effective active site on IL-1 beta content in rat serum, hippocampus

As shown in fig. 10, both serum and hippocampus IL-1 β content was higher in the lipopolysaccharide-induced model group than in the blank group, indicating that lipopolysaccharide induced inflammation and pro-inflammatory cytokine release. The serum of the rat in the low-dose group of the n-butanol and the expression level of the IL-1 beta in the sea horse are increased compared with the blank group, and the expression level of the IL-1 beta in the sea horse in the low-dose group of the n-butanol is higher than the blank group.

Compared with the model group, the piracetam tablet group, the n-butanol high-dose group and the n-butanol medium-dose group can reduce the content of IL-1 beta in rat serum and sea horse, which indicates that the n-butanol high-dose group and the n-butanol medium-dose group can improve the rat neuroinflammation caused by lipopolysaccharide, thereby improving the learning and memory capacity of rats. Compared with the piracetam tablet group, the serum and the IL-1 beta level content in the sea horse of the n-butanol low-dose group are obviously increased, and the IL-1 beta content in the sea horse of the n-butanol low-dose group is higher. Serum of rats in the low-dose group and IL-1β content in the hippocampus are significantly increased compared with the high-dose group of n-butanol, and the IL-1β content in serum of the low-dose group of n-butanol is slightly higher than that of the high-dose group of n-butanol.

3.6.2 Effect of effective active site on IL-6 content in rat serum, hippocampus

The changes in IL-6 content in the serum and hippocampus of each group of rats are shown in FIG. 11. The serum and the IL-6 content in the hippocampus of the rats in the model group are obviously increased compared with those in the blank group. Serum from rats in the low dose group and serum from rats in the normal butanol group, and IL-6 content in the hippocampus were also significantly higher than those in the blank group. The high dose group of n-butanol was the hippocampus with slightly higher IL-6 content than the blank group. The remaining groups were not statistically different from the blank groups. Compared with the model group, the serum of rats with high and medium doses of n-butanol and the IL-6 content in the sea horse are obviously reduced, the lower dose group with lower IL-6 content in the serum of the high dose group is further reduced, and the lower dose group with lower IL-6 content in the sea horse is also reduced. The serum and the IL-6 content in the hippocampus of the piracetam tablet group are obviously reduced compared with the model group, the IL-6 content in the serum of rats is obviously increased in the normal butanol group and the low-dose group compared with the piracetam tablet group, and the IL-6 content in the hippocampus of the rats in the low-dose group is higher.

Effects of 3.6.3 effective active site on TNF- α content in rat serum, hippocampus

The changes in TNF- α levels in the serum and hippocampus of rats from each experimental group are shown in FIG. 12. The results showed a significant increase in TNF- α content in the serum and hippocampus of rats in the model group compared to the blank group. The TNF-alpha content in the serum of rats and in the sea horse of the low-dose group of n-butanol is also significantly higher than that of the blank group. Whereas the TNF-alpha content in the hippocampus was also significantly higher in the piracetam tablet group and the high dose group than in the blank group.

Compared with the model group, the piracetam tablet group, the n-butanol high-dose group and the n-butanol medium-dose group can reduce the TNF-alpha content in rat serum and hippocampus, and the TNF-alpha content in the rat serum of the n-butanol low-dose group is also reduced. Compared with the piracetam tablet group, the serum TNF-alpha content of rats in the n-butanol dose group is obviously increased, the TNF-alpha content in the hippocampus is slightly increased, the serum of rats in the n-butanol low-dose group and the TNF-alpha content in the hippocampus are also obviously increased, and the rest groups have no obvious statistical significance. The serum TNF-alpha content of rats in the n-butanol-dosed group was significantly increased compared to the n-butanol-dosed group, the TNF-alpha content in the hippocampus was slightly increased, whereas the TNF-alpha content in the serum of rats and in the hippocampus was significantly increased compared to the higher-dose group of the n-butanol-dosed group.

Effects of 3.6.4 active site on NO content in rat serum, hippocampus

The changes in serum and hippocampal NO levels of rats in each experimental group are shown in figure 13. Compared with the blank group, the serum and the in-hippocampal NO content of the rats in the model group, the normal butanol medium-low dose group and the normal butanol high-dose group are obviously increased, the serum and the in-hippocampal NO content of the rats in the normal butanol high-dose group are also increased, and the serum NO content of the rats in the piracetam tablet group is slightly increased. Compared with the model group, the serum and the NO content in the hippocampus of the piracetam tablet group, the high-and medium-dose group of the n-butanol are obviously reduced, and the serum and the NO content in the hippocampus of the low-dose group of the n-butanol are slightly reduced. Compared with the piracetam tablet group, the serum and the hippocampal NO content of rats in the normal butanol group and the low dose group are obviously increased. The serum NO content of rats in the n-butanol medium dose group is increased compared with that in the n-butanol high dose group, and the serum NO content of rats in the n-butanol low dose group is obviously increased.

Effects of 3.6.5 effective active site on IL-10 content in rat serum, hippocampus

The present study tested the amount of anti-inflammatory cytokine IL-10 in lipopolysaccharide-induced neuroinflammation in rats and the results are shown in FIG. 14. As shown in the table, the IL-10 content in the serum of the rats in the model group and the sea horse is obviously lower than that in the blank group, the IL-10 content in the serum of the rats in the normal butanol group and the low dose group and the IL-10 content in the sea horse are also obviously lower than that in the blank group, the IL-10 content in the serum of the rats in the normal butanol high dose group is lower than that in the blank group, the IL-10 content in the sea horse is obviously lower than that in the blank group, the IL-10 content in the serum of the rats in the piracetam tablet group is obviously lower than that in the blank group, and the IL-10 content in the sea horse is lower than that in the blank group.

Compared with the model group, the serum and hippocampal IL-10 content of the piracetam tablet group, the high and medium dose groups of the n-butanol are obviously increased, and the low dose group of the n-butanol is slightly increased. Compared with the piracetam tablet group, the serum IL-10 content of the rats in the low-dose group of the n-butanol is reduced, and the IL-10 content in the sea horse is obviously reduced. Serum IL-10 content of rats in the n-butanol low dose group was reduced compared to the n-butanol high dose group, and hippocampal IL-10 content of rats in the n-butanol low dose group was significantly reduced.

3.7 Effect of effective active site on IL-1 beta, TNF-alpha, IL-10, BDNFmRNA expression levels in rat hippocampus

3.7.1 Effect of effective Activity site on IL-1 beta, TNF-alpha mRNA expression levels in rat hippocampus

The effective active site is shown in FIG. 15 for IL-1. Beta. And TNF-. Alpha.mRNA expression levels in rat hippocampus. The results show that compared with a blank group, the expression level of IL-1 beta and TNF-alpha mRNA in the rat hippocampus in the model group and the low-dose group of the n-butyl alcohol are obviously increased, the expression level of IL-1 beta mRNA in the rat hippocampus in the low-dose group of the n-butyl alcohol is obviously increased, and the expression level of TNF-alpha mRNA in the high-dose group of the n-butyl alcohol is slightly increased. Compared with the model group, the expression level of IL-1 beta and TNF-alpha mRNA in the sea horse of rats in the high, medium and low dose groups of the piracetam tablet group and the n-butanol is obviously reduced. The IL-1β mRNA expression level in the hippocampus of rats was significantly elevated in the n-butanol low dose group compared to the piracetam tablet group and the n-butanol high dose group. The expression level of TNF-alpha mRNA in the hippocampus of the rat in the n-butanol low-dose group is obviously increased compared with that in the piracetam tablet group, the expression level of TNF-alpha mRNA in the rat in the higher-dose group is slightly increased, and the difference is not statistically significant compared with the other groups.

3.7.2 Effect of effective active site on IL-10 and BDNFmRNA expression levels in rat hippocampus

IL-10 and BDNFmRNA expression levels in the hippocampus of each group of rats are shown in FIG. 16. Compared with the blank group, the expression level of IL-10 and BDNFmRNA in the hippocampus of the rats in the model group, the high, medium and low dose groups of n-butanol is obviously reduced, and the expression level of BDNFmRNA in the hippocampus of the rats in the piracetam tablet group is also obviously reduced compared with the blank group. It is suggested that lipopolysaccharide-induced learning and memory disorder results in reduced expression of anti-inflammatory factors and neurotrophic factors in the hippocampus of rats, and that the learning and memory ability of rats is reduced.

Compared with the model group, the IL-10 and BDNFmRNA expression level in the hippocampus of the piracetam tablet group, the n-butanol high-dose group and the medium-dose group is obviously increased, the IL-10mRNA expression level in the hippocampus of the n-butanol low-dose group is slightly increased compared with the model group, but no statistical difference exists, and the BDNFmRNA expression level in the hippocampus of the n-butanol low-dose group is increased compared with the model group. IL-10 and BDNFmRNA expression levels were significantly reduced in the hippocampus of rats in the high, medium and low dose groups of n-butanol, and IL-10mRNA expression levels were reduced in the hippocampus of rats in the higher dose group of n-butanol, as compared to the piracetam tablet group. The above results suggest that the effective active site (n-butanol group) can improve the levels of anti-inflammatory factors and neurotrophic factors in the hippocampus of rats to exert the effect of improving learning and memory disorders.

4. Results

From the above experiments, the most effective part of the ganoderma lucidum cordyceps militaris fermented gastrodia elata product for improving learning and memory impairment rats is the n-butanol component. The escape latency of the rat in the water maze experiment can be reduced by each dosage group of the n-butanol component, the escape latency of the rat in the escape experiment can be prolonged, the number of the escape errors of the rat can be reduced, and the dosage group with high and medium n-butanol is most obvious (P < 0.01). Pathological section results show that: neurons in the CA1 region of the hippocampus of rats in the blank group have complete structures and are orderly arranged and clear in level, while neurons in the hippocampus of rats in the model group are loose and disordered, and pathological phenomena such as apoptosis, abnormal arrangement and the like are shown. The arrangement of neurons in CA1 region of hippocampus of rats in high and medium dose intervention groups of n-butanol is relatively orderly, and the malformation phenomenon is obviously improved. The results of ELISA showed: the content of IL-1 beta, IL-6, TNF-alpha and NO in the serum of the rat in the model group and the sea horse is obviously increased compared with that in the blank group, the content of IL-10 is obviously reduced compared with that in the blank group (P is less than 0.01), and the content of IL-1 beta, IL-6, TNF-alpha and NO in the serum of the rat in the model group and the sea horse can be reduced in a dose-dependent manner by the n-butanol component, so that the content of IL-10 in the serum and the sea horse of the rat is increased; qRT-PCR results showed: compared with a blank group, the expression level of IL-1 beta and TNF-alpha mRNA in the hippocampus of a rat in a model group is obviously increased, the expression level of IL-10 and BDNFmRNA is obviously reduced, the n-butanol component can effectively reverse the trend, the expression level of IL-1 beta and TNF-alpha mRNA in the hippocampus of a rat with learning and memory disorder is reduced in a dose-dependent manner, and the expression level of IL-10 and BDNFmRNA in the hippocampus of the rat with learning and memory disorder is increased. The above ELISA and qRT-PCR results are strong evidence of memory improvement by the n-butanol fraction.

Claims

1. The preparation method of the gastrodia elata fermentation product polar component for assisting in improving memory is characterized by comprising the following steps of:

(2) Activating strains: respectively transferring Cordyceps militaris strain B1528 and Ganoderma strain B1.4 onto PDA plate, and culturing at 25deg.C in dark for 7 d; the ganoderma lucidum strain B1.4 is preserved in China center for type culture collection, and the preservation number is as follows: cctcm 2019790, cordyceps militaris strain B1528 is preserved in the chinese collection of typical cultures, accession number: ccccccm 2019789;

(5) Preparing n-butanol polar part of fermentation product of Ganoderma lucidum, cordyceps militaris and rhizoma Gastrodiae: taking the lucid ganoderma cordyceps militaris gastrodia elata fermentation product alcohol extract in the step (4), adding water, uniformly mixing to obtain a suspension, transferring the suspension into a separating funnel, adding petroleum ether according to the ratio of 1:1, slightly shaking the separating funnel, fully and uniformly mixing the petroleum ether and the fermentation product alcohol extract, and then placing the mixture on a funnel stand to be layered; after layering, the upper layer of liquid similar to transparent oil is the petroleum ether extraction part, the lower layer is the part left after petroleum ether extraction, and the upper layer part and the lower layer part are respectively collected into a beaker; transferring the lower layer part left by extraction into a separating funnel, respectively adding ethyl acetate and n-butanol with the same proportion according to the method, sequentially extracting each solvent for 6 times and 0.5h each time, and finally extracting the rest water part; concentrating the solution obtained by n-butanol extraction under reduced pressure until no smell is present, to obtain extract of n-butanol polar part of fermentation product of Ganoderma Cordyceps militaris and rhizoma Gastrodiae.

2. The use of the alcoholic extract of the fermentation product of ganoderma lucidum, cordyceps militaris and gastrodia elata in preparing a medicament for treating Alzheimer's disease and dysmnesia.

3. The use of the alcoholic extract of the fermentation product of ganoderma lucidum, cordyceps militaris and gastrodia elata in preparing functional foods and health-care products for assisting in improving memory.

4. The use of the n-butanol polar part of the fermentation product of ganoderma lucidum, cordyceps militaris and gastrodia elata according to claim 1 in preparing medicines for treating Alzheimer's disease and dysmnesia.

5. The application of the ganoderma lucidum, cordyceps militaris and gastrodia elata fermentation product n-butanol polar part in preparing functional foods and health-care products for assisting in improving memory.

6. A use according to claim 2 or 3, characterized in that: taking the ethanol extract of the fermentation product of the lucid ganoderma cordyceps militaris and the tall gastrodia tuber, and preparing the ethanol extract into pharmaceutically acceptable dosage forms including granules, capsules, tablets, injections, oral liquid and dripping pills.

7. Use according to claim 4 or 5, characterized in that: taking extract of n-butanol polar part of fermentation product of Ganoderma, cordyceps militaris and rhizoma Gastrodiae, and making into pharmaceutically acceptable dosage forms including granule, capsule, tablet, injection, oral liquid and dripping pill.