TW201720453A - Active substance for treating dementia, preparation method thereof, pharmaceutical composition containing the same, and preparation method of pharmaceutical composition which treats dementia by increasing enzyme to make the protein decomposed - Google Patents

Abstract

The present invention provides an active substance for treating dementia, a preparation method thereof, a pharmaceutical composition containing the same, and a preparation method of the pharmaceutical composition. The preparation method of the active substance is prepared through manners of plate culture, flask culture and fermentation tank culture to obtain active substance of mycelium of Hericium erinaceus in powdery form. The mycelium powder of Hericium erinaceus can be further extracted with alcohol solvent to obtain alcohol extract, which is further purified and separated to obtain erinacines A and S.

Description

Active substance for treating dementia, preparation method thereof, pharmaceutical composition containing the same, and preparation method of the same

The present invention relates to an active substance for treating dementia, a preparation method thereof, a pharmaceutical composition containing the same, and a preparation method of the pharmaceutical composition, and more particularly to a pharmaceutical composition comprising an active substance of Hericium erinaceus mycelium and Preparation.

Alzheimer’s disease (AD)

As the proportion of the population ageing in society increases, the population suffering from dementia also increases. Dementia is not specific to a disease, but about half of the proportion is Alzheimer's disease. Patients with Alzheimer's disease lose their ability to recognize, lose their memory, lose their sense of direction, change their behavioral habits, delusions, loss of self-control, and so on. The most widely accepted cause of the disease is the accumulation of amyloid proteins in the brain causing neurodegeneration. The disease is divided into two major categories, one is familial Alzheimer's disease and the other is sporadic Alzheimer's disease. Details are as follows: Familial Alzheimer's disease is a hereditary disease, and its condition is on chromosome 21 amyloid precursor protein (APP), chromosome 14 Presenilin 1 (PS1), and chromosome 1 Presenilin 2 (PS2). There are mutations. The above-mentioned gene mutations increase the synthesis amount of the amyloid-like protein Aβ42, thereby increasing the probability of suffering from Alzheimer's disease in those who have mutations in these genes.

Sporadic Alzheimer's disease is more common, usually occurs in older people over the age of 65, but a small number of patients are prematurely Alzheimer's before the age of 65. disease.

Amyloid beta (Aβ)

The starch-like protein is mainly produced by the continuous translocation of the membrane protein-based amyloid precursor protein (APP) via β-secretase and γ-secretase. . The metabolites are commonly Aβ40 and Aβ42. Among them, the structure of Aβ42 is hydrophobic and easily aggregates with each other. Therefore, when the amount of Aβ42 is reduced or the amount of Aβ42 is increased, the formation of toxic oligomers (oligomer) is increased, thereby increasing the oxidative stress of nerve cells. Nerve cells produce inflammatory reactions, abnormal neuronal transmission, and imbalance of calcium ion regulation. Eventually, nerve cells initiate apoptosis and cause impairment of cognitive memory and other functions.

Brain starch protein degradation pathway

Under normal physiological conditions, Aβ is immediately decomposed by enzymes once it is formed to avoid excessive accumulation of Aβ, which causes damage to brain neurons. The enzymes that degrade Aβ are insulin-degrading enzyme (IDE) and enkephalinase (Nepriral endopeptidase (NEP) or membrane metallo-endopeptidase (MME)).

Insulin-degrading enzymes were discovered in 1994. In the high-insulin physiological environment, insulin competes with Aβ for IDE, causing the degradation pathway of Aβ to be blocked, causing excessive accumulation of Aβ and producing neurotoxicity, indicating that insulin-degrading enzymes not only degrade insulin. In addition to ability, it can also degrade Aβ (Kurochkin & Goto, 1994).

Enkephalinase encodes an enzyme of the MME gene (hence also known as membrane metallo-endopeptidase, MME), which catalyzes the activation of specific peptide hormones by hydrolyzing the hydrophobic end of the amino acid of the peptide chain. Previous studies have indicated that reducing the production of somatostatin in the brains of older people can result in inhibition of Neprilysin activity, which in turn contributes to the accumulation of untreated Aβ. Neprilysin's performance in the brain is not high, but it is related to synaptic plasticity and memory, so it has a certain impact on the physiological functions and substances of the brain.

Alzheimer's treatment

The current treatment of Alzheimer's disease, with different medications for the control of etiology, cognitive function, or emotional symptoms, can only temporarily improve the symptoms of Alzheimer's disease, but these drugs are not effective. Healing Alzheimer's disease. Details are as follows: The purpose of etiological treatment is to prevent or reduce the formation of nerve fiber tangles and amyloid-like proteins, but there is currently no effective control of drugs or methods.

The cognitive function drug is anti-acetylcholine hydrolase. For patients with moderate to severe disease, the drugs approved by the Department of Health are aricept, exelon, reminyl, and Yi Bijia. (ebixa).

Because most people with dementia are accompanied by behavioral or emotional conditions such as insomnia, irritability, hallucinations, delusions, etc., they have treatments for behavioral and emotional symptoms. These therapeutic drugs are hypnotics and anti-depressants. , emotional stabilizers and antipsychotics, anti-epileptic drugs. However, its benefits are not high and there is a risk of increased mortality, so application is not recommended.

Hericium

According to the "Chinese medicinal fungi" records: "Herbal-headed mushrooms are sweet, flat, can benefit the five internal organs, help digestion, nourish, have good effects on indigestion, neurodegenerative and duodenal ulcers and gastric ulcers." It is known in ancient medicine that the Hericium erinaceus has the effect of treating diseases and is a dual-use medicinal diet. Hericium erinaceus , which is classified under the fungus (Fungi), Eumycota, Basidiomycotina, Basidiomycetes, Aphyllophorales, Hydnaceae ), Hericium in the family Hericioideae. On the appearance of Hericium erinaceus, the shape of its fruit body is composed of long and rough protrusions, which are soft round shape, white when fresh, and turn yellow brown after drying. The extracts from the fruiting bodies or mycelia of Hericium erinaceus contain sugars (Wang et al., 2001; Yang et al., 2003) and Erinacines (Saito et al., 1998; Kenmoku et al. , 2002; Kenmoku et al., 2004; Watanabe et al., 2007; Watanabe and Nakada, 2008; Lee et al., 2014; Li et al., 2014), dilinoleoyl-phosphatidylethanolamine ; DLPE) (Nagai et al., 2006), amino acids, proteins and trace elements (Jia et al., 2004). In the past literature, there is no shortage of the effect of the polysaccharide of Hericium erinaceus on immune mediation, hypolipidemic, hypoglycemic or inhibition of gastric inflammation and gastric cancer. Hericium erinaceus is a dual-use medicinal diet. Studies have confirmed that Hericium erinaceus polysaccharide can inhibit the growth of tumor cells. RAW264.7 macrophages are used as immunoregulation. It is found that the water extract of Hericium erinaceus 10g/ml can induce NO(nitric oxide) and IL1 (interleukin). -1) performance, and confirmed by increasing NF-Kb (nuclear factor kB) binding activity to achieve immunomodulatory effects (Son, CG, et al. Int. Immunol., 1363-1369 2006), and Hericium erinaceus water Extract concentrations of 10 and 100 g / ml can also increase the activity of spleen cells, induce the expression of INF-γ (interferon-gamma), IL-12 (interleukin-12), and then activate natural killer cells (Yim, MH et al ., Acta Pharmacol 901-907 2007). Hericium erinaceus contains multi-peptides, phenols and their derivatives, in addition to increase immune function in the body, increase lymphocyte activity, induce cytokines and produce antibodies. In clinical trials, the monkey head mushroom tablets were given to chronic gastritis, peptic ulcer patients three times a day, three tablets for 60 days, the rate of cure for chronic gastritis patients was 88.89%, and the cure rate for patients with peptic ulcer was 84.62%. 87.5%. (Pan Chaoxiong et al. Journal of Hainan Medical College, Vol. 27, No. 6, 260-261 2004). At present, there is no literature pointing out that the mechanism of the treatment of Alzheimer's disease can be studied by the polysaccharide of Hericium erinaceus or its secondary metabolites.

It is an object of the present invention to provide a method for preparing an active material of Hericium erinaceus which can be used for the preparation of an active substance for treating dementia.

To achieve the foregoing objective, the method for preparing the active material of the Hericium erinaceus provided by the present invention comprises the following steps: (a) inoculation of a mycelium of Hericium erinaceus on a plate and culturing at a temperature of 15-32 ° C for 8-16 days; (b) inoculating the mycelium of the cultured Hericium erinaceus in step (a) in a flask The medium is cultured for 3-5 days at a temperature of 20-30 ° C and a pH of 4.5-6.5; (c) the mycelium of the cultured Hericium erinaceus in step (b) is inoculated into a fermentation tank medium at a temperature 24-32 ° C and pH 4.5-5.5, culture for 8-16 days, to obtain a Hericium erinaceus mycelium lysate; (d) Step (c) of the Hericium erinaceus mycelium lysate Dry to obtain a mycelium powder of Hericium erinaceus.

Preferably, the culture of the step (b) in the above preparation method is an oscillating culture, and the oscillation rate is 100 to 250 rpm.

Preferably, in the step (c) of the above preparation method, the tank pressure of the fermentation tank is 0.8-1.2 kg/cm ² , the stirring rate is 10-150 rpm, and a gas is introduced into the gas at aeration rate of 0.5-1 vvm. Fermentation tank.

Preferably, the gas in the aforementioned preparation method is air, oxygen, carbon dioxide, nitrogen, or a combination thereof.

Preferably, the same medium is used in the step (b) and the step (c) in the above preparation method.

Preferably, the medium in the above preparation method comprises a comprehensive carbon and nitrogen source, an animal or plant derived protein or a hydrolyzate thereof, an inorganic salt, a saccharide, a yeast, a malt extract, an antifoaming agent or a combination thereof.

Preferably, the synthetic carbon and nitrogen source in the foregoing preparation method is cereal or legume; The inorganic salts are sulfates or phosphates.

Preferably, the mycelium mycelium powder of the step (d) in the preparation method is further extracted with an alcohol solvent to obtain a mycelium alcohol extract of Hericium erinaceus.

Preferably, the alcohol solvent in the above preparation method is 30-100 v/v% ethanol or 30-100 v/v% methanol.

Preferably, the mycelial extract of Hericium erinaceus in the preparation method is further extracted with water and ethyl acetate, and then analyzed by column chromatography to obtain a cephalosporin S, a cephalosporin A or a combination thereof. .

Preferably, the Mycelium of the Hericium erinaceus active substance in the preparation method is Hericium S, Hericidin A or a combination thereof.

The invention further provides a mycelium mycelium active substance for treating dementia, which is prepared by the method as described above.

Preferably, the active substance is prepared as described in part of the foregoing method, wherein the active material of the Hericium erinaceus is in the form of a powder.

Preferably, the active substance is prepared as described in part of the foregoing method, wherein the active material of the Hericium erinaceus is in the form of an alcohol extract.

Preferably, the aforementioned active substance is prepared by a method as described in part, wherein the active material of the Hericium erinaceus is Hericium S, Hericidin A or a combination thereof.

Preferably, the aforementioned active substance is used to treat the dementia by increasing the enzyme to break down the protein.

Preferably, the enzyme is an insulin degrading enzyme (IDE).

Preferably, the protein is a starch-like protein.

Preferably, the starch protein is Aβ40, Aβ42, Aβ36, Aβ37, Aβ38, Aβ39, Aβ41, or Aβ43.

Preferably, the dementia is Alzheimer's disease.

The invention further provides a pharmaceutical composition for treating dementia comprising the Mycelium of the Hericium erinaceus active as previously described, and a biologically acceptable carrier, excipient, diluent or adjuvant.

The invention further provides a method of preparing a pharmaceutical composition for treating dementia comprising an effective amount of a mycelium active material of a Hericium erinaceus, and a biologically acceptable carrier, excipient, diluent or adjuvant .

Preferably, the Hericium erinaceus mycelium active material is prepared by a partial method as described above.

Preferably, the Hericium erinaceus mycelium active substance comprises Hericium S, Hericidin A or a combination thereof.

The first panel shows the results of HPLC analysis of the sample containing the cephalosporin A in Example 3.

The second panel shows the results of HPLC analysis of the sample containing the cephalosporin S in Example 4.

The third panel shows the ratio of IDE and NEP in the mouse brain homogenate by immunoinfiltration in Example 6.

The fourth panel shows the cross-sectional area of the amyloid-like protein plaque in mouse brain tissue analyzed by immunohistochemistry in Example 7.

An object of the present invention is to provide a pharmaceutical composition and a preparation method thereof, wherein the pharmaceutical composition obtained by the preparation method can contain various types of Hericium erinaceus mycelium active substances, and the pharmaceutical composition can improve insulin degradation Decomposition Powder protein, for the purpose of treating dementia.

experimental method

Source of bacteria:

The Hericium erinaceus strain used in the examples of the present invention was purchased from the Food Industry Research (BCRC 35669), but the active material of the Hericium erinaceus described in the present invention is not limited to the strain.

Liquid culture:

The liquid culture method of the mycelium of Hericium erinaceus is as follows, which comprises inoculating the mycelium on a plate and culturing at a suitable temperature such as 15-32 ° C for about 14 days. Next, the hyphae were scraped off and inoculated into the flask, and cultured under shaking at 20-30 ° C, pH 4.5-6.5, and shaking rate of 100-250 rpm until the beginning of the log period, 3-5 days, using the following medium. Finally, the flask culture is inoculated into the fermentation tank medium (with the flask medium) at a pressure of 0.5-1.2 v/cm 2 at 24-32 ° C, and a pH of about 4.5-5.5, at a vapour rate of 0.5-1 vvm. Air, or a mixture of air and oxygen, carbon dioxide or nitrogen, preferably air, is cultured for 8-16 days at a stirring rate of 10-150 rpm to obtain a mycelium fermentation mycelium, including mycelium and Clarification solution.

The medium formula is as follows:

The comprehensive carbon and nitrogen source may be cereals (such as wheat flour, bran) or beans (such as soybean powder, mung bean powder, soy flour, etc.); wherein the inorganic salt may be magnesium sulfate or phosphoric acid. Dihydrogen potassium, potassium dihydrogen phosphate, iron sulfate, zinc sulfate, etc.; wherein the sugar may be glucose, fructose, maltose, sucrose, etc.; wherein, in addition to the above components, the remainder is water.

An antifoaming agent may be additionally added to the fermentation tank medium to inhibit the formation of a large amount of foam during the cultivation, wherein the defoaming agent may be a commercially available conventional defoaming agent, such as 0.01% antifoaming agent such as eucalyptus oil or enamel resin. An aqueous defoamer. The culture method of the specific examples is as described later.

Preparation and analysis of active substances

The lyophilized solution obtained above is dried to obtain a powdery active material of the mycelium of Hericium erinaceus, and the drying method may, for example, be spray drying, hot air drying, drum drying, freeze drying, or the like, which is applicable to the present invention. The drying method of the invention. Preferably, the freeze-dried method is adopted, so that the obtained powdery mycelium mycelium active substance is a mycelium freeze-dried powder. Further, the lyophilized powder is extracted with an alcohol solvent to obtain an alcohol extract of mycelium of Hericium erinaceus. The ethanol extracts were extracted in a different ratio by means of water and ethyl acetate, and the resulting ethyl acetate layer was further analyzed by column chromatography to obtain a sample containing cephalosporin A and cephalosporin S. The obtained sample containing the cephalosporin A or the cephalosporin S was qualitatively analyzed by HPLC and standard comparison.

Animal experiment

Gene-transferred mice with a specific pattern were purchased, and after feeding to a specific month of age, the mice were grouped and the monkey active tissues were dosed continuously for a fixed period of time. After the feeding period, the mice were sacrificed, and the brain sections were obtained by cryosectioning, and the distribution of the amyloid-like proteins in the brain slices was observed by immunohistochemical detection. On the other hand, the brain homogenate of the mouse after sacrifice is subjected to the Western blotting method, and the substance in the homogenized liquid is separated by electrophoresis, and then the antibody is combined with the specific protein to be detected, thereby quantifying the content contained in the homogenized liquid. Specific protein.

Example 1: Culture of Mycelium of Hericium erinaceus and Preparation of Active Substance

Plate culture:

The mycelium was inoculated on a plate medium, and cultured at 25 ° C for about 7 days using potato dextrose Agar (PDA).

Flask culture:

The hyphae on the plate were scraped and inoculated into the flask, and shaken on a shaker at about 26 ° C, pH 5.0, and a rotation speed of 120 rpm for 5 days;

Medium formula:

Fermentation tank culture:

The culture medium used in the fermentation tank culture is the same as the flask culture step, and the flask culture is inoculated into the fermentation tank medium at a stirring rate of 26-1.0 kg/cm 2 , pH 5.0, 10-150 rpm at 26 ° C or not. With air lift, air was introduced at a ventilation rate of 0.5-1.0 vvm and cultured for 12 days. After 12 days, the mashing solution was obtained, which included mycelium and clear liquid, and contained the active substance of Hericium erinaceus having the effect of treating dementia.

Example 2: Preparation of freeze-dried powder of mycelium of Hericium erinaceus

After the lyophilized solution is freeze-dried, the Hericium erinaceus mycelium lyophilized powder (abbreviated as lyophilized powder) can be obtained, and 20 metric tons of the lyophilized broth can be lyophilized after being freeze-dried to obtain about 80 kg. powder.

Example 3: Extraction and analysis of Hericium A

The lyophilized powder of Hericium erinaceus mycelium was added to the lyophilized powder by 10 times by weight of methanol for the first extraction, followed by ultrasonic wave extraction for one hour, the suspension was centrifuged, and the supernatant was taken after centrifugation. The residue was subjected to a second extraction with methanol, and the above extraction step was repeated to obtain a supernatant. Finally, the supernatant was concentrated under reduced pressure to obtain a creamy mycelium extract of Hericium erinaceus.

The mycelial extract of Hericium erinaceus was extracted by liquid-liquid partitioning with water and ethyl acetate at a ratio of 1:1, and the obtained ethyl acetate layer was analyzed by a color column of ruthenium tube column, and n-hexane/ethyl acetate was used. Gradient elution was carried out to obtain 12 stratifications, and the ninth stratification was analyzed by chrome column chromatography and gradient elution with n-hexane/acetone to obtain a sample containing the compound erinacine A. Qualitative and quantitative analysis by HPLC was carried out as follows: Reversed chromatography column Cosmosil 5C18-AR-II was used at 40 ° C with 2% (v/v) acetic acid and methanol at a starting ratio of 40:60. The methanol was gradually increased to 90% by volume in 20 minutes, the flow rate was 1 ml/min, the UV detection wavelength was 340 nm, and the retention time of the cephalosporin A was 17.7 minutes. The results of this HPLC analysis are shown in the first panel (labeled as the monkey head A standard). In the first figure, the above curve is the cephalosporin A standard (the standard of the cephalosporin A is the standard prepared by the applicant in the above manner, as a quantitative basis for the cephalosporin A in the extract component), The curve below the first figure is a sample containing cephalosporin A. As can be seen from the results of the first graph, the sample containing the cephalosporin A showed peaks at 3 and 17.7 minutes, and the standard of the cephalosporin A showed a peak at 17.7 minutes. After comparison of the two phases, the peak appearing at 17.7 minutes was confirmed. It belongs to Hericium A, so the mycelium freeze-dried powder alcohol extract of Hericium erinaceus does have the active substance of Hericium A, and the quantitative result of Hericium A in the sample containing Hericium A is 223 ppm.

Example 4: Extraction and Analysis of Hericium S

The lyophilized powder of Hericium erinaceus mycelium was added to the lyophilized powder by 25 times by weight of 95v/v% ethanol for the first extraction, and then extracted by ultrasonic shock at an oscillation rate of 120 rpm for one hour, and the suspension was centrifuged and centrifuged. After taking the supernatant. The supernatant was subjected to a second extraction with 85 v/v% ethanol, and the above extraction step was repeated to obtain a supernatant. Finally, the supernatant was concentrated under reduced pressure to obtain a creamy mycelium extract of Hericium erinaceus.

The mycelial extract of Hericium erinaceus was extracted by liquid-liquid partitioning with water and ethyl acetate at a ratio of 1:4, and the obtained ethyl acetate layer was analyzed by tannin gel and LH-20 gel column color layer. The alkane/ethyl acetate was subjected to gradient elution to obtain 7 layers, and the third layer (n-hexane/ethyl acetate was 3:2) was analyzed by LH-20矽 rubber column chromatography to obtain a compound-containing monkey head. The sample of S (erinacine S) was qualitatively and quantitatively analyzed by HPLC. The analysis method was as follows: 60% extraction with acetonitrile at 40 ° C with Cosmosil 5C ₁₈ -AR-II column, within 20 minutes The acetonitrile was gradually increased to 65%, the flow rate was 1 ml/min, the wavelength was 290 nm, and the cephalosporin S appeared at about 14 minutes. The results of this HPLC analysis are shown in the second figure. The upper curve in the second figure is the standard of the monkey head (the standard product of the monkey head S is the standard prepared by the applicant himself, as the basis for judging whether or not the monkey head S is included in the subsequent extract component). The curve below the second graph is a sample containing the monkey head S. As can be seen from the results of the second graph, the sample containing the cephalosporin S showed a peak at 14.827 minutes, and the peak of the standard of the cephalosporin S appeared at the same time point, so it can be confirmed that the peak appearing at 14.827 minutes is the monkey head. Therefore, the lyophilized powder of the mycelium of the Hericium erinaceus does have the active substance of the cephalosporin S, and the quantitative result of the cephalosporin S in the sample containing the cephalosporin S is 32 ppm.

The compound described in the preceding paragraph is the compound newly discovered by the applicant. For the chemical structure and preparation method of the compound, please refer to Patent Document: Taiwan Patent Application No.: 104121632.

The hydrogen spectrum and carbon spectrum of the cephalosporin S were obtained by NMR, as shown in the following table:

Example 5: Experimental animal model and feeding of Hericium erinaceus mycelium active substance

A gene-transfected mouse of APP/PS1 was purchased from Jackson Laboratory (No. 005864), which has a human-type chimeric APP gene (Swedish KM594/595NL). /Hu APP695) and the human PS1 gene with the ninth exon (dE9) mutation. Under the joint action of the above two genes, the production and accumulation of amyloid-like protein will be accelerated, and the mouse will have abnormalities such as cognitive dysfunction in the old age, and can simulate the pathological characteristics and course of Alzheimer's disease. It is a model of experimental mice commonly used to study Alzheimer's disease.

The breeding and experimental procedures of the transgenic mice were carried out according to the Animal Use Procedures (IACUC No: 100-A-04 and 102-417-3) by the Laboratory Animal Care and Use Committee of the National Institute of Traditional Chinese Medicine of the Ministry of Health and Welfare. The mouse was housed in a transparent plastic cage with a length, width and height of 30 (W) × 20 (D) × 10 (H) cm, and the plastic cage was at room temperature. 20-25 ° C and humidity 60-70% in the dust-free automatic control room. The automatic control room controls the illumination period with an automatic timer, the dark period is from 07:00 to 19:00, and the light period is from 19:00 to 07:00. Diet and water are normally supplied during this feeding period.

It is known that the brain-like amyloid plaques of the 6-month-old APP/PS1 gene-transferred mouse model in the above environment are clearly visible, and therefore, after the mice are reared to 5 months of age, the active substances of the monkey-headed mushrooms are randomly grouped. experiment of. The mice were fed to the mice by the gastric tube in the same manner as in the fourth embodiment and the fourth embodiment. The feeding dose was 30 mg per kg per mouse. Feed for 30 days.

Example 6: Effect of active material of mycelium of Hericium erinaceus on enzyme

The 5-month-old mice fed the active substance of Hericium erinaceus in the above procedure were taken out after 30 days of feeding, and the brain tissue was taken out in Dounce Tissue Grinders in a homogenization buffer (320 mM sucrose, 2 mM EDTA, 20 mM Tris-HCl). (pH 7.4), 1 mM PMSF, 5 g/ml aprotinin). Prepare brain homogenate. The brain homogenate (containing 30 μg of protein) was subjected to Western blotting to determine whether the active substance of the feeding of the Hericium erinaceus would affect the enzyme content in the mouse brain. First, the protein in the brain homogenate is separated by colloidal electrophoresis, and then the protein on the colloid is transferred to a polyvinylidene fluoride (PVDF) membrane. The transfected protein-coated PVDF membrane was immersed in blocking buffer (PBS containing 3% normal donkey serum, 1% BSA and 0.3% Triton X-100) to block non-specific binding. The PVDF membrane was further immersed in a rabbit anti-NEP antibody (Millipore) and a rabbit anti-IDE antibody (Millipore) as primary antibodies overnight. The PVDF membrane was washed with PBS buffer, the primary antibody was washed away, and then immersed in the anti-rabbit IgG antibody conjugated with horseradish peroxidase (HRP) (GE Healthcare) as a secondary antibody. ) anti-mouse IgG antibody (anti-mouse) conjugated with wasabi peroxidase IgG antibody conjugated with HRP (Jackson ImmunoResearch)) for 2 hours. Finally, the signal of the protein was detected by the enhanced chemiluminescence detection reagents (GE Healthcare) and the Fujifilm LAS-3000 Luminescent Image Analyzer (Tokyo, Japan), and the Bonferroni multiple was determined by the post hoc test. The analysis of variance (ANOVA) with post-hoc Bonferroni multiple comparisons tests was performed to analyze the protein signals, and the results of the analysis were expressed as mean ± standard deviation (SD).

The above experimental results are shown in the third figure, in which the third picture A is the fluorescence signal detected by the fluorescence image analyzer, and the third picture B and C are the content of the IDE and NEP after the software is analyzed by the software. . In the third panel A, there are three groups from left to right, which are the control group, the feeding of cephalosporin A, and the feeding of cephalosporin S: three groups of three experimental mice are detected respectively. In the figure, from the top to the bottom are enkephalinase (NEP), insulin degrading enzyme (IDE), and actin. As can be seen from the third panel A, the signal intensity of NEP was similar to that of the control group, indicating that the NEP content in the brain of the mouse was not significantly different from that of the monkey A and S. The signal intensity of the IDE of mice fed with cephalosporin A and S was significantly stronger than that of the control group, indicating that the feeding of cephalosporin A and S had a positive relationship with the content of IDE in the brain of mice. In the third panel B, the IDE content of the control group was set to 100%, and the IDE content of the feeding of the cephalosporin A and S was significantly improved compared with the control group. After deducting the IDE content of 100% in the control group, The increase ratios were 141.1±63.7% and 130.5±68.9%, respectively. In contrast, the third panel C shows the content of NEP in the mouse brain, and the content of the control group is also set to 100%, but the NEP content of the group fed with the monkey A and S is not compared with the control phase group. There are significant differences. The results of this experiment indicate that the feeding of cephalosporin A and S has no significant effect on the content of NEP in the brain, and the main effect is to improve the IDE content.

Example 7: Accumulation analysis of amyloid-like protein in mouse brain

The 5-month-old mice fed the active substance of Hericium erinaceus in the above steps were perfused with a physiological saline solution having a pH of 7.4 (pH 7.4) after 30 days of feeding, and the brain of the mouse was taken out for immunohistochemistry. Analysis to understand the distribution of amyloid in mouse brain. First, the removed mouse brain pieces were immersed in 4% formaldehyde aqueous solution at 4 ° C for 18 hours, and then immersed in a sucrose solution to protect the frozen tissue, and then the brain was cut by an automatic floating blade to obtain a thickness of 30 μm brain sections. The sections were immersed in blocking buffer (PBS buffer containing 3% normal donkey serum, 1% bovine serum albumin (BSA) and 0.3% Triton X-100) for 6 minutes. To block non-specific binding. The above sections were immersed in mouse anti-A β 1-16 antibody (AB10, 1:300, Millipore) for 16 hours, and conjugated anti-mouse antibody with fluorescent isothiocyanate (Fluorescein isothiocyanate- The conjugated donkey anti-mouse IgG) was reacted for 2 hours in a dark room at room temperature. The sections were finally washed with PBS buffer containing 0.01% Triton X-100, and the mouse brain tissue patch was completed with a sealant Aqua Poly/Mount (Polyscience Inc., Warrington, PA, USA). The tissue patch was analyzed with a Zeiss LSM 780 conjugated focal fluorescence microscope (Jena, Germany) for immunohistochemical fluorescence images, and Image J software was used to analyze the amount of amyloid-like protein deposited in the brain.

The above experimental results are shown in the fourth figure, in which the fourth picture A is a fluorescent image (green highlight) of amyloid plaque load in the mouse brain detected by fluorescence microscopy, and it can be observed that the mouse is fed. After amyloid A (HE-A) and cephalosporin S (HE-S), the amyloid plaques were significantly less than those fed the drug vehicle. Figure 4B shows the percentage of amyloid plaques in the image of the fluorescent signal of the image as a percentage of the area of the brain slice. The three groups were followed by a control group (vehicle), a feeding of cephalosporin A (HE-A), and a feeding of cephalosporin S (HE-S). The percentage of area fed with cephalosporin A and cephalosporin S was significantly reduced, which was 40.2±15.2% and 38.1±19.7%, respectively, indicating that after feeding cephalosporin A and cephalosporin S, The amyloid protein in the brain of the mouse is decomposed, so that the effect of treating Alzheimer's disease can be achieved.

In summary, the active substances of various types of Hericium erinaceus provided by the present invention include the lyophilized powder of Hericium erinaceus mycelium, the mycelial extract of Hericium erinaceus, the cephalosporin S, or the monkey head. A has been experimentally proven to increase the efficacy of IDE, so that the accumulated amyloid protein can be degraded, and the effect of treating Alzheimer's disease can be achieved. Pharmaceutical compositions containing the active substance of Hericium erinaceus can be prepared in accordance with the method of the present invention for use in a wide variety of applications for the treatment of dementia.

Claims (24)

A method for preparing a mycelium active material of Hericium erinaceus for treating dementia comprises the following steps: (a) injecting a mycelium of Hericium erinaceus on a plate and cultivating at a temperature of 15-32 ° C for 8-16 (b) inoculation of the mycelium of Hericium erinaceus cultured in step (a) in a flask medium, and culturing for 3-5 days at a temperature of 20-30 ° C and a pH of 4.5-6.5; (c) the step (b) The cultured mycelium of Hericium erinaceus is inoculated into a fermentation tank medium and cultured for 8-16 days at a temperature of 24-32 ° C and a pH of 4.5-5.5 to obtain a mycelium fermentation of Hericium erinaceus. (d) drying the Hericium erinaceus mycelium broth in step (c) to obtain a mycelium mycelium powder. The preparation method of the first aspect of the patent application, wherein the culture of the step (b) is an oscillating culture, and the oscillation rate is 100-250 rpm. The preparation method of the first aspect of the patent application, wherein the tank pressure of the fermentation tank in the step (c) is 0.8-1.2 kg/cm ² , the stirring rate is 10-150 rpm, and the aeration rate is 0.5-1 vvm. Gas to the fermentation tank. The preparation method of claim 3, wherein the gas is air, oxygen, carbon dioxide, nitrogen, or a combination thereof. The preparation method of claim 1, wherein the same medium is used in the step (b) and the step (c). The preparation method of claim 5, wherein the medium comprises comprehensive A carbon and nitrogen source, an animal or plant derived protein or a hydrolyzate thereof, an inorganic salt, a saccharide, a yeast, a malt extract, an antifoaming agent, or a combination thereof. The preparation method of claim 6, wherein the comprehensive carbon and nitrogen source is cereal or legume; and the inorganic salt is a sulfate or a phosphate. The preparation method of claim 1, wherein the mycelium mycelium powder of the step (d) is further extracted with an alcohol solvent to obtain a mycelium alcohol extract of Hericium erinaceus. The preparation method of claim 8, wherein the alcohol solvent is 30-100 v/v% ethanol or 30-100 v/v% methanol. The preparation method of claim 8 , wherein the mycelial extract of Hericium erinaceus is further extracted with water and ethyl acetate, and then analyzed by column chromatography to obtain a cephalosporin S and a cephalosporin. A or a combination thereof. The preparation method of claim 1, wherein the mycelium mycelium active substance is cephalosporin S, cephalosporin A or a combination thereof. A mycelium mycelium active substance for treating dementia is prepared by the method of any one of claims 1-10. The active material of claim 12, wherein the active material of the Hericium erinaceus is in the form of a powder when prepared by the method of any one of claims 1-7. The active substance of claim 12, wherein the active material of the Hericium erinaceus is in the form of an alcohol extract, when prepared by the method of any one of claims 8 or 9. The active substance of claim 12, wherein the active material of the Hericium erinaceus comprises Hericium S, Hericidin A or a combination thereof when prepared according to the method of claim 10 of the patent application. For example, in the active substance of claim 12, the protein is decomposed by adding an enzyme to treat the dementia. The active substance of claim 16, wherein the enzyme is an insulin degrading enzyme (IDE). The active substance of claim 16, wherein the protein is a starch-like protein. The active substance of claim 18, wherein the starch protein is Aβ40, Aβ42, Aβ36, Aβ37, Aβ38, Aβ39, Aβ41, or Aβ43. For example, in the active substance of claim 16, wherein the dementia is Alzheimer's disease. A pharmaceutical composition for treating dementia, comprising the mycelium active material of the genus Hericium erinaceus according to any one of claims 12 to 20, and a biologically acceptable carrier, excipient, diluent or auxiliary Agent. A method of preparing a pharmaceutical composition for treating dementia comprising an effective amount of a mycelium active material of Hericium erinaceus, and a biologically acceptable carrier, excipient, diluent or adjuvant. The method of claim 22, wherein the Hericium erinaceus mycelium active material comprises the active material of any one of claims 12 to 20. Such as the method of claim 22, wherein the Hericium erinaceus mycelium active substance The system comprises Hericium S, Hericidin A or a combination thereof.