TWI552755B - A mycelia of mycelia, a method for preparing the same, a pharmaceutical composition comprising the same, and a use thereof - Google Patents

Description

Anthraquinone mycelium active substance, preparation method thereof, pharmaceutical composition containing the same, and use thereof

The present invention relates to an anthrax mycelium active substance, particularly to an anthrax mycelium active substance for preventing and/or treating a dry eye disease, a preparation method thereof, a pharmaceutical composition comprising the same, and use thereof .

Antrodia type

Antrodia is also known as burdock mushroom, scorpion, and scorpion scorpion. Taiwan has a yin and yang counterpart. The body of A. camphorata is perennial and has a strong aroma of eucalyptus, which is quite different from general ganoderma lucidum. Its appearance is plate-shaped or bell-shaped. In the plate shape, the surface is orange-red (yellow) color, the whole surface has micropores, and the bottom layer of the plate has a pale yellow-white cork, whereby the cork is attached to the inner wall of the hollow material in the burdock tree. The bell-shaped type, the fruit body (clock face) is also orange (yellow) color, full of micropores (4-5 bacteria / mm), the spore taste is extremely bitter, fresh orange-red, then become Orange-brown or brown, the bell body is dark green-brown leather. The spores were observed under a microscope, and the shape was a smooth, colorless transparent micro-bend shape.

The current status of production of Anzhi

Natural Antrodia camphorata grows only in the hollow trunk of the unique burdock tree in Taiwan. Based on the concept of ecological protection, the government has listed burdock as a conservation tree species, and the locusts that grow on it are also restricted from being collected, so the current marketers Most of the oysters sold are produced by artificial cultivation. The artificial culture of Antrodia camphora mainly includes eucalyptus cultivation, space bag culture, dish culture and liquid culture, among which eucalyptus plants The method of producing the mycelium is the closest to the natural Antrodia camphorata, and the method of liquid fermentation to produce mycelium from the natural fruiting body is short-lived and easy to be cultivated on a large scale. The mainstream of apron production.

樟芝 efficacy

Traditionally, it has been said that natural anthraquinone has the effects of detoxification, anti-inflammatory, analgesic, anti-cancer, and liver protection, but it has not been confirmed by scientific methods. In recent years, the market of Antrodia camphora has flourished, and Zhizhi has become a hot research target. The paper has reached hundreds of articles. The comprehensive results show that Antrodia can prevent inflammation, anti-oxidation, liver protection, anti-tumor, enhance immunity, lower blood pressure, etc. The main physiological active components are polysaccharide, triterpenoid and superoxide disproportionation. Enzymes and adenosine. The artificially cultured mycelium of Antrodia camphorata can induce the content and composition of the metabolites different from the natural Antrodia camphorata by adjusting the composition of the liquid medium and the culture conditions, so the efficacy is not exactly the same as the fruiting body, and this part is to be carried out by scientists. Systematic discussion. The applicant has been conducting research on the efficacy of Antrodia camphorata for a long time. The produced Astragalus mycelium capsule products have passed the two national health food certifications for protecting the liver and regulating blood pressure (Daily Health Food No. A00182). Continue to work towards the development of new functional ingredients.

Introduction to dry eye syndrome

Keratoconjunctivitis sicca is a fairly common disease (15-20% of the adult population), mainly due to lack of tears on the surface of the eyeball or poor quality of tears. Patients may have sore eyes, foreign body sensation, and burning. Uncomfortable symptoms such as increased sensation or secretion, temporary blurred vision. There are many reasons for the abnormal secretion of tears, including excessive use of the eye, wearing contact lenses for a long time, inflammation of the ocular surface, aging, imbalance of hormones, autoimmune diseases and some external factors causing damage to the ocular surface. Many factors can cause dry eye symptoms, so dry eye syndrome can also be called Dry eye syndrome.

Introduction to tear film

The tear film is a liquid film covering the cornea and the conjunctiva, which has the function of moisturizing and protecting. The structure of the tear film can be easily divided into three layers:

(1) Lipid layer: the outermost layer of the tear film, mainly composed of wax, cholesterol, phospholipids, triglycerides, etc., which are meibomian glands or tarsal glands, Zeiss glands, Moll glands Secreted, in a liquid state at normal body temperature, can prevent tears from evaporating.

(2) Aqueous layer: It is the middle layer of the tear film. It is the thickest layer in the three-layer structure. It is mainly composed of water, inorganic salts, organic matter, tear lipocalin, lactoferrin, lysozyme and lacritin. , the lacrimal gland cells (lacrimal gland) secreted.

(3) Mucous layer: the inner layer of the tear film. Covering the surface of the corneal epithelium, it is composed of mucin, etc., mainly secreted by conjunctival goblet cells, and partly secreted by the main lacrimal gland. The mucus is semi-solid and highly hydrophilic, forming a hydrophilic interface between the corneal epithelial layer and the tear layer of the tear film, which serves to nourish, protect the cornea and smooth the surface of the cornea.

Any cause of decreased tear secretion, decreased mucus secretion, decreased lipid secretion, corneal epithelial roughness and changes in tear composition, can lead to the destruction of the physiological characteristics of the tear film, causing dryness of the corneal conjunctiva, which in turn leads to the occurrence of dry eye syndrome.

Dry eye treatment

The treatment of dry eye syndrome is mainly to find the cause, the right medicine, to eliminate the disease because of the first weight, if the cause can not be ruled out to slow down the symptoms. Simple dry eye symptoms caused by poor daily habits such as excessive use of eyes, long-term wear of contact lenses, etc., usually recommended by physicians to improve their sleep by adequate sleep, hot compressing the eyes, blinking the eye and rubbing the physique. If you cannot slow down Symptoms, further treatment methods can be divided into medical therapy and surgical therapy: internal medicine therapy will give artificial tears, mucolytic agents, anti-inflammatory drugs or antibiotics according to the patient's condition, the function is to moisturize the ocular surface and reduce abnormal secretions. Or reduce inflammatory conditions to reduce ocular surface discomfort; and surgical treatment is usually used when the symptoms are very serious, including tear tube cauterization surgery, tear tube filling and eyelid suture, etc. Permanent or temporary closure of the lacrimal canal can reduce the patient's tears The loss of the tear film on the ocular surface, and suturing the eyelids can avoid further damage to the cornea in patients with severe dry eye syndrome.

Therefore, the present invention artificially cultures the mycelium of Antrodia camphorata by a specific method, and extracts the active substance thereof. It has been proved by animal experiments that the active substance of Antrodia camphorata prepared by this method has a good protective effect on physical or chemical ocular surface damage, and can be used for new purposes such as eye care or dry eye prevention and/or treatment.

According to an embodiment of the present invention, a method for preparing an anthrax mycelium active substance for preventing and/or treating dry eye is provided. The preparation method comprises the steps of: (a) cultivating a mycelium of A. glabra at a predetermined temperature and a predetermined pH for 5-7 days; (b) cultivating the mycelium of A. glabra after the step (a) Inoculation into a fermenting tank, stirring and cultivating at the predetermined temperature and the predetermined pH value for about 7-20 days; (c) freeze-drying the mycelium of the genus Antrodia camphorata after the step (b), and grinding to form a crucible a lyophilized powder of mycelium; (d) mixing a predetermined amount of the solvent with the lyophilized powder of the mycelium mycelium, and removing the lyophilized powder of the mycelium of the genus Antrodia camphorata after the shock extraction, thereby forming a Phytophthora a silk extract; (e) concentrating and drying the mycelium mycelium extract under reduced pressure to obtain the mash Mycelium active substance.

In one embodiment, the above-mentioned Antrodia sinensis mycelium system is deposited in the Biological Resources Preservation and Research Center of the Hsinchu Food Industry Development Research Institute, and the registered Astragalus mycelium is registered as CCRC (BCRC) 35398.

In one embodiment, the predetermined temperature is 20-30 ° C and the predetermined pH is 3-8.

In one embodiment, the culture of step (a) of the above preparation method is an oscillating culture, and the oscillation rate is 50-250 rpm.

In one embodiment, the fermentation tank of step (b) of the above preparation method has a tank pressure of 0.5 to 1.0 kg per square centimeter, aeration rate of 0.05 to 2 vvm, and a stirring rate of 5 to 250 rpm.

In one embodiment, the same medium is used in steps (a) and (b) of the above preparation method.

In one embodiment, the medium used in the above preparation method comprises: a cereal, a sulfate compound, a phosphate compound, a sugar, a yeast extract, a bean, or a combination thereof.

In one embodiment, the predetermined amount in the step (d) of the above preparation method is 10-50 times the weight of the lyophilized powder of the mycelium mycelium.

In one embodiment, the solution in step (d) of the above preparation method is an alcohol. In still another embodiment, the alcohol is methanol or ethanol.

According to another embodiment of the present invention, there is provided an anthrax mycelium active substance for preventing and/or treating dry eye, which is prepared by the aforementioned preparation method.

According to still another embodiment of the present invention, the use of the above-described Antrodia camphorata active substance is for the preparation of a medicament for preventing and/or treating dry eye.

According to still another embodiment of the present invention, there is provided a method for preventing and/or treating dry eyes Medical composition. The pharmaceutical composition comprises the above-described Antrodia camphorata active substance, and a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

According to still another embodiment of the present invention, the use of the above pharmaceutical composition is for the preparation of a medicament for preventing and/or treating dry eye.

In order to make the features and advantages of the above-described embodiments of the present invention more apparent, the embodiments of the present invention will be described below in conjunction with the drawings.

Figure 1 shows the grading criteria for the Corneal opacity test, where level 0 represents normal corneal transparency; level 1 represents mild opacity; level 2 represents moderate opacity; level 3 represents moderate opacity and The iris is unclear; grade 4 represents severe opaque degeneration, and significant white opacity is observed with corneal ulceration.

Figures 2A to 2D show corneal appearance analysis test results in the UVB mouse mode, wherein Figure 2A is the Corneal smoothness test; Figure 2B is the Corneal opacity test; Figure 2C For the Corneal topography test; Figure 2D is the Corneal staining test.

Figure 3 is a photograph of the central cornea region of HE staining of mouse eyeball tissue in UVB mouse mode.

4A to 4D are graphs showing the corneal appearance analysis test results of the BAC mouse model, wherein FIG. 4A is a corneal smoothness test; FIG. 4B is a corneal clearness test; and FIG. 4C is a corneal map test; FIG. 4D For corneal damage staining test.

Figure 5 is a photograph of the central corneal area of HE staining of mouse eyeball tissue in BAC mouse mode.

Experimental principle

Ultraviolet (UV) is ubiquitous on the earth. It can be divided into UVA (315-380nm), UVB (280-315nm) and UVC (100-280nm) depending on its wavelength range. UVC energy is the strongest, but most of it is absorbed by the ozone layer. It has little effect on the human body, and UVA has strong penetrating power, which will cause aging of eye lens disease, which is one of the causes of cataract. UVB penetration is weaker than UVA, which mainly leads to Ocular surface and Light damage to the cornea of the eye, long-term exposure will cause foreign body sensation and stinging, corneal epithelial shedding and other symptoms. In general, in addition to wearing sunglasses, the general public does not have eye protection. Therefore, ignoring UVB damage to the ocular surface unconsciously leads to chronic inflammation or even dry eye syndrome.

Benzalkonium chloride (BAC, BAK) is a cationic surfactant. It is a non-oxidizing broad-acting fungicide. It can be used for sterilization, disinfection, antisepsis, emulsification, descaling and solubilization. Early use has often been added to eye drops for antiseptic use, but recent studies have shown that this compound can cause tear film instability, loss of goblet cells, squamous metaplasia (Aetapatia) and apoptosis (Apoptosis), corneal epithelium Destruction of the barrier and damage to deeper eye tissue, mild symptoms can cause eye irritation and blinking, and severe cases can even cause permanent damage to the ocular surface and affect vision. The mechanism by which BAC causes these effects has not been clarified, but current studies have confirmed that it causes the release of proinflammatory cytokine, apoptosis, and Oxidative stress, which leads to immune inflammatory reactions. It has a direct interaction with the lipid components on the tear film and cell membrane.

Therefore, this experiment designed two mouse animal models (UVB according to the above principle). And BAC mode) to observe the preventive and/or therapeutic effect of the active substance of Antrodia camphorata on dry eye caused by physical or chemical induced ocular surface damage.

Experimental procedure

Mycelium culture and active substance preparation of Antrodia camphorata

The active substance of the mycelium mycelium used in this example is prepared in the following manner:

(a) Scrape fresh mycelium mycelium from the plate (strain source: Bioresource Conservation and Research Center of Hsinchu Food Industry Development Research Institute, registration number: BCRC/CCRC 35398, which can be purchased on BCRC website) In the conical flask, the medium components are listed in Table 1, and cultured at a rate of 50-250 rpm at 30 ° C and pH 4.5 to the beginning of the logarithmic reproduction period (log period) (about 5-7 days). The conditions of the culture temperature, pH and the like can be adjusted according to actual conditions, preferably the temperature is between 20 and 30 ° C, and the pH is between 3 and 8.

(b) Next, the culture in the Erlenmeyer flask is inoculated into the fermentation tank medium, and the composition of the medium is also the formulation of Table 1, at 30 ° C, the tank pressure is 0.5-1.0 kg/cm ² , pH 4.5, The 1.0 vvm aeration rate was passed through the air and harvested at a stirring rate of 200 rpm for about 10 days. The actual culture time, aeration rate and agitation rate can be adjusted according to requirements, preferably for 7 to 20 days, the aeration rate is between 0.05 and 2.0 vvm, and the agitation rate is between 5 and 250 rpm.

(c) The lyophilized powder of the mycelium of A. angustifolia is freeze-dried after being harvested. It can be freeze-dried together with the culture solution (liquid medium) in the fermentation tank to form a whole liquid lyophilized powder of the mycelium mycelium.

(d) The lyophilized powder of the mycelium of A. camphorata is placed in 10-50 times by weight of ethanol or methanol for shock extraction. The extraction time is about 24 hours. The freeze-dried powder is removed by suction filtration to obtain the Phytophthora. Silk extract.

(e) The mycelium mycelium extract is concentrated to dryness under reduced pressure, that is, the mycelium mycelium active substance used in this example.

In particular, the medium formulation used in the present invention may be adjusted as needed, or may be used in combination with a commercially available culture solution formulation, and is not particularly limited.

Example 1 UVB mouse animal model

This experiment used female mice of the ICR strain, purchased from Lesco Biotech Co., Ltd. (Taiwan), 7-10 weeks old, weighing 25-33 g, feeding conditions of 12 hours light and dark cycle, 20 ± 2 ° C and 50 ±5% relative humidity and provide clean feed and water.

Before the experiment, the mice were randomly divided into 4 groups, 6 in each group, which were control group (feeding the same amount of normal saline, no UVB treatment), UVB treatment group (treated by UVB), and low dose test group (fed 10 mg). /kg‧bw active substance of the invention, treated with UVB) and high dose test group (feeding 100 mg/kg ‧ bw of the active substance of the invention, treated with UVB). Here mg/kg. The bw unit represents how many mg of drug is administered per kg of body weight, and bw represents the body weight.

The test was carried out for 10 days. The test group was given the test design dose of the mycelium mycelium active substance daily. The UVB treatment group and the test group started from the 4th day, and the mice were anesthetized with 2.5% Avertin daily. In the dark box, the eyeball was irradiated with UVB of 0.72 J/cm ² intensity for 90 seconds, causing damage to the ocular surface of the mouse. All groups were evaluated for corneal appearance and sacrificed eye tissue staining on the 10th day of the experiment to evaluate whether the active substance of Antrodia camphorata can improve the ocular surface damage caused by UVB. The test evaluation method is detailed in the "Implementation Method" in the latter paragraph.

Example 2 BAC mouse animal model

This experiment used female mice of the ICR strain, purchased from Lesco Biotech Co., Ltd. (Taiwan), 7-10 weeks old, feeding conditions of 12 hours light and dark cycle, 20 ± 2 ° C and 50 ± 5% relative humidity And provide clean feed and water.

Before the experiment, the mice were randomly divided into 4 groups, 6 in each group, which were control group (feeding the same amount of normal saline, no BAC treatment), BAC treatment group (via BAC treatment), low-dose test group (feeding 10 mg). /kg‧bw active substance of the invention, treated by BAC) and high-dose test group (feeding 100 mg/kg ‧ bw of the active substance of the invention, treated by BAC).

The test was carried out for a total of 14 days. The test group was fed daily from the first day to the 13th day of the test design dose of the mycelium mycelium active substance, from the 4th day to the 13th day, the BAC treatment group and the test tissue In mice, 5 μL of 0.2% BAC was applied to the eyeball daily to damage the ocular surface of the mouse. All groups were evaluated for corneal appearance and tissue staining on the 14th day of the experiment to evaluate whether the active substance of the mycelium can improve the ocular surface damage caused by BAC. The test evaluation method is detailed in the later section "Implementation Method".

Method of implementation - assessment of ocular surface damage

In this experiment, two mouse animal models (Example 1-UVB and Example 2-BAC) were used to observe the protective effect of the active substance of Antrodia camphorata on the ocular surface. According to the experimental design, Samples were taken for the degree of ocular surface damage and dry eye assessment during the trial and after the end of the trial.

The evaluation included corneal appearance analysis and HE stain histological analysis; corneal appearance analysis included four assessments of corneal smoothness, corneal clarity, corneal map, and corneal damage staining.

Corneal appearance analysis was divided into Corneal smoothness, Corneal opacity, Corneal topography and Corneal staining. The higher the score, the higher the corneal damage. .

(1) Corneal smoothness analysis: the surface of the eyeball is illuminated by a ring-shaped light source, and is classified according to the integrity of the annular image of the corneal reflection source, and is classified into 0 to 5 levels; the 0-level image is a complete non-twisted ring, 1- The 3rd order is 1/4, 1/2, 3/4 part of the ring appears to be distorted, the 4th level is distorted for the entire ring, and the most serious 5th level is extremely distorted to the unrecognizable circular line.

(2) Corneal opacity: The eyeball is illuminated by a light source to observe the clarity of the cornea. It is classified into 0 to 4 grades according to the degree of opacity; 0 is the normal corneal transparency, and 1-3 grades are mild. Moderate, moderate (unclear iris) opacity, while grade 4 is severe opaque degeneration, visible white opacity with corneal ulcers, detailed grading criteria as shown in Figure 1.

(3) Corneal topography: Corneal map analysis is based on a five-ring circular pattern to project the ocular surface, allowing a wide range of corneal smoothness to be observed. The evaluation method is to divide the ocular surface into 4 zones by cross, and each zone has 5 circular arcs of five rings. Whenever one circular arc is distorted or cannot be read, it counts 1 point, and the whole eye has 20 points; Higher means that the cornea is not smooth The higher the degree is divided into 0-4 levels, the score 0 points is set to 0 level, 1-4 is divided into 1 level, 5-9 is divided into 2 levels, 10-14 is divided into 3 levels, and 15-19 is divided into 4 levels. Level, the most serious 20 points are set to level 5.

(4) Corneal staining: Since the damaged cornea is colored by the dye, the degree of corneal damage can be evaluated by the stained area. According to the size of the area, the analysis results are set to 0-5, those who are not dyed are grade 0, 25% are grade 1, 25-50% are grade 2, 50-75% are grade 3, and 75%-99% are At grade 4, the whole cornea is stained to a grade of 5.

Hematoxylin and eosin stain (H&E stain) is one of the most commonly used staining methods for histology. Hematoxylin dyes basophilic structures into blue-violet, such as intranuclear chromatin and ribosome. Etc., Eosin is an acid dye, which can make the cytoplasm and extracellular matrix appear red for histological interpretation. At the end of the last day of the experiment, the mice were sacrificed, their eyeballs were removed, paraffin-embedded, sectioned and HE stained. The number, shape and thickness of the central cornea were observed, and the mycelium activity of Antrodia camphorata was evaluated. The effect of matter.

The above experimental results were analyzed by Mann-Whitney U without parental number test in the 18th edition SPSS software. The experimental results are listed in Fig. 2A to Fig. 5. In the experimental results shown in the figure, the asterisk (*) indicates p<0.05, that is, there is a significant difference between the two.

Experimental result

Example 1 Results UVB mouse animal model

Corneal appearance analysis

The test results are as shown in Fig. 2A to Fig. 2D. In each figure, the X axis is from left to right, respectively, the control group (Blank), the treatment group (UVB treatment), and the low dose test group (10 mg/kg ‧ bw of the active substance of the present invention) , UVB treatment) and high dose test group (100 mg / kg ‧ bw active substance of the invention Quality, UVB treatment), the bw omission in the low-dose test group and the high-dose test group has made the schema clearer; the number of samples in each group is n=6, and the asterisk * in the figure indicates p<0.05, that is, there is Significant difference. In the four tests of corneal smoothness (corresponding to Figure 2A), corneal clarity (corresponding to Figure 2B), corneal map (corresponding to Figure 2C) and corneal damage staining (corresponding to 2D), the UVB-treated group Significant corneal damage (highest score score) was observed, while the degree of damage was improved in the group fed the active substance of the present invention (reduced score fraction), and dose effects were observed, high dose test group (100 mg/ The score of kg.bw) was lower than that of the low-dose test group; in the two tests of corneal smoothness (Fig. 2A) and corneal injury staining (Fig. 2D), high doses of active substance (100 mg/kg ‧ were administered Bw the active substance of the invention) can significantly reduce corneal damage in mice.

HE staining

On the 10th day after sacrifice of the mice, the corneal tissue was evaluated by HE staining with Central cornea. As shown in Fig. 3, several layers of structure were observed in the normal corneal epithelium, while the UVB treatment group, the low dose group and the high The number of cells in the dose group and the structure were not much different from the control group, indicating that UVB caused ocular surface damage not to the corneal epithelium.

Example 2 Results BAC Mouse Animal Model

Corneal appearance analysis

The test results are shown in Figures 4A to 4D. In each figure, the X-axis from left to right is the control group (Blank), the treatment group (BAC treatment), and the low-dose test group (10 mg/kg ‧ bw of the active substance of the present invention) , BAC treatment) and high-dose test group (100 mg / kg ‧ bw active substance of the invention, BAC treatment); the number of samples in each group n = 6, in the figure, the asterisk * indicates p < 0.05, there is a significant difference between the two . Treatment of BAC will result in corneal smoothness (corresponding to Figure 4A), corneal clarity (corresponding to Figure 4B), corneal map (corresponding to Figure 4C) and corneal damage staining (corresponding to Figure 4D). The numerical value of the trial was improved, with significant differences in corneal clarity (Fig. 4B) and corneal damage staining (Fig. 4D). The high dose of active substance administered to mice (feeding 100 mg/kg ‧ bw of the active substance of the present invention) significantly reduced the degree of corneal damage caused by BAC treatment in all four evaluations.

HE staining

After the mice were sacrificed on the 14th day, the corneal tissue was evaluated by HE staining with Central cornea. As shown in Fig. 5, several layers of the corneal epithelial layer were observed in the normal corneal epithelial layer, and the corneal epithelial cell layer was significantly affected by the BAC treatment group. In the low-dose group and the high-dose group, the low-dose group and the high-dose group administered with the mycelium active substance of Antrodia camphorata were improved.

The above test proves that the active substance of the mycelium produced by the preparation method of the present embodiment has an effect on the physical and chemical corneal damage, and can improve the damage thereof.

The above embodiments are intended to be illustrative of specific embodiments of the invention, and are not intended to limit the invention. It is to be understood by those skilled in the art that the scope of the present invention is intended to be limited by the scope of the appended claims.

Claims (9)

The use of an active substance of an anthraquinone mycelium for preparing a medicament for preventing and/or treating dry eye, wherein the anthrax mycelium active substance is prepared by the following steps: (a) taking an anthraquinone The mycelium is cultured for 5-7 days at a predetermined temperature and a predetermined pH; (b) inoculating the mycelium of the anthocyanin cultured in the step (a) into a fermentation tank at the predetermined temperature and the predetermined pH The culture is stirred for about 7 to 20 days; (c) the mycelium of the anthraquinones after the step (b) is freeze-dried and then ground to form a lyophilized powder of the mycelium; (d) a predetermined amount The solvent is mixed with the lyophilized powder of the mycelium mycelium, and the lyophilized powder of the mycelium mycelium is removed by filtration to form an extract of the mycelium mycelium; and (e) concentrated under reduced pressure and dried. Anthocyanin mycelium extract to obtain the active substance of the mycelium mycelium. A pharmaceutical composition for the preparation of a medicament for the prevention and/or treatment of dry eye, the pharmaceutical composition comprising a pharmaceutically acceptable carrier, excipient, diluent or adjuvant, and according to the following steps The prepared active substance of Antrodia camphorata mycelium: (a) cultivating the mycelium of A. glabra L. at a predetermined temperature and a predetermined pH for 5-7 days; (b) cultivating the Phytophthora infestans after the step (a) The filament is inoculated into a fermentation tank, and the culture is stirred and cultured at the predetermined temperature and the predetermined pH for about 7 to 20 days; (c) the mycelium of the anthraquinone cultured in the step (b) is freeze-dried and ground to form a lyophilized powder of mycelium; (d) mixing a predetermined amount of the solvent with the lyophilized powder of the mycelium mycelium, after the shock extraction Removing the lyophilized powder of the mycelium of the genus Aster, to form an extract of the mycelium of the genus Zhizhi; and (e) concentrating and drying the extract of the mycelium of the genus Zhizhi, under reduced pressure, to obtain the mycelium activity of the genus substance. The use according to claim 1 or 2, wherein the predetermined temperature in the preparation step (a) of the anthrax mycelium active substance is 20-30 ° C, and the predetermined pH value is 3-8 . The use according to the first or second aspect of the patent application, wherein the culture system in the preparation step (a) of the mycelium mycelium active substance is a shock culture, and the oscillation rate is 50-250 rpm. The application according to claim 1 or 2, wherein the tank pressure of the fermentation tank in the preparation step (b) of the anthrax mycelium active substance is 0.5-1.0 kg per square centimeter, and the aeration rate is 0.05- 2 vvm and agitation rate of 5-250 rpm. The use of the first or second aspect of the patent application, wherein the same medium is used in the steps (a) and (b) of preparing the anthrax mycelium active substance. The use according to the first or the second aspect of the patent application, wherein the predetermined amount in the preparation step (d) of the mycelium mycelium active substance is 10-50 of the weight of the lyophilized powder of the mycelium mycelium Times. The use according to the first or second aspect of the invention, wherein the solvent of the anthrax mycelium active substance preparation step (d) is an alcohol. The use of the mycelium mycelium active material as described in claim 8 wherein the alcohol is methanol or ethanol.