CN106265768B - Cordyceps cicadae active substance, preparation method thereof, pharmaceutical composition containing cordyceps cicadae active substance and application of cordyceps cicadae active substance - Google Patents
Cordyceps cicadae active substance, preparation method thereof, pharmaceutical composition containing cordyceps cicadae active substance and application of cordyceps cicadae active substance Download PDFInfo
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Abstract
The invention relates to a cordyceps sobolifera active substance, a preparation method thereof, a medical composition containing the same and application thereof, wherein the cordyceps sobolifera active substance is used for preventing and/or treating xerophthalmia and can be prepared by the following method: culturing Cordyceps cicadae mycelium on plate culture medium at 15-35 deg.C for 5-14 days; inoculating cultured Cordyceps cicadae mycelium into flask, and culturing at 15-35 deg.C and pH of 2-8 for several days; inoculating cultured Cordyceps cicadae mycelium into fermentation tank, at 15-35 deg.C, pH 2-8, and tank pressure 0.5-1.0kg/cm2And culturing at aeration rate of 0.01-1.5VVM for 3-5 days to obtain Cordyceps cicadae mycelium fermentation broth; freeze-drying the fermentation liquor, and grinding into powder to form cordyceps sobolifera mycelium freeze-dried powder; extracting the freeze-dried powder with a solvent to form cordyceps sobolifera mycelium extract liquid; drying the extract to obtain the cordyceps sobolifera active substance. The cordyceps sobolifera active substance can be used for preparing a medical composition for preventing and/or treating dry eye.
Description
Technical Field
The invention relates to the development of cordyceps sobolifera active substances, in particular to the technical field of application of the cordyceps sobolifera active substances in prevention of dry eye related pathological changes induced by physical injury or chemical injury.
Background
Dry eye syndrome (Xerophthalma)
"xerophthalmia" is one of the quite common diseases (accounting for 10-15% of the adult population) in the outpatient department of ophthalmology, and is mainly caused by the lack of tears on the eyeball surface or poor tear quality, and common symptoms comprise dry eyes, easy fatigue, sleepiness, itching, foreign body sensation, burning sensation, tight and heavy eyelids, sticky and thick secretion, fear of wind, photophobia, sensitivity to external stimulation, temporary blurred vision and the like; sometimes the eyes are too dry, the basic tears are insufficient, and reflective tears secretion is stimulated to cause the symptoms of frequent tears; in more serious patients, the eyes are red, swollen, congested and keratinized, the corneal epithelium is broken and filiform substances are adhered, and long-term damage can cause keratoconjunctival diseases and influence the vision. There are many possible causes of abnormal tear secretion, including excessive eye use, prolonged contact lens wear, ocular surface inflammation, aging, hormone imbalance, autoimmune diseases, diabetes, and damage to the ocular surface due to some external factors. Dry eye syndrome (Dry eye syndrome) is also known as Dry eye syndrome because of the Dry eye symptoms caused by many factors.
Composition and function of tear fluid
A layer of lacrimal fluid layer is uniformly distributed on the normal eye surface through the blinking action, covers the cornea and the conjunctiva of the eye to form a lubricating protective film, is finally removed through the nasolacrimal duct, and has the functions of moistening and protecting; the lacrimal fluid layer can be divided into 3 layers from outside to inside:
(1) grease layer: secreted by the sebaceous glands of the eyelids, in the outermost layer, the main functions are to increase the surface tension of the tear film, delay the evaporation of aqueous layer liquid, and lubricate the eyelids and eyeball surfaces.
(2) A water liquid layer: secreted by lacrimal gland, in the middle layer, it occupies most part of tear film, and contains nutrients, vitamins and antibacterial substances; the main functions are to provide the eye with a smooth and clear surface, to provide corneal oxygen, to have a bactericidal effect, and to scavenge metabolites.
(3) Mucin layer: secreted by conjunctival goblet cells, in the innermost layer, the main function is to contact with keratoconjunctival epithelial cells to make them hydrophilic, so that the aqueous layer of tears can be uniformly distributed on the surface of the keratoconjunctiva.
The composition of these 3 tears, if one is hypo-secreted or unevenly distributed, can cause dry eye symptoms.
Dry eye is a chronic disease and not easy to cure. Current therapies can be divided into medical and surgical therapies:
medical treatment of xerophthalmia patients
1. Artificial tears: artificial tears provide moistening and moisture-adding functions, and also have the effects of diluting inflammatory substances, reducing the osmotic pressure of tears, and the like, and are therefore the most commonly used therapy for dry eye patients. If allergic to preservatives or the frequency of use is high (more than 4 to 6 eye drops in 1 day, other drugs containing non-artificial tears), it is advisable to use lubricating eye drops without preservatives.
2. Autologous serum: serum has tear-like components and is also rich in growth factors, and thus can be used to treat severe dry eye conditions. The serum after each preparation can be stored for 3-6 months in a freezing way, so that patients do not need to draw blood too frequently.
3. Expectorants (mucolytic agents) mucolytic agents for the treatment of respiratory diseases are used topically to reduce the formation of filiform patches, mucus, or mucus plaques on the ocular surface.
4. Implantable artificial tears, which are solid artificial tears placed deep in the conjunctiva of the lower eye, slowly release a lubricant to keep the eye moist for 24 hours. But has the disadvantages of difficult placement (for demonstration by ophthalmologists), thick ocular surface and slightly affected vision.
5. Anti-inflammatory therapy, dry eye disease is not only insufficient lacrimal secretion, but also rapid volatilization or abnormal lacrimal composition; it is now known that the inflammatory response of the ocular surface also plays an important role. Topical steroids, or Restasis (ciclosporin-surface cyclosporine ophthalmic solutions), are therefore also another guideline for treatment. Restasis has been approved by the united states drug and food inspection office (FDA) for the treatment of chronic dry eye in 2002, which is the only drug that has been demonstrated to have an increasing effect on the amount of lacrimal fluid secretion among all dry eye drugs currently on the market.
6. Antibiotics: oral tetracycline or its related drugs (doxyccine) are particularly effective in patients with inflammation of the eyelid plates, especially in conditions of rosacea. However, it is desirable for such patients to be able to attend to eyelid cleaning and thermotherapy assistance simultaneously. Topical vitamin a ointment: can be used before sleep, but the effect is still controversial at present.
7. If the patient is accompanied with systemic immune system diseases, the patient needs to be treated together with a special medical doctor for rheumatoid immunity.
Surgical treatment of dry eye patients
Surgical treatment of dry eye is generally used for severe, ineffectual medical treatments of patients. The usual methods can be divided into the following categories:
1. lacrimal canaliculus packing: the opening of the lacrimal canaliculus is blocked by an embolization method, the action principle of the embolization method is similar to that of a water tank, only a small amount of tears flows out, more tears can be remained on the surface of the eye, and the stability of the tear film on the eyeball is maintained. Typically, an ophthalmologist will first use a temporary, dissolvable plug to test the treatment effect; if temporary embolization ameliorates the symptoms of dry eye, the physician will not place a permanent embolization. Patients receiving the lacrimal duct filling need to pay attention to whether serious inflammation occurs before operation, so as to avoid the injury of the ocular surface caused by the long-term retention of inflammatory substances on the ocular surface after the operation.
2. Eyelid suturing: patients with very severe dry eye, patients with other corneal pathologies or eyelid disunion, may undergo "eyelid sutures" to avoid further deterioration.
Diagnosis of dry eye
Ophthalmologists can diagnose dry eye from clinical symptoms and some examinations; including tear secretion tests, keratoconjunctival staining tests, tear layer disruption time, and examination of other lacrimal glands, examination of conjunctival function, and the like; in addition, some immune disease-related examinations can help to find out the cause.
Cordyceps sobolifera (Cordyceps cicadae)
The pattern and distribution of cordyceps sobolifera
Cicada fungus is also called Cicada fungus, entomophyte, Cicada grass, Cicada fungus, Cicada pupa grass, Cicada fungus or silkworm antler, and is called Cicada fungus, which is fungus of Ascomycotina (Ascomycotina), Claricipitales (Claricipitales), Clavicitaceae (Clavicipitaceae), Cordyceps (Cordyceps), and is formed by infecting larva of Cicada pupa or Cicada (Cicada flumate) of cicadae, mole cricket (Platyuraceae), black locust (Cryptotympata) and bamboo Cicada (Platylomia pili) to die, and then forming a bud-shaped stroma at the front end of the Cicada pupa or the head of the polypide. Is a bacterium and insect complex. The cicada fungus can be classified into three types, namely big cicada fungus or cicada grass (C.cicada), small cicada fungus (C.sobolifera) and cicada grass or cicada-grown cordyceps sinensis (C.cicada dicola) according to different hosts and infected strains. Cicada fungus is mostly produced in tropical and subtropical areas of the south of the Yangtze river, and is produced in Fujian, Zhejiang, Sichuan, Yunnan and Jiangsu provinces in China. There are also wild cicada fungus body traces in Taiwan mountain area.
The sexual stage of Paecilomyces cicadae is known as Cordyceps cicadae (Cordyceps cicadae), which is commonly known as Dujiaolong, with a rod-like or horn-like stroma, emerging from the head of the host as a single or cluster brown color. Paecilomyces cicadae (cicada fungus) is widely distributed in nature, and large cicada grass is rare.
Cicada fungus efficacy
The cicada fungus is a rare traditional Chinese medicinal material, has cold property, sweet taste and no toxicity, can be used as a medicine after being dried in the sun, and has the effects of dispelling wind and heat, relieving convulsion, improving eyesight, removing nebula and promoting eruption. It is mainly used for treating infantile convulsion, palpitation and night cry in compendium of materia Medica.
The cordyceps sobolifera has been used as a medicine for more than one thousand years, and the history of the wild cordyceps sobolifera is 800 years earlier than that of cordyceps sinensis. The name of cicada fungus is first found in Legong processing treatise of Liu Song times in North and south China, wherein the record is as follows: all the cordyceps sobolifera should be white. Drying under room, removing soil, decocting with serous fluid for one day, baking to dry, and grinding.
According to the records of traditional Chinese medical books "herbal from the picture of Song dynasty Su song," shan shu with one corner like crown of flower on cicada, which is called cicada flower. "; recorded in the syndrome herbal book of Ming dynasty in northern Song Tang Dynasty, the Changhua has sweet taste, cold nature and no toxicity, has the effects of dispelling wind and heat, arresting convulsion and relieving spasm, and is mainly used for treating symptoms such as nocturnal fretfulness in infants, palpitation and the like. "; ben Cao gang mu records that Chan Hua has the same action as Chan tui and can stop malaria. "; there are also traditional Chinese medicine books which record the main indications of nebula blinding, such as: "cicada flower powder" Jingyue quan Shu (Jingyue quan Shu): it is indicated for wind-heat in liver channel, up-attacking of toxic gas, conjunctival congestion and pain, and all nebula. "however, there is no scientific demonstration or publication at present.
Other conventional prescriptions such as Wanyingchanhua powder, Chanhua Mingmu prescription and Chanhua Qingre prescription can be recorded in Zhonghua Yao Da quan and Zhonghua Yao Hai.
However, there are also reports in the literature that "Changchuawuweisan" and "Wanyichanhuasan" are applied to the research of eye related diseases. The yingguanghua and the like in the first subsidiary hospital of Henan medical university use the traditional Chinese medicine raw liquid powder and the cordyceps sobolifera five-flavor powder to treat traumatic low intraocular pressure in combination with western medicines. Traumatic ocular hypotension is a common complication of ocular trauma that severely affects visual function. The results show that 14 people in the Chinese and western medicine treatment group reach normal intraocular pressure (>1.33kPa), the effective rate accounts for 46.67%, and the average intraocular pressure is increased by 0.76kPa, 8 people in the western medicine treatment group 30 reach normal intraocular pressure, the effective rate accounts for 26.67%, and the average intraocular pressure is increased by 0.41 kPa; observation of Xuda Mei of traditional Chinese medicine institute in Xinxiang City of China on 100 cases of clinical observation of treating conjunctivitis in spring by adding and subtracting oral and external washing, and comparison observation with western medicine treatment. Results the cure rate in the treatment group was 78% and in the control group 26%. After one year, the relapse rate was 22% in the treated group and 88% in the control group. The two cases are all compound applications of adding cordyceps sobolifera sporocarp, and clinical observation of intraocular pressure and spring conjunctivitis respectively does not clearly show the preparation method of the active substance of the liquid fermentation mycelium of the single cordyceps sobolifera or the cordyceps sobolifera and the prevention application and effect of the active substance on corneal injury or xerophthalmia symptoms.
The cordyceps sobolifera and cordyceps sinensis belong to the same genus entomogenous fungi complex, and the function and the application of the cordyceps sobolifera are not inferior to those of the cordyceps sinensis and cordyceps militaris, so that the cordyceps sobolifera has similar medical and health-care effects and contains similar chemical components, and is often used as a substitute of the cordyceps sinensis. The yield of the natural cordyceps sinensis is gradually reduced, the number of sporocarp of the natural cordyceps sobolifera is small, the natural cordyceps sobolifera is limited to be used in large quantities, the cordyceps sinensis can be artificially cultured to be used as a substitute of the natural cordyceps sobolifera, and the main bioactive components and the pharmacological effects of the artificial culture are similar to or superior to those of the natural cordyceps sobolifera. The generation of natural cicada fungus is dependent on host, and the host is restricted by natural environment, especially influenced by climate factor or artificial factor. The artificial culture product is used for replacing increasingly exhausted natural resources, and is an ideal way, so that the cordyceps sobolifera liquid fermentation mycelium has high economic application value.
Disclosure of Invention
The invention aims to provide a cordyceps sobolifera active substance, a preparation method of the active substance and application of the active substance in preventing or treating corneal injury or dry eye disease induced by physical injury or chemical injury and pathological changes caused by the corneal injury or the dry eye disease. Compared with the common western medicine treatment or eye drop, the preparation method of the invention is safer, simpler and more convenient, the active substances are more natural and safer, and the effect of preventing and/or treating the eye pathological changes is achieved.
The present invention provides a method for preparing an active cicada fungus material for preventing and/or treating dry eye, comprising the steps of:
(a) culturing Cordyceps cicadae mycelium on plate culture medium at 15-35 deg.C for 5-14 days;
(b) inoculating the cordyceps sobolifera mycelium cultured in the step (a) into a flask, and culturing for several days at the temperature of 15-35 ℃ and under the condition of pH 2-8;
(c) inoculating the cultured Cordyceps cicadae mycelia in step (b) into a fermentation tank, and culturing at 15-35 deg.C, pH 2-8 and tank pressure 0.5-1.0kg/cm2And culturing at aeration rate of 0.01-1.5VVM for 3-5 days to obtain Cordyceps cicadae mycelium fermentation broth;
(d) freeze-drying the cicada fungus mycelium fermentation liquor and then grinding the cicada fungus mycelium fermentation liquor into powder to form cicada fungus mycelium freeze-dried powder;
(e) extracting the cordyceps sobolifera mycelium freeze-dried powder with at least one solvent to form cordyceps sobolifera mycelium extract liquid; and
(f) drying the cordyceps sobolifera mycelium extract to obtain the cordyceps sobolifera active substance.
In one embodiment, the flask culture of the step (b) is performed by shaking culture at a shaking speed of 10-250 rpm.
In one embodiment, the gas introduced into the fermentation tank in the step (c) comprises air, oxygen, carbon dioxide, helium or a combination thereof.
In one embodiment, the incubation temperature of steps (a), (b) and (c) is 25 ℃ and the pH of steps (b) and (c) is 4-7.
In one embodiment, the incubation pH of steps (b) and (c) is 4.5.
In one embodiment, the same culture medium is used in the steps (b) and (c), and the culture medium comprises cereals, beans, inorganic salts, saccharides, Yeast extract (Yeast extract), malt extract, or a combination thereof.
In one embodiment, the number of the solvents in the step (e) is two, and the two solvents include water and alcohols.
In one embodiment, the alcohol in step (e) is methanol and/or ethanol.
In one embodiment, in the step (f), the cordyceps sobolifera mycelium extract liquid with water as a solvent is dried to form a water extract, the cordyceps sobolifera mycelium extract liquid with alcohol as a solvent is dried to form an alcohol extract, and the cordyceps sobolifera active substance is formed by mixing the water extract and the alcohol extract.
In one embodiment, the Cordyceps cicadae active substance is prepared by mixing equal weight of water extract of Cordyceps cicadae mycelium and alcohol extract of Cordyceps cicadae mycelium.
In another aspect, the present invention provides a cordyceps sobolifera active substance for treating and/or preventing dry eye, which is prepared by the aforementioned preparation method.
In another aspect, the cordyceps sobolifera active substance can be used for preparing a medicine for treating and/or preventing dry eye. Namely, the invention provides the application of the cordyceps sobolifera active substance in preparing the medicine for treating and/or preventing the dry eye syndrome.
In another aspect, the present invention provides a pharmaceutical composition for treating and/or preventing dry eye. The pharmaceutical composition comprises the cordyceps sobolifera active substance and a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. In another aspect, the pharmaceutical composition of the present invention can be used for preparing a medicament for treating and/or preventing dry eye. Namely, the present invention provides an application of the pharmaceutical composition in the preparation of a medicament for treating and/or preventing dry eye.
In order to make the features and advantages of the present invention clearer, embodiments of the present invention are described below with reference to the accompanying drawings.
Drawings
FIGS. 1A to 1D are schematic views (tables 3-1 to 3-4, respectively) drawn based on the results of a tear test for mice. FIG. 1A is a schematic representation of the change in tear secretion in a group of UVB mice; FIG. 1B is a graph plotting changes in tear secretion in a group of BAC mice; FIG. 1C is a graph plotting tear secretion amounts of UVB mouse groups; FIG. 1D is a graph plotting tear secretion amounts of the BAC mouse group.
FIGS. 2A and 2B are graphs (corresponding to tables 4-1 and 4-2, respectively) plotted based on the results of the corneal smoothness analysis in mice. FIG. 2A is a schematic representation of an average grading plot of corneal smoothness in UVB mice; figure 2B is a schematic drawing plotted against the average grade of BAC mouse corneal smoothness.
FIGS. 3A and 3B are graphs (Table 5-1 and Table 5-2, respectively) plotted based on the results of the corneal clarity analysis in mice. FIG. 3A is a schematic representation plotted against the mean grading of UVB mouse corneal clarity; figure 3B is a schematic drawing plotted against the average grading of BAC mouse corneal clarity.
FIGS. 4A and 4B are graphs (Table 6-1 and Table 6-2, respectively) drawn based on the results of corneal map analysis of mice. FIG. 4A is a schematic representation of an average graded rendering from a UVB mouse cornea map; figure 4B is a schematic drawing drawn from the mean rank of BAC mouse corneal maps.
FIGS. 5A and 5B are schematic diagrams (corresponding to tables 7-1 and 7-2, respectively) drawn based on the results of corneal lesion staining analysis in mice. FIG. 5A is a schematic representation of a mean grade plot of staining based on UVB mouse corneal lesions; figure 5B is a schematic drawing drawn according to the mean grade of BAC mouse corneal lesion staining.
Fig. 6A and 6B are schematic diagrams drawn based on the results of HE staining analysis of mice. FIG. 6A is a schematic drawing drawn from UVB mouse HE staining; figure 6B is a schematic drawing drawn based on BAC mouse corneal HE staining.
Fig. 7A to 7D are schematic diagrams (corresponding to tables 8-1 to 8-4, respectively) plotted according to the results of the tear film break-up time test for mice. FIG. 7A is a graph plotting corneal disruption time as a function of UVB mice; FIG. 7B is a schematic graph plotted against corneal disruption time for BAC mice; FIG. 7C is a graph plotting corneal disruption versus time for UVB mice; figure 7D is a graph plotting corneal disruption versus time for BAC mice.
FIGS. 8A and 8B are schematic diagrams (Table 9-1 and Table 9-2, respectively) drawn based on the results of the corneal sensitivity analysis in mice. FIG. 8A is a schematic representation plotted according to the results of UVB mouse corneal sensitivity analysis; fig. 8B is a schematic drawing based on the results of BAC mouse corneal sensitivity analysis.
Microbial preservation for patent procedure:
the preservation date is as follows: 04 months on 2015 05;
the preservation unit: china general microbiological culture Collection center (CGMCC);
the address of the depository: xilu No.1 Hospital No. 3 of Beijing, Chaoyang, China academy of sciences, institute of microbiology, 100101
The preservation number is: CGMCC No. 10486;
and (3) classification and naming: cordyceps cicadae.
Detailed Description
Principle of experiment
The cornea is one of the most dense and sensitive tissues in the human body, is the foremost part of the eye, is in direct contact with air, has no vascular tissue, obtains nutrients and oxygen through tears and aqueous humor, and is easy to damage and cause diseases. Therefore, some embodiments of the present invention are to prevent or treat dry eye and other eye diseases caused by physically or chemically induced corneal damage and to evaluate the effect and use of cordyceps sobolifera active substance.
Ultraviolet (UV) is ubiquitous in the earth and can be divided into UVA (315 + 380nm), UVB (280 + 315nm) and UVC (100 + 280nm) according to the wavelength range. Excessive exposure to ultraviolet light can cause photochemical damage, with UVB being the most significant damage to the eye. It can trigger the formation of free radicals, and inactivate antioxidant enzymes on the cornea of the eye. UVB is absorbed by the cornea, which mainly causes Ocular surface (Ocular surface) and corneal photodamage, resulting in conjunctival connective tissue hyperplasia, and eyelid skin also causes keratinized hyperplasia due to exposure, resulting in corneal conjunctival and eyelid tissue damage and accelerated aging. Prolonged exposure can cause eye irritation and foreign body sensation, as well as corneal inflammation, epithelial detachment, and degenerative damage to the cornea of the eye. The general public has less eye protection than wearing sunglasses and therefore unconsciously ignores the damage of UVB to the ocular surface resulting in chronic inflammation and even the development of dry eye.
Benzalkonium chloride (BAC, BAK) is a cationic surfactant, belongs to non-oxidative broad-spectrum bactericide, and can be used for sterilizing, disinfecting, antisepsis, emulsifying, removing scale, solubilizing, etc. Early additions to ophthalmic water were often used as a preservative, but recent studies have shown that this compound causes tear film instability, loss of goblet cells, conjunctival squamous Metaplasia (Metaplasia) and Apoptosis (Apoptosis), disruption of the corneal epithelial barrier and damage to deeper ocular tissues, mild symptoms can lead to ocular inflammation and dry eye, and severe individuals can even cause permanent damage to the ocular surface and affect vision. The mechanism by which BAC contributes to these effects has not been clarified, but current studies have demonstrated that it causes the release of pro-inflammatory cytokines (pro-inflammatory cytokines), apoptosis, and Oxidative stress (Oxidative stress) leading to an immunoinflammatory response, and in addition, it interacts directly with the lipid components of the tear film and cell membrane (Interaction).
Therefore, according to the principle, two mouse animal modes (UVB and BAC modes) are designed to observe the prevention effect of the cordyceps sobolifera active substance on dry eye caused by physical or chemical induced eye surface damage and the related application of the cordyceps sobolifera active substance to eye health care.
Experimental procedure
Preparation of cordyceps sobolifera active substance
Cordyceps cicadae mycelium source
The mycelium of the cicada fungus (Cordyceps cicadae) used in the embodiment of the invention is obtained by separating the collected fruiting body of the wild cicada fungus in Taiwan, the mycelium is obtained and is stored on a plate culture medium for successive transfer, the gene sequence of the mycelium is the cicada fungus (Cordyceps cicadae) which is proved by the identification of the development research institute of food industry in Taiwan, the strain is published and deposited in the biological resource research center (BCRC) of the development research institute of food industry of the financial institute of legal Engineers, and the deposit number is MU 30106. The strain is also preserved in 2015 at 04 th month 05 to China general microbiological culture Collection center (CGMCC), and the preservation unit address: west road No.1, north zhou zhao yang district, beijing, china academy of sciences, microbial research institute, 100101, accession number: CGMCC No. 10486; the strains are classified and named: cordyceps cicadae. However, the cordyceps sobolifera active substance is not limited to that obtained by the strain.
Liquid culture
Preparing cordyceps sobolifera liquid fermentation mycelium active substances and culturing cordyceps sobolifera mycelium liquid, namely inoculating the mycelium onto a plate culture medium, culturing for 5 days to 2 weeks at a proper temperature of 15-35 ℃ (preferably 25 ℃), scraping the mycelium and inoculating the mycelium into a flask; culturing at 15-35 deg.C (preferably 25 deg.C), pH 2-8, preferably pH 4-7, more preferably pH 4.5, and shaking speed of 10-250rpm for about 3 days, wherein the culture time can be adjusted according to culture conditions and mycelium growth; then inoculating the flask culture in a fermenter medium (see Table 1 below) (same flask culture medium) at 15-35 deg.C (preferably 25 deg.C) under a tank pressure of 0.5-1.0kg/cm2Introducing air or a mixture of air and oxygen, carbon dioxide and nitrogen, preferably air, at a ventilation rate of 0.01-1.5VVM at a stirring speed of 10-150rpm or without stirring (air lift) at a pH of 2-8 for 3-5 days to obtain cordyceps sobolifera mycelium liquid culture fermentation liquid comprising mycelium and clarified liquid. The fermentation liquor contains the cordyceps sobolifera active substance. The cordyceps sobolifera mycelium liquid culture fermentation broth can be further prepared into fermentation broth freeze-dried powder through a drying step.
The media formulations are as follows in table 1:
TABLE 1 culture Medium formulation
| Composition (I) | Content (wt%) |
| Comprehensive carbon-nitrogen source | 0.01～5 |
| Animal and plant derived protein and hydrolysate thereof | 0.01～2 |
| Yeast or malt extract (powder, paste) | 0.001～2 |
| Inorganic salts | 0.0001～0.05 |
| Saccharides and their use as anti-inflammatory agents | 0.01～10 |
In the above culture medium formula, the comprehensive carbon and nitrogen source can be cereals (such as wheat flour) or beans (such as semen glycines powder, semen Phaseoli Radiati powder, semen glycines powder, etc.); the inorganic salt can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferric sulfate, etc.; the saccharide can be glucose, fructose, maltose, sucrose, etc. Specifically, the above-mentioned culture medium formula is only an example, and the components can be adjusted according to the requirements when used, or can be used with a commercially available culture medium, and is not particularly limited.
Drying
Drying includes, but is not limited to: spray drying, hot air drying, roller drying, freeze drying, vacuum concentrating or other suitable drying method, and making into fermented liquid lyophilized powder.
Extraction of
1. Water extraction
Adding distilled water into lyophilized powder of fermentation liquid prepared by drying method of fermentation liquid for redissolving, heating at 90-121 deg.C for several minutes, cooling, and drying by reduced pressure concentration method or any one of the above drying methods to obtain Cordyceps cicadae mycelium water extract.
2. Alcohol extraction
Adding alcohol solvent into lyophilized powder of fermentation broth prepared by drying Cordyceps cicadae mycelium liquid culture fermentation broth
(1-100% by weight or volume of methanol and/or ethanol), extracting for several minutes (including but not limited to soaking, stirring, shaking or ultrasonic extraction), and drying by vacuum concentration or any drying method to obtain alcoholic extract of Cordyceps cicadae mycelium.
3. Extract mixture (any ratio)
The mixture of the above water extract and alcohol extract may contain any mixing ratio, and is not particularly limited.
Example 1 liquid fermentation culture of Cordyceps sobolifera and preparation of active substance
Mycelium strain: plate culture: the cordyceps sobolifera mycelia were inoculated on a plate medium, which is Potato dextrin medium (PDA), and cultured at 25 ℃ for about 5 days.
Flask culture:
scraping the hyphae on the plate, inoculating into a flask, and culturing at 25 deg.C and pH 4.5 with shaking at 120rpm for 3 days in a shaking machine with the culture medium of Table 2;
TABLE 2 culture Medium formulation
And (3) fermentation tank culture:
the culture medium is as shown in Table 2, and the flask culture is inoculated into a fermentation tank culture medium at 25 deg.C under a tank pressure of 0.5-1.0kg/cm2Introducing air at a ventilation rate of 0.5-1.0VVM at pH 4.5 and at a stirring speed of 10-150rpm or without stirring (air lift), and culturing for 3 days to obtain mycelium and clarified liquid, which is called cordyceps sobolifera mycelium fermentation liquid. The fermentation liquor contains the cordyceps sobolifera active substance. The cordyceps sobolifera mycelium fermentation liquid is frozen and dried to obtain the fermentation liquid freeze-dried powder.
Preparing an extract:
water extraction
Adding 20 times volume of distilled water into Cordyceps cicadae mycelium fermentation liquid lyophilized powder for dissolving, heating at 100 deg.C for thirty minutes, cooling, and lyophilizing to obtain Cordyceps cicadae mycelium water extract.
Alcohol extraction
Adding 20 times volume of ethanol into the cordyceps sobolifera mycelium fermentation broth freeze-dried powder for redissolving, extracting for 1 hour by using ultrasonic oscillation, centrifuging the extraction suspension, taking supernatant, and concentrating under reduced pressure to obtain the cordyceps sobolifera mycelium ethanol extract.
Extract mixture
Mixing the equal weight water extract and alcohol extract, and freeze drying to obtain water/alcohol extract mixture.
As a result:
about 110 kg of freeze-dried fermentation broth powder can be obtained after 20 tons of cordyceps sobolifera mycelium liquid culture fermentation broth cultured in the fermentation tank is freeze-dried. Higher concentrations of cordyceps sobolifera active substance can be obtained through the extraction step to prevent and/or treat dry eye and resulting lesions induced by physical or chemical injury. The Cordyceps cicadae active substance comprises Cordyceps cicadae mycelium fermentation liquid (mycelium and clarified liquid), fermentation liquid lyophilized powder, water/alcohol extractive solution mixture or other dosage forms. In example 2 below, a water/alcohol extract mixture was used as the cordyceps sobolifera active substance.
Example 2 analysis of animal models and associated pointers for Dry eye syndrome
1. Establishment of animal model of xerophthalmia mice
Experimental animals:
the strain ICR female mice are purchased from Lescow corporation (Taiwan of China), are 7-10 weeks old and 25-33 g in weight, are raised in the experimental animal center of Zhongshan medical university, provide normal clean feed and drinking water, are raised in 12-hour illumination and 12-hour dark circulating illumination, are controlled in temperature of 20 +/-2 ℃ and humidity of 50 +/-5%.
(1) Ultraviolet lamp UVB induced xerophthalmia
The ultraviolet lamp is purchased from Vilber lourmat company, the model of the lamp tube is VL-6MC, the wavelength is set to be in the range of 280nm to 320nm, and the main peak is 312 nm.
The test was performed by randomly dividing the mice into 4 groups of 6 mice each, a control group (fed with physiological saline, not fed with cordyceps sobolifera active substance, no UVB treatment), a UVB treated group (subjected to UVB treatment), a low dose test group (fed with 10mg/kg bw cordyceps sobolifera active substance, subjected to UVB treatment) and a high dose test group (fed with 100mg/kg bw cordyceps sobolifera active substance, subjected to UVB treatment). Wherein, the unit mg/kg · bw refers to the number of milligrams of the drug given per kilogram of body weight, and bw represents the body weight.
The experiment was carried out for 10 days, and the experimental groups were tube-fed daily with the experimental design dose of cordyceps sobolifera active substance (water/alcohol extract mixture), and the UVB-treated groups and experimental groups started on day 4, mice were anesthetized with 2.5% Avertin daily and placed in a dark box with 0.72J/cm eyeball illumination upward2UVB at intensity for 90 seconds damaged the ocular surface of the mice. All groups were subjected to tear testing in mice (days 4, 7 and 10), tear film break-up time and corneal sensitivity testing (days 4, 7 and 10), corneal appearance assessment and post-sacrifice eye tissue staining analysis (day 10) to assess whether the cicada fungus active could ameliorate ocular surface damage caused by UVB and dry eye symptoms. The data show (as detailed later), that the cordyceps sobolifera active substance has a protective effect on dry eye caused by the damage of the ocular surface induced by the physical damage of ultraviolet lamp UVB.
(2) BAC induced xerophthalmia
The test was performed by randomly dividing the mice into 4 groups of 6 mice each, namely a control group (fed with physiological saline, not fed with the cordyceps sobolifera active substance, no BAC treatment), a BAC treatment group (treated with BAC), a low dose test group (fed with the cordyceps sobolifera active substance at 10mg/kg bw, treated with BAC) and a high dose test group (fed with the cordyceps sobolifera active substance at 100mg/kg bw, treated with BAC) respectively.
The test is carried out for 14 days, the test group is fed with the cicada fungus active substance with the test design dose every day from day 1 to day 13, and from day 4 to day 13, the BAC treatment group and the mice of the test group take 5 mu L of 0.2% BAC to be spotted on the eyeballs every day, so that the eye surfaces of the mice are damaged. All groups were subjected to tear test in mice (days 4, 7, 10 and 13), tear film break-up time and corneal sensitivity test (days 4, 7, 10 and 13), corneal appearance evaluation and post-sacrifice staining of eye ball tissue analysis (day 14) to evaluate whether the cicada fungus active substance could improve ocular surface damage and dry eye symptoms caused by BAC. The data results show (detailed below) that the cordyceps sobolifera active substance has a protective effect on dry eye caused by eye surface injury induced by chemical injury of chemical substance BAC.
2. Eye surface damage degree detection item and dry eye evaluation related pointer analysis
Two mouse animal models (UVB and BAC lesion model) were used in this experiment to observe the protective effect of cordyceps sobolifera actives on the ocular surface. According to the test design, samples are taken respectively during the test and after the test is finished to evaluate the ocular surface damage degree and the xerophthalmia.
The related index analysis adopts items similar to the clinical dry eye evaluation and diagnosis, including tear test, corneal appearance analysis, HE staining histology analysis, tear film rupture time and corneal sensitivity; wherein the corneal appearance analysis was performed with 4 assessments of corneal smoothness, corneal clarity, corneal map, and corneal damage staining.
(1) Tear test (Tear production)
The tear test reflects the basal secretion of tears. The amount of lacrimal fluid secreted from the mice was tested using litmus paper. The test paper is put into an eye socket on the side close to the ear of an anesthetized mouse to suck the tear, and the test paper is taken out after a few seconds and the wet length of the test paper is measured to evaluate whether the mouse has the condition that the tear secretion quantity is changed.
(2) Corneal appearance analysis
The test was performed in 4 items of Corneal smoothness (Corneal smoothness), Corneal clarity (Corneal opacity), Corneal map (Corneal topograph) and Corneal damage staining (Corneal staining), and a higher rating score indicates a higher degree of Corneal damage.
(2.1) corneal smoothness analysis: irradiating the surface of an eyeball by using an annular light source, and grading according to the integrity of an annular image of a cornea reflection light source into 0 grade to 5 grades; the image at level 0 is a complete and undistorted ring, the ring at levels 1-3, 1/4, 1/2, and 3/4 in sequence, is distorted at level 4, and the most severe level 5 is a ring that is extremely distorted to be indistinguishable.
(2.2) corneal clarity analysis: irradiating the eyeball with a light source, observing the clarity of the cornea, and classifying into 0 grade to 4 grades according to the opacity degree; grade 0 is normal corneal transparency, grades 1-3 are mild, moderate, and moderate (unclear iris) opacity changes, respectively, while grade 4 is severe opacity changes, with a clear white turbidity observed with corneal ulceration.
(2.3) corneal map analysis: corneal map analysis projects the ocular surface in a 5-fold annular pattern, allowing a wide range of corneal smoothness to be observed. The evaluation method comprises dividing the ocular surface into 4 zones equally by cross, wherein each zone has 5 circular arcs with 5 rings, and each time 1 circular arc is distorted or cannot be judged, counting 1 point, and the total eyes are 20 points; higher score indicates higher degree of corneal unevenness, and is classified into 0-5 grades according to degree, score 0 is classified into 0 grade, 1-4 grades are classified into 1 grade, 5-9 grades are classified into 2 grades, 10-14 grades are classified into 3 grades, 15-19 grades are classified into 4 grades, and the most serious 20 grade is classified into 5 grades.
(2.4) corneal lesion staining analysis: since the damaged cornea is stained with a stain, the degree of corneal damage can be assessed by the stained area. The analysis results were rated as 0-5, those not stained were rated as 0, those below 25% were rated as 1, those 25-50% were rated as 2, those 50-75% were rated as 3, those 75% -99% were rated as 4, and all corneas were stained were rated as 5.
(3) HE staining (Hematoxylin and eosin stain, H)&E stain)
One of the most commonly used staining methods in histology is Hematoxylin (Hematoxylin), which stains basophilic structures as bluish purple, such as intranuclear chromatin and intracellular chromatin, while Eosin (Eosin), which is an acidic dye, stains cytoplasm and extracellular matrix red, thereby facilitating histological identification. After the last day of the experiment, the mice were sacrificed, the eyeball tissues were taken out and soaked in 3% formalin and dehydrated by n-butanol, paraffin embedding, sectioning and HE staining were performed, the number of cell layers, the shape and the thickness of the central cornea were observed, and the effect of administering the cordyceps sobolifera active substance was evaluated.
(4) Tear film break-up time (TBUT)
Tear film quality is also one of the causes affecting whether dry eye occurs. Tear film break-up time can assess tear quality, which can reflect tear film stability. 1 μ L of 0.1% liquid fluorescer sodium was instilled into the conjunctival sac, and tear film rupture time was recorded in seconds after three blinks. After 90 seconds, the corneal epithelial damage degree was graded by slit lamp microscopy through a cobalt blue filter. The cornea was divided into four quadrants, which were scored separately, and the four scores were added to obtain a total score (16 points for full score). 0 point is obtained after undyed; slight punctate staining and 1 point less than 30 points; the point dyeing exceeds 30 points and no diffusion phenomenon is divided into 2 points; severe punctate staining and spreading, but no apparent plaques scored 3; there were distinct fluorescent plaques scored 4.
(5) Corneal Sensitivity (CS)
Corneal sensitivity was evaluated using a Cochet and Bonnet aesthesiometer, and corneal central perception and sensitivity were measured. Non-anesthetized mice after physical or chemical injury were held by the nape of the neck at various time points and touched the corneal surface perpendicularly with monofilament fibers of adjustable length until the line was bent to an observable curve (approximately 5 ° deflection). If the mouse has no blinking reaction, the length of the monofilament fiber is shortened in sequence until the mouse has blinking reaction. The monofilament fiber unit interval is 0.5 cm, and the longer the monofilament fiber is, the easier it is to bend, and the smaller the tip pressure born by the cornea surface is, the longer the monofilament fiber is, the longer the test that the monofilament fiber with the length of 6.0-0.5 cm is sequentially used for touching the cornea surface. Blink responses were normal with monofilament fibers that did not differ by more than 5 mm. Each monofilament fiber of different length was touched perpendicular to the corneal surface at least 4 times to be recorded as a non-blinking reaction. The blink number scored 0 on a 0.5 centimeter monofilament touch. Each operational measurement must be by the same operator.
The results of the above experiments were all statistically analyzed by the mother-number-free assay Mann-Whitney U using SPSS software version 18. In the test results below, asterisks (—) indicate p <0.05, i.e., there is a significant difference between the two.
Example 3 evaluation of Effect of Cordyceps sobolifera active Material on prevention of Dry eye and related indices
1. Tear testing
In this test, litmus paper (Toyo Roshi Kaisha, Ltd) cut to a width of 1mm was used, and placed in the eye socket on the side of the ear of an anesthetized mouse to suck up tears, and after 20 seconds, the paper was taken out and the wet length (mm) of the paper was measured to test the amount of tears secreted from the mouse, thereby evaluating whether the mouse had a change in the amount of tears secreted. The test results are shown in tables 3-1 to 3-4 below (corresponding to FIGS. 1A to 1D, respectively). This result shows that, whether physically or chemically induced damage, the cordyceps sobolifera active substance can reduce the change of the lacrimal secretion amount and increase the lacrimal secretion amount, which helps prevent the generation of dry eye symptoms and/or treat dry eye.
TABLE 3-1 change in tear secretion (mm) in UVB mouse group (corresponding to FIG. 1A)
TABLE 3-2 tear secretion Change (mm) in BAC mouse group (FIG. 1B)
TABLE 3-3 tear secretion (mm) in UVB mice (FIG. 1C)
TABLE 3-4 tear secretion (mm) in BVC mouse group (FIG. 1D)
2. Corneal appearance analysis
(1) Corneal smoothness analysis:the annular light source is used for irradiating the surface of the eyeball, and the annular image is sequentially classified into 0 grade to 5 grades according to the completeness of the annular image of the corneal reflection light source from no distortion to serious distortion. The test results are shown in tables 4-1 and 4-2 below (FIG. 2A and FIG. 2B, respectively). The results show that damage, whether physically or chemically induced, is fedThe average distortion degree of the annular image of the reflecting light source of the cordyceps sobolifera active substance group is lower than that of the contrast group, so that the corneal smoothness damage is effectively reduced, and the generation of symptoms of dry eye is prevented and/or the dry eye is treated.
TABLE 4-1 UVB average corneal smoothness rating Table (FIG. 2A)
TABLE 4-2 average grading Table for corneal smoothness of BAC mice (FIG. 2B)
(2) Corneal clarity analysis:the light source is used to irradiate the eyeball, and the clarity of the cornea is observed and is divided into 0 grade to 4 grades according to the opacity degree. The test results are shown in tables 5-1 and 5-2 below (FIG. 3A and FIG. 3B, respectively). The results show that the group fed with the cordyceps sobolifera active substance has lower average degree of turbid degeneration than the control group, thereby effectively reducing turbid degeneration injury of cornea, and being helpful for preventing the symptom of dry eye and/or treating dry eye.
TABLE 5-1 UVB mouse cornea clarity average rating Table (corresponding to FIG. 3A)
TABLE 5-2, BAC mouse cornea clarity average rating Table (corresponding to FIG. 3B)
(3) Cornea map analysis:the ocular surface was projected in a quintuple annular pattern and a greater range of corneal smoothness was observed. The evaluation method comprises dividing the ocular surface into 4 zones equally, each zone having five circular 5 arc lines, counting 1 point when 1 arc line is distorted or cannot be judged, and totally sharing eyes20 points, which are classified into 0-5 grades according to the degree of corneal unevenness. The test results are shown in tables 6-1 and 6-2 below (FIG. 4A and FIG. 4B, respectively). The results show that the group fed with the cordyceps sobolifera active substance has lower average grade of the distortion degree of the circular arc line than the control group, thereby effectively reducing the unsmooth degree of the cornea and being beneficial to preventing the symptom of the dry eye and/or treating the dry eye.
TABLE 6-1 UVB mouse cornea map average rating Table (corresponding to FIG. 4A)
TABLE 6-2, BAC mouse cornea map average rating Table (corresponding to FIG. 4B)
(4) Corneal lesion staining analysis:since the damaged cornea is stained with a stain, the degree of corneal damage can be assessed by the stained area. The results were rated as 0-5, depending on the extent of the area stained. The test results are shown in tables 7-1 and 7-2 below (FIG. 5A and FIG. 5B, respectively). The results show that the groups fed with the cordyceps sobolifera active substance have lower average level of the dyed area size than the control group, and effectively reduce the degree of corneal damage, and are helpful for preventing the symptom of dry eye and/or treating the dry eye.
TABLE 7-1 average grading Table for UVB mouse corneal staining (FIG. 5A)
TABLE 7-2 average grading Table for corneal staining of BAC mice (FIG. 5B)
HE staining (Histology and H-E stain)
The number of cell layers, morphology and thickness of the cornea were observed by HE staining. The test results are shown in fig. 6A and fig. 6B, which show that the number of cell layers, morphology and thickness of the cornea were all greater than those of the group induced by injury after HE staining the group fed with cordyceps sobolifera active substance, regardless of physical UVB or chemical BAC induced injury, which effectively reduces the degree of corneal damage, and helps to prevent the symptoms of dry eye and/or treat dry eye.
4. Tear film break-up time TBUT (tear film break-up time)
And (4) dripping a fluorescent agent into the conjunctival sac, and observing the tear film rupture time by using a slit lamp to evaluate the tear film quality. The test results are shown in tables 8-1 to 8-4 below (corresponding to FIGS. 7A to 7D, respectively). The results show that the tear film rupture time and the tear film rupture relative time of the group fed with the cordyceps sobolifera active substance are higher than those of the group fed with the cordyceps sobolifera active substance, so that the tear film rupture time is effectively prolonged, the degree of corneal damage is reduced, and the preparation method is helpful for preventing symptoms of dry eye and/or treating the dry eye.
TABLE 8-1 UVB mice tear film break-up time (seconds) (corresponding to FIG. 7A)
TABLE 8-2 time (seconds) to tear film disruption in BAC mice (FIG. 7B)
TABLE 8-3 UVB mice tear film break-up relative time in seconds (corresponding to FIG. 7C)
TABLE 8-4 tear film disruption relative time (seconds) for BAC mice (corresponding to FIG. 7D)
5. Corneal sensitivity CS (corneal sensitivity)
Corneal sensitivity was assessed using a Coco-Boehringer tester. The test results are shown in tables 9-1 and 9-2 below (FIG. 8A and FIG. 8B, respectively). The results show that the group fed with the cordyceps sobolifera active substance has lower corneal sensitivity than the group induced with injury, and effectively reduces the degree of corneal injury, namely the corneal sensitivity, and is helpful for preventing the symptom of dry eye and/or treating dry eye.
TABLE 9-1 UVB mouse corneal sensitivity (mm) (corresponding to FIG. 8A)
TABLE 9-2 corneal sensitivity (mm) in BAC mice (FIG. 8B)
The above experiment of example 3 proves that the cordyceps sobolifera active substance prepared by the preparation method of example 1 has a therapeutic effect on physical UVB and chemical BAC-induced corneal damage, and can improve the damage. Therefore, the cordyceps sobolifera active substance can be applied to the fields of eye health care and dry eye disease treatment and/or prevention.
The above embodiments are specific illustrations of several possible embodiments of the present invention, but these embodiments are not intended to limit the present invention. Those skilled in the art will recognize that equivalent implementations or modifications can be made to these embodiments without departing from the spirit of the present invention.
Claims (12)
1. A method for preparing an active cicada fungus material for preventing and/or treating dry eye, comprising the steps of:
(a) culturing Cordyceps cicadae (Cordyceps cicadae) mycelium on a plate culture medium at 15-35 deg.C for 5-14 days, wherein the Cordyceps cicadae mycelium system is preserved in China general microbiological culture Collection center (CGMCC) with preservation number of CGMCC No. 10486;
(b) inoculating the cordyceps sobolifera mycelium cultured in the step (a) into a flask, and culturing for 3 days at the temperature of 15-35 ℃ and under the condition of pH 2-8;
(c) inoculating the cordyceps sobolifera mycelium cultured in the step (b) into a fermentation tank, and culturing for 3-5 days at the temperature of 15-35 ℃ and the pH value of 2-8 to form cordyceps sobolifera mycelium fermentation liquor;
(d) freeze-drying the cordyceps sobolifera mycelium fermentation liquor and then grinding the cordyceps sobolifera mycelium fermentation liquor into powder to form cordyceps sobolifera mycelium freeze-dried powder;
(e) respectively extracting the cordyceps sobolifera mycelium freeze-dried powder by using water and ethanol as solvents to form two cordyceps sobolifera mycelium extraction liquid; and
(f) drying the cordyceps sobolifera mycelium extract liquid with water as a solvent to form a water extract, drying the cordyceps sobolifera mycelium extract liquid with ethanol as a solvent to form an alcohol extract, and mixing the water extract and the alcohol extract to obtain the cordyceps sobolifera active substance.
2. The production method according to claim 1, wherein the flask culture of the step (b) is a shaking culture at a shaking rate of 10 to 250 rpm.
3. The method of claim 1, wherein the gas introduced into the fermentation tank in step (c) comprises air, oxygen, carbon dioxide, helium, or a combination thereof.
4. The method according to claim 1, wherein the fermentation tank of the step (c) has a tank pressure of 0.5 to 1.0kg/cm2And the aeration rate is 0.01-1.5 VVM.
5. The method of claim 1, wherein the same medium is used in step (b) and step (c), and the medium comprises cereals, beans, inorganic salts, sugars, yeast extract, malt extract, or a combination thereof.
6. The method of claim 1, wherein the cicada fungus active substance is prepared by mixing equal weight of water extract and alcohol extract.
7. An active cicada fungus material for the treatment and/or prevention of dry eye, which is prepared by the method of any one of claims 1 to 6.
8. Use of a cordyceps sobolifera active substance according to claim 7 for the preparation of a medicament for the treatment and/or prevention of dry eye.
9. The use of cicada fungus active substance according to claim 8 wherein the medicament for the treatment and/or prevention of dry eye is administered orally.
10. A pharmaceutical composition for the treatment and/or prevention of dry eye comprising an active substance of cordyceps sobolifera as claimed in claim 7, and a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.
11. Use of the pharmaceutical composition of claim 10 for the preparation of a medicament for the treatment and/or prevention of dry eye.
12. The use of the pharmaceutical composition according to claim 11, wherein the medicament for treating and/or preventing dry eye is administered orally.
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| CN109266556B (en) * | 2018-10-15 | 2021-08-17 | 贵州大学 | Artificial cultivation method of paecilomyces cicadae and analgesic aqueous extract cultivated by same |
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| CN110791497B (en) * | 2019-11-06 | 2022-04-29 | 江苏医药职业学院 | Enzyme composition and preparation method thereof |
| TWI788083B (en) * | 2021-11-03 | 2022-12-21 | 葡萄王生技股份有限公司 | Use of cordyceps cicadae mycelia active substance for preventing or curing macular degeneration |
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