TW202024331A - Method of manufacturing and the use of cordyceps cicadae mycelia active substance for preventing and/or improving acute lung injury - Google Patents

Abstract

A method for manufacturingCordyceps Cicadae mycelia active substance for preventing and/or improving acute lung injury is provided. The methodcomprisesfollowing steps: （a）culturing aCordyceps Cicadae mycelium in a plate media between 15℃ and 30℃ for 1 to 2 weeks; （b）inoculating the mycelium of step （a） to a flask and culturing it between 15℃ and 30℃ with a pH of 2 to 6 for 3 to 14 days; （c）inoculating the mycelium of step （b） to a fermentation tank and culturing it between 15℃ and 30℃ with a pH of 2 to 6 for 3 to 21 days, so as to obtain aCordyceps Cicadae mycelium fermentation liquid containing saidCordyceps Cicadae mycelium active substance.

Description

Cicada flower mycelium active substance for preventing and/or improving acute lung injury, preparation method and use thereof

The invention relates to a cicada flower mycelium active substance, its preparation method and application. Specifically, it relates to a cicada flower mycelium active substance for preventing and/or ameliorating acute lung injury, its preparation method, and its use in food or medicine.

The Ministry of Health and Welfare announced that among the top ten causes of death in 2017, trachea, bronchus and lung cancer were the first cause of death among malignant tumors, the third cause of death was pneumonia, and the seventh cause of death was chronic lower respiratory disease. The top ten causes of death are inextricably related to lung inflammation.

Among the ten major causes of death announced in the United States, there are also three diseases related to lung inflammation, including lung and bronchial cancer, chronic respiratory diseases, influenza and pneumonia. Further statistical analysis found that, on average, 79 and 59 people per 100,000 people in the United States suffer acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) each year. Among them, the death rate caused by ALI/ARDS is about 43%, causing about 75,000 deaths in the United States each year. As air pollution is getting worse, the environment and the average remaining life are increasing year by year, it is speculated that the incidence of ALI/ARDS in the United States will increase more than twice in the next 25 years.

ALI and more severe ARDS are the representative diseases of common clinical acute lung inflammation, which can cause respiratory failure and lead to death, and are related to many derived respiratory diseases.

The risk factors that cause ALI are divided into two categories. Direct factors refer to the risk factors that originate from the lungs, including bacteria or viruses that cause infectious pneumonia, inhalation of gastric acid or substances, lung contusions, etc.; indirect factors refer to risk factors Not derived from the lungs, including sepsis, long-term alcohol and drug abuse, transfusion of artificial plasma, etc. Among them, bacterial infection is the main risk factor for ALI, and gram-negative bacteria are one of the major categories. The main component of its mantle is endotoxin, also known as lipopolysaccride (LPS).

Due to the complex mechanism and high fatality rate of ALI, there is no clinically effective drug to control the fatality rate. However, the commonly used treatment methods include mechanical ventilation, β2 adrenergic receptor stimulators, anticoagulation, thrombolysis, surfactants, and surgery. Therefore, research on effective treatment of ALI is still an important development direction.

Cordyceps cicadae , also known as insect flower, soil cicada flower, cicada and cicada chrysalis, refers to the dead body of cicada larvae parasitized by the spores of Cordyceps fungi of the family Clavicipitaceae ( Clavicipitaceae ). On the head of the body of the cicada, a sub seat formed by mycelium grows, which looks like a flower bud. In the Compendium of Materia Medica, Syndrome Materia Medica, and Chinese Medicine Sea, it is pointed out that the cicada flower mainly treats children's night crying, heart palpitations, stops malaria, and has the effects of dispelling wind-heat and suppressing convulsions. After component analysis, it is found that the fruit body of natural cicada flower is similar to Cordyceps sinensis.

Modern pharmacological research and analysis have found that the cicada flower has biological activities of immune regulation, anti-oxidation, neuroprotection and anti-cancer. However, there is no research on the improvement of ALI by cicada flower.

The present invention provides a cicada flower mycelium active substance and a preparation method thereof, which can be used to prepare a composition for preventing and/or improving acute lung injury. Compared with general western medicine and treatment methods, the preparation method of liquid fermented cicada flower mycelium active substance disclosed in the present invention is safer and simpler, and the prepared cicada flower mycelium active substance is more natural, safe, and can be effectively improved ALI.

According to an embodiment of the present invention, there is provided a preparation method for preparing an active substance of cicada flower mycelium for improving ALI, which comprises the following steps:

(A) Take Cordyceps Cicadae mycelia ( Cordyceps Cicadae mycelia) on a plate medium and culture it at a temperature of 15 to 30 ℃ for 1 to 2 weeks;

(B) Inoculate the cicada flower mycelium cultured in step (a) into a flask, and cultivate for 3 to 14 days in an environment of 15 to 30°C and pH 2 to 6;

(C) Inoculate the cicada flower mycelium cultured in step (b) in a fermentation tank, stir and cultivate for 3 to 21 days under an environment of 15 to 30 ℃ and pH 2 to 6, to form an activity containing cicada flower mycelium Substance of fermentation broth of cicada flower mycelium.

In one embodiment, the method for preparing the active substance of the mycelium of the cicada flower further includes the step (d): freeze-drying the fermentation liquid of the mycelium of the cicada flower and then pulverize it to form a cicada containing the active substance of the cicada flower Mycelium freeze-dried powder.

In one embodiment, the fermentation tank in step (c) is further vented with a gas, the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof, the tank pressure of the fermentation tank is 0.5 to 1.0 kg/cm ² and the aeration rate It is 0.01 to 1.5 VVM.

Another embodiment of the present invention provides a cicada flower mycelium active substance, which is manufactured by the above-mentioned preparation method.

Another aspect of the present invention is to provide a composition for preventing and/or ameliorating acute lung injury, which contains the above-mentioned active substance of cicada flower mycelium, and pharmaceutically acceptable carriers and excipients , Diluent or adjuvant.

Another aspect of the present invention is to provide a use of the above active substance from the mycelium of cicada flower, which is used to prepare a composition for preventing and/or ameliorating acute lung injury.

In one embodiment, the improvement of acute lung injury refers to the alleviation of pathological symptoms of lung inflammation.

In one embodiment, the pathological reduction of lung inflammation means that the structure of the alveoli tends to be complete from destruction or fusion.

In one embodiment, the improvement of acute lung injury refers to the reduction of protein exudation response.

In one embodiment, the improvement of acute lung injury refers to a decrease in the degree of infiltration of inflammatory cells.

In one embodiment, the inflammatory cell refers to white blood cells, phagocytes and/or neutrophils.

In order to make the above and other aspects of the present invention clearer and easier to understand, the following specific embodiments are described in detail in conjunction with the accompanying drawings.

Example 1: Cultivation of cicada flower mycelium

The Cordyceps cicadae hypha system used in the examples of the present invention is obtained by separating the fruiting bodies of the wild cicada flowers from Taiwan to obtain the mycelium, which is then stored on a plate medium. After identifying its gene sequence by Taiwan Food Industry Development Research, it was confirmed to be Cordyceps cicadae . This strain has been publicly deposited in the Bioresources Research Center (BCRC) of the Food Industry Development Research Institute, and its deposit number is MU30106 . However, the source of the active substance of the mycelium of cicada flower in the present invention is not limited to that obtained from the strain.

(1) Plate culture: Inoculate the cicada flower mycelium on the plate, and culture it at 15-30°C for 1 to 2 weeks (in this example, culture at 25°C for 7 days). The composition of the plate medium may include Potato Dextrose Agar (PDA), carbon source and nitrogen source, and there is no particular limitation.

(2) Flask culture: (1) Scrape the mycelium on the plate and inoculate it in a flask, and shake culture for 3 to 14 days under the conditions of 15 to 30 ℃, pH 2 to 6 and rotation speed of 110 to 130 rpm (this implementation In the example, culture was shaken for 7 days at 25°C, pH 5, and rotating speed 120 rpm). This shaking culture was cultured in the medium shown in Table 1 below.

Table 1. Medium formula ingredient The content used in this embodiment (wt%) Preferred content range (wt%) Comprehensive carbon and nitrogen source 1 0.01 to 5 carbohydrate 2.5 0.01 to 10 Yeast or malt extract 0.8 0.001 to 2 Animal and vegetable protein and its hydrolysate 0.5 0.01 to 2 Inorganic salts 0.05 0.0001 to 0.05

In the above medium formula, the comprehensive carbon and nitrogen sources can be selected from cereals (such as wheat flour) or beans (such as soybean powder, mung bean powder, soybean powder, cinnamon powder, etc.); sugars can be glucose, fructose, maltose, sucrose Etc.; inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, iron sulfate and the like. In particular, the medium formula in Table 1 is only one example, and the ingredients can be adjusted according to requirements during use, or used with commercially available medium, and there is no special restriction.

(3) Fermentation tank culture: inoculate the mycelium cultured in the flask in (2) into the fermentation tank at 15 to 30 ℃, tank pressure 0.5 to 1.0 kg/cm ² , pH 2 to 6, and stirring speed 50 to 150 Cultivate for 3 to 21 days at an aeration rate of 0.1 to 1.5 VVM under rpm conditions to form a cicada mycelium fermentation broth (in this example, it is at 25°C, tank pressure 0.5 kg/cm ² , pH 5, and stirring Cultivate for 14 days at a speed of 80 rpm and 1.0 VVM (air) to obtain a fermentation broth of cicada flower mycelium. The culture medium used in the fermentation tank culture can be the same as the culture medium used in the flask culture in step (2), or an appropriate medium can be additionally configured (this embodiment uses the same culture medium as that in step (2)). This cicada flower mycelium fermentation broth contains the cicada flower mycelium active substance of the present invention. The fermentation broth of cicada flower mycelium can be further prepared as a freeze-dried powder of the fermentation broth of cicada flower mycelium through a freeze-drying step. In this example, about 3 kg of freeze-dried powder can be obtained from 100 L of the fermentation broth of cicada flower mycelium.

The active substance of the mycelium of the cicada flower can be contained in the fermentation broth (mycelium and clarified liquid) of the cicada flower mycelium, the freeze-dried powder of the fermentation broth, and the form that the freeze-dried powder is re-dissolved in solvent or other formulation . In a preferred embodiment, the solvent of the lyophilized powder is water, ethanol or a combination thereof. In a preferred embodiment, the ratio of water to ethanol used as the solvent of the re-dissolved lyophilized powder is 1:1. In the following example two, the lyophilized powder of the fermented broth of cicada flower mycelium was used as the active substance of the cicada flower mycelium for subsequent relevant experimental analysis. Example 2: Analysis of improving ALI by freeze-dried powder of cicada flower mycelium

Bacterial infection is the main risk factor for ALI. Gram-negative bacteria are one of the major categories. The main component of its mantle is endotoxin, also known as lipopolysaccride (LPS). Among many pathogenic sources, LPS is widely accepted as the best inducer to cause acute lung inflammation, and it is also the closest model to acute inflammation in bed. Therefore, if the lung inflammation and immune mediation effects and mechanisms caused by LPS can be inhibited, the effect of improving ALI can be achieved. There have been experiments using animal models of ALI disease induced by endotoxin. Refer to Yunhe Fu et al. (2017), Protective effect of TM6 on LPS-induced acute lung injury in mice, SCIENTIFIC REPORTS , 7:572. According to this paper, LPS is used to establish the acute lung inflammation model in mice, and LPS 50 μg / 20 μL (intranasally, in) LPS is introduced into the nasal cavity to cause acute lung injury, and then lung pathology, alveolar washing fluid total cells 數, protein concentration 度And the analysis of changes in various cytokines to evaluate synthetic peptides such as: cell-permeable TIR domain-derived decoy peptide derived from the TIR domain to improve the results of ALI. In this experiment, LPS was also used to establish the acute lung inflammation model in mice, and the changes in lung pathology, alveolar washing fluid total cells, protein concentration, and various cytokines were analyzed to evaluate the improvement results of the active substances from the mycelium of cicada flowers on ALI. .

BALB/c mice were used for animal experiments. According to the route of administration, they were divided into two groups: intraperitoneal injection and oral administration. Each group was divided into a control group (Control group) without administration of any substance (Control group) according to different substances administered. LPS was administered as a negative control group (LPS group), LPS was also administered to the lyophilized powder group of cicada flower mycelia active substance (CC mycelia group), and LPS was also administered to Dexamethasone as a positive control Group (DEX group), a total of 8 groups are shown in Table 2. There are 6 mice in each group, 48 mice in total.

Table 2. Experimental grouping Giving way Group Give substance Intraperitoneal injection Control group no LPS group LPS CC mycelia group LPS +CC mycelia 0.25mg/g mouse body weight (the mouse body weight is 25g, the dose of CC mycelia is about 6.25 mg) DEX group LPS + DEX 5μg/g mouse body weight (the mouse body weight is 25g, the DEX dose is about 125 μg) oral Control group no LPS group LPS CC mycelia group LPS +CC mycelia 0.25mg/g mouse body weight (the mouse body weight is 25g, the dose of CC mycelia is about 6.25 mg) (a total of three times once a day) DEX group LPS + DEX 5μg/g mouse body weight (the mouse body weight is 25g, the DEX dose is about 125 μg)

Regarding the intraperitoneal injection group, the endotoxin group (LPS) used nasal inhalation to administer 20 μl, and the LPS concentration was 50 μg per mouse. After 24 hours, the mice were taken from their specimens for the following analysis. The CC mycelia group first re-dissolved the lyophilized powder of cicada flower mycelia in a solvent prepared with a ratio of water and ethanol at a ratio of 1:1, then homogenized by ultrasonic vibration, and confirmed that there was no precipitation, as the sample to be administered. Intraperitoneal injection (i.p.) was used to administer 50 μl of cicada flower samples to mice. After 30 minutes, endotoxin (LPS) was administered through the nasal cavity. The methods of administration and sacrifice were the same as above. In the DEX group, DEX was dissolved in saline, and 50 μl was administered to mice by intraperitoneal injection (i.p.). After 30 minutes, endotoxin (LPS) was administered through the nasal cavity. The methods of administration and sacrifice were the same as above.

Regarding the oral administration group, the endotoxin group (LPS) was administered 20 μl by nasal inhalation, and the LPS concentration was 50 μg per mouse. After 24 hours, the mice were taken from the mice for the following analysis. The CC mycelia group first re-dissolved the lyophilized powder of cicada flower mycelia in a solvent prepared with a ratio of water and ethanol at a ratio of 1:1, then homogenized by ultrasonic vibration, and confirmed that there was no precipitation, as the sample to be administered. Using an oral tube, 200 μl of cicada flower samples were administered to mice for three days, once a day, three days later, endotoxin (LPS) was administered through the nasal cavity, and the methods of administration and sacrifice were the same as above. In the DEX group, DEX was dissolved in saline, and 50 μl was administered to mice by intraperitoneal injection (i.p.). After 30 minutes, endotoxin (LPS) was administered through the nasal cavity. The methods of administration and sacrifice were the same as above.

Observation of lung histopathology: Take the right lung of the mouse without alveolar lavage, fix it with formalin immediately, make paraffin sections, and then stain with hematoxylin and eosin stain (H&E stain). Methods Stain and observe the histopathological changes under an optical microscope.

Bronchoalveolar lavage (BAL): The left lung of the mouse was lavaged three times with 1ml PBS through the tracheal intubation, and the contents in the bronchoalveolar cavity were flushed out to obtain the BAL fluid (BALF). After centrifugation of the rinsing solution, the part of the precipitated cells is classified and counted; the part of the supernatant is subjected to the Bradford protein assay (Bradford protein assay) for protein concentration.

White blood cell count and type analysis: analysis by flow cytometry: an analysis method with high sensitivity and low human error. The blood and BALF specimens are used for white blood cell surface with specific antigen characteristics to perform white blood cell typing, including (1 ) Analysis of the total number of cells in BALF; (2) the total number of white blood cells (CD45); (3) the total number of phagocytes (CD45+/CD11b+); (4) the total number of neutrophils (CD45+/Ly6G+); (5) the total number of macrophages [( CD45+/CD11b+)-(CD45+/Ly6G+)] total.

Statistical method: The experimental data are all expressed in Mean ± standard deviation (SD), and statistics are analyzed by one-way ANOVA, and the differences between groups are compared by LSD post-test. If the analysis result is less than 0.05, it is regarded as statistically significant .

The results of intraperitoneal injection as shown in Figure 1 show that the lyophilized powder of cicada flower mycelium can effectively improve the pathological changes of lung inflammation. There was no significant change in the Control group, and most of the alveolar structure remained intact. The lung tissue of the LPS group had obvious pathological changes, and the alveolar structure was destroyed and fused, while the lung disease of the CC mycelia group and DEX group was significantly reduced.

After intraperitoneal injection, the protein exudation reaction caused by endotoxin-induced pulmonary inflammation was analyzed, and it was found that the lyophilized powder of the active substance of cicada flower mycelium can effectively reduce the protein exudation reaction as shown in Figure 2 and Table 3 below. # p <0.05 compared with the control group, *p <0.05 compared with the LPS group.

Table 3. Protein exudation results of intraperitoneal injection Group Protein exudation (mg/mL) Control group 2.87±1.02 LPS group 9.93±1.52# CC mycelia group 5.39±1.27* DEX group 3.83±1.72* (N = 6) #Indicating statistically significant difference from Control group (p＜0.05) *Indicating statistically significant difference from LPS group (p＜0.05)

After intraperitoneal injection, the infiltration reaction of (A) white blood cells (CD45), (B) phagocytes (CD45+/CD11b+), and (C) neutrophils (CD45+/Ly6G+) caused by endotoxin-induced pulmonary inflammation was analyzed. The lyophilized powder of active substances from cicada flower mycelium can effectively improve the infiltration of white blood cells, phagocytes and neutrophils (PMN) as shown in Figure 3 (A to C) and Tables 4 to 6 below. #p <0.05 compared with the control group; * p <0.05 compared with the LPS group.

Table 4. Results of white blood cell infiltration by intraperitoneal injection Group Leukocyte infiltration (10 ⁵ ) Control group 0.14±0.06 LPS 3.29±1.95# CC mycelia group 0.46±0.34* DEX group 0.56±0.29* (N = 6) #Indicating statistically significant difference from Control group (p＜0.05) *Indicating statistically significant difference from LPS group (p＜0.05)

Table 5. Results of phagocytic cell infiltration by intraperitoneal injection Group Phagocyte infiltration (10 ⁵ ) Control group (Control group) 0.07±0.03 LPS group 1.62±0.67# CC mycelia group 0.25±0.14* DEX group 0.27±0.26* (N = 6) #Indicating statistically significant difference from Control group (p＜0.05) *Indicating statistically significant difference from LPS group (p＜0.05)

Table 6. Results of neutrophil infiltration by intraperitoneal injection Group Neutrophil infiltration (10 ⁵ ) Control group 0.04±0.02 LPS group 1.12±0.80# CC mycelia group 0.15±0.10* DEX group 0.16±0.16* (N = 6) #Indicating statistically significant difference from Control group (p＜0.05) *Indicating statistically significant difference from LPS group (p＜0.05)

The results of oral administration as shown in Figure 4 show that the lyophilized powder of the active substance from the mycelium of cicada flower can effectively improve the pathological changes of lung inflammation. There was no significant change in the Control group, and most of the alveolar structure remained intact. The lung tissue of the LPS group had obvious pathological changes, and the alveolar structure was destroyed and fused, while the lung disease of the CC mycelia group and DEX group was significantly reduced.

After oral administration, the protein exudation reaction caused by endotoxin-induced pulmonary inflammation was analyzed, and it was found that the lyophilized powder of the active substance of cicada flower mycelium can effectively reduce the protein exudation reaction as shown in Figure 5 and Table 7 below. # p <0.05 compared with the control group, *p <0.05 compared with the LPS group.

Table 7. Protein exudation results of oral administration Group Protein exudation (mg/mL) Control group 2.60±0.24 LPS group 6.65±1.18# CC mycelia group 2.48±0.35* DEX group 3.11±0.64* (N = 6) #Indicating statistically significant difference from Control group (p＜0.05) *Indicating statistically significant difference from LPS group (p＜0.05)

After oral administration, analysis of (A) white blood cells (CD45), (B) phagocytes (CD45+/CD11b+), and (C) neutrophil (CD45+/Ly6G+) infiltration reactions caused by endotoxin-induced lung inflammation, found The lyophilized powder of active substances from cicada flower mycelium can effectively improve the infiltration of white blood cells, phagocytes and neutrophils (PMN) as shown in Figure 6 (A to C) and Tables 8 to 10 below. #p <0.05 compared with the control group; *p <0.05 compared with the LPS group.

Table 8. Leukocyte infiltration results of oral administration Group Leukocyte infiltration (10 ⁵ ) Control group) 0.38±0.22 LPS group 3.17±0.82# CC mycelia group 0.70±0.46* DEX group 0.88±0.25* (N = 6) #Indicating statistically significant difference from Control group (p＜0.05) *Indicating statistically significant difference from LPS group (p＜0.05)

Table 9. Results of infiltration of phagocytes by oral administration Group Phagocyte infiltration (10 ⁵ ) Control group) 0.06±0.04 LPS group 0.62±0.35# CC mycelia group 0.11±0.10* DEX group 0.08±0.05* (N = 6) #Indicating statistically significant difference from Control group (p＜0.05) *Indicating statistically significant difference from LPS group (p＜0.05)

Table 10. The results of oral administration of neutrophil infiltration Group Neutrophil infiltration (10 ⁵ ) Control group) 0.02±0.01 LPS group 0.29±0.09# CC mycelia group 0.04±0.02* DEX group 0.04±0.02* (N = 6) #Indicates that there is a statistically significant difference from the control group (p<0.05) *Indicating a statistically significant difference from the negative control group (LPS) (p<0.05)

The active substance of the cicada flower mycelium of the present invention has been proved to have the effect of improving ALI through the above experiments.

Example 3: Preparation of the composition

The present invention provides a composition comprising active substances of cicada flower mycelium, which is prepared as a medicinal composition and can also be used as a health-care nutritional food.

The composition further contains additives. In a preferred embodiment, the additives may be excipients, preservatives, diluents, fillers, absorption enhancers, sweeteners, lubricants, thickeners, or combinations thereof. The excipient can be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose or a combination thereof. The preservative can extend the shelf life of the pharmaceutical composition, such as benzyl alcohol, parabens, silicon dioxide or a combination thereof. The diluent can be selected from water, ethanol, propylene glycol, glycerin or a combination thereof. The filler can be selected from lactose, nougat, high molecular weight ethylene glycol or a combination thereof. The absorption enhancer can be selected from dimethyl sulfide (DMSO), azodipine, propylene glycol, glycerin, polyethylene glycol or a combination thereof. The sweetener can be selected from Acesulfame K, aspartame, saccharin, sucralose, neotame or a combination thereof. The lubricant can be selected from magnesium stearate or gum arabic. The thickener may be corn starch. In addition to the additives listed above, other suitable additives can be selected according to requirements without affecting the medical effect of the composition.

The composition can be developed into different commodities in the medical field. In a preferred embodiment, the composition is a medicine, feed, beverage, nutritional supplement, dairy product, food or health food.

The composition can adopt different forms according to the needs of the recipient. In a preferred embodiment, the composition is in the form of powder, lozenge, granulation, suppository, microcapsule, ampoule (ampoule/ampule), liquid spray or suppository.

The composition of the present invention can be used in animals or humans. Under the premise of not affecting the effect, the composition can be prepared into any pharmaceutical form, and administered to the animal or human by an applicable route according to the pharmaceutical form.

If the cicada flower mycelium active material of the present invention is applied to food use, the following aspect of composition 1 is taken as an illustrative example.

Composition 1: Take the lyophilized powder (20 wt%) of cicada flower mycelium active substance, magnesium stearate (8 wt%) as a lubricant, and silicon dioxide (7 wt%) as a preservative, and mix thoroughly, and Dissolve in pure water (65 wt%) and store at 4℃ for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

If the cicada flower mycelium active substance of the present invention is applied in a liquid dosage form for medical purposes, the following composition 2 is taken as an illustrative example.

Composition 2: Take the lyophilized powder (20 wt%) of the active substance of the cicada flower mycelium, sucralose (8 wt%) as a sweetener, gum arabic (7 wt%) as a lubricant, and an excipient Sucrose (10 wt%) is mixed thoroughly, dissolved in pure water (55 wt%), and stored at 4°C for later use. The aforementioned wt% refers to the proportion of each component to the total weight of the composition.

Although the present invention has been disclosed in the above embodiments, it is not intended to limit the present invention. Those of ordinary skill in the art, after considering the above teachings, can properly modify the content of the above embodiments, and still achieve the effects claimed in this case. Therefore, the protection scope of the present invention should be subject to the scope of the patent application attached thereafter.

Figure 1 shows that lyophilized powder of cicada flower mycelium is injected into the abdominal cavity, and then endotoxin is inhaled through the nasal cavity to induce acute lung inflammation, which can effectively improve the pathological changes of ALI.

Figure 2 shows that the lyophilized powder of cicada flower mycelium is injected through the abdominal cavity, and then endotoxin is inhaled through the nasal cavity to induce acute lung inflammation, which can effectively improve the destruction of the alveolar microvascular barrier of ALI.

Figure 3 shows that the lyophilized powder of cicada flower mycelium is injected into the abdominal cavity, and then endotoxin is inhaled through the nasal cavity to induce acute lung inflammation, which can effectively improve the white blood cell infiltration (A), phagocytes (B), and neutrophils of ALI White blood cells (PMN) (C).

Figure 4 shows that the lyophilized powder of cicada flower mycelium is administered orally, and then endotoxin is used to induce acute lung inflammation through nasal cavity inhalation, which can effectively improve the pathological changes of ALI.

Figure 5 shows that the lyophilized powder of cicada flower mycelium is administered orally, and then endotoxin is inhaled through the nasal cavity to induce acute lung inflammation, which can effectively improve the destruction of the alveolar microvascular barrier of ALI.

Figure 6 shows that the lyophilized powder of cicada flower mycelium is administered orally, and then endotoxin is inhaled through the nasal cavity to induce acute lung inflammation, which can effectively improve the white blood cell infiltration (A), phagocytes (B), and mesophilia of ALI Sexual white blood cells (PMN) (C).

Claims (11)

A method for preparing an active substance of cicada flower mycelia for preventing and/or ameliorating acute lung injury, which comprises the following steps: (a) Take a cicada flower mycelia ( Cordyceps Cicadae mycelia) on a plate medium, Cultivate for 1 to 2 weeks at a temperature of 30 ℃; (b) Inoculate the mycelium of cicada flower cultured in step (a) into a flask, and cultivate 3 to 14 in an environment of 15 to 30 ℃ and pH 2 to 6 (C) Inoculate the mycelium of the cicada flower cultured in step (b) in a fermentation tank, stir and cultivate for 3 to 21 days under an environment of 15 to 30 ℃ and pH 2 to 6 to form the cicada flower The mycelium active substance is a cicada flower mycelium fermentation broth. The preparation method as described in item 1 of the scope of the patent application, which further includes step (d): freeze-drying the fermentation broth of the cicada flower mycelium and then grinding to form a cicada flower containing the active substance of the cicada flower mycelium Mycelium freeze-dried powder. The preparation method as described in item 1 of the scope of patent application, wherein in step (c), the fermentation tank is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof, and the tank pressure of the fermentation tank is 0.5 to 1.0 kg/cm ² and a ventilation rate of 0.01 to 1.5 VVM. A cicada flower mycelium active substance, which is manufactured by the preparation method described in any one of items 1 to 3 in the scope of patent application. A composition for preventing and/or ameliorating acute lung injury, which contains the active substance of cicada flower mycelium as described in item 4 of the scope of patent application, and pharmaceutically acceptable carriers, excipients, and dilutions Agent or adjuvant. A use of the active substance of cicada flower mycelium as described in item 4 of the scope of patent application, which is used to prepare a composition for preventing and/or ameliorating acute lung injury. The use as described in item 6 of the scope of patent application, wherein the improvement of acute lung injury refers to the alleviation of pathological symptoms of lung inflammation. The use described in item 7 of the scope of patent application, wherein the pathological reduction of lung inflammation means that the structure of the alveoli tends to be intact from destruction or fusion. The use as described in item 6 of the scope of patent application, wherein the improvement of acute lung injury refers to the reduction of protein exudation response. The use as described in item 6 of the scope of patent application, wherein the improvement of acute lung injury refers to the reduction of the degree of infiltration of inflammatory cells. The use according to item 10 of the scope of patent application, wherein the inflammatory cells refer to white blood cells, phagocytes and/or neutrophils.

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