CA3040916A1

CA3040916A1 - Method of manufacturing and the use of cordyceps cicadae mycelia active substance for preventing and/or improving acute lung injury

Info

Publication number: CA3040916A1
Application number: CA3040916A
Authority: CA
Inventors: Yu-Hsiang Kuan; Chin-Chu Chen; I-Chen Li; Jui-Hsia Hsu
Original assignee: Grape King Bio Ltd
Current assignee: Grape King Bio Ltd
Priority date: 2018-12-25
Filing date: 2019-04-23
Publication date: 2020-06-25
Anticipated expiration: 2039-04-23
Also published as: KR20200080100A; CA3040916C; CN111434342B; JP6789339B2; TWI701335B; JP2020105159A; KR102223608B1; TW202024331A; CN111434342A

Abstract

A method for manufacturing Cordyceps cicadae mycelia active substance for preventing and/or treating acute lung injury is provided. The method comprises the following steps: (a) culturing a Cordyceps cicadae mycelia in a plate media between 15 to 30°C for 1 to 2 weeks; (b) inoculating the mycelia of step (a) to a flask and culturing it between 15 to 30°C at a pH of 2 to 6 for 3 to 14 days; (c) inoculating the mycelia of step (b) to a fermenter tank and culturing it between 15 to 30°C at a pH of 2 to 6 for 3 to 21 days, so as to obtain a Cordyceps cicadae mycelium fermentation liquid containing said Cordyceps cicadae mycelia active substance.

Description

Method of manufacturing and the use of Cordyceps cicadae mycelia active substance for preventing and/or improving acute lung injury BACKGROUND
Technical Field [0001] The present invention relates to a Cordyceps cicadae mycelia active substance, a method for manufacturing said Cordyceps cicadae mycelia active substance, and the use thereof. In particular, the present invention relates to a Cordyceps cicadae mycelia active substance for preventing and/or improving acute lung injury, a method for manufacturing said Cordyceps cicadae mycelia active substance, and the use thereof in food or pharmaceuticals.
Description of Related Art

[0002] Among the ten leading causes of death in 2017 according to Taiwan's Ministry of Health and Welfare, four of them are closely associated with lung inflammation, namely, malignant neoplasms (ranked no. 1) including cancers of trachea, bronchus and lung, pneumonia (ranked no. 3), and chronic lower respiratory diseases (ranked no. 7).

[0003] Among the ten leading causes of death in the United States, three of them are associated with lung diseases, namely, lung cancer and bronchogenic carcinoma, chronic respiratory diseases, influenza, and pneumonia. Statistical analyses further showed that in the U.S., 79 out of 100,000 were inflicted with acute lung injury (ALI), 59 out of 100,000 were with acute respiratory distress syndrome (ARDS), and that 43% of all deaths were caused by ALI/ARDS, that is, 75,000 deaths per year. As air pollution levels continue to increase and life expectancy continues to rise every year, it is estimated that the incidence of ALI/ARDS in the U.S. will at least double in 25 years.

[0004] ALT and ARDS (more severe than ALI) are acute inflammatory lung diseases that are frequently seen clinically, both of which may cause respiratory failure and may be fatal. They are also associated with many respiratory diseases.

[0005] ALI-causing risk factors fall into two major groups: direct and indirect risk factors. Direct risk factors are those which originate in the lungs, such as infective pneumonia caused by bacteria or viruses, aspiration of gastric acid or foreign objects, or lung contusion. On the other hand, indirect risk factors are those which do not originate in the lungs, such as sepsis, chronic use of alcohol and drugs, or transfusion of artificial blood plasma.
Bacterial infection is one of the above-mentioned major risk factors, an example being infection by Gram-negative bacteria whose envelope is mainly composed of endotoxins, also known as lipopolysaccharide (LPS).

[0006] Due to the complex mechanism and high case fatality rate of ALT, there is still a lack of clinically effective drugs that can be used to manage ALI's case fatality rate, while common treatments of ALT include mechanical ventilation, beta-2 adrenergic receptor agonist, anticoagulation, thrombolysis, surfactant therapy, and surgery. Therefore, research on effective treatments of ALT remains essential for development.

[0007] Cordyceps cicadae, also known as chong hua, tit chan hua, hil chan, and chan yong cao, is a flower bud-shaped stroma formed with strains at the front end of a cicada larva, which is parasitized by a genus of Cordyceps, a family of Clavicipitaceae fungal spores, and turned into a bacteria-carrying carcass. It is written in Compendium of Materia Medica, Herbology of Classified Syndromes and Zhong hua yao heti that Cordyceps cicadae is mainly used for treating morbid night crying of babies, palpitation, and malaria, as well as dispelling wind and heat from the body and relieving convulsion. It has been found through component analysis that natural Cordyceps cicadae fruit bodies and Ophiocordyceps sinensis are composed in a similar way.

[0008] Recent pharmaceutical research has found that Cordyceps cicadae is bioactive with effects of immune response regulation, anti-oxidation, anti-inflammation, neuroprotection, and anti-cancer. However, there has been no research done on improving ALI using Cordyceps cicadae mycelia.
SUMMARY

[0009] Provided herein is a Cordyceps cicadae mycelia active substance and a method of manufacturing said Cordyceps cicadae mycelia active substance, which can be used for manufacturing a composition effective in preventing and/or improving acute lung injury. Compared to conventional drugs and treatments, the method for manufacturing the fermentation liquid of the Cordyceps cicadae mycelia active substance disclosed herein is safer and simpler, and the Cordyceps cicadae mycelia active substance manufactured is more natural and safer, as well as effective in improving ALT.

[0010] According to one embodiment of the present invention, a method is provided for manufacturing a Cordyceps cicadae mycelia active substance for improving AL!. The method comprises the following steps:

[0011] (a) culturing Cordyceps cicadae mycelia in a plate medium between 15 and 30 C for 1 to 2 weeks;

[0012] (b) inoculating the Cordyceps cicadae mycelia of step (a) to a flask and culturing the mycelia between 15 and 30 C and at a pH of 2 to 6 for 3 to 14 days; and

[0013] (c) inoculating the Cordyceps cicadae mycelia of step (b) to a fermenter tank and culturing the mycelia by stirring between 15 and 30 C
and at a pH of 2 to 6 for 3 to 21 days, so as to obtain a Cordyceps cicadae mycelium fermentation liquid containing Cordyceps cicadae mycelia active substance.

[0014] In one embodiment, the method for manufacturing a Cordyceps cicadae mycelia active substance further includes steps of (d): freeze-drying the Cordyceps cicadae mycelium fermentation liquid and grinding the freeze-dried product, so as to obtain a Cordyceps cicadae mycelium powder containing Cordyceps cicadae mycelia active substance.

[0015] In one embodiment, a gas is further fed into the fermenter tank of step (c), and the gas comprises air, oxygen, carbon dioxide, helium or a combination thereof; the pressure of the fermenter tank is 0.5 to 1.0 kg/cm2, and a gas flow rate is 0.01 to 1.5 VVM.

[0016] In another embodiment of the present invention, a Cordyceps cicadae mycelia active substance is provided, which is manufactured using the said method.

[0017] In yet another embodiment of the present invention, a composition for preventing and/or improving acute lung injury is provided, which comprises said Cordyceps cicadae mycelia active substance, and a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

[0018] In yet another embodiment of the present invention, A use of said Cordycep cicadae mycelia active substance is provided, which is used for manufacturing a composition for preventing and/or improving acute lung injury.

[0019] In one embodiment, improving acute lung injury as mentioned above includes alleviating pathological symptoms of lung inflammation.

[0020] In one embodiment, alleviating pathological symptoms of lung inflammation as mentioned above includes restoring the integrity of damaged or fused alveoli.

[0021] In one embodiment, improving acute lung injury as mentioned above includes reducing protein leakage response.

[0022] In one embodiment, improving acute lung injury as mentioned above includes reducing the level of infiltration in inflamed cells.

[0023] In one embodiment, said inflamed cells includes leukocytes, phagocytes and/or neutrophils.

[0024] For the purpose of further illustrating the above and other aspects of the present invention, several exemplary embodiments will be described with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Fig. 1 shows that pathological conditions of ALI can be improved by administering with freeze-dried powder of Cordyceps cicadae mycelia by intraperitoneal injection and then administering endotoxins by nasal inhalation to induce acute lung inflammation.

[0026] Fig. 2 shows that ALI-caused damage of alveolar-capillary barrier can be improved by administering with freeze-dried powder of Cordyceps cicadae mycelia by intraperitoneal injection and then administering endotoxins by nasal inhalation to induce acute lung inflammation.

[0027] Fig. 3 shows that ALI-caused infiltration in (A) leukocytes, (B) phagocytes and (C) neutrophils can be improved by administering with freeze-dried powder of Cordyceps cicadae mycelia by intraperitoneal injection and then administering endotoxins by nasal inhalation to induce acute lung inflammation.

[0028] Fig.
4 shows that pathological conditions of ALT can be improved by administering with freeze-dried powder of Cordyceps cicadae mycelia orally and then administering endotoxins by nasal inhalation to induce acute lung inflammation.

[0029] Fig.
5 shows that ALT-caused damage of the alveolar-capillary barrier can be improved by administering freeze-dried powder of Cordyceps cicadae mycelia orally and then administering endotoxins by nasal inhalation to induce acute lung inflammation.

[0030] Fig.
6 shows that ALT-caused infiltration in (A) leukocytes, (B) phagocytes and (C) neutrophils can be improved by administering freeze-dried powder of Cordyceps cicadae mycelia by orally and then administering endotoxins by nasal inhalation to induce acute lung inflammation.
DETAILED DESCRIPTION
Example 1: Culturing Cordyceps cicadae mycelia

[0031]
Cordyceps cicadae mycelia of the present invention are obtained through the following steps: gathering a natural Taiwanese Cordyceps cicadae strain, separating its mycelium from the fruit body and storing the mycelia in a plate medium by subculture. The gene sequence of the strain is confirmed as Cordyceps cicadae by Taiwan Food Industry Research and Development Institute. This strain of Cordyceps cicadae strains are now publicly deposited in the Bioresource Collection and Research Center (BCRC) of Taiwan Food Industry Research and Development Institute with BCRC number: MU30106, but the Cordyceps cicadae mycelia active substance of the present invention is not limited to the substance prepared from such strains.

[0032] (1) Plate culture: A Cordyceps cicadae mycelium was inoculated onto a plate medium and cultured between 15 to 30 C for 1 to 2 weeks (at 25 C for 7 days in this example). The recipe for the medium may include Potato Dextrose Agar (PDA), carbon sources and nitrogen sources, and is not specifically limited.

[0033] (2) Culture in a flask: Cordyceps cicadae mycelia were scraped from the plate of step (1) and inoculated to a flask. The culture was grown at 110 to 130 rpm in a shaker incubator between 15 to 30 C and at a pH of 2 to 6 for 3 to 14 days (at 120 rpm, 25 C and pH 5 for 7 days in this example).
The recipe of the shaking culture was shown in Table 1:

[0034] Table 1: Recipe for the culture medium Amount used in this Preferred range of Ingredient example (weight%) amount (weight%) Mixed carbon and nitrogen 1 0.01 to 5 sources Carbohydrates 2.5 0.01 to 10 Yeast or malt extract 0.8 0.001 to 2 Animal and plant proteins and 0.5 0.01 to 2 hydrolysates thereof Inorganic salts 0.05 0.0001 to 0.05

[0035] Among the above ingredients, mixed carbon and nitrogen sources can be cereals (such as wheat flour) or legumes (such as soya bean powder, mung bean powder, Glycine max powder or cinnamon powder);
carbohydrates can be glucose, fructose, maltose, sucrose and the like; and inorganic salts can be magnesium sulfate, dipotassium phosphate, potassium dihydrogen phosphate, ferric sulfate and the like. It should be noted that the recipe as shown in Table 1 is only exemplary, and that the ingredients therein are not specifically limited and can be adjusted according to actual needs or used in combination with commercially available culture media.

[0036] (3) Culture in a fermenter tank: The culture in the flask of step (2) was further inoculated into a fermenter tank and was stirred between 50 to 150 rpm in temperature between 15 to 30 C, the tank pressure between 0.5 to 1.0 kg/cm2 and the pH between 2 to 6. Meanwhile, gas was fed into the tank at a gas flow rate of 0.1 to 1.0 VVM. The resulting culture was incubated for 3 to 21 days to obtain a Cordyceps cicadae mycelium fermentation liquid (in this example, the culture conditions were 25 C, 0.5 kg/cm2, pH 5, 80 rpm and 1.0 VVM (air) for 14 days). The medium used in the fermenter tank may contain either the same recipe as the culture in a flask of step (2) or other appropriate media (the same recipe as step (2) was used in this example). This Cordyceps cicadae mycelia fermentation liquid includes Cordyceps cicadae mycelia active substance of the present invention. The Cordyceps cicadae mycelium fermentation liquid can be further freeze-dried to obtain a freeze-dried powder of Cordyceps cicadae mycelia. In this example, 100 L of Cordyceps cicadae mycelium fermentation liquid was freeze-dried into 3 kg of freeze-dried powder.

[0037] Cordyceps cicadae mycelia active substance can take various forms including Cordyceps cicadae mycelium fermentation liquid (strains and clarified liquid), a freeze-dried powder made from fermentation liquid, freeze-dried powder dissolved in a solvent, or in other formulations. In a preferred embodiment, the solvent for dissolving the freeze-dried powder is water, ethanol or a combination thereof In a preferred embodiment, the ratio of water to ethanol used as the solvent for dissolving the freeze-dried powder is 1:1. In the following Example 2, subsequent experiments and analyses were carried out with the Cordyceps cicadae mycelia active substance in the form of freeze-dried powder made from the fermentation liquid.
Example 2: Analyzing the use of Cordyceps cicadae mycelium freeze-dried powder to improve AL!

[0038] One of the major risk factors leading to ALT is Gram-negative bacterial infection, whose envelope is mainly composed of endotoxins, also known as lipopolysaccharide (LPS). Among various pathogens that cause ALT, LPS is widely considered as the most effective inducer of acute lung inflammation and the model of this induction is the closest to the acute inflammation that occurs clinically. Therefore, suppressing LPS-caused lung inflammation as well as immune-regulatory functions and mechanisms shall achieve the effect of improving ALT. There have been experiments in which animal models of endotoxin-induced ALT are employed. See Yunhe Fu et al.
(2017), Protective effect of TM6 on LPS-induced acute lung injury in mice, SCIENTIFIC REPORTS, 7:572. A mouse model of acute lung inflammation was established by LPS following the description in the above publication.
LPS 50 lig/ 20 [iL was administered intranasally to induce acute lung injury.
Analyses of changes in pulmonary pathology, total bronchoalveolar lavage fluid cell counts, protein concentration and various cytokines were then carried out, so as to assess the improvements that synthesized peptides, such as cell-permeable TIR domain-derived decoy peptide, had done to ALT. In this experiment, a mouse model of acute lung inflammation was also established using LPS. Analyses of changes in pulmonary pathology, total bronchoalveolar lavage fluid cell counts, protein concentration and various cytokines were then carried out to assess the improvements that the Cordyceps cicadae mycelia active substance had done to ALT.

[0039] BALB/c mice were used in this animal experiment and were divided into two major groups according to the route of administration:
intraperitoneal administration and oral administration. The two major groups were further divided into four groups: a control group to which no substance was administered (Control group), a group to which only LPS was administered (LPS group), a group to which both LPS and Cordyceps cicadae mycelia active substance were administered (CC mycelia group), and a positive control group to which both LPS and Dexamethasone were administered (DEX group), making a total of eight groups as shown in Table 2 below. There were six mice in each group with a total of 48 mice.

[0040] Table 2: Group description Route of Group Substance administered administration Intraperitoneal Control N/A
LPS LPS
CC mycelia LPS + CC mycelia 0.25 mg/g weight of mice (6.25 mg of CC
mycelia for a mouse weighing 25g) DEX LPS + DEX 5 [ig/g weight of mice (125 pg of DEX for a mouse weighing 25g) Oral Control N/A
LPS LPS
CC mycelia LPS + CC mycelia 0.25 mg/g weight of mice (6.25 mg of CC

mycelia for a mouse weighing 25g) (once per day, three times in total) DEX LPS + DEX 5 p.g/g weight of mice (125 1.1g of DEX for a mouse weighing 25g) (once per day, three times in total)

[0041] In the intraperitoneal administration group, 20 1_, of LPS
was administered intranasally, and the concentration of LPS is 50 lig per mouse.
Mice were sacrificed 24 hours after administration, and samples were collected to be analyzed as described below. In the CC mycelia group, freeze-dried powder of Cordyceps cicadae mycelia was dissolved in a water/ethanol solvent at a ratio of 1:1. The resulting mixture was homogenized using sonication and used as the sample to be administered when it was confirmed that no precipitation was formed. 50 [EL of Cordyceps cicadae sample was administered intraperitoneally in mice and endotoxins (LPS) were administered intranasally 30 minutes after the administration of the Cordyceps cicadae sample using the same methods of administration and sacrifice as described above. In the DEX group, DEX was dissolved in saline and 50 lit of the resulting mixture was administered intraperitoneally in mice. Endotoxins (LPS) were administered intranasally 30 minutes after the administration of the DEX sample using the same methods of administration and sacrifice as described above.

[0042] In the oral administration group, 20 tiL of endotoxins (LPS) were administered intranasally, and the concentration of LPS is 50 i.tg per mouse. Mice were sacrificed 24 hours after administration, and samples were collected to be analyzed as described below. In the CC mycelia group, freeze-dried powder of Cordyceps cicadae mycelia was dissolved in a water/ethanol solvent at a ratio of 1:1. The resulting mixture was homogenized using sonication and used as the sample to be administered when it was confirmed that no precipitation was formed. 200 IlL of Cordyceps cicadae sample was administered in mice via a feeding tube for three days, once per day. Endotoxins (LPS) were administered intranasally three days after using the same methods of administration and sacrifice as described above. In the DEX group, DEX was dissolved in saline and the resulting mixture was administered in mice via a feeding tube, once per day.
Endotoxins (LPS) were administered intranasally three days after using the same methods of administration and sacrifice as described above.

[0043] Histopathological observation of lung tissues: the right lung of the mice that was not treated with alveolar lavage was taken out and fixated immediately in formalin. The fixated product was made into paraffin sections and dyed with conventional hematoxylin and eosin stain (H&E
stain). Histopathological changes were observed using light microscopy.

[0044] Bronchoalveolar lavage (BAL): lung lavage was performed on the left lung of the mice with 1 mL of PBS three times using endotracheal intubation to wash out the contents in the alveolar cavity and to obtain BAL
fluid (BALF). The BALF was centrifuged and the precipitated cells were measured using differential blood cell count. The protein concentration in the supernatant was determined using Bradford protein assay.

[0045] Measurement of leukocytes count and type using flow cytometry: flow cytometry is a highly sensitive quantification method causing only small personal errors for determining white blood cell differentials in blood and BALF samples, based on antigens with specificity that are present on the surface of leukocytes. Targets of measurement include: (1) cell count in BALF; (2) leukocytes (CD45) count; (3) phagocytes (CD45+/CD1 lb) count; (4) neutrophils (CD45+/Ly6G+) count;
and (5) macrophage [(CD45+/CD1 lb+) - (CD45+/Ly6G+)] count.

[0046] Statistical method: data are expressed as Mean + standard deviation (SD) and analyzed using one-way ANOVA. Group differences are analyzed using an LSD post-hoc test. Results with a p value less than 0.05 are considered statistically significant.

[0047] As shown in Fig. 1, intraperitoneal injection of freeze-dried powder of Cordyceps cicadae mycelia proved to be effective in improving inflammatory lung diseases. No significant changes were observed in the Control group with most alveolar structure remaining intact. In the LPS
group, pathological changes in the lung were clearly observed with the alveolar structure being damaged and fused. On the other hand, the lung diseases of the mice in the CC mycelia and DEX groups clearly improved.

[0048] After intraperitoneal injection, protein leakage response caused by endotoxin-induced lung inflammation was analyzed. It was found that freeze-dried powder of Cordyceps cicadae mycelia was effective in reducing protein leakage response, as shown in Fig. 2 and Table 3 below. # p <0.05 compared with Control group; * p <0.05 compared with LPS group.

[0049] Table 3: Protein leakage response after intraperitoneal injection Group Protein leakage (mg/mL) Control 2.87+1.02 LPS 9.93+1.52#
CC mycelia 5.39+1.27*
DEX 3.83+1.72*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0050] After intraperitoneal injection, infiltration responses of (A) leukocytes (CD45) (B) phagocytes (CD45+/CD1 lb+) and (C) neutrophils (CD45+/Ly6G+) caused by endotoxin-induced lung inflammation were analyzed. It was found that freeze-dried powder of Cordyceps cicadae mycelia was effective in reducing infiltration response of leukocytes, phagocytes, and neutrophils, as shown in Fig. 3 (A to C) and Tables 4-6 below. # p < 0.05 compared with Control group; * p <0.05 compared with LPS group.

[0051] Table 4: leukocytes infiltration response after intraperitoneal injection Group leukocytes infiltration (105) Control 0.14 0.06 LPS 3.29 1.95#
CC mycelia 0.46 0.34*
DEX 0.56 0.29*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0052] Table 5: phagocytes infiltration response after intraperitoneal injection Group phagocytes infiltration (105) Control 0.07 0.03 LPS 1.62 0.67#
CC mycelia 0.25 0.14*

DEX 0.27 0.26*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0053] Table 6: Neutrophils infiltration response after intraperitoneal injection Group Neutrophils infiltration (105) Control 0.04 0.02 LPS 1.12 0.80#
CC mycelia 0.15 0.10*
DEX 0.16 0.16*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0054] As shown in Fig. 4, oral administration of freeze-dried powder of Cordyceps cicadae mycelia active substance proved to be effective in improving inflammatory lung diseases. No significant changes were observed in the Control group with most alveolar structure remaining intact.
In the LPS group, pathological changes in the lung were clearly observed with the alveolar structure being damaged and fused. On the other hand, the lung diseases of the mice in the CC mycelia and DEX groups clearly improved.

[0055] After oral administration, protein leakage response caused by endotoxin-induced lung inflammation was analyzed. It was found that freeze-dried powder of Cordyceps cicadae mycelia active substance was effective in reducing protein leakage response, as shown in Fig. 5 and Table 7 below. # p < 0.05 compared with Control group; * p <0.05 compared with LPS group.

[0056] Table 7: Protein leakage response after oral administration Group Protein leakage (mg/mL) Control 2.60 0.24 LPS 6.65 1.18#
CC mycelia 2.48 0.35*
DEX 3.11 0.64*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0057] After oral administration, infiltration responses of (A) leukocytes (CD45) (B) phagocytes (CD45+/CD1 1 b+) and (C) neutrophils (CD45+/Ly6G+) caused by endotoxin-induced lung inflammation were analyzed. It was found that freeze-dried powder of Cordyceps cicadae mycelia active substance was effective in reducing infiltration responses of leukocytes, phagocytes and neutrophils, as shown in Fig. 6 (A to C) and Tables 8-10 below. # p < 0.05 compared with Control group; * p <0.05 compared with LPS group.

[0058] Table 8: leukocytes infiltration response after oral administration Group leukocytes infiltration (105) Control 0.38 0.22 LPS 3.17 0.82#
CC mycelia 0.70 0.46*
DEX 0.88 0.25*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0059] Table 9: phagocytes infiltration response after oral administration Group phagocytes infiltration (105) Control 0.06 0.04 LPS 0.62 0.35#
CC mycelia 0.11 0.10*
DEX 0.08 0.05*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0060] Table 10: Neutrophils infiltration response after oral administration Group Neutrophils infiltration (105) Control 0.02 0.01 LP S 0.29 0.09#
CC mycelia 0.04 0.02*
DEX 0.04 0.02*
(n=6) # Statistical significance compared to the Control group (p<0.05) * Statistical significance compared to the LPS group (p<0.05)

[0061] The Cordyceps cicadae mycelia active substance of the present invention have been proved through the above experiment to be effective in improving ALT.
Example 3: Preparing composition

[0062] The present invention provides a composition comprising a Cordyceps cicadae mycelia active substance to be prepared into a pharmaceutical composition as well as a health food.

[0063] The composition further includes an additive. In a preferred embodiment, the additive can be an excipient, preservative, diluent, filler, absorption enhancer, sweetener, lubricant, thickener or a combination thereof The excipient can be selected from sodium citrate, calcium carbonate, calcium phosphate, sucrose or a combination thereof. The preservative, such as benzyl alcohol, parabens, silicon or a combination thereof, can prolong the shelf life of pharmaceutical compositions. The diluent can be selected from water, ethanol, propylene glycol, glycerol or a combination thereof The filler can be selected from lactose, high molecular weight polyethylene glycol or a combination thereof. The absorption enhancer can be selected from dimethyl sulfoxide (DMSO), laurocapram, propylene glycol, glycerol, 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 can be cornstarch.
In addition to the additives listed above, other ones may be selected according to actual needs provided that the pharmaceutical effects of the composition are not affected.

[0064] The composition can be developed into various products in the pharmaceutical industry. In a preferred embodiment, the composition is a drug, feed, drink, nutritional supplement, dairy product or health food.

[0065] The composition can take various forms to meet the subject's needs. In a preferred embodiment, the composition can be in powder, tablet, granule, suppository, microcapsule, ampoule/ampule, liquid spray or suppository form.

[0066] The composition of the present invention can be administered to an animal or a human. Provided that the effects of the composition are not affected, it can be made into any dosage forms and administered via an appropriate route to the animal or human depending on the dosage form.

[0067] When the Cordyceps cicadae mycelia active substance of the present invention is of dietary use, the form of the composition 1 as described below shall be an illustrative example of the Cordyceps cicadae mycelia active substance.

[0068] Composition 1: Freeze-dried powder of the Cordyceps cicadae mycelia active substance (20 wt%) was well-mixed with magnesium stearate used as a lubricant (8 wt%) and silicon used as a preservative (7 wt%). The resulting mixture was dissolved in pure water (65 wt%) and stored at 4 C

for future use. The notation "wt%" refers to the proportion of the weight of each ingredient relative to the total weight of the composition.

[0069] When the Cordyceps cicadae mycelia active substance of the present invention is of medical use in a liquid form, the form of the composition 2 as described below shall be an illustrative example of the Cordyceps cicadae mycelia active substance.

[0070] Composition 2: Freeze-dried powder of the Cordyceps cicadae mycelia active substance (20 wt%) was well-mixed with sucralose used as a sweetener (8 wt%), gum Arabic used as a lubricant (7 wt%) and sucrose used as an excipient (10 wt%). The resulting mixture was dissolved in pure water (55 wt%) and stored at 4 C for future use. The notation "wt%" refers to the proportion of the weight of each ingredient relative to the total weight of the composition.

[0071] These examples, though disclosed in the above description, do not intend to limit the present invention. A person ordinarily skilled in the art should be able to make appropriate changes to the features described therein with reference to the above teachings and still achieve the effects as claimed by the present application. Therefore, the scope of claims protecting the present invention shall be determined by the appended claims.

Claims

WHAT IS CLAIMED IS:

1. A method of preparing Cordyceps cicadae mycelia active substance for preventing and/or improving acute lung injury, the method comprising the following steps:
(a) culturing a Cordyceps cicadae mycelia in a plate medium between 15 and 30°C for 1 to 2 weeks;
(b) inoculating the Cordyceps cicadae mycelia of step (a) to a flask and culturing the mycelia between 15 and 30°C and at a pH of 2 to 6 for 3 to 14 days; and (c) inoculating the Cordyceps cicadae mycelia of step (b) to a fermenter tank and culturing the mycelia by stirring between 15 and 30°C at a pH of 2 to 6 for 3 to 21 days, so as to obtain a Cordyceps cicadae mycelium fermentation liquid containing Cordyceps cicadae mycelia active substance.

2. The method of claim 1, wherein the method further comprises steps of (d): freeze-drying the Cordyceps cicadae mycelium fermentation liquid and grinding the freeze-dried product, so as to obtain a Cordyceps cicadae mycelium powder containing Cordyceps cicadae mycelia active substance.

3. The method of claim 1, wherein a gas is further fed into the fermenter tank of step (c), and the gas comprises air, oxygen, carbon dioxide, helium or a combination thereof; pressure of the fermenter tank is 0.5 to 1.0 kg/cm2, and a gas flow rate is 0.01 to 1.5 VVM.

4. A Cordyceps cicadae mycelia active substance, which is manufactured using the method of claims 1.

5. A composition for preventing and/or treating acute lung injury, which comprises the Cordyceps cicadae mycelia active substance of claim 4, and a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

6. Use of the Cordycep cicadae mycelia active substance of claim 4 or the composition of claim 5, for manufacturing a medicine for preventing and/or treating acute lung injury.

7. Use of the Cordycep cicadae mycelia active substance of claim 4 or the composition of claim 5 for preventing and/or treating acute lung injury.

8. The use of claim 6 or claim 7, wherein said improving acute lung injury includes alleviating pathological symptoms of lung inflammation.

9. The use of claim 8, wherein said alleviating pathological symptoms of lung inflammation includes restoring the integrity of damaged or fused alveoli.

10.The use of claim 6 or claim 7, wherein said improving acute lung injury includes reducing protein leakage response.

11.The use of claim 6 or claim 7, wherein said improving acute lung injury includes reducing the level of infiltration in inflamed cells.

12.The use of claim 11, wherein said inflamed cells includes leukocytes, phagocytes and/or neutrophils.