# US 10,865,377 B2 · Cordyceps cicadae mycelium active substances, preparation method, pharmaceutical composition and application thereof

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- Number: US 10,865,377 B2
- Document ID: US10865377B2
- Status: Granted
- Kind: B2
- Application number: 15/896,752
- Filed: 2018-02-14
- Published: 2018-06-21
- Granted: 2020-12-15
- Earliest claimed: 2015-04-22
- Assignees: Grape King Bio Ltd.
- Applicants: Grape King Bio Ltd.
- Inventors: Pei-Cheng Lin; Han-Hsin Chang; Chin-Chu Chen; Shu-Hsing Yeh; Li-Ya Lee; Jui-Hsia Hsu
- Web page: https://patentgrep.com/patent/US10865377B2

## CPC

- C12N 1/14: Fungi (culture of mushrooms A01G18/00; as new plants A01H15/00); Culture media therefor
- A61K 35/66: Microorganisms or materials therefrom (fungi, yeasts or candida A61K36/06)
- A61K 36/068: Cordyceps
- A61P 27/02: Ophthalmic agents
- C12P 1/02: by using fungi
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

A method of preparing Cordyceps cicadae mycelium active substances for preventing and/or treating xerophthalmia is provided. The method comprises: (a) culturing a Cordyceps cicadae mycelium in a plate media at 15 to 35° C. for 5 to 14 days; (b) inoculating the mycelium of step (a) to a flask containing liquid media and culturing it at 15 to 35° C. with a pH of 2 to 8 for 3 days; (c) inoculating the mycelium of step (b) to a fermenter tank and culturing it at 15 to 35° C. with a pH of 2 to 8 for 3 days, so as to obtain a Cordyceps cicadae mycelium fermentation liquid; (d) freeze-drying and grating the fermentation liquid, so as to obtain a Cordyceps cicadae mycelium powder; (e) extracting the powder with solvent, so as to obtain a Cordyceps cicadae mycelium extract; and (f) drying the extract, so as to obtained the Cordyceps cicadae mycelium active substances.

## Claims

### Claim 1

A method of preparing a mixture of Cordyceps cicadae mycelium active substances for preventing and/or treating xerophthalmia, wherein the method comprises following steps:
(a) culturing a Cordyceps cicadae mycelium in a plate media at 15 to 35° C. for 5 to 14 days;
(b) inoculating the Cordyceps cicadae mycelium of step (a) to a flask containing liquid media and culturing the mycelium at 15 to 35° C. with a pH of 2 to 8 for 3 days;
(c) inoculating the Cordyceps cicadae mycelium of step (b) to a fermenter tank and culturing the mycelium at 15 to 35° C. with a pH of 2 to 8 for 3 days, so as to obtain a Cordyceps cicadae mycelium fermentation liquid;
(d) freeze-drying and grating the Cordyceps cicadae mycelium fermentation liquid, so as to obtain a Cordyceps cicadae mycelium powder;
(e) dividing the Cordyceps cicadae mycelium powder into two parts, extracting one part of the Cordyceps cicadae mycelium powder with water and extracting the other part of the Cordyceps cicadae mycelium powder with alcohol separately, so as to obtain a Cordyceps cicadae mycelium water extract and a Cordyceps cicadae mycelium alcohol extract; and
(f) drying the Cordyceps cicadae mycelium water extract and the Cordyceps cicadae mycelium alcohol extract, and mixing the water extracts and the alcohol extracts, so as to obtain the mixture of Cordyceps cicadae mycelium active substances.

### Claim 2 (depends on 1)

The method of claim 1, wherein the culturing process of step (b) is shake flask cultivation, and the flask is shaking at a speed range between 10 and 250 rpm.

### Claim 3 (depends on 1)

The method of claim 1, wherein in step (c), the gas feed in the fermenter tank comprises air, oxygen, carbon dioxide, helium or a combination thereof.

### Claim 4 (depends on 1)

The method of claim 1, wherein in step (c), the pressure of the fermenter tank is at 0.5 to 1.0 kg/cm2, and the gas flow rate of fermenter tank is 0.01 to 1.5 VVM.

### Claim 5 (depends on 1)

The method of claim 1, wherein the liquid media used in step (b) and step (c) are the same, and the liquid media comprises grains, beans, inorganic salts, carbohydrates, yeast extract, malt extract or a combination thereof.

### Claim 6 (depends on 1)

The method of claim 1, wherein the alcohol is methanol or ethanol.

### Claim 7 (depends on 1)

The method of claim 1, wherein the mixture of Cordyceps cicadae mycelium active substances comprise the same weight of the water extracts and the alcohol extracts from the Cordyceps cicadae mycelium powder.

### Claim 8

A mixture of Cordyceps cicadae mycelium active substances for preventing and/or treating xerophthalmia, wherein the mixture is prepared by a method comprising:
(a) culturing a Cordyceps cicadae mycelium in a plate media at 15 to 35° C. for 5 to 14 days;
(b) inoculating the Cordyceps cicadae mycelium of step (a) to a flask containing liquid media and culturing the mycelium at 15 to 35° C. with a pH of 2 to 8 for 3 days;
(c) inoculating the Cordyceps cicadae mycelium of step (b) to a fermenter tank and culturing the mycelium at 15 to 35° C. with a pH of 2 to 8 for 3 days, so as to obtain a Cordyceps cicadae mycelium fermentation liquid;
(d) freeze-drying and grating the Cordyceps cicadae mycelium fermentation liquid, so as to obtain a Cordyceps cicadae mycelium powder;
(e) dividing the Cordyceps cicadae mycelium powder into two parts, extracting one part of the Cordyceps cicadae mycelium powder with water and extracting the other part of the Cordyceps cicadae mycelium powder with alcohol separately, so as to obtain a Cordyceps cicadae mycelium water extract and a Cordyceps cicadae mycelium alcohol extract and
(f) drying the Cordyceps cicadae mycelium water extract and the Cordyceps cicadae mycelium alcohol extract, and mixing the water extracts and the alcohol extracts, so as to obtain the mixture of Cordyceps cicadae mycelium active substances.

### Claim 9 (depends on 8)

A pharmaceutical composition for preventing and/or treating xerophthalmia, which comprises a therapeutically effective amount of the mixture of Cordyceps cicadae mycelium active substances of claim 8 and a pharmaceutically acceptable carrier, excipient, diluents or adjuvant.