LU502181B1

LU502181B1 - Method of producing antrodia cinnamomea mycelium and use thereof for treating cancers

Info

Publication number: LU502181B1
Application number: LU502181A
Authority: LU
Inventors: Pang-Kuei Hsu; Chia-Feng Wu; Yu-Cheng Lin
Original assignee: Greenyn Biotechnology Co Ltd
Priority date: 2022-05-27
Filing date: 2022-05-27
Publication date: 2023-05-15
Also published as: LU502181A1

Abstract

The present application discloses a method of producing antrodia cinnamomea mycelium and use thereof for treating cancers, wherein the method of producing antrodia cinnamomea mycelium comprises culturing antrodia cinnamomea in a plate and a triangular flask successively, then inoculating the antrodia cinnamomea in a predetermined solid medium to carry out solid culture, and collecting antrodia cinnamomea mycelium obtained from the culture. The antrodia cinnamomea mycelium produced from the above method is capable of inhibiting the proliferation of cancer cells and angiogenesis. Therefore, the antrodia cinnamomea mycelium disclosed in the present application can be prepared into an anti-cancer composition.

Description

Specification ad

METHOD OF PRODUCING ANTRODIA CINNAMOMEA MYCELIUM AND

USE THEREOF FOR TREATING CANCERS

TECHNICAL FIELD

The present application relates to a method of preparing a precious Chinese herbal medicine and use thereof and in particular to a method of producing antrodia cinnamomea mycelium and use thereof for treating cancers.

BACKGROUND

Antrodia cinnamomea is a unique medicinal fungus in Taiwan that has antioxidant, anti-inflammatory, anti-allergic, and neuroprotective properties. Its main active ingredients include polysaccharides, triterpenes, sterols, benzene, and benzoquinone derivatives. Antrodia cinnamomea in the wild can only be parasitic on the trunks of withered cinnamomum kanehirae. However, since cinnamomum kanehirae is listed as a conservation plant in Taiwan, it is not easy to obtain wild antrodia cinnamomea. Therefore, antrodia cinnamomea is precious.

In order to legally obtain antrodia cinnamomea or its internal ingredients for subsequent utilization, antrodia cinnamomea is currently cultured by artificial culture methods of liquid culture or solid culture. Although the artificial culture methods can achieve the effect of reducing the cost of collecting antrodia cinnamomea and avoiding the felling of conservation trees, the content of active ingredients in antrodia cinnamomea may be affected due to differences in the culture and extraction techniques used. Currently, there is a lack of technology in the prior art to ensure the content of active ingredients in antrodia cinnamomea.

SUMMARY

The primary object of the present application is to provide a method of producing antrodia cinnamomea mycelium. Through special culture steps, the antrodia cinnamomea mycelium can be mass-produced in a standardized manner and the content of active ingredients in the antrodia cinnamomea mycelium can be increased, which ensure that the cultured antrodia cinnamomea mycelium has a stable quality and physiological activity, thereby achieving effective control of the 1 production cost and increase in the economic value.

Another object of the present application 1s to provide a use of antrodia cinnamomea mycelium for treating cancers. In particular, the antrodia cinnamomea mycelium disclosed in the present application has the effect of inhibiting angiogenesis of endothelial cells and growth of cancer cells.

Therefore, the antrodia cinnamomea mycelium disclosed in the present application can be used as an active ingredient in an anti-cancer composition.

In order to achieve the above objects, the present application discloses a method of producing antrodia cinnamomea mycelium, which mainly comprises expanding antrodia cinnamomea and mycelium thereof by plate culture and shake flask culture, culturing a fungus liquid containing the antrodia cinnamomea in a solid medium having a special composition by a solid culture method, and obtaining the antrodia cinnamomea mycelium after culturing.

Wherein, the above method of producing antrodia cinnamomea mycelium further comprises performing drying and grinding procedures under a predetermined condition on the obtained antrodia cinnamomea mycelium, thereby producing an antrodia cinnamomea mycelium powder.

In one embodiment of the present application, the method of producing antrodia cinnamomea mycelium comprises the following steps:

Step a): carrying out shake flask culture on a mother piece of antrodia cinnamomea with an

ME culture solution, wherein the ME culture solution comprises malt extract, soy peptone and dextrose.

Step b): inoculating a fungus liquid obtained from the culture of Step a) in a solid medium and culturing it under a predetermined culture condition, wherein the solid medium comprises at least one of coarse cereals; and the culture condition comprises a culture temperature of 23-27°C, a humidity of 55%, and a culture time of 75-105 days.

Step c): collecting the antrodia cinnamomea mycelium and performing a grinding procedure after a drying procedure under a predetermined temperature, thereby obtaining an antrodia cinnamomea mycelium powder.

Wherein, the mother piece of antrodia cinnamomea in Step a) is obtained from plate culture of antrodia cinnamomea in a plate medium; and the plate medium comprises malt extract, soy peptone, dextrose and agar.

Wherein, the solid medium is prepared by mixing of buckwheat, brown rice and barley in a predetermined ratio. 2

Wherein, the drying procedure in Step c) is performed under a temperature of 70-80°C. PSE

In another embodiment of the present application, provided is an anti-cancer composition comprising an effective amount of antrodia cinnamomea mycelium or powders thereof, wherein the antrodia cinnamomea mycelium or powders thereof is prepared by the above method, and has the effect of inhibiting angiogenesis and proliferation of cancer cells; that is, by administering the anti-cancer composition to a cancer patient, the effect of treating cancer or slowing down the progression of cancer can be effectively achieved.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows the results of analyzing cell viability of different cancer cells treated by the antrodia cinnamomea mycelium disclosed in the present application for 24 hours.

FIG. 2 shows the results of analyzing cell viability of different cancer cells treated by the antrodia cinnamomea mycelium disclosed in the present application for 48 hours.

FIG. 3 is the results of microscope observation of the cell morphology of different cancer cells treated by the antrodia cinnamomea mycelium disclosed in the present application for 48 hours.

FIG. 4 is a microscope observation of tube formation of endothelial cells in each group.

FIG. 5 is the results of quantitative analysis of tube formation of endothelial cells in each group after treating with different concentrations of the antrodia cinnamomea mycelium powder disclosed in the present application.

DETAILED DESCRIPTION

The present application discloses a method of producing antrodia cinnamomea mycelium and use thereof for treating cancers. Specifically, the method of producing antrodia cinnamomea mycelium disclosed in the present application comprises culturing antrodia cinnamomea in a plate and a triangular flask successively, then inoculating the antrodia cinnamomea in a predetermined solid medium to carry out solid culture, and collecting antrodia cinnamomea mycelium obtained from the culture. Thereafter, for ease of preservation and transportation, the antrodia cinnamomea mycelium is subjected to drying and grinding procedures to obtain an antrodia cinnamomea mycelium powder. The method of producing antrodia cinnamomea mycelium disclosed in the present application can not only mass-produce antrodia cinnamomea mycelium, but also effectively increase the active ingredients, such as triterpenoids, in the antrodia cinnamomea mycelium. 3

The present application further verifies that the antrodia cinnamomea mycelium obtained by. the above method is capable of inhibiting the growth of cancer cells and angiogenesis of endothelial cells. In another word, the antrodia cinnamomea mycelium disclosed in the present application has the effect of treating cancer or slowing down the progression of cancer. Therefore, the antrodia cinnamomea mycelium or powders thereof disclosed in the present application can be prepared into an anti-cancer composition for treating cancers or inhibiting the metastasis of cancer cells.

Wherein, the antrodia cinnamomea mycelium disclosed in the present application can be used to treat or improve various cancers, including liver cancer, colon cancer, and melanoma.

The term “anti-cancer composition” as used herein refers to a composition comprising 0.1%-100% of the antrodia cinnamomea mycelium or powders thereof disclosed in the present application as an active ingredient; the anti-cancer composition may further comprise other components according to the purpose of use and the subject of use. For example, when the anti-cancer composition is used as a medicine, it may further include a pharmaceutically acceptable excipient or carrier, and when the anti-cancer composition is used as a nutritional supplement, it may further comprise a food acceptable component. In addition, the anti-cancer composition can be formulated into different dosage forms, such as lozenges, powders, pills, and liquids, according to needs or purposes.

Hereinafter, in order to illustrate the technical features and effects of the present application, some embodiments are given and described with the drawings as follows.

One embodiment of the present application discloses a method of producing antrodia cinnamomea mycelium, comprising the following steps:

Step 101: carrying out plate culture on antrodia cinnamomea in a plate medium to obtain a mother piece of antrodia cinnamomea; and collecting antrodia cinnamomea mycelium from the mother piece of antrodia cinnamomea and inoculating it in a triangular flask containing a predetermined culture solution to carry out shake flask culture; wherein: the plate medium comprises malt extract, soy peptone, dextrose and agar; and the predetermined culture solution is an ME culture solution comprising malt extract, soy peptone and dextrose.

Step 102: preparing a solid medium, wherein the solid medium comprises a matrix resulting from mixing of buckwheat, brown rice and barley in a predetermined ratio. inoculating a fungus liquid of antrodia cinnamomea obtained from the culture in Step a) in the 4 solid medium and culturing it under a culture temperature of 23-27°C and a humidity of 55% for a ° culture time of 75-105 days, to produce the antrodia cinnamomea mycelium.

Step 103: collecting the antrodia cinnamomea mycelium and performing a grinding procedure after drying under a temperature of 70-80°C, thereby obtaining an antrodia cinnamomea mycelium powder.

By comprising the above steps, the method of producing antrodia cinnamomea mycelium disclosed in the present application is capable of mass-producing antrodia cinnamomea mycelium in batches with a unified and standardized process and increasing the content of active ingredients therein, thereby ensuring the produced antrodia cinnamomea mycelium maintains its physiological activity. Moreover, through the subsequent drying and grinding procedures, the antrodia cinnamomea mycelium is powdered, which can not only effectively reduce the transportation cost, but also increase the convenience of operation and storage for subsequent use.

The antrodia cinnamomea mycelium disclosed in the present application has the effect of inhibiting growth of cancer cells and angiogenesis of endothelial cells. Therefore, in one embodiment of the present application is an anti-cancer composition, comprising at least an effective amount of the antrodia cinnamomea mycelium powder prepared by the above method, wherein the anti-cancer composition is administered to a cancer patient, which cancer includes liver cancer, colon cancer, melanoma and the like.

In order to confirm the anti-cancer efficacy of the antrodia cinnamomea mycelium disclosed in the present application, some experiments are described below.

Experiment 1: Analysis of Cancer Cell Viability

The following cancer cells were used: liver cancer cells Hep3B, melanoma cells B16-F10, cervical cancer cells HeLa, breast cancer cells MCF7, ovarian cancer cells SKOV3, colon cancer cells Colo205, prostate cancer cells PC3, gastric cancer cells AGS, and endometrial cancer cells

HEC-1A. Each of the cancer cells was seeded in a culture plate in a number of 2x10* cells/well.

After the cells adhered, the culture medium was replaced with culture media containing antrodia cinnamomea mycelium at a concentration of 0 and 400 ug/ml, respectively, and cultured for 24 and 48 hours, respectively. After the culture was completed, MTT reagent was added to each group of cells (20 pL of MTT reagent was added to each 100 pL of culture medium) for reaction. Then, the absorbance at 490 nm was read by an ELISA reader for analysis to obtain the cell viability of each group of cells cultured for different durations. The results are shown in FIG. 1 and FIG. 2. The cell 5 morphology of each group of cells cultured for 48 hours was observed under a microscope, rad shown in FIG. 3;

As can be seen from FIG.1 to FIG. 3, among the cancer cells treated by the antrodia cinnamomea mycelium disclosed in the present application for 24 hours, only the melanoma cells were shown to be significantly inhibited. However, when the treatment duration was prolonged to 48 hours, it was found that the antrodia cinnamomea mycelium disclosed in the present application significantly inhibited the proliferation of liver cancer cells Hep3B, melanoma cells B16-F10, and colon cancer cells Colo205. The ICso of the antrodia cinnamomea mycelium disclosed in the present application to the three types of cancer cells was further calculated, as shown below in Table 1.

Table 1: the concentration of antrodia cinnamomea mycelium for inhibiting the growth of cancer cells by 50% (IC50)

Experiment 2: Inhibition of angiogenesis 10 ul of Matrigel solution was added to the culture plate, and then a total volume of 50 ul of culture solution was added to each well. The culture solution comprised 5x10* of endothelial cells and solution of the antrodia cinnamomea mycelium disclosed in the present application under different concentrations: 0, 6.25, 12.5, 25, and 50 pg/ml. After culturing for 24 hours, the tube formation of cells in each well was observed with a microscope and analyzed with software. The results are shown in FIG 4 and FIG 5.

As can be seen from the results of FIG 4 and FIG 5, when the culture solution did not comprise the antrodia cinnamomea mycelium disclosed in the present application, the reticular structure formed by the endothelial cells was denser. When the culture solution was added with different concentrations of the antrodia cinnamomea mycelium, the inhibition of tube formation of the endothelial cells was improved with the increase of the concentrations added. As can be seen from the results, the antrodia cinnamomea mycelium disclosed in the present application can indeed inhibit the angiogenesis of endothelial cells, and thus can achieve the effect of treating cancer or slowing down the progression of cancer. 6

[Description of Component Symbol]

None 7

Claims

CLAIMS WHAT IS CLAIMED IS:

1. À method of producing antrodia cinnamomea mycelium, comprising the following steps: Step a): carrying out shake flask culture on a mother piece of antrodia cinnamomea with a predetermined culture solution; Step b): preparing a solid medium, inoculating a fungus liquid obtained from the culture in Step a) in the solid medium and culturing it under a predetermined culture condition, wherein the culture condition comprises a culture temperature of 23-27°C, a humidity of 55%, and a culture time of 75-105 days; and Step c): collecting the antrodia cinnamomea mycelium.

2. The method of claim 1, wherein the mother piece of antrodia cinnamomea in Step a) is obtained from plate culture of antrodia cinnamomea in a plate medium, and the plate medium comprises malt extract, soy peptone, dextrose and agar.

3. The method of claim 1, wherein the predetermined culture solution in Step a) is an ME culture solution comprising malt extract, soy peptone and dextrose.

4. The method of claim 1, wherein the solid medium in Step b) comprises coarse cereals.

5. The method of claim 4, wherein the solid medium comprises buckwheat, brown rice and barley.

6. The method of claim 1, wherein the Step c) further comprises performing a grinding procedure on the antrodia cinnamomea mycelium after a drying procedure under a temperature of 70-80°C, thereby obtaining an antrodia cinnamomea mycelium powder.

7. An anti-cancer composition, comprising an effective amount of antrodia cinnamomea mycelium, wherein the antrodia cinnamomea mycelium is prepared by the method of any one of claims 1-6.

8. Use of the anti-cancer composition of claim 7 for inhibiting growth of cancer cells.

9. Use of the anti-cancer composition of claim 7 for inhibiting angiogenesis of endothelial cells. 8