BE1029918B1 - PROCESS FOR MANUFACTURING AND USE OF ANTRODIA CINNAMOMEA MYCELIUM IN THE TREATMENT OF CANCER - Google Patents

Abstract

The present application discloses a method for producing Antrodia cinnamomea mycelium and its use for treating cancer, the method for producing Antrodia cinnamomea mycelium comprising culturing Antrodia cinnamomea sequentially in a plate and a triangular flask, then inoculating the Antrodia cinnamomea in a predetermined solid medium for conducting solid culture and collecting Antrodia cinnamomea mycelium obtained from the culture. The Antrodia cinnamomea mycelium produced by 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 made into an anti-cancer composition.

Description

PROCESS FOR PRODUCTION OF ANTRODIA CINNAMOMEA MYCELIUM

AND USE THEREOF FOR THE TREATMENT OF CANCER

technical field

The present application relates to a method for producing a valuable Chinese herbal medicine and its use, and more particularly to a

Process for the production of Antrodia cinnamomea mycelium and its use in the treatment of cancer.

State of the art

Antrodia cinnamomea is a unique medicinal mushroom in Taiwan that has antioxidant, anti-inflammatory, anti-allergic and neuroprotective properties. The main active ingredients include polysaccharides, triterpenes, sterols, benzene and

benzoquinone derivatives. In the wild, Antrodia cinnamomea can only be found on the

Parasitize stems of wilted 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 valuable.

In order to legally obtain Antrodia cinnamomea or its inner contents for later use, 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 felling of conservation trees, the content of active ingredients in Antrodia cinnamomea may be affected due to differences in culture and extraction techniques used. There is currently a lack of technology in the state of the art to determine the level of active ingredients in

Antrodia cinnamomea.

Content of the present utility model

The main object of the present application is to provide a method for

to provide production of Antrodia cinnamomea mycelium. through special

Culture steps, the Antrodia cinnamomea mycelium can be mass-produced _standardized and the content of active ingredients in the Antrodia cinnamomea mycelium can be increased, thereby ensuring that the cultured Antrodia cinnamomea mycelium has stable quality and physiological activity to the

Effectively control production costs and increase economic value.

Another object of the present application is to provide a

Use of Antrodia cinnamomea mycelium to treat cancer.

In particular, the Antrodia BE2022/5410 cinnamomea mycelium disclosed in the present application has the effect, the angiogenesis of endothelial cells and the

inhibit the growth of cancer cells. Therefore, in the present

Application disclosed Antrodia cinnamomea mycelium as an active ingredient in a

anti-cancer composition are used.

In order to achieve the above objects, the present application discloses a

Process for the production of Antrodia cinnamomea mycelium, which mainly

expanding Antrodia cinnamomea and mycelium thereof by plate culture and

Shake flask culture, culturing a fungal liquid containing Antrodia cinnamomea in a solid medium having a specific composition by a

Solid culture method, and recovery of Antrodia cinnamomea mycelium

cultivation, includes.

Here, the above method for producing Antrodia cinnamomea mycelium further comprises subjecting the obtained Antrodia cinnamomea mycelium to drying and milling under a predetermined condition, thereby producing an Antrodia cinnamomea mycelium powder.

In one embodiment of the present application, the method for

Preparation of Antrodia cinnamomea mycelium the following steps:

Step a): performing a shake flask culture on a mother of Antrodia cinnamomea with an ME broth, the ME broth being malt extract,

soy peptone and dextrose.

Step b): Inoculating a fungal 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 conditions a culture temperature of 23-27°C, a

humidity of 55% and a culture period of 75-105 days.

Step c): Collecting the Antrodia cinnamomea mycelium and performing a

milling after drying at a predetermined temperature, whereby an Antrodia cinnamomea mycelium powder is obtained.

Here, the mother piece of Antrodia cinnamomea in step a) from a

Obtain plate culture of Antrodia cinnamomea in a plate medium; and the

Plate medium includes malt extract, soy peptone, dextrose and agar.

Here, the solid medium is prepared by mixing buckwheat, brown rice and

Barley produced in a predetermined ratio.

The drying process in step c) is carried out at a temperature of 70-80°C. BE2022/5410

In another embodiment of the present application, a

Provided 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 process and the

has effect to inhibit angiogenesis and proliferation of cancer cells; that is, by administering the anticancer composition to a cancer patient, the effect of treating cancer or slowing down the progress of cancer can be effectively achieved.

Brief description of the characters

Figure 1 shows the results of analysis of cell viability of various

Cancer cells that are 24 hours with the disclosed in the present application

Antrodia cinnamomea mycelium were treated.

Figure 2 shows the results of analysis of cell viability of various

Cancer cells infected for 48 hours with the disclosed in the present application

Antrodia cinnamomea mycelium were treated.

Fig. 3 is the results of microscopic observation of cell morphology of various cancer cells observed for 48 hours with the in the present

Application disclosed Antrodia cinnamomea mycelium treated.

Figure 4 is a microscopic observation of endothelial cell tubing in each group.

Fig. 5 is the results of the quantitative analysis of the tube formation of

Endothelial cells in each group after treatment with different

Concentrations of Antrodia cinnamomea mycelium powder disclosed in the present application.

Detailed Embodiments

The present application discloses a process for the production of Antrodia cinnamomea mycelium and its use in the treatment of cancer.

In particular, the method disclosed in the present application for

Preparation of Antrodia cinnamomea mycelium: Culturing Antrodia cinnamomea in a plate and a triangular flask one by one, then

inoculating the Antrodia cinnamomea in a predetermined solid medium to carry out solid culture and collecting obtained from the culture

Antrodia cinnamomea mycelium. Thereafter, the Antrodia cinnamomea mycelium is desiccated and dried for ease of storage and transportation

subjected to milling processes to obtain an Antrodia cinnamomea mycelium powder.

The process disclosed in the present application for the production of BE2022/5410

Antrodia cinnamomea mycelium can not only Antrodia cinnamomea mycelium in

Mass production, but also the active ingredients, such as triterpenoids, in the

Effectively increase Antrodia cinnamomea mycelium.

The present application further verifies that the Antrodia cinnamomea mycelium obtained by the above method is capable of supporting the growth of

inhibit cancer cells and endothelial cell angiogenesis. With others

Words, 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 disclosed in the present application or powder thereof can be made into an anti-cancer composition

treatment of cancer or inhibition of metastasis of cancer cells.

Therein, the Antrodia cinnamomea mycelium disclosed in the present application can be used to treat or ameliorate various types of cancer, including liver cancer, colon cancer and melanoma.

The term "anticancer composition" as used herein refers to a composition comprising 0.1% to 100% of the Antrodia cinnamomea myceliums or powder thereof disclosed in the present application as an active ingredient; the anti-cancer composition may also vary depending on the intended use and

Object of use include other components. If the

For example, when the anti-cancer composition is used as a medicament, it may further contain a pharmaceutically acceptable excipient or carrier; and when the anti-cancer composition is used as a dietary supplement, it may further comprise a food-acceptable component. In addition, the anticancer composition can be prepared in various forms depending on the need or purpose

dosage forms are formulated, such as lozenges, powders,

pills and liquids.

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.

An embodiment of the present application discloses a method for

Preparation of Antrodia cinnamomea mycelium comprising the following steps:

Step 101: Plate culture on Antrodia cinnamomea in a

plating medium to obtain a mother piece of Antrodia cinnamomea; and

collecting Antrodia cinnamomea mycelium from the mother of Antrodia cinnamomea and inoculating the same into a triangular flask containing a predetermined culture solution to perform shake flask culture;

wherein BE2022/5410 - the plating medium comprises malt extract, soy peptone, dextrose and agar; and 5 the predetermined broth is an ME broth comprising malt extract, soy peptone and dextrose.

Step 102: preparing a solid medium, the solid medium comprising a matrix resulting from mixing buckwheat, brown rice and barley in a predetermined ratio.

Inoculating a fungal liquid of Antrodia cinnamomea obtained from the culture in

step a) into the solid medium and culturing it at 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: Collect the Antrodia cinnamomea mycelium and perform a

Grinding process after drying at a temperature of 70-80 ° C, creating a

Antrodia cinnamomea mycelium powder is obtained.

By comprising the above steps, the method for the production of Antrodia cinnamomea-mycelium disclosed in the present application is able to

Antrodia cinnamomea mycelium in batches with a uniform and standardized method in mass production and the content of

To increase active ingredients in it, to ensure that the produced Antrodia camphorata mycelium maintains its physiological activity, the Antrodia camphorata mycelium can be pulverized through the subsequent drying and grinding process, which can not only effectively reduce transportation costs, but also increase the convenience of storage for the later use.

The Antrodia cinnamomea mycelium disclosed in the present application has the effect of inhibiting the growth of cancer cells and the angiogenesis of

inhibit endothelial cells. Therefore, in one embodiment of the present

Application for an anti-cancer composition comprising at least one active

Amount of Antrodia cinnamomea mycelium powder prepared by the above

A method wherein the anti-cancer composition is administered to a patient with cancer, the cancer including liver cancer, colon cancer, melanoma, and the like.

To the anti-cancer efficacy of the disclosed in the present application

To confirm Antrodia cinnamomea mycelium, some are listed below

experiments described.

Experiment 1: Analysis of cancer cell viability

The following cancer cells were used: liver cancer cells Hep3B, BE2022/5410

Melanoma cells B16-F10, cervical cancer cells HeLa, breast cancer cells MCF7,

SKOV3 ovarian cancer cells, Colo205 colon cancer cells, prostate cancer cells

PC3, gastric cancer cells AGS and endometrial cancer cells HEC-1A. Each of the

Cancer cells were seeded in a culture plate at a rate of 2 x 10* cells/well.” After the cells had adhered, the culture medium was through

Culture media replaced containing Antrodia cinnamomea-mycelium at a concentration of

0 and 400 pg/ml, respectively, and cultured for 24 and 48 hours, respectively. after the

Cultivation was completed, MTT reagent was added to each group of cells (20uL MTT reagent was added to each 100uL culture medium) for the

reaction added. Then the absorbance at 490 nm was measured with a

ELISA reader for analysis to obtain the cell viability of each group of cells cultured for different durations. The

Results are in FIG. 1 and FIG. 2 shown. The cell morphology of each cell group cultured for 48 hours was observed under a microscope as shown in FIG

FIG: 3 shown;

As can be seen from FIG. 1 to FIG. 3, it was shown that among the cancer cells treated with the Antrodia cinnamomea mycelium disclosed in the present application for 24 hours, only the melanoma cells were significantly inhibited. However, when the treatment period was extended to 48 hours, it was found that the Antrodia cinnamomea mycelium disclosed in the present application inhibited the proliferation of liver cancer cells Hep3B, melanoma cells B16-F10 and

Colon cancer cells significantly inhibited Colo205. The ICso of the present

Application disclosed Antrodia cinnamomea myceliums against the three types of cancer cells was further calculated as shown in Table 1 below.

Table 1: the concentration of Antrodia cinnamomea mycelium to inhibit the

Growth of cancer cells by 50% (ICso)

Experiment 2: Inhibition of angiogenesis 10uL of Matrigel solution was added to the culture plate, and then a

Total volume of 50 UL culture solution added to each well. The

Culture solution comprised 5 x 10° endothelial cells and a solution of Antrodia cinnamomea myceliums disclosed in the present application in different concentrations: 0, 6.25, 12.5, 25 and 50 wg/ml. After 24 hours of cultivation, tube formation of cells in each well was observed with a microscope and analyzed with software. The results are in FIG:

4 and FIG: 5 shown. BE2022/5410

As can be seen from the results of FIG. 4 and FIG

Endothelial cells formed reticular structure denser when the culture solution did not include Antrodia cinnamomea mycelium disclosed in the present application.

When different concentrations of Antrodia cinnamomea mycelium were added to the culture solution, the inhibition of tube formation of the endothelial cells improved with increasing concentration addition. How from the

Results can be seen, that disclosed in the present application

Antrodia cinnamomea mycelium actually inhibit angiogenesis of endothelial cells, thus achieving the effect of treating cancer or slowing down the progression of cancer.

Claims (9)

Expectations A method for producing Antrodia cinnamomea mycelium, comprising the steps of: step a): performing shake flask culture on a mother of Antrodia cinnamomea with a predetermined culture solution; Step b): preparing a solid medium, inoculating a fungal liquid obtained from the culture in step a) into the solid medium and culturing under a predetermined culture condition, the culture condition being 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. The method of claim 1, wherein the Antrodia cinnamomea mother in step a) is obtained from a plate culture of Antrodia cinnamomea in a plate medium, and the plate medium comprises malt extract, soy peptone, dextrose and agar. 3. The method according to claim 1, wherein the predetermined broth in step a) is an ME broth 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. The method according to claim 1, wherein the step c) further comprises subjecting the Antrodia cinnamomea mycelium to a grinding process after a drying process at a temperature of 70-80°C, thereby obtaining an Antrodia cinnamomea mycelium powder. An anti-cancer composition comprising an effective amount of Antrodia BE2022/5410 cinnamomea mycelium, wherein the Antrodia cinnamomea mycelium is prepared by the method of any one of claims 1 to 6. 8. Use of the anti-cancer composition according to claim 7 for inhibiting the growth of cancer cells. 9. Use of the anticancer composition of claim 7 for inhibiting angiogenesis of endothelial cells.