TW201439318A - A method for culturing Antrodia cinnamomea, a method for manufacturing an Antrodia cinnamomea alcoholic extract and the use of the Antrodia cinnamomea alcoholic extract thereof - Google Patents

Abstract

A method for culturing Antrodia cinmamomea improving content of antcin C and zhankuic acid C is disclosed. The method comprises: providing a Cinnamomum kanehirai wood segment; inoculating a strain of Antrodia cinnamomea to the wood segment with dry weight of 0.162 g of hyphae and mycelium of the strain of Antrodia cinnamomea per kg wood segment; and irradiating the wood segment containing the strain of Antrodia cinnamomea with a light of wavelength of 600 to 700 nm to, to obtain a fruiting body. Specifically, the irradiation time is 8 to 12 hrs per day within 30 to 40 days.

Description

Method for cultivating Antrodia camphorata, extraction method of extract of Antrodia camphorata and extraction of the extract of Antrodia camphorata Use

The invention relates to a method for cultivating Antrodia camphorata, in particular to a method for cultivating Antrodia camphorata C, which can enhance the content of Antrodia camphoric acid C and oyster mushroom C, and the invention relates to a method for extracting Antrodia camphorata, which can obtain a high content of Antrodia camphorata The extract of the Antrodia camphorata wine having the acid C and the yoghurt C content, and the extract of the Antrodia camphorata wine for inhibiting the growth of tumor cells.

Antrodia cinnamomea is a unique and precious medicinal material in Taiwan. It is grown only on the inner wall of the hollow core material of Cinnamomum kanehirai , which is rich in triterpenoids. The triterpenoids have tumor suppressor cells. The function of growing, repairing the liver and promoting liver function, lowering blood lipids and blood pressure, and improving immunity, so that the price of wild Antrodia camphora is high.

However, since the amount of wild Antrodia camphora is small and difficult to obtain, the industry obtains the mycelium or fruit body of Antrodia camphorata by artificially cultivating Antrodia camphorata to further extract physiological active substances in the mycelium or fruit body of the Antrodia camphorata (such as Sancha Class of compounds).

The method of cultivating A. sinensis comprises: liquid fermentation method, solid culture method and eucalyptus cultivation method, wherein the conventional liquid fermentation method has a faster growth rate, but the biochemical and physiological metabolic pathway of the fermented mycelium is different from that of wild A. sinensis. The nutrients produced are also far from the wild Antrodia camphorata; although the solid-state culture method can be used to produce a similar fruit to the wild Antrodia camphorata Body, but the nutrients of the fruiting bodies produced by the conventional solid culture method are still slightly different from the wild A. angustifolia fruiting bodies.

The conventional eucalyptus cultivation method utilizes the dead burdock eucalyptus, and inoculates the burdock bacillus in the burdock eucalyptus, so that the burdock bacillus can grow in the burdock eucalyptus, thereby taking the fruit body, and the advantages thereof. It is that the same ingredients as the wild A. angustifolia fruit body can be produced. However, the conventional eucalyptus cultivation method has to undergo a culture time of 2 to 3 years, and the cultivation process must maintain an appropriate environmental temperature. Therefore, the conventional eucalyptus cultivation method has problems such as high cultivation cost and long cultivation time, and thus is disadvantageous. Mass production.

The main object of the present invention is to provide a method for cultivating Antrodia camphorata, which can produce a large amount of ricinic acid C and ricinoleic acid C to enhance the nutritional value of Antrodia camphorata.

A further object of the present invention is to provide a method for cultivating Antrodia camphorata, which can shorten the cultivation time of Antrodia camphorata, reduce the cultivation cost, and further facilitate the mass production of Antrodia camphorata.

Another object of the present invention is to provide an extraction method of an extract of Antrodia camphorata, which has a high content of ricinic acid C and ricinoleic acid C, which can reduce the survival rate of tumor cells.

Still another object of the present invention is to provide an extract of Antrodia camphorata wine using the extract of Antrodia camphorata as an active ingredient to inhibit growth of tumor cells.

In order to achieve the foregoing object, the technical means and the effects that can be achieved by the technical method include: a method for cultivating anthraquinone C which increases the content of yoghurt C and yoghurt C, comprising: providing a cow Eucalyptus eucalyptus; providing a burdock bacterium, inoculating the burdock bacillus in the burdock eucalyptus, so that each kilogram of the burdock eucalyptus contains 0.162 grams of hyphae and hyphae; and the wavelength is 600 One of the 700 nm light sources irradiates the burdock eucalyptus inoculated with the burdock bacillus to obtain a burdock fruit body; wherein the burdock eucalyptus eucalyptus The light source is irradiated for 8 to 12 hours per day and continuously irradiated for 30 to 40 days.

The method for culturing an Antrodia camphorata according to the present invention, wherein the Antrodia camphorata system is in a liquid state, and the larvae of the Antrodia camphorata contains 8.1 g of dry weight hyphae and hyphae per liter.

In the method for culturing Antrodia camphorata according to the present invention, the wavelength of the light source is preferably selected to be 660 nm.

In the method for cultivating Antrodia camphorata according to the present invention, the light source is preferably derived from a light-emitting diode.

In the method for cultivating Antrodia camphorata according to the present invention, the time for illuminating the burdock eucalyptus eucalyptus with the light source per day is preferably 8 hours.

In the method for cultivating Antrodia camphorata according to the present invention, it is preferable that the light source is selected to continuously irradiate the burdock eucalyptus inoculated with the burdock bacillus for 30 days.

An extraction method of an extract of Antrodia camphorata wine having a high content of ricinic acid C and ricinoleic acid C, comprising: providing a raw body of Antrodia camphorata as described above; and extracting the Antrodia camphorata by ultrasonic vibration using 95% ethanol at 25 ° C The fruiting body to obtain an extract of the burdock wine.

The use of the extract of Antrodia camphorata for inhibiting the growth of liver tumor cells, wherein the extract of Antrodia camphorata as an active ingredient is used for preparing a pharmaceutical composition for treating liver tumors.

The use of the extract of Antrodia camphorata for inhibiting the growth of lung tumor cells, which is used as an active ingredient for preparing a pharmaceutical composition for treating lung tumors.

The use of the extract of Antrodia camphorata for inhibiting the growth of colorectal tumor cells, which is used as an active ingredient to prepare a pharmaceutical composition for treating colorectal tumors.

The use of extract of Antrodia camphorata for inhibiting the growth of sarcoma cells, wherein the extract of Antrodia camphorata as described above is used as an active ingredient for preparing a drug composition for treating sarcoma Minute.

The use of the extract of Antrodia camphorata for inhibiting the growth of breast tumor cells, which is used as an active ingredient to prepare a pharmaceutical composition for treating breast tumors.

The method for cultivating the Antrodia camphorata of the present invention can produce a large amount of ricinic acid C and oyster mushroom acid C, thereby improving the nutritional value of Antrodia camphorata.

The method for cultivating the Antrodia camphorata of the invention can shorten the cultivation time of the Antrodia camphorata, thereby improving the economic value of the Antrodia camphorata.

The extract of Antrodia camphorata wine prepared by the extraction method of the extract of Antrodia camphorata wine of the invention has high content of ricinic acid C and ricinoleic acid C, which can reduce the survival rate of tumor cells and can be used as an active ingredient for inhibiting the growth of tumor cells. , is the effect of the invention.

The extract of the Antrodia camphorata wine of the invention can be used as an active ingredient, and can effectively inhibit the growth of tumor cells such as liver tumor cells, lung tumor cells, colorectal tumor cells, sarcoma cells and breast tumor cells, and has the function of killing tumor cells. The effect of hyperplasia or spread.

Figure 1 is a chemical structure diagram of oxalic acid C.

Figure 2 is a chemical structure diagram of oyster mushroom acid C.

The above and other objects, features and advantages of the present invention will become more <RTIgt; And the method for cultivating the Astragalus sinensis C content, comprising the following steps: providing a burdock eucalyptus; providing a burdock bacterium, the burdock The bacillus is inoculated to the burdock eucalyptus, so that each kilogram of the burdock eucalyptus contains 0.162 g of hyphae and hyphae; and the light source is irradiated with one of the wavelengths of 600 to 700 nm. The burdock eucalyptus is obtained to obtain a burdock fruit body.

Providing a burdock eucalyptus which can be cultured in an incubator with water, maintaining the temperature of the incubator at 28 to 30 ° C and a humidity of 80% or more; preferably, to avoid other microorganisms The competition can be carried out after the burdock eucalyptus and the incubator are sterilized, and then the subsequent steps are carried out.

A burdock bacillus is provided, and the burdock bacillus is inoculated to the burdock eucalyptus. For example, the Antrodia camphorata system used in the present invention may be selected from a strain of Burdock bacteria purchased from the Bioresource Conservation and Research Center of the Republic of China Food Industry Development Research Institute, such as the Bacillus ssp. strain No. BCRC 35396, but not In this preferred embodiment, in the case of inoculation, the liquid Astragalus membranaceus cells can be selected and 1 liter of the A. sinensis cells (containing about 8.1 grams of dry weight of hyphae and bacteria per liter) The spheroid is inoculated into 50 kg of the burdock eucalyptus, so that the burdock bacillus can obtain sufficient growth space and nutrients from the burdock eucalyptus; in addition, the inoculation method of the present embodiment can select the burdock bacillus Sprinkle evenly on the surface of the burdock eucalyptus.

The bovine eucalyptus eucalyptus inoculated with the genus Antrodia camphorata is irradiated with a light source having a wavelength of 600 to 700 nm. Preferably, the light source is selected to be red light, and the wavelength of the light source is 660 nm, and the light source is preferably The choice is from a light-emitting diode, which has many advantages such as high photoelectric conversion efficiency, fixed wavelength and low heat generation, and is easy to adjust its light quantity and light quality (red/blue light ratio), so it is suitable for a closed growth environment; In a preferred embodiment, the burdock eucalyptus inoculated with the burdock bacillus is irradiated with a light source having a wavelength of 660 nm every day for 8 to 12 hours, and continuously irradiated for 30 to 40 days, so that the inoculated burdock bacillus can be used. The burdock eucalyptus grows into a burdock fruit body; according to this, the content of yoghurt C and edodenic acid C contained in the body of the burdock can be improved.

In addition, the present invention also provides an extraction method of an extract of Antrodia camphorata, which can increase the content of Antrodia C and Aceric acid C of the extract of Antrodia camphorata, and comprises: providing a body of A. angustifolia as described above; The burdock fruit body was extracted by ultrasonic vibration using 95% ethanol at °C.

In detail, in the preferred embodiment, 5 grams of Antrodia camphorata fruit body is taken, placed in 600 ml of 95% ethanol, and subjected to shock extraction at a frequency of 40 KHz at 25 ° C. The active ingredient (such as triterpenoids) in the fruit body of Antrodia camphorata can be dissolved in ethanol; preferably, the extraction time is 8 hours, and the extraction is repeated 3 times, the total extraction amount can be increased to obtain the extract of the Antrodia camphorata wine; Alternatively, a concentrated concentrated extract of Antrodia camphorata can be obtained by lyophilization.

In order to confirm that the method for cultivating Antrodia camphorata in the present embodiment can increase the content of oxalic acid C and edodenic acid C, and can enhance the inhibitory effect of Antrodia camphorata on tumor, the following test is carried out:

(A) Triterpenoid content detection

In this test, the extract of Antrodia camphorata as shown in Table 1 may be taken, the group A1 is the extract of Antrodia camphorata obtained by the method of culturing the Antrodia camphorata in the preferred embodiment, and the group A2 is in the process of cultivating the Antrodia camphorata. The obtained extract of Antrodia camphorata is irradiated with a light source having a wavelength of 600 to 700 nm. Weigh 0.2 g of the extract of the burdock wine into a spiral test tube, add 5 ml of methanol, vortex for 15 minutes with ultrasonic wave, centrifuge for 10 minutes at 3000 rpm, take 5 ml of the supernatant, and place a clean test tube. Then, it was heated to dryness in a 100 ° C water bath.

Then, the Purospher STAR (Merck) RP-18e (5 μm) 250 mm × 4 mm column was used for analysis. The flow ratio of the acetonitrile (Acetonitrile, ACN for short) and 0.085% phosphoric acid solution was used as the mobile phase at a volume ratio of 47:53. For 1 ml/min, the absorbance at a wavelength of 254 nm was detected for subsequent analysis.

Referring to Tables 2 and 3, compared with Group A2, the extract of Antrodia camphorata in Group A1 has a higher content of ricinic acid C and ricinoleic acid C, showing the culture method of Antrodia camphorata of the preferred embodiment. It can indeed increase the content of oxalic acid C and edodenic acid C.

(B) Effect on survival rate of tumor cell lines

In this test, a tumor cell line as shown in Table 4 is selected, which includes liver tumors (HepG2 cells, groups B1-1 and B1-2) and lung tumors (A549) purchased from the Food Industry Development Research Institute. Cells, groups B2-1 and B2-2), colorectal tumors (HCT-116 cells, groups B3-1 and B3-2), sarcomas (S-180 cells, groups B4-1 and B4-2) and Breast tumor (MDA-MB-231 cells, groups B5-1 and B5-2) cell lines, all of which were cultured in 10% fetal bovine serum (FBS, purchased from Biological Industries, Kibbutz beit haemek), 2 mmol/L L-Glutamine (purchased from HyClone, USA), 1 x non-essential amino acid (purchased from HyClone, USA), 100 μg/ml streptomycin and 100 U/ Million penicillin (Peniccilin, purchased from HyClone, USA) culture medium (Dulbecco's Modified Eagle Medium; DMEM), the tumor cell lines were placed in an incubator at 37 ° C, 5% carbon dioxide, 95% humidity, every two days Replace the fresh medium once.

When the above-mentioned tumor cell strain is subcultured, the culture solution and the cells can be centrifuged at 1000 rpm for 5 minutes, the supernatant is removed, then the fresh culture solution is added, and the number of cells maintained in the 10 cm culture dish is about 1 × 10 ⁵ to 1 × 10 ⁶ cells/ml.

When the test is carried out, the 10 cm culture dish of the above tumor cell line which has been 8 to 9 minutes old may be taken out, the discolored culture medium is removed, the cells are washed by adding 8 ml of PBS buffer solution, and trypsin/ethylenediamine acetic acid is added. Trypsin/EDTA), after 1 to 3 minutes of reaction, gently shake the culture dish to detach the cells from the wall, add the warmed culture medium, gently disperse the cells, and then place the cell-containing culture solution into the centrifuge tube. After centrifugation at 1500 rpm for 10 minutes, after removing the supernatant, the serum-containing culture solution was added to uniformly disperse the cells, and 20 μl of the tumor cells were taken out, and 20 μl of 0.04% trypan blue (Trypan Blue) was added. Stain, place in a cell counter, and count the number of viable cells under the microscope. The cell viability must be greater than 85% before proceeding. Continued testing.

The concentration of the above tumor cell strain was adjusted to 1×10 ⁵ cell/ml with a serum-containing culture solution, and 100 μl of each of the tumor cell lines was inoculated into a 96-well plate, so that each well contained 1×10 ⁴ cells, and Incubate overnight at 37 ° C in an incubator containing 5% carbon dioxide.

After 24 hours of incubation, the extract of Antrodia camphorata (concentration: 125 μg/ml) as shown in Table 4 was added, mixed uniformly, and cultured overnight at 37 ° C in a 5% carbon dioxide incubator for 24 hours.

After 24 hours, the culture solution was removed, rinsed with phosphate buffer, and then 100 μl of fresh culture medium containing CCK-8 was added, placed at 37 ° C, reacted for 2 hours in a 5% carbon dioxide incubator, and shaken for 5 minutes. After that, the absorbance of each slot at a wavelength of 450 nm is detected.

Please refer to Table 4 for liver tumors (groups B1-1 and B1-2), lung tumors (groups B2-1 and B2-2), and colorectal tumors (groups B3-1 and B3-2). , sarcoma (groups B4-1 and B4-2) or breast tumors (groups B5-1 and B5-2), providing extracts of Antrodia camphorata (Group A1 or A2) can reduce the survival of the tumor cells Rate, and the extract of Antrodia camphorata (Group A1) irradiated by the light source has a better effect of reducing the survival rate of tumor cells (Comparative Groups B1-1 and B1-2, Groups B2-1 and B2-2, Groups B3-1 and B3-2, Groups B4-1 and B4-2 or Groups B5-1 and B5-2).

(C) Evaluation of tumor suppressive efficacy in tumor mice

This trial used Balb/C male mice purchased from the Animal Center of the National Cheng Kung University School of Medicine. The mice were all over 8 weeks old and weighed between 20-25 g. The mice were housed in the SPF Animal Center of the University of Stanley, and maintained in an animal room at room temperature of 25 ± 1 °C for 12 hours each. Moreover, mice were free to eat and drink; feed (purchased from LabDiet, Inc., USA) and drinking water were supplied by the Animal Center of the University of Science and Technology.

The sarcoma cell strain (such as the aforementioned S-180 cell strain, purchased from the Food Industry Development Research Institute) was diluted with physiological saline to a concentration of 5×10 ⁶ cells/ml, and the sarcoma cell strain was placed in the subgingival area of the mouse. Subcutaneous injection.

Next, as shown in the fifth table, the extract of the Antrodia camphorata wine of the preferred embodiment is fed to the mouse via a gastric tube, and is administered twice a day at a fixed time for 30 consecutive days, and is x. Light exposure records the tumor area.

Referring to Table 5, the tumor volume of the mice fed the extract of Antrodia camphorata (Group A1) of the preferred embodiment was reduced by 53.4 compared to the mice not fed the extract of Antrodia camphorata (Group C0). ±6.7% (Group C1), and the tumor volume of the mice fed the Antrodia camphorata extract (Group A2) without the light source was reduced by 31.6±8.7% (Group C2); it can be known that the preferred embodiment is The cultured Aster sinensis has a better effect of inhibiting tumor growth.

In summary, the method for cultivating the Antrodia camphorata of the present invention can produce a large amount of ricinic acid C and ricinoleic acid C, thereby improving the nutritional value of Antrodia camphorata.

Furthermore, the method for cultivating the Antrodia camphorata of the present invention can shorten the cultivation time of Antrodia camphorata, thereby improving the economic value of Antrodia camphorata.

Moreover, the extract of Antrodia camphorata prepared by the extraction method of the extract of Antrodia camphorata wine of the present invention has a high content of ricinic acid C and ricinoleic acid C, which can reduce the survival rate of tumor cells and can be used as a tumor cell growth inhibiting. The active ingredient is the efficacy of the present invention.

In addition, the extract of the Antrodia camphorata wine of the present invention can be used as an active ingredient, and can effectively inhibit liver tumor cells, lung tumor cells, colon cancer cells, sarcoma cells, and breast tumors. The growth of tumor cells, such as cells, has the effect of causing the tumor cells to be poisoned and unable to proliferate or spread.

While the invention has been described in connection with the preferred embodiments described above, it is not intended to limit the scope of the invention. The technical scope of the invention is protected, and therefore the scope of the invention is defined by the scope of the appended claims.

Claims (12)

The invention relates to a method for cultivating anthraquinone C and anchoic acid C content, which comprises: providing a burdock eucalyptus; providing a burdock bacterium, inoculating the burdock bacillus in the burdock eucalyptus, so that the kilogram per kilogram Anthocyanin eucalyptus contains a hyphae and a hyphae ball with a dry weight of 0.162 g; and the bovine eucalyptus eucalyptus inoculated with the boletus edulis cells is irradiated with a light source having a wavelength of 600 to 700 nm to obtain a burdock fruit body; The burdock eucalyptus which is inoculated with the burdock fungus body is irradiated with the light source for 8 to 12 hours per day, and continuously irradiated for 30 to 40 days. The method for cultivating Antrodia camphorata according to Item 1, wherein the Antrodia camphorata system is in a liquid state, and each liter of the Antrodia camphorata contains 8.1 g of dry weight hyphae and hyphae. The method for cultivating Antrodia camphorata according to Item 1, wherein the wavelength of the light source is 660 nm. The method for cultivating Antrodia camphorata according to Item 1, wherein the light source is from a light-emitting diode. The method for cultivating Antrodia camphorata according to the first aspect of the invention, wherein the time period in which the light source is inoculated with the burdock eucalyptus is 8 hours. The method for cultivating Antrodia camphorata according to the first aspect of the invention, wherein the arborvitae eucalyptus inoculated with the burdock bacillus is continuously irradiated with the light source for 30 days. An extraction method of an extract of Antrodia camphorata wine having a high content of ricinic acid C and ricinoleic acid C, comprising: providing an Antrodia camphorata fruit body as described in claim 1; and using 95% ethanol at 25 ° C The burdock fruit body was extracted by ultrasonic vibration to obtain a burdock wine extract. The use of the extract of Antrodia camphorata for inhibiting the growth of liver tumor cells, which is used as an active ingredient for preparing a pharmaceutical composition for treating liver tumors, as an active ingredient of the extract of Antrodia camphorata as described in claim 7. The use of the extract of Antrodia camphorata for inhibiting the growth of lung tumor cells, which is used as an active ingredient for preparing a pharmaceutical composition for treating lung tumors, as an active ingredient of the extract of Antrodia camphorata as described in claim 7. The use of the extract of Antrodia camphorata for inhibiting the growth of colorectal tumors, which is an active ingredient of the extract of Antrodia camphorata as described in claim 7 of the patent application, for the preparation of a pharmaceutical composition for treating colorectal tumors. An extract of Antrodia camphorata for inhibiting the growth of sarcoma, which is used as an active ingredient for preparing a pharmaceutical composition for treating sarcoma as an active ingredient as described in claim 7 of the patent application. The use of an extract of Antrodia camphorata for inhibiting the growth of a breast tumor, which is used as an active ingredient for preparing a pharmaceutical composition for treating a breast tumor, as an active ingredient of the Antrodia camphorata wine described in claim 7 of the patent application.