TW202003004A - Use of Antrodia cinnamomea mycelia active substance for ameliorating chronic obstructive pulmonary disease - Google Patents

Abstract

A use ofAntrodia cinnamomea mycelia active substance is provided. TheAntrodia cinnamomea mycelia active substance is for the treatment of chronic obstructive pulmonary disease, and is prepared by the following steps: (a)culturing aAntrodia cinnamomea mycelium in a plate media at 15 to 30℃ for 4 to 10 days; (b)inoculating the mycelium of step(a) to a flask and culturing it at 15 to 30℃ with a pH of 2 to 6 for 3 to 14 days; (c)inoculating the mycelium of step(b) to a fermenter tank and culturing it at 15 to 30℃ with a pH of 2 to 6 for 3 to 21 days, so as to obtain aAntrodia cinnamomea mycelium fermentation liquid containing saidAntrodia cinnamomea mycelium active substance.

Description

Use of Antrodia camphorata mycelium active substance for improving chronic obstructive pulmonary disease

The invention relates to a preparation method and application of active substances of Antrodia camphorata mycelium. Specifically, it relates to the preparation of an active substance of Antrodia camphorata mycelia for improving chronic obstructive pulmonary disease, and its use in food or medicine.

Chronic Obstructive Pulmonary Disease (COPD) is currently the fourth leading cause of death in the world. It is a common, frequent, high disability rate and high mortality rate of chronic respiratory diseases. There are more than 300 million people with chronic obstructive pulmonary disease worldwide.

Smoking is the main cause of COPD. However, in recent years, due to the increased emissions of locomotives and automobiles in China and the increased use of power generation and fuel coal in industry, the air pollution of Particulate Matter (PM) 2.5 has increased, and this disease is more important. . Long-term exposure to a polluted environment can cause an inflammatory reaction in the lungs, resulting in narrowing of airflow channels and resulting in lung tissue dysfunction.

COPD treatment methods currently include smoking cessation, lung rehabilitation, inhaled bronchodilators, corticosteroid use, long-term oxygen supply, or lung transplantation. The newly approved new drug series in 2015 merged the two muscle relaxants used in COPD in the past, and so far there has been no breakthrough new drug development. Therefore, research on effective treatment of chronic obstructive pulmonary disease is still an important development direction.

Antrodia cinnamomea ( Antrodia cinnamomea ), also known as camphor, camphor, camphor mushroom, cinnamomum , cinnamomea , red camphor, red camphor, only grows in the decayed trunk of Antrodia camphorata at an altitude of 450-1500 meters in the mountainous area of Taiwan The inner wall of the heartwood or the dark and damp surface of the withered and fallen camphor wood belongs to the precious medicinal fungi of Taiwan national treasure level. At present, there is still a lack of research on Zhangzhi to improve chronic obstructive pulmonary disease.

The invention provides an active substance of Antrodia camphorata mycelium and a preparation method thereof, which can be used for preparing a composition with improved chronic obstructive pulmonary disease. Compared with the general western medicine and the treatment method, the preparation method of the liquid fermented Antrodia camphorata mycelium active substance disclosed by the present invention is safer and simpler, and the prepared Antrodia camphorata mycelium active substance is more natural and safe, and can be effectively improved COPD.

According to an embodiment of the present invention, there is provided a use of an active substance of Antrodia camphorata mycelium for preparing a composition for improving chronic obstructive pulmonary disease, wherein the active substance of Antrodia camphorata mycelium is prepared by the following steps:

(a) Take Antrodia cinnamomea on a plate medium and incubate at a temperature of 15-30℃ for 4-10 days;

(b) Inoculate the Antrodia camphorata mycelium cultured in step (a) into the flask, and cultivate it for 3-14 days in an environment of 15-30°C and pH 2-6;

(c) Inoculate the mycelium of Antrodia camphorata after cultivation in step (b) in a fermentation tank, and stir and cultivate for 3-21 days at an environment of 15-30°C and pH 2-6 to form an activity containing Antrodia camphorata mycelia Substance of Antrodia camphorata mycelium fermentation broth.

In one embodiment, the step of preparing the active substance of Antrodia camphorata mycelia further includes step (d): freeze-drying the fermentation broth of Antrodia camphorata mycelia and grinding it to form antrodia camphorata mycelium containing the active substance of Antrodia camphorata mycelia Body lyophilized powder.

In one embodiment, the step of preparing the active substance of Antrodia camphorata mycelium further includes step (e): extracting the freeze-dried powder of Antrodia camphorata mycelium with at least one solvent to form Antrodia camphorata containing the active substance of Antrodia camphorata mycelium Silk extract.

In one embodiment, the step of preparing the active substance of Antrodia camphorata mycelia further includes step (f): drying the extract of the mycelium of Antrodia camphorata to obtain the active substance of the mycelium of Antrodia camphorata.

In one embodiment, the solvent in step (e) is selected from water or alcohols.

In one embodiment, when the solvent in step (e) is water, the effective concentration of the active substance of Antrodia camphorata mycelium is between 0.25-1 mg/ml.

In one embodiment, when the solvent in step (e) is an alcohol, the effective concentration of the active substance of Antrodia camphorata mycelium is between 25-100 μg/ml.

In one embodiment, the flask culture in the above step (b) is shaking culture, and the rotation speed is 110-130 rpm.

In one embodiment, the fermentation tank in step (c) is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium, or a combination thereof, the fermentation tank has a tank pressure of 0.5-1.0 kg/cm ² and aeration rate It is 0.01-1.5VVM.

In one embodiment, the composition is a pharmaceutical composition, and the pharmaceutical composition further includes a pharmaceutically acceptable carrier, excipient, diluent or adjuvant.

In one embodiment, the composition is a food additive.

In order to make the above and other aspects of the present invention clearer and more understandable, the embodiments are specifically described below in conjunction with the accompanying drawings.

Example 1 　 Antrodia camphorata mycelium culture

The Antrodia cinnamomea used in this example was purchased from the Biological Resource Preservation and Research Center (BCRC, formerly CCRC) of the Food Industry Development Institute (FIRDI) of the consortium, and the number is CCRC-35396. However, the Antrodia camphorata varieties that can be used in the present invention are not limited to this single one, but can also include BCRC numbers 35396, 35398, 35716, 36401, 36711, 36795, 37848, 37849, 37850, 37889, 37890, 37891, 37893, The species of Antrodia camphorata strains of 37894, 37941 or other biological material storage databases are not limited by the present invention.

(1) Plate culture: Antrodia camphorata mycelium is inoculated on the plate and cultivated at 15-30°C for 4-10 days (in this example, it is cultivated at 25°C for 7 days). The components of the plate medium may include potato dextrin medium (Potato Dextrose Agar, PDA), carbon source and nitrogen source, and are not particularly limited.

(2) Flask culture: scrape (1) inoculate the mycelium on the plate into the flask, and culture with shaking at 15-30°C, pH 2-6 and rotation speed 110-130 rpm for 3-14 days (this implementation In the example, it was cultured with shaking at 25°C, pH 5, and rotation speed of 120 rpm for 10 days). This shaking culture was carried out with the medium shown in Table 1 below.

Table 1 Medium formula

In the above medium formula, soybean powder and cinnamon powder are comprehensive carbon and nitrogen sources, which can be selected from cereals (such as wheat flour) or beans (such as soybean powder, mung bean powder, soybean powder, cinnamon powder, etc.); sugars can be It is glucose, fructose, maltose, sucrose, etc.; inorganic salts can be magnesium sulfate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, ferric sulfate, etc. In particular, the formula of the medium in Table 1 is just one example, and the ingredients can be adjusted according to needs or used with commercially available medium without special restrictions.

(3) Fermentation tank culture: inoculate the mycelium cultured in the flask in (2) into the fermentation tank at 15-30 ℃, tank pressure 0.5-1.0 kg/cm ² , pH 2-6 and stirring speed 50-150 Under the condition of rpm, incubate at aeration rate of 0.1-1.5 VVM for 3-21 days to form a fermentation liquid of Antrodia camphorata mycelium (in this example, it was stirred at 25°C, tank pressure 0.5 kg/cm ² , pH 4 and stirred Under the conditions of speed 80 rpm and 1.0 VVM (air), culture for 14 days), the fermentation liquid of Antrodia camphorata mycelium is obtained. The culture medium used for fermentation tank culture may be the same as the culture medium used for flask culture in step (2). The Antrodia camphorata mycelium fermentation liquid contains the active substance of Antrodia camphorata mycelium according to the present invention. The fermentation liquid of Antrodia camphorata mycelium can be further prepared as a freeze-dried powder of the fermentation liquid of Antrodia camphorata mycelium by a freeze-drying step;

(4) Preparation of extract: The lyophilized powder of the fermentation solution of Antrodia camphorata mycelium is divided into two parts, and 20 times the weight of distilled water and ethanol are added to extract. The extract using water as a solvent is heated at a temperature of 100°C for thirty minutes. After cooling, the supernatant is taken by centrifugation and dried by freeze-drying to obtain an aqueous extract of fermented mycelia of Antrodia camphorata. The ethanol-extracted extract was shaken with ultrasound for 1 hour. After centrifugation, the supernatant was taken, and the supernatant was concentrated under reduced pressure to obtain an alcohol extract of Antrodia camphorata fermentation mycelium;

Through the extraction step, water extracts and alcohol extracts containing higher concentrations of active substances of Antrodia camphorata mycelium can be obtained. The active substances of Antrodia camphorata mycelia include the fermentation liquid of mycelium of Antrodia camphorata (mycelium and clarified liquid), lyophilized powder of fermentation broth, water extract, alcohol extract, water extract and mixture of alcohol extract Or other dosage forms. In the second embodiment below, the water extract and the alcohol extract are used as the active substance of Antrodia camphorata mycelium. Example 2 Analysis of water extract and alcohol extract of Antrodia camphorata mycelia to improve COPD

There have been experiments using Cigarette smoke extract (CSE) and human lung cancer epithelial cells A549 to successfully establish a COPD model. Refer to Miaomiao Chen et al. (2015). Azithromycin attenuates cigarette smoke extract-induced oxidative stress injury in human alveolar epithelial cells. MOLECULAR MEDICINE REPORTS , 11: 3414-3422. This paper uses human lung cancer epithelial cells A549 to CSE MTT assay was performed to evaluate the improvement of COPD by antibiotic Azithromycin. CSE damage to the alveolar epithelium is an important process in the mechanism of chronic obstructive pulmonary disease. Therefore, if it can suppress the damage of CSE to alveolar epithelium, it can achieve the effect of improving chronic obstructive pulmonary disease. In this experiment, the human lung cancer epithelial cell A549 was also used to perform cell survival analysis (MTT assay) on CSE to evaluate the improvement of COPD by the active substances of Antrodia camphorata mycelium.

(1) Collect human lung cancer epithelial A549 cells, adjust the concentration of the cell suspension, and plant them in 96-well dishes at a density of 50,000-100,000/mL;

(2) Place the 96-well plate of (1) in a 37°C, 5% CO ₂ incubator for 16-24 hours, and obtain the water extract of Antrodia camphorata mycelium with different doses obtained in Example 1 (0, 0.25, 0.5, 1 mg/ml) and alcohol extracts (0, 25, 50, 100 μg/ml). Antrodia camphorata mycelium extract is dissolved in Dimethyl sulfoxide (DMSO), the concentration of DMSO does not exceed 0.1% when tested to avoid its toxicity affecting cell growth; the control group is treated with 0.1% DMSO, each concentration Three replicates were performed and the results were placed in a 37°C, 5% CO ₂ incubator for 24 hours;

(3) The result of (2) above was first centrifuged at 400 × g for 10 min, the supernatant was removed, and then the cigarette smoke extract CSE 12.5% was added to induce the cytotoxicity of A549, the control group was not treated. Then put it in 37℃, 5% CO ₂ incubator for 6 hours;

(4) Remove the supernatant from (3), add 100 μl MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution to each well, and react at 37℃ for 2 hours After that, stop the culture and carefully remove the MTT solution in the well;

(5) Add 100 μl DMSO to each well in (4), shake for 2 min, and measure the absorbance at 570 nm. This absorbance represents the ability of cells to reduce MTT (formation of formazan), that is, the activity of mitochondria (number of viable cells). The higher the absorbance at 570 nm, the higher the cell survival rate.

Statistical analysis: The experimental result data is expressed as mean (Mean) ± standard deviation (SD). The test data was analyzed by Student's t-test statistics to analyze the differences between groups. If p <0.05, it is regarded as having statistically significant difference.

The experimental results of the water extract are shown in Figure 1 and Table 2 below. Cigarette smoke extract CSE can induce A549 cytotoxicity, resulting in decreased cell survival rate. However, the addition of water extracts of fermented mycelia of Antrodia camphorata can significantly inhibit cell damage and improve survival rate, and the higher the concentration added, the higher the survival rate. Using the water extract of Antrodia camphorata mycelium in Example 1 of the present invention has the effect of improving COPD, and the effective concentration is between 0.5-1 mg/ml.

(n = 3) #表示與對照組(control)統計上具顯著差異(p＜0.05) *表示與負對照組(CSE)統計上具顯著差異(p＜0.05)Table 2 Test results of water extract of Antrodia camphorata mycelium

(n = 3) # indicates statistically significant difference from the control group (control) (p<0.05) * indicates statistically significant difference from the negative control group (CSE) (p<0.05)

The experimental results of alcohol extracts are shown in Figure 2 and Table 3 below. The results also show that the addition of the Antrodia camphorata mycelium alcohol extract in the first embodiment of the present invention can improve the COPD, and the effective concentration is between 50-100 μg/ml.

(n = 3) #表示與對照組(control)統計上具顯著差異(p＜0.05) *表示與負對照組(CSE)統計上具顯著差異(p＜0.05)Table 3 Test results of alcohol extract of Antrodia camphorata mycelium

(n = 3) # indicates statistically significant difference from the control group (control) (p<0.05) * indicates statistically significant difference from the negative control group (CSE) (p<0.05)

The active substance of Antrodia camphorata according to the present invention has been confirmed to have the effect of improving COPD through the above experiments. It can be combined with pharmaceutically acceptable carriers, excipients, diluents or adjuvants to prepare pharmaceutical compositions and can also be used as food additives.

Although the present invention has been disclosed as above using embodiments, it is not intended to limit the present invention. Those of ordinary knowledge in the art, after referring to the above teachings, should be able to make appropriate modifications to the content of the above-mentioned embodiments and still achieve the effect claimed in this case. Therefore, the scope of protection of the present invention shall be subject to the appended amendment to the scope of the patent application.

Figure 1 shows the results of the water extract of Antrodia camphorata mycelium improving the ability of cigarette smoke extract to induce the injury of alveolar epithelial A549 cells, where AcH(L), AcH(M) and AcH(H) refer to Antrodia camphorata respectively The concentration of silk extracts was 0.25 mg/ml, 0.5 mg/ml and 1.0 mg/ml.

Figure 2 shows the results of the mycelium alcohol extract of Antrodia camphorata improving the ability of cigarette smoke extract to induce damage to alveolar epithelial A549 cells, where AcMe(L), AcMe(M) and AcMe(H) refer to Antrodia camphorata respectively Serum alcohol extract concentration was 25 μg/ml, 50 μg/ml and 100 μg/ml.

Claims (11)

An active substance of Antrodia camphorata mycelium is used for preparing a composition for improving chronic obstructive pulmonary disease, wherein the active substance of Antrodia camphorata mycelium is prepared by the following steps: (a) Take an Antrodia camphorata mycelium ( Antrodia cinnamomea ) cultured on a plate medium at a temperature of 15-30℃ for 4-10 days; (b) Inoculate the mycelium of Antrodia camphorata after cultivation in step (a) into a flask at 15-30℃, Cultivation in pH 2-6 environment for 3-14 days; (c) Inoculate the Antrodia camphorata mycelium cultured in step (b) in a fermentation tank, and stir at 15-30℃, pH 2-6 Within 3-21 days, a fermentation liquid of Antrodia camphorata mycelia containing one of the active substances of the Antrodia camphorata mycelia was formed. The use as described in item 1 of the scope of the patent application, wherein the step of preparing the active substance of the mycelium of Antrodia camphorata further includes step (d): freeze-drying the fermentation liquid of the mycelium of Antrodia camphorata to form a powder containing the camphor Antrodia camphorata mycelium freeze-dried powder, one of the active substances of Zhi mycelium. The use as described in item 2 of the patent application scope, wherein the step of preparing the active substance of the mycelium of Antrodia camphorata further includes step (e): extracting the freeze-dried powder of the mycelium of Antrodia camphorata with at least one solvent to form At least one extract of Antrodia camphorata mycelium active substance. The use as described in Item 3 of the scope of the patent application, wherein the step of preparing the active substance of the Antrodia camphorata mycelium further includes step (f): drying the extract of the Antrodia camphorata mycelium to obtain the mycelium of Antrodia camphorata Active substance. The use as described in item 3 or 4 of the patent application scope, wherein in step (e), the solvent is selected from water or alcohols. The use as described in item 5 of the patent application scope, wherein when the solvent in step (e) is water, the effective concentration of the active substance of the mycelia of Antrodia camphorata is between 0.5-1 mg/ml. The use as described in item 5 of the patent application scope, wherein when the solvent in step (e) is an alcohol, the effective concentration of the active substance of the mycelia of Antrodia camphorata is between 50-100 μg/ml. The use as described in item 1 of the patent application scope, wherein the flask culture in step (b) is shaking culture and the rotation speed is 110-130 rpm. The use as described in item 1 of the patent application scope, wherein in step (c) the fermentation tank is further fed with a gas, the gas includes air, oxygen, carbon dioxide, helium or a combination thereof, and the fermentation tank has a tank pressure of 0.5 -1.0 kg/cm ² and the ventilation rate is 0.01-1.5 VVM. The use as described in item 1 of the patent application scope, wherein the composition is a pharmaceutical composition, and the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, excipient, diluent or adjuvant. The use as described in item 1 of the patent application scope, wherein the composition is a food additive.

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