TWI527586B - Use of an antrodia cinnamomea (antrodia camphorata or taiwanofungus camphoratus) extract for preparing a drug for modulating th17 cells - Google Patents

Description

Use of extract of Antrodia camphorata for the preparation of drugs for regulating Th17 cells way

The present invention relates to the use of an extract of Antrodia camphorata, and in particular to the use of an extract of Antrodia camphorata for the preparation of a medicament for regulating Th17 cells.

Th 17 cells are a group of unique CD4+ T cells that were originally thought to produce IL-17 and IL-17F T cells (Harrington et al., 2005; Park et al., 2005; Langrish et al., 2005). Unlike Th1 and Th2 cells, the development of Th17 cells is regulated by TGF-β and other inflammatory cytokines such as IL-6, IL-21, IL-1β and IL-23 (Bi and Yang, 2012). In addition to IL-17A and IL-17F, Th17 cells also secrete other indicator cytokines such as IL-21, IL-22, IFN-γ, IL-4, IL-10, IL-9, IL-26, CXCL8 and CCL20, etc. (Marwaha et al., 2012), thus acting on other cells such as fibroblasts, keratinocytes, endothelial cells, neutrophils, and memory T cells (Gaffen, 2011; Ghoreschi et al., 2011; Wilke et al., 2011a). It has been found that Th17 cells and their action molecules such as IL-17, IL-21, IL-22, GM-CSF and CCL20 are involved in the pathogenesis of autoimmune diseases and inflammatory diseases, such as: systemic Lupus erythematosus, dryness, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, allergies, asthma, etc. (Maddur et al., 2012).

Antrodia chinensis (known as Antrodia cinnamomea, Antrodia camphorata or Taiwanofungus camphoratus ) is a traditional Chinese herbal medicine, a unique fungus in Taiwan, which is only parasitic on the solid wood or moist surface of Cinnamomum kanehirae Hayata (Lauraceae) (Ao et al) .2009; Lu et al. 2009). Nowadays, Antrodia camphorata has been used for the treatment of hepatitis, cancer, abdominal pain, diarrhea, hypertension or liver protection, detoxification, etc. However, it is still unclear whether or not Antrodia camphorata has the function of regulating Th17 cells.

The invention provides a pharmaceutical composition for regulating Th17 cells, comprising: an extract of Antrodia camphorata as an active ingredient, wherein the extract of Antrodia camphorata has the function of regulating Th17 cells.

The present invention also provides a use of an extract of Antrodia camphorata for preparing a medicament for regulating Th17 cells, wherein the extract of Antrodia camphorata has the effect of regulating Th17 cells.

The present invention further provides a use of an extract of Antrodia camphorata for the preparation of a medicament for treating a disease associated with the regulation of Th17 cells, wherein the extract of Antrodia camphorata has an effect of inhibiting the differentiation of Th17 cells.

Figure 1 shows the effect of Antrodia camphorata extract ACFB03 on the differentiation of Th17 cells, **: p <0.01, ***: p <0.001.

Figure 2 shows the effect of Antrodia camphorata extract ACFB06 on the differentiation of Th17 cells, *: p < 0.05, **: p < 0.01.

Figure 3 shows the effect of Antrodia camphorata extract ACFB12 on the differentiation of Th17 cells, *: p < 0.05.

Figure 4 shows the effect of Antrodia camphorata extract AC06-EA on the differentiation of Th17 cells, *: p < 0.05.

Figure 5 shows the effect of Antrodia camphorata extract AC06-A on the differentiation of Th17 cells, **: p <0.01.

Figure 6 shows the effect of Antrodia camphorata extract ACFB12 on Th17 cell activity in patients with systemic lupus erythematosus, ***: p < 0.001.

Figure 7 shows the effect of Antrodia camphorata extract ACFB12 on Th17 cell activity in Graves patients, *: p < 0.05.

Figure 8 shows the effect of Antrodia camphorata extract ACFB12 on the activity of Th17 cells in the imiquimod-induced dry animal model, ***: p < 0.001.

In one embodiment of the invention, the invention provides a pharmaceutical composition for modulating Th17 cells. The pharmaceutical composition for regulating Th17 cells of the present invention may include an extract of Antrodia camphorata as an active ingredient, but is not limited thereto.

In the pharmaceutical composition for regulating Th17 cells of the present invention, the extract of Antrodia camphorata may have an effect of regulating Th17 cells. The above-described effects of regulating Th17 cells may include, for example, inhibiting or promoting Th17 cell differentiation, proliferation, and the like, but are not limited thereto. In one embodiment, the above-described efficacy of modulating Th17 cells comprises inhibiting Th17 cell differentiation.

Further, in the pharmaceutical composition for regulating Th17 cells of the present invention, the extract of Antrodia camphorata may include an extract of an extract of Antrodia camphorata or a cow An extract of the mycelium of Antrodia camphorata.

The extract of Antrodia camphorata can be obtained by performing an extraction process on the fruit body or mycelium of Antrodia camphorata, but is not limited thereto. The extraction process described above may use at least one extraction solvent.

In one embodiment, the extract of Antrodia camphorata is an extract of a fruit body of Antrodia camphorata. In this embodiment, the extract of the body complex of the Antrodia camphorata can be obtained by performing an extraction process on the body of the Antrodia camphorata, but is not limited thereto. The extraction process described above may use at least one extraction solvent.

In this embodiment, the at least one extraction solvent may include, but is not limited to, an alcohol and/or water. Examples of the above alcohols may include methanol, ethanol, isopropanol, butanol, and the like, but are not limited thereto. In one embodiment, the above alcohol is ethanol, and the above ethanol may include 10-95% aqueous ethanol solution, but is not limited thereto. Further, in a particular embodiment, the above ethanol may be a 25%, 50% or 95% aqueous solution of ethanol.

Furthermore, the above extraction process for obtaining an extract of the body complex of Antrodia camphorata may include the following steps, but is not limited thereto.

First, the Astragalus lucidum fruiting body is heated to reflux with at least one of the above extraction solvents to form a mixture. In one embodiment, the time for heating to reflux may be from about 0.5 to about 8 hours. In a particular embodiment, the time to heat reflux can be about one hour.

Then, the above mixture is filtered to obtain a filtrate and a filter residue, and the obtained filtrate is obtained from an extract of Antrodia camphorata fruit body.

In another embodiment, the extract of Antrodia camphorata is an extract of Mycelium of Antrodia camphorata. In this embodiment, the extract of the mycelium of Antrodia camphorata can be borrowed It is obtained by performing an extraction process on the mycelium of Antrodia camphorata, but is not limited thereto. The extraction process described above may use at least one extraction solvent.

In this embodiment, the at least one extraction solvent may include, but is not limited to, ethyl acetate or acetone, etc., but is not limited thereto. In one embodiment, at least one extraction solvent is ethyl acetate, and in another embodiment, at least one extraction solvent is acetone.

Further, the above extraction process for obtaining an extract of Antrodia camphorata mycelium may include the following steps, but is not limited thereto.

First, the mycelium of Antrodia camphorata is heated and refluxed with at least one of the above extraction solvents to form a mixture. In one embodiment, the time for heating to reflux may be from about 0.5 to about 8 hours. In a particular embodiment, the time to heat reflux can be about one hour.

Then, the above mixture is filtered to obtain a filtrate and a filter residue, and the obtained filtrate is obtained from the extract of A. serrata.

Further, in one embodiment, the above pharmaceutical composition for regulating Th17 cells of the present invention may further comprise a pharmaceutically acceptable carrier or salt.

In the pharmaceutical composition for regulating Th17 cells of the present invention, the above pharmaceutically acceptable carrier may include, but is not limited to, a solvent, a dispersion medium, a coating, an antibacterial and an antifungal agent, and the like. The osmotic pressure and absorption delaying reagents and the like are compatible with pharmaceutical administration. For different modes of administration, the pharmaceutical compositions can be formulated into a dosage form using conventional methods.

Further, the above pharmaceutically acceptable salts may include, but are not limited to, salts including inorganic cations such as alkali metal salts such as sodium, potassium or amine salts. Alkaline earth metal salts, such as magnesium, calcium salts, salts containing divalent or tetravalent cations, such as zinc, aluminum or zirconium salts. In addition, it may also be an organic salt such as dicyclohexylamine salt, methyl-D-glucosamine, an amine salt such as arginine, lysine, histidine, glutamine. .

The pharmaceutical administration prepared by the present invention can be administered orally, parenterally, via inhalation spray or by implantation of an implanted reservoir. Non-oral may include subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal (intrasternal), intrathecal (intrathecal), intralesional injection and perfusion techniques.

Forms of oral ingredients can include, but are not limited to, tablets, capsules, emulsions, aqueous suspensions, dispersions, and solutions.

Furthermore, in one embodiment, the pharmaceutical composition for regulating Th17 cells of the present invention can be used for the treatment of diseases associated with Th17 cell regulation, but is not limited thereto. Examples of the above diseases associated with Th17 cell regulation may include, but are not limited to, systemic lupus erythematosus, griffith disease, cognac, rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis, multiple sclerosis Disease, Parkinson's disease, inflammatory bowel disease, allergic disease, acquired immunodeficiency disease, chronic obstructive pulmonary disease, atherosclerosis, myocarditis, dilated cardiomyopathy and cancer.

In another embodiment, the pharmaceutical composition of the present invention for modulating Th17 cells can be used for the treatment of systemic lupus erythematosus, griffith disease or cognac Treatment. The above systemic lupus erythematosus may include active systemic lupus erythematosus, but is not limited thereto.

In another embodiment of the present invention, the present invention provides a use of an extract of Antrodia camphorata for preparing a medicament for regulating Th17 cells, wherein the extract of Antrodia camphorata has the function of regulating Th17 cells. The above-described effects of regulating Th17 cells may include, but are not limited to, inhibiting or promoting Th17 cell differentiation, proliferation, and the like. In one embodiment, the above-described efficacy of modulating Th17 cells comprises inhibiting Th17 cell differentiation.

In the use of the extract of Antrodia camphorata for the preparation of a medicament for regulating Th17 cells, the extract of Antrodia camphorata may comprise an extract of an extract of Antrodia camphorata or an extract of Mycelium of Antrodia camphorata.

The extract of Antrodia camphorata can be obtained by performing an extraction process on the fruit body or mycelium of Antrodia camphorata, but is not limited thereto. The extraction process described above may use at least one extraction solvent.

In one embodiment, the extract of Antrodia camphorata is an extract of a fruit body of Antrodia camphorata. In this embodiment, the extract of the body complex of the Antrodia camphorata can be obtained by performing an extraction process on the body of the Antrodia camphorata, but is not limited thereto. The extraction process described above may use at least one extraction solvent.

The at least one extraction solvent may include, but is not limited to, an alcohol and/or water. Suitable alcohols may include, for example, methanol, ethanol, isopropanol, butanol, and the like, but are not limited thereto. In one embodiment, the alcohol is ethanol, and the ethanol may include 10-95% aqueous ethanol solution, but is not limited thereto. Further, in a particular embodiment, the above ethanol may be a 25%, 50% or 95% aqueous solution of ethanol.

The above extraction process for obtaining the extract of the body complex of Antrodia camphorata The following steps may be included, but are not limited thereto.

First, the Astragalus lucidum fruiting body is heated to reflux with at least one of the above extraction solvents to form a mixture. In one embodiment, the time for heating to reflux may be from about 0.5 to about 8 hours. In a particular embodiment, the time to heat reflux can be about one hour.

Next, the above mixture is filtered to obtain a filtrate and a filter residue, wherein the obtained filtrate is an extract of Antrodia camphorata fruit body.

In another embodiment, the extract of Antrodia camphorata is an extract of Mycelium of Antrodia camphorata. In this embodiment, the extract of the mycelium of Antrodia camphorata can be obtained by performing an extraction process on the mycelium of Antrodia camphorata, but is not limited thereto. The extraction process described above may use at least one extraction solvent.

In this embodiment, the at least one extraction solvent may include, but is not limited to, ethyl acetate or acetone, etc., but is not limited thereto. In one embodiment, at least one extraction solvent is ethyl acetate, and in another embodiment, at least one extraction solvent is acetone.

Further, the above extraction process for obtaining an extract of Antrodia camphorata mycelium may include the following steps, but is not limited thereto.

First, the mycelium of Antrodia camphorata is heated and refluxed with at least one of the above extraction solvents to form a mixture. In one embodiment, the time for heating to reflux may be from about 0.5 to about 8 hours. In a particular embodiment, the time to heat reflux can be about one hour.

Then, the above mixture is filtered to obtain a filtrate and a filter residue, and the obtained filtrate is obtained from the extract of A. serrata.

Also, in one embodiment, the extract of Antrodia camphorata is used in the present invention. In the use of the drug for regulating Th17 cells, the above-described drug for regulating Th17 cells can be used for the treatment of diseases associated with the regulation of Th17 cells, but is not limited thereto.

Diseases associated with Th17 cell regulation may include systemic lupus erythematosus, griffith disease, cognac, rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis, multiple sclerosis, Parkinson's disease, inflammatory Enteric disease, allergic disease, acquired immunodeficiency disease, chronic obstructive pulmonary disease, atherosclerosis, myocarditis, dilated cardiomyopathy or cancer, etc., but are not limited thereto.

Further, in the use of the extract of Antrodia camphorata for the preparation of a medicament for regulating Th17 cells, the above-described drug for regulating Th17 cells can be used for the treatment of systemic lupus erythematosus, Graves' disease or cognac, but is not limited thereto. The above systemic lupus erythematosus may include, for example, active systemic lupus erythematosus.

In still another embodiment of the present invention, the present invention provides a use of an extract of Antrodia camphorata for the preparation of a medicament for treating a disease associated with the regulation of Th17 cells, wherein the extract of Antrodia camphorata has an effect of inhibiting the differentiation of Th17 cells. In one embodiment, the Antrodia camphorata extract is the aforementioned Antrodia camphorata extract.

Examples of diseases associated with Th17 cell regulation may include, limited to systemic lupus erythematosus, griffith disease, cognac, rheumatoid arthritis, type 1 diabetes, Hashimoto's thyroiditis, multiple sclerosis, Parkinson's disease Disease, inflammatory bowel disease, allergic disease, acquired immunodeficiency disease, chronic obstructive pulmonary disease, atherosclerosis, myocarditis, dilated cardiomyopathy and cancer.

In one embodiment, the above-described disease associated with Th17 cell regulation is systemic lupus erythematosus, griffith disease or cognac. Further, the above systemic lupus erythematosus may include, for example, active systemic lupus erythematosus.

Example Example 1: Preparation of Antrodia camphorata extract

1. Preparation of Astragalus lucidum fruiting body extract ACFB03

The preparation method of the ACFB03 sample is described as follows:

(1) 10 g of the body mass of Antrodia camphorata was added to 100 ml of a 95% aqueous solution of ethanol to form a mixture, and then the mixture was heated under reflux for 1 hour.

(2) The mixture was centrifuged to remove impurities (6000 rpm; 20 minutes) to collect the supernatant, followed by concentration under reduced pressure to remove the solvent in the supernatant to obtain a dried extract.

(3) 20 ml of pure water was added and vortexed by ultrasonic waves to dissolve the above extract and form a suspension.

(4) The suspension is freeze-dried to remove moisture.

(5) A total of 1.67 g of the freeze-dried sample was collected. Code name ACFB03.

2. Preparation of Astragalus lucidum fruiting body extract ACFB06

The preparation method of the ACFB06 sample is described as follows:

(1) 10 g of A. sylvestris fruit body powder was added to 100 ml of a 25% aqueous solution of ethanol to form a mixture, and then the mixture was heated under reflux for 1 hour.

(2) The mixture was centrifuged to remove impurities (6000 rpm; 20 minutes) to collect the supernatant, followed by concentration under reduced pressure to remove the solvent in the supernatant to obtain a dried extract.

(3) 20 ml of pure water was added and vortexed by ultrasonic waves to dissolve the above extract and form a suspension.

(4) The suspension is freeze-dried to remove moisture.

(5) A total of 0.59 g of the freeze-dried sample was collected. Code name ACFB06.

3. Preparation of Astragalus lucidum fruiting body extract ACFB12

The preparation method of the ACFB12 sample is described as follows:

(1) 80 g of A. sylvestris fruit body powder was added to 800 ml of a 50% aqueous solution of ethanol to form a mixture, and then the mixture was heated under reflux for 1 hour.

(2) The mixture was filtered under reduced pressure with a filter paper (Toyo, N0.2) to collect the filtrate and obtain a residue of the body of the Antrodia camphorata (resist residue). The extract of the body complex of Antrodia camphorata was drained by continuous vacuum filtration for 30 minutes.

(3) The above-mentioned residue of the body complex of Antrodia camphorata was recovered, and 1600 ml of pure water was added thereto to form a mixture, and then the mixture was heated under reflux for 1 hour.

(4) The mixture was filtered under reduced pressure using a filter paper (Toyo, N0.2), and the filtrate was collected.

(5) The filtrate collected twice is concentrated under reduced pressure, and the filtrate of the step 2 is concentrated first, and the filtrate obtained in the step 4 is further concentrated under reduced pressure during the step of concentrating the filtrate of the step 2, concentrated to a suitable concentration, and then freeze-dried. To remove moisture.

(6) A total of 20.34 g of the freeze-dried sample was collected. Code name ACFB12.

4. Preparation of Antrodia camphorata mycelium extract AC06-EA (ethyl acetate extract)

The preparation method of the AC06-EA sample is described as follows:

(1) 2 g of the mycelium of Antrodia camphorata was added to 30 ml of ethyl acetate to form a mixture, and then the mixture was heated under reflux for 1 hour.

(2) The mixture was centrifuged to remove impurities (6000 rpm; 10 minutes), and then filtered through a filter paper (Toyo, N0.2) to collect the filtrate, followed by concentration under reduced pressure to remove the solvent in the filtrate to obtain an extract. .

(3) A total of 0.32 g of the extract sample was collected. Code AC06-EA.

5. Preparation of Astragalus membranaceus mycelium extract AC06-A (acetone extract)

The preparation method of AC06-A sample is described as follows:

(1) 2 g of A. angustifolia mycelium was added to 30 ml of acetone to form a mixture, and then the mixture was heated under reflux for 1 hour.

(2) The mixture was centrifuged to remove impurities (6000 rpm; 10 minutes), and then filtered through a filter paper (Toyo, N0.2) to collect the filtrate, followed by concentration under reduced pressure to remove the solvent in the filtrate to obtain an extract. .

(3) A total of 0.30 g of the extract sample was collected. Code AC06-A.

Example 2: Evaluation of the ability of extract of Antrodia camphorata fruit body to inhibit the differentiation of Th17 cells

In the present example, the extract of the extract of A. angustifolia (ACFB12, ACFB03, ACFB06) obtained from Example 1 and the mycelium extract of Antrodia camphorata (AC06-EA and AC06-) were evaluated by the Th17 cell differentiation system constructed by mouse spleen cells. A) Effect on Th17 cell differentiation.

1. Evaluation of the ability of ACFB03 extract from Antrodia camphorata fruit body to inhibit the differentiation of Th17 cells

The spleens of C57BL/6 mice (6-8 weeks old) were taken out and ground using a sieve, and the cell suspension was collected as primary spleen cells. CD4+ T cells were then isolated using IMag magnetic system (BD Pharmingen, San Jose, CA), and the cells were added to a 24-well cell culture dish at a density of ⁵ × ^{10 5} cells/well, and the cells were divided into control group and negative control group. , experimental group A, experimental group B, experimental group C five groups, and the detailed treatment of each group is shown in Table 1.

See Table 1. The control group was CD4+ T cells without any treatment reagent; the negative control group was Th17 differentiation group, and treated with cytokines and neutralizing antibodies to promote differentiation of CD4+ T cells into Th17 cells; experimental group A, experimental group B In the experimental group C, different concentrations of the extract of Antrodia camphorata fruit body (ACFB03) were added simultaneously to observe the effect on the differentiation of Th17 cells. The above cells were cultured at 37 ° C in a 5% CO ₂ incubator. After three days of culture, 10 ul of PMA (0.05 mg/ml) (Sigma) and 1 ul of brefeldin A (eBioscience) were added to each well for 4 hours, after which cells and supernatant liquid were separately collected. The cells were fixed and penetrated by Fixation & Permeabilization Buffers (eBioscience), followed by staining with anti-mouse IL-17 FITC antibody (0.25 ug/test) (eBioscience) and analyzed by flow cytometry (Partec). The percentage of Th17 cells, the results are shown in Figure 1.

Figure 1 shows that the experimental group can inhibit the differentiation of Th17 cells after treatment with the extract of A. nigra, and it has a good inhibitory effect at 10ug/ml and 20ug/ml. Inhibition of differentiation of Th17 cells.

2. Evaluation of the ability of A. nigrum fruiting body extract ACFB06 to inhibit the differentiation of Th17 cells

The spleens of C57BL/6 mice (6-8 weeks old) were taken out and ground using a sieve, and the cell suspension was collected as primary spleen cells. CD4+ T cells were then isolated using an IMag magnetic system (BD Pharmingen, San Jose, CA), and the cells were added to a 24-well cell culture dish at a density of ⁵ × ^{10 5} cells/well, and the cells were divided into a control group and a negative control group. Experimental group D, experimental group E, and experimental group F were five groups, and the detailed treatment of each group is shown in Table 1.

See Table 2. The control group was CD4+ T cells without any treatment reagent; the negative control group was Th17 differentiation group, and treated with cytokines and neutralizing antibodies to promote differentiation of CD4+ T cells into Th17 cells; experimental group D, experimental group E In the experimental group F, different concentrations of the extract of A. angustifolia fruit body ACFB06 were added simultaneously to observe the effect on the differentiation of Th17 cells. The above cells were cultured at 37 ° C in a 5% CO ₂ incubator. After three days of culture, 10 ul of PMA (0.05 mg/ml) (Sigma) and 1 ul of brefeldin A (eBioscience) were added to each well for 4 hours, after which cells and supernatant liquid were separately collected. The cells were fixed and penetrated by Fixation & Permeabilization Buffers (eBioscience), followed by staining with anti-mouse IL-17 FITC antibody (0.25 ug/test) (eBioscience) and analyzed by flow cytometry (Partec). The percentage of Th17 cells, the results are shown in Figure 2.

Figure 2 shows that the treatment of ACFB06 in the experimental group can also inhibit the differentiation of Th17 cells.

3. Evaluation of the ability of ACFB12 extract from Antrodia camphorata to inhibit the differentiation of Th17 cells

The spleens of C57BL/6 mice (6-8 weeks old) were taken out and ground using a sieve, and the cell suspension was collected as primary spleen cells. CD4+ T cells were then isolated using an IMag magnetic system (BD Pharmingen, San Jose, CA), and the cells were added to a 24-well cell culture dish at a density of ⁵ × ^{10 5} cells/well, and the cells were divided into a control group and a negative control group. Experimental group G, experimental group H, and experimental group I five groups, and the detailed treatment of each group is shown in Table 3.

See Table 3. The control group was CD4+ T cells without any treatment reagent; the negative control group was Th17 differentiation group, and treated with cytokines and neutralizing antibodies to promote differentiation of CD4+ T cells into Th17 cells; experimental group G, experimental group H In the experimental group I, different concentrations of the extract of A. angustifolia fruit body ACFB12 were added simultaneously to observe the effect on the differentiation of Th17 cells. The above cells were cultured at 37 ° C in a 5% CO ₂ incubator. After three days of culture, 10 ul of PMA (0.05 mg/ml) (Sigma) and 1 ul of brefeldin A (eBioscience) were added to each well for 4 hours, after which cells and supernatant liquid were separately collected. The cells were fixed and penetrated by Fixation & Permeabilization Buffers (eBioscience), followed by staining with anti-mouse IL-17 FITC antibody (0.25 ug/test) (eBioscience) and analyzed by flow cytometry (Partec). The percentage of Th17 cells, the results are shown in Figure 3.

Figure 3 shows that the treatment of ACFB12 in the experimental group can inhibit the differentiation of Th17 cells, and the inhibitory effect tends to increase with increasing dose, especially at concentrations of 10 ug/ml and 20 ug/ml. The inhibition effect is more significant.

According to the results shown in Figures 1-3, the inhibitory effect of the extract of A. angustifolia fruit body ACFB06 on the differentiation of Th17 cells was much weaker than that of the ACFB12 and ACFB03 extracts.

Example 3: Evaluation of the ability of Antrodia camphorata mycelium extract to inhibit the differentiation of Th17 cells

In the present example, the effect of the extract of A. angustifolia mycelium (AC06-EA and AC06-A) obtained in Example 1 on the differentiation of Th17 cells was evaluated by a Th17 cell differentiation system constructed by mouse spleen cells.

1. Evaluation of the ability of Astragalus membranaceus mycelium extract AC06-EA to inhibit the differentiation of Th17 cells

The spleens of C57BL/6 mice (6-8 weeks old) were taken out and ground using a sieve, and the cell suspension was collected as primary spleen cells. CD4+ T cells were then isolated using an IMag magnetic system (BD Pharmingen, San Jose, CA), and the cells were added to a 24-well cell culture dish at a density of ⁵ × ^{10 5} cells/well, and the cells were divided into a control group and a negative control group. Experimental group J, experimental group K, and experimental group L were five groups, and the detailed treatment of each group is shown in Table 4.

See Table 4. The control group was CD4+ T cells without any treatment reagent; the negative control group was Th17 differentiation group, and treated with cytokines and neutralizing antibodies to promote differentiation of CD4+ T cells into Th17 cells; experimental group J, experimental group K Different from the experimental group L, different concentrations of the mycelium extract of Aconitum sinensis AC06-EA were added to observe the effect on the differentiation of Th17 cells. The above cells were cultured at 37 ° C in a 5% CO ₂ incubator. After three days of culture, 10 ul of PMA (0.05 mg/ml) (Sigma) and 1 ul of brefeldin A (eBioscience) were added to each well for 4 hours, after which cells and supernatant liquid were separately collected. The cells were fixed and penetrated by Fixation & Permeabilization Buffers (eBioscience), followed by staining with anti-mouse IL-17 FITC antibody (0.25 ug/test) (eBioscience) and analyzed by flow cytometry (Partec). The percentage of Th17 cells, the results are shown in Figure 4.

Fig. 4 shows that the treatment of AC06-EA of A. angustifolia in the experimental group can inhibit the differentiation of Th17 cells, and the inhibitory effect has a relatively increasing tendency with increasing dose, especially at a concentration of 20 ug/ml. More significant inhibition effect.

2. Evaluation of the ability of Astragalus membranaceus mycelium extract AC06-A to inhibit the differentiation of Th17 cells

The spleens of C57BL/6 mice (6-8 weeks old) were taken out and ground using a sieve, and the cell suspension was collected as primary spleen cells. CD4+ T cells were then isolated using an IMag magnetic system (BD Pharmingen, San Jose, CA), and the cells were added to a 24-well cell culture dish at a density of ⁵ × ^{10 5} cells/well, and the cells were divided into a control group and a negative control group. Experimental group M, experimental group N, and experimental group O were five groups, and the detailed treatment of each group is shown in Table 5.

Table 5, detailed processing of each group

See Table 5. The control group was CD4+ T cells without any treatment reagent; the negative control group was Th17 differentiation group, and treated with cytokines and neutralizing antibodies to promote differentiation of CD4+ T cells into Th17 cells; experimental group M, experimental group N Different from the experimental group O, different concentrations of the mycelium extract of Aconitum sinensis AC06-A were added to observe the effect on the differentiation of Th17 cells. The above cells were cultured at 37 ° C in a 5% CO ₂ incubator. After three days of culture, 10 ul of PMA (0.05 mg/ml) (Sigma) and 1 ul of brefeldin A (eBioscience) were added to each well for 4 hours, after which cells and supernatant liquid were separately collected. The cells were fixed and penetrated by Fixation & Permeabilization Buffers (eBioscience), followed by anti-mouse IL-17 FITC antibody (0.25 ug/test) (eBioscience) for staining and analysis by flow cytometry (Partec). The percentage of Th17 cells, the results are shown in Figure 5.

Fig. 5 shows that the treatment of AC06-A of Antrodia camphorata in the experimental group also inhibited the differentiation of Th17 cells and its inhibitory effect was similar to that of AC06-EA.

Example 4: Effect of Astragalus lucidum fruiting body extract ACFB12 on Th17 cells in patients with systemic lupus erythematosus

In this experiment, peripheral blood mononuclear cells from patients with systemic lupus erythematosus were used to evaluate the effect of ACFB12 extract from A. nigrum on the activity of Th17 cells. The procedure was as follows: The peripheral blood of 21 patients with active systemic lupus erythematosus and 20 healthy subjects were treated with ACFB12 (20 ng/ml) (experimental group) or DMSO (control group) for 6 hours, and then separated. Its monocytes. Then, intracellular staining was performed with IL-17 antibody, and finally the percentage of Th17 cells was analyzed by flow cytometry. The experimental results are shown in Figure 6.

The results of the experiment in Figure 6 show that the ACFB12 extract of A. annuum can effectively reduce Th17 cells in patients with systemic lupus erythematosus (mean: 0.95% vs. 0.48%; p < 0.001), but ACFB12 Th17 cells in healthy people have no effect.

Example 5: Effect of ACFB12 of Antrodia camphorata fruit body extract on Th17 cells of Graves' patients

In this example, the effect of the extract of A. angustifolia fruit body ACFB12 on Th17 cells was evaluated by peripheral blood mononuclear cells of the Graves patient. The implementation steps are as follows:

The peripheral blood of 1 Graves patient and 1 healthy person was collected, and then peripheral blood mononuclear cells were isolated and treated with ACFB12 (20ug/ml) (experimental group) and DMSO (control group) for 16 hours. The IL-17 antibody was subjected to intracellular staining, and finally the percentage of Th17 cells was analyzed by flow cytometry. The experimental results are shown in Figure 7.

The results of the experiment in Figure 7 show that the ACFB12 extract of A. annuum can be reduced in the concentration of 20ug/ml, in addition to reducing the Th17 cells of the Graves patient (mean: 0.30% vs. 0.21%). Th17 cells (mean: 0.08% vs. 0.03%; p < 0.05).

Example 6: Effect of A. angustifolia fruiting body extract ACFB12 on imiquimod (IMQ)-induced dry sputum model Th17 cells

In this example, the effect of ACFB12 of A. angustifolia fruiting body extract on Th17 cells was evaluated by imiquimod (IMQ) induced dry animal model. The implementation steps are as follows:

The back hair of Balb/c mice (6-8 weeks old) was first shaved, and the mice were divided into three groups: control group, control group and experimental group. No treatment was given to the mice in the control group; the control group would be 62.5 mg. Imiquimod cream (Aldara; 3M Pharmaceuticals) was applied to the back of the mice, and imiquimod cream was given once a day for 10 days to induce dry skin symptoms in mice; the experimental group would have 62.5 mg of mice. The quinote cream was applied to the back of the mouse, and then the ACFB12 (10 mg/kg) extract of the body extract of Antrodia camphorata was administered by intraperitoneal injection, and the imiquimod cream and the body extract of A. angustifolia ACFB12 (10 mg/kg) were administered once a day. Give ten days in a row. The mice were then sacrificed and their spleens removed and the spleens passed through a 70-μm grid of iron mesh to obtain a single cell suspension. The primary spleen lymphocytes are obtained by knocking out the red blood cells in the above single cell suspension using Red blood cell lysis buffer, and then washing them twice with PBS. Further, spleen lymphocytes (2 × 10 ⁶ ) were implanted into a 24-well cell culture plate coated with anti-CD3 monoclonal antibody (1 μg/ml) (BD Pharmingen), and the cells were stimulated for 24 hours, and the cells were taken out and dissolved. 0.2% BSA/PBS, stained with anti-mouse CD4 PE antibody for 30 minutes, then the cells were washed three times with PBS. Further fixation and penetration were performed using 2% paraformaldehyde with 0.5% saponin, followed by addition of anti-mouse IL-17 FITC antibody for intracellular staining. Finally, the percentage of CD4+/IL-17+ double positive cells was analyzed by flow cytometry (FACSCalibur; BD Biosciences) and CellQuest software (BD Biosciences).

Figure 8 shows that the control group was able to significantly enhance CD4+/IL-17+ double positive cells in the spleen of mice after administration of imiquimod, whereas in the experimental group, imiquimod and burdock were simultaneously administered. After the solid extract ACFB12, CD4+/IL-17+ double positive cells were reduced (mean: 9.3% vs. 3.6%; p < 0.001).

Claims (10)

The use of an extract of Antrodia camphorata for preparing a medicament for regulating Th17 cells, wherein the extract of Antrodia camphorata has the function of regulating Th17 cells. The use of the extract of Antrodia camphorata according to claim 1 for the preparation of a medicament for regulating Th17 cells, wherein the effect of regulating Th17 cells comprises inhibiting the differentiation of Th17 cells. The use of the extract of Antrodia camphorata according to claim 1 for the preparation of a medicament for regulating Th17 cells, wherein the extract of Antrodia camphorata comprises an extract of an extract of Antrodia camphorata or an extract of Mycelium of Antrodia camphorata. The use of the extract of Antrodia camphorata according to Item 3 of the patent application for the preparation of a medicament for regulating Th17 cells, wherein the extract of the Antrodia camphorata fruit body is obtained by performing an extraction process on the A. angustifolia fruiting body, the extraction process At least one extraction solvent is used, the at least one solvent comprising an alcohol and/or water. Use of an extract of Antrodia camphorata as described in claim 4 for the preparation of a medicament for modulating Th17 cells, wherein the alcohol comprises methanol, ethanol, isopropanol or butanol. The use of the extract of Antrodia camphorata according to claim 5, for the preparation of a medicament for regulating Th17 cells, wherein the ethanol comprises an aqueous solution of 10-95% ethanol. The use of the extract of Antrodia camphorata as described in claim 4 for the preparation of a medicament for modulating Th17 cells, wherein the extraction process comprises: heating the burdock fruit body with the at least one solvent to form a mixture; and The mixture is filtered to obtain a filtrate, and the filtrate is the burdock Fruiting body extract. The use of the extract of Antrodia camphorata according to claim 3, for extracting a drug for regulating Th17 cells, wherein the extract of the mycelium of A. angustifolia is obtained by performing an extraction process on the mycelium of A. angustifolia. The extraction process uses at least one extraction solvent, and the at least one solvent includes ethyl acetate or acetone. The use of the extract of Antrodia camphorata according to claim 1 for the preparation of a medicament for regulating Th17 cells, wherein the medicament for regulating Th17 cells is for treating a disease associated with Th17 cell regulation. The use of the extract of Antrodia camphorata according to claim 9 for the preparation of a medicament for regulating Th17 cells, wherein the disease associated with the regulation of Th17 cells is Graves' disease or cognac.