CN113855713A

CN113855713A - Application of antrodia camphorata composition in immunoregulation

Info

Publication number: CN113855713A
Application number: CN202010616051.2A
Authority: CN
Inventors: 林进忠; 赖宗贤; 陈婉菁; 李政墩
Original assignee: Taiwan Leader Biotech Corp
Current assignee: Taiwan Leader Biotech Corp
Priority date: 2020-06-30
Filing date: 2020-06-30
Publication date: 2021-12-31

Abstract

A new application of Antrodia camphorata composition in immunoregulation is provided, wherein the Antrodia camphorata composition comprises: 50 to 100 wt% of antrodia cinnamomea mycelium; and 0 wt% to 50 wt% of an Antrodia camphorata fruiting body extract.

Description

Application of antrodia camphorata composition in immunoregulation

Technical Field

The invention relates to a new application of an antrodia camphorata composition, which is used for immunoregulation.

Background

The therapeutic potential of polysaccharide and triterpene derivatives in the immunological field has been widely studied. Among them, antrodia camphorata is one of the unique fungi in taiwan, and is known to be used for treating inflammatory reaction, infection and cancer. For example, it is known that Antrodia camphorata extract can inhibit secretion of interleukin-1 β (IL-1 β) and interleukin 18(IL-18), reduce expression of inflammasome NLRP3, and inhibit ATP-induced ROS production and activation of caspase-1; thus, Antrodia camphorata extract can be used in the treatment of inflammatory response (Journal of Ethnopharmacology, Volume 155, Issue 1, 8 August 2014, Pages 154-. In addition, it has been reported that when mice were administered with antrodia camphorata polysaccharide, the expression of interferon gamma (IFN-. gamma.) and tumor necrosis factor alpha (TNF-. alpha.) was increased, and spleen dendritic cells and macrophages were presentCell, CD8 alpha⁺Surface appearance, major histocompatibility complex (major histocompatibility) class II I-A/I-E on dendritic cells also increased, and CD4 in spleen cells⁺/CD8⁺The proportion and the number of B cells are also improved; meanwhile, when antrodia camphorata polysaccharide was administered, the infection of mice was also suppressed (International Immunopharmacology, Volume 8, Issue 3, March 2008, Pages 458-. In addition, it has been shown that the administration of Antrodia camphorata extract can significantly inhibit the growth and metastasis of lung cancer tumor without affecting the biological parameters of serum or body weight, so Antrodia camphorata extract can be used as a candidate drug for treating lung cancer (Scientific Reports, Volume 9, anticancer number: 5145 (2019)).

Furthermore, the antigen-presentation mechanism is associated with dendritic cell, B cell and MHC II molecule interactions. Studies have shown that monocytes are associated with acquired immunity, such as antigen presentation and promotion of proliferation of regulatory T cells (Treg cells). Regulatory T cells and T cells can help maintain autoantigen tolerance and down regulate regulatory T cells. Thus, MHC II⁺Dendritic cells can be another opportunity for immunological studies.

In immunotherapy, programmed death molecule 1 (PD-1)/programmed death molecule 1 ligand (PD-1 ligand, PD-L1) axis is regarded as an important index. The PD1 receptor is expressed on the surface of activated T cells. The PD-L1 receptor is over-expressed in tumor cells. The PD1 receptor on T cells can be linked to the PD-L1 receptor of tumor cells, inhibiting cytotoxic T cells. The PD-1/PD-L1 axis is one of the novel targets common immunologically.

Therefore, if a novel application of the antrodia camphorata composition in regulating immune response can be provided, the composition will be beneficial to subsequent immunological research or immunotherapy (e.g., immunotherapy).

Disclosure of Invention

The invention mainly aims to provide a new application of an antrodia camphorata composition, which is used for immune regulation.

The antrodia camphorata composition comprises: 50 to 100 wt% of antrodia cinnamomea mycelium; and 0 wt% to 50 wt% of an Antrodia camphorata fruiting body extract. In one embodiment of the present invention, the antrodia camphorata composition comprises: 50 to 99 wt% of antrodia cinnamomea mycelium; and 1 wt% to 50 wt% of an Antrodia camphorata fruiting body extract. In another embodiment of the present invention, the antrodia camphorata composition is prepared from 50 wt% to 100 wt% of antrodia camphorata mycelium; and 0 wt% to 50 wt% of Antrodia camphorata fruiting body extract. In yet another embodiment of the present invention, the antrodia camphorata composition is prepared from 50 wt% to 99 wt% of antrodia camphorata mycelium; and 1 wt% to 50 wt% of Antrodia camphorata fruiting body extract.

When the antrodia camphorata composition is used, the effect of immunoregulation can be achieved. Therefore, the invention provides an application of the antrodia camphorata composition for preparing a medicinal composition with immunoregulatory property. In addition, the invention also provides an immune regulation method, which comprises the following steps: providing an effective amount of the above Antrodia camphorata composition to a subject in need thereof to modulate the immune response of the subject. Wherein the subject may be a human or other mammal.

In the present invention, the Antrodia camphorata mycelium can be a solid culture of Antrodia camphorata mycelium. Wherein, the Antrodia Camphorata mycelium can be prepared by solid-state culture of edible coarse cereals, drying after harvesting, grinding, and making into powder.

In the present invention, the extract of the fruiting body of Antrodia camphorata may be an extract of water and ethanol. Wherein the Antrodia camphorata fruiting body extract can be an extract extracted by 50 wt% to 95 wt% of ethanol.

In the present invention, each gram (i.e., gram) of antrodia camphorata composition contains active ingredients including: 4, 7-dimethoxy-5-methyl-1, 3-benzodioxole (2.9 mg-17 mg), 4-hydroxy-2, 3-dimethoxy-6-methyl-5- [3, 7, 11-trimethyl-2, 6, 10-dodecatriene ] -2-cyclohexenone (0.35 mg-21 mg), coenzyme Q3(0.23 mg-10 mg), camphorflavin C (0.24 mg-9 mg), camphoric acid B (0.09 mg-5 mg), camphoric acid H (0.25 mg-13 mg), camphoric acid K (0.15 mg-8 mg), camphoric acid A (0.005 mg-2 mg), camphoric acid C (0.03 mg-2 mg), dehydrothiochromic acid (0.14 mg-11 mg) and dehydroodontic acid (0.15 mg-15 mg).

In the present invention, the immunomodulatory property may be a reduction in PD-L1⁺B cell (PD-L1)⁺B cells), reduced PD-L1⁺Mononuclear cells (PD-L1)⁺monocytes), MHC II reduction⁺Dendritic cells (MHC II)⁺dendritic cells), increasing acting CD4 α β T cells (Effector CD4 α β T cells) and/or increasing acting CD8 α β T cells (Effector CD8 α β T cells). In particular, when a subject is administered with the composition of Antrodia camphorata of the present invention or the pharmaceutical composition comprising the composition of Antrodia camphorata, the PD-L1 in the peripheral blood is greater than before the subject is not administered with the composition of Antrodia camphorata or the pharmaceutical composition comprising the composition of Antrodia camphorata⁺B cell depletion, PD-L1⁺Monocyte depletion, MHC II⁺Dendritic cell depletion, increased active CD4 α β T cells, and/or increased active CD8 α β T cells. Among them, PD-L1 in peripheral blood⁺B cell, PD-L1⁺Monocyte, MHC II⁺Dendritic cells, effector CD4 α β T cells, and/or effector CD8 α β T cells can be obtained by flow cytometry, Enzyme-linked Immunospot Assay (ELISPOT), or immunohistochemical staining (IHC) analysis.

In the present invention, the pharmaceutical composition or antrodia camphorata composition may be administered non-orally, nasally, topically, or via an implantable reservoir (implantable reservoir) or the like.

In addition, the pharmaceutical composition of the present invention may comprise 300mg to 1500mg of antrodia camphorata composition, for example, 300mg to 1400mg, 300mg to 1300mg, 400mg to 1200mg, 400mg to 1100mg, 500mg to 1000mg or 800mg to 1000 mg. Further, in the pharmaceutical composition of the present invention, the pharmaceutical composition may be in the form of a solution, capsule, tablet, granule, spray, syrup, or other suitable form. When the pharmaceutical composition is in the form of a capsule or a tablet, the above-mentioned dosage of the antrodia camphorata composition may be included in one or more capsules or tablets.

In the present invention, the pharmaceutical composition or the antrodia camphorata composition may be administered in an amount of 300mg to 4000mg per day of the antrodia camphorata composition. For example, a composition of antrodia camphorata may be 800mg to 4000mg, 1200mg to 4000mg, 1300mg to 4000mg, 1400mg to 3800mg, 1400mg to 3500mg, 1500mg to 3300mg, 1600mg to 3000mg, 1600mg to 2800mg, 1600mg to 2500mg, 1600mg to 2300mg, or 1600mg to 2000mg daily.

In the present invention, the pharmaceutical composition or the antrodia camphorata composition may be administered 1 to 3 times per day. In addition, the administration timing is not particularly limited, and may be before meal, after meal, before sleep or at other times.

In the present invention, the pharmaceutical composition or the Antrodia camphorata composition is administered for at least 7 days, so that the effect of regulating immunity can be achieved.

As used herein, the term "effective amount" refers to the amount required to produce the desired effect in a desired subject, and will vary depending on the route of administration, the excipient employed, and the possibility of co-use with other agents.

In the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, stabilizer, diluent, dispersant, suspending agent, thickener or excipient, or a combination thereof.

As used herein, the term "acceptable" is used in that it must be compatible with (preferably, stabilize) the antrodia composition and not cause harm to the intended subject.

Other objects, advantages and novel features of the invention will become apparent from the following detailed description of the invention when considered in conjunction with the drawings.

Drawings

FIG. 1 is PD-L1 of a subject⁺B cell profiling;

FIG. 2 is PD-L1 of a subject⁺Monocyte cell distribution maps;

FIG. 3 is MHC II of a subject⁺(ii) a dendritic cell profile;

FIG. 4 is a graph of the profile of CD4 α β T cells acting in a subject;

figure 5 is a graph of the effect CD8 α β T cell profile of a subject.

Detailed Description

Various embodiments of the invention are provided below. These examples are intended to illustrate the technical content of the present invention, and are not intended to limit the scope of the present invention. Features of one embodiment may be applied to other embodiments by appropriate modification, substitution, combination, or separation.

Preparation of antrodia camphorata composition

The antrodia camphorata composition is composed of 50-99 wt% of antrodia camphorata solid culture mycelium, 1-50 wt% of antrodia camphorata fruiting body water and ethanol extract. Wherein, the antrodia camphorate mycelium is cultured in a solid state (the inoculation ratio is 5-15% (v/w)) by edible grains, cultured for 3-5 months at 18-30 ℃, collected and processed to obtain the antrodia camphorate mycelium powder. In addition, the extract of the fruiting body of Antrodia camphorata is obtained in the following way: harvesting sporophores from the cinnamomum kanehirae section wood subjected to fungus planting, drying the harvested sporophores, extracting the dried sporophores with water (50-100 ℃) and ethanol (35-50 ℃) according to the proportion of 1: 8 to 1: 12(W/V) for 1-10 hours respectively, repeatedly extracting for one to two times, and concentrating the extract for later use.

Mixing the obtained solid culture powder of Antrodia Camphorata mycelium with water and ethanol extract of Antrodia Camphorata fruiting body at ratio of 99: 1 to 1: 1 to obtain Antrodia Camphorata composition. The first combination example is: 80 g of Antrodia camphorata mycelium solid culture powder, and 20 g of Antrodia camphorata fruiting body water and ethanol extract. The second combination example is: 90 g of Antrodia camphorata mycelium solid culture powder, 10 g of Antrodia camphorata fruiting body water and ethanol extract.

The following examples used the antrodia camphorata active composition, "LEAC 102 test sample", which was the antrodia camphorata composition described in the first example of the combination, and was developed and produced by taiwan, littoral biotechnology, ltd. Through composition analysis, the antrodia camphorata active composition comprises the following active ingredients: 4, 7-dimethoxy-5-methyl-1, 3-benzodioxole (2.9 mg-17 mg), 4-hydroxy-2, 3-dimethoxy-6-methyl-5- [3, 7, 11-trimethyl-2, 6, 10-dodecatriene ] -2-cyclohexenone (0.35 mg-21 mg), coenzyme Q3(0.23 mg-10 mg), camphorflavin C (0.24 mg-9 mg), camphoric acid B (0.09 mg-5 mg), camphoric acid H (0.25 mg-13 mg), camphoric acid K (0.15 mg-8 mg), camphoric acid A (0.005 mg-2 mg), camphoric acid C (0.03 mg-2 mg), dehydrothiochromic acid (0.14 mg-11 mg) and dehydroodontic acid (0.15 mg-15 mg).

Experimental methods

In this example, a test was performed on healthy subjects. Flow cytometry was used as one of the means to evaluate immune modulatory responses for 4 assays. In this example, all subjects orally took the LEAC102 test sample twice a day, each time corresponding to 800-1000 mg of Antrodia camphorata intake, and took the test sample for 3 months. The relationship between immune cells and immune regulatory function index was analyzed by paired student T test.

In this example, the reagents used included: cell separation medium Ficoll-Paque PREMIUM (GE Healthcare cat #17-5442), staining buffer solution, intracellular staining Foxp3 perforation reagent kit (eBioscience)^TMFoxp3) (eBioscience cat #00-5523-0), pasteur dropper (Volac cat # D812), 1.5ml microcentrifuge tube (SSI cat #1260-00), 15ml and 50ml plastic centrifuge tube (Corning CentriStar cat #4307941/#430829), sterile plastic Pipette (Thermo Scientific Nunc sequential Pipette) (cat #170355/#170356/#170357), 96 well plate (V base) (for cell count) (ic Life cat # BL6001) and FACS^TMTest tube (flow tube) (Corning cat # FLCON 352008).

Ficoll density gradient centrifugation technology (Ficoll-Paque) is used^TMPREMIUM; GE Healthcare, USA), human peripheral blood mononuclear cells were isolated from buffy coat (buffy coat). After centrifugation, peripheral blood mononuclear cells were washed with 1-fold PBS buffer solution, and then with staining buffer solution (2% FBS + 0.02% NaN)₃In 1 × PBS) to perform cell surface marker (cell surface marker) staining or intracellular staining.

Peripheral blood mononuclear cells were obtained from peripheral blood and immunostained with cell surface markers. Wherein, the antibody used by the cell surface marker comprises human T cell receptor alpha/beta (TCR alpha/beta) and CD33 which are marked by Fluorescein Isothiocyanate (FITC); human labeled with Phycoerythrin (PE)CD4, CD14, CD25, CD39, PD-L-1 and PD-1; human CD45 labeled with a coupling Dye (ECD); APC-tagged human T cell receptor gamma/8 (TCR gamma/delta) and CD11 c; and with

488 APC-labeled human CD25 and PD-1. Data were taken on a flow cytometer (Navios flow cytometer) (Beckman Coulter) and analyzed by Kaluza software version 2.1(Beckman Coulter).

Immune subpopulations (immune subset) were phenotypically screened (comprehensive screening phenotyping) and analyzed for depth (in-depth) data, while comparing differences at different time points with kinetics. The data at different time points are used to qualitatively compare student's T test and to check statistical differences.

Results of the experiment

Antrodia camphorata active composition for treating PD-L1 in peripheral blood mononuclear cells⁺Effect of cell subsets (subset)

The PD-1/PD-L1 axis (PD-1/PD-L1 axis) is highly correlated with immune balance (immunebalance) regulation. PD-L1 is a protein on the upper surface of normal and tumor cells that transmits a negative signal to PD-1 surface T cells (PD-1 expressing T cells) inhibiting T cell activation. To evaluate the modulating effect of the antrodia camphorata active composition of this example on PD-1/PD-L1 axis, peripheral blood mononuclear cells from the subjects were collected and analyzed by flow cytometry and antibodies to specific antigens using the above analysis software (P < 0.05, P < 0.01, P < 0.001). The results are shown in FIGS. 1 and 2. Wherein, FIG. 1 shows PD-L1 of the subject⁺B cell distribution map, and FIG. 2 is PD-L1 of the subject⁺Monocyte cell profile.

As shown in FIG. 1, PD-L1 at week 12 and week 16 compared to week 0⁺B cells showed a significant decrease. As shown in FIG. 2, PD-L1 at week 4 and week 12 compared to week 0⁺Mononuclear sphere performance was also significantly reduced. This result shows that PD-L1 shows reduced expression in B cells and monocytes; the active composition of Antrodia camphorata of this example is representative of oral administrationCan effectively inhibit the functions of PD-L1 and PD-1.

Antrodia camphorata active composition for MHC II in peripheral blood mononuclear cells⁺Effect of dendritic cells

Dendritic cells are efficient antigen presenting and processing cells (antigen presenting and processing cells) that help provide antigens suitable for T cells and B cells. Among human dendritic cells, all dendritic cells exhibit a large number of MHC II associated markers. To evaluate the modulating effect of the active composition of Antrodia camphorata of this example on MHC-II positive cell subsets (MHC-II positive cell subsets), peripheral blood mononuclear cells from the subjects were collected and analyzed by flow cytometry and antibodies to specific antigens using the aforementioned analysis software (P < 0.05, P < 0.01, P < 0.001). The results are shown in FIG. 3, which is the MHC II of the subject⁺Dendritic cell profiling.

As shown in FIG. 3, MHC II at week 16 compared to week 0⁺Dendritic cell performance was significantly reduced. This result shows that oral administration of the antrodia camphorata active composition of this example is effective in helping to inhibit cross-presentation activity in the subject.

Effect of antrodia camphorata active composition on acting CD4 alpha beta T cells and acting CD8 alpha beta T cells in peripheral blood mononuclear cells

T cells are one of the necessary immune subsets against extracellular and intracellular pathogens. T cells can be divided into two major sub-populations, CD4 and CD 8T cells, which are helper T cells and cytotoxic T cells, respectively. In addition, T cells are stimulated to activate and up-regulate surface antigen expression. To evaluate the modulating effect of the antrodia camphorata active composition of this example on the effector CD4 α β T cells and effector CD8 α β T cells, peripheral blood mononuclear cells from the subjects were collected and analyzed by flow cytometry and antibodies to specific antigens using the above-described analysis software (P < 0.05, P < 0.01, P < 0.001). The results are shown in fig. 4 and 5, which are profiles of acting CD4 α β T cells and acting CD8 α β T cells, respectively, in the subject.

As shown in fig. 4, the active CD4 α β T cells exhibited significant increases at week 12 and week 16 compared to week 0. As shown in fig. 5, the expression of the active CD8 α β T cells was also significantly increased at week 12 and week 16 compared to week 0. The results show that the expression of the active CD4 α β T cells and the active CD8 α β T cells is increased, which indicates that the active composition of antrodia camphorata of the present embodiment can effectively enhance T cell activity by oral administration.

The above experimental results show that the antrodia camphorata active composition has the effect of regulating immunity, and can reduce PD-L1 of healthy subjects⁺B cell, PD-L1⁺Mononuclear sphere and MHC II⁺Dendritic cells, and increasing the active CD4 α β T cells and increasing the active CD8 α β T cells in healthy subjects. In particular, the antrodia camphorata active composition can negatively regulate PD-L1/PD-1 axis and positively regulate active CD4 alpha beta T cells and active CD8 alpha beta T cells. In addition, the antrodia camphorata active composition can be used for regulating the immunity in the future. In the experimental results of cell proliferation, after the antrodia camphorata active composition is taken for 16 weeks, the MHC-II of the subject is significantly negatively regulated⁺Dendritic cell expression with concomitant reduction in PD-L1⁺Expression of mononuclear cells. The results show that the antrodia camphorata active composition of the present invention can exhibit effective therapeutic efficacy in the regulation of the acquired immune response.

The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of an antrodia composition for the preparation of a pharmaceutical composition with immunomodulatory properties, wherein the antrodia composition comprises:

50 to 100 wt% of antrodia cinnamomea mycelium; and

0 to 50 wt% of Antrodia camphorata fruiting body extract.

2. The use of claim 1, wherein the Antrodia camphorata mycelium is a solid culture of Antrodia camphorata mycelium.

3. The use according to claim 1, wherein the extract of Antrodia camphorata fruiting body is an extract extracted with 50 wt% to 95 wt% ethanol.

4. The use of claim 1, wherein the immunomodulatory property is reduced PD-L1⁺B cell (PD-L1)⁺B cells)。

5. The use of claim 4, wherein the immunomodulatory property is PD-L1 in peripheral blood after a subject takes the pharmaceutical composition compared to the subject without the pharmaceutical composition⁺B cells were decreased.

6. The use of claim 5, wherein PD-L1 in peripheral blood is analyzed by flow cytometry, enzyme-linked immunospot, or immunohistochemical staining⁺B cells.

7. The use of claim 1, wherein the immunomodulatory property is reduced PD-L1⁺Mononuclear cells (PD-L1)⁺monocytes)。

8. The use of claim 7, wherein the immunomodulatory property is PD-L1 in peripheral blood after a subject takes the pharmaceutical composition compared to the subject without the pharmaceutical composition⁺Monocyte depletion.

9. The use of claim 8, wherein PD-L1 in peripheral blood is analyzed by flow cytometry, enzyme-linked immunospot, or immunohistochemical staining⁺A monocyte cell.

10. The use of claim 1, wherein the immunomodulatory property isFor reducing MHC II⁺Dendritic cells (MHC II)⁺dendritic cells)。

11. The use of claim 10, wherein the immunomodulatory property is MHC II in peripheral blood after a subject takes the pharmaceutical composition compared to the subject without the pharmaceutical composition⁺The dendritic cells are reduced.

12. Use according to claim 11, wherein peripheral blood is analyzed for MHC II by flow cytometry, enzyme-linked immunospot or immunohistochemical staining⁺A dendritic cell.

13. The use of claim 1, wherein the immunomodulatory property is an increasing effect of CD4 α β T cells (Effector CD4 α β T cells).

14. The use of claim 13, wherein the immunomodulatory property is an increase in CD4 α β T cells in peripheral blood after a subject takes the pharmaceutical composition compared to their onset prior to not taking the pharmaceutical composition.

15. The use of claim 14, wherein the affected CD4 α β T cells in peripheral blood are analyzed by flow cytometry, enzyme-linked immunospot, or immunohistochemical staining.

16. The use of claim 1, wherein the immunomodulatory property is an increasing effect of CD8 α β T cells (Effector CD8 α β T cells).

17. The use of claim 16, wherein the immunomodulatory property is an increase in CD8 α β T cells in peripheral blood after a subject takes the pharmaceutical composition compared to their onset prior to not taking the pharmaceutical composition.

18. The use of claim 17, wherein the affected CD8 α β T cells in peripheral blood are analyzed by flow cytometry, enzyme-linked immunospot, or immunohistochemical staining.

19. The use of claim 1, wherein the pharmaceutical composition comprises 300mg to 1500mg of the antrodia camphorata composition.

20. The use according to claim 1, wherein the pharmaceutical composition is administered in an amount of 300mg to 4000mg per day of the antrodia camphorata composition.