USE OF ANTRODIA CINNAMOMEA COMPOSITION FOR IMMUNE MODULATION

Abstract

A method for modulating an immune system in a subject in need thereof, comprising: administering to a subject an effective amount of a pharmaceutical composition comprising an Antrodia cinnamomea composition, wherein the Antrodia cinnamomea composition comprises: 50 wt % to 100 wt % of Antrodia cinnamomea mycelium; and 0 wt % to 50 wt % of an extract of fruiting body of Antrodia cinnamomea.

Claims

1. A method for modulating an immune system in a subject in need thereof, comprising: administering to a subject an effective amount of a pharmaceutical composition comprising an Antrodia cinnamomea composition, wherein the Antrodia cinnamomea composition comprises: 50 wt % to 100 wt % of Antrodia cinnamomea mycelium; and 0 wt % to 50 wt % of an extract of fruiting body of Antrodia cinnamomea.

2. The method of claim 1, wherein the Antrodia cinnamomea mycelium is solid-state cultivated Antrodia cinnamomea mycelium.

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

4. The method of claim 1, wherein PD-L1.sup.+ B cells in the subject are decreased.

5. The method of claim 4, wherein the PD-L1.sup.+ B cells in peripheral blood of the subject are decreased after administering the effective amount of the pharmaceutical composition, compared with the PD-L1.sup.+ B cells in the peripheral blood of the subject before administering the effective amount of the pharmaceutical composition.

6. The method of claim 5, wherein the PD-L1.sup.+ B cells in the peripheral blood are detected by flow cytometry, Enzyme-linked Immunospot Assay or immunohistochemistry.

7. The method of claim 1, wherein PD-L1.sup.+ monocytes in the subject are decreased.

8. The method of claim 7, wherein the PD-L1.sup.+ monocytes in peripheral blood of the subject are decreased after administering the effective amount of the pharmaceutical composition, compared with the PD-L1.sup.+ monocytes in the peripheral blood of the subject before administering the effective amount of the pharmaceutical composition.

9. The method of claim 8, wherein the PD-L1.sup.+ monocytes in the peripheral blood are detected by flow cytometry, Enzyme-linked Immunospot Assay or immunohistochemistry.

10. The method of claim 1, wherein MHC II.sup.+ dendritic cells in the subject are decreased.

11. The method of claim 10, wherein the MHC II.sup.+ dendritic cells in peripheral blood of the subject are decreased after administering the effective amount of the pharmaceutical composition, compared with the MHC II.sup.+ dendritic cells in the peripheral blood of the subject before administering the effective amount of the pharmaceutical composition.

12. The method of claim 11, wherein the MHC II.sup.+ dendritic cells in the peripheral blood are detected by flow cytometry, Enzyme-linked Immunospot Assay or immunohistochemistry.

13. The method of claim 1, wherein effector CD4 αβ T cells in the subject are increased.

14. The method of claim 13, wherein the effector CD4 αβ T cells in peripheral blood of the subject are increased after administering the effective amount of the pharmaceutical composition, compared with the effector CD4 αβ T cells in the peripheral blood of the subject before administering the effective amount of the pharmaceutical composition.

15. The method of claim 14, wherein the effector CD4 αβ T cells in the peripheral blood are detected by flow cytometry, Enzyme-linked Immunospot Assay or immunohistochemistry.

16. The method of claim 1, wherein effector CD8 αβ T cells in the subject are increased.

17. The method of claim 16, wherein the effector CD8 αβ T cells in peripheral blood of the subject are increased after administering the effective amount of the pharmaceutical composition, compared with the effector CD8 αβ T cells in the peripheral blood of the subject before administering the effective amount of the pharmaceutical composition.

18. The method of claim 17, wherein the effector CD8 αβ T cells in the peripheral blood are detected by flow cytometry, Enzyme-linked Immunospot Assay or immunohistochemistry.

19. The method of claim 1, wherein the pharmaceutical composition comprises 300 mg to 1500 mg of the Antrodia cinnamomea composition.

20. The method of claim 1, wherein 300 mg to 4000 mg of the Antrodia cinnamomea composition is administered to the subject per day.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a diagram showing the profiling results of PD-L1.sup.+ B cells in the subjects.

[0024] FIG. 2 is a diagram showing the profiling results of PD-L1.sup.+ monocytes in the subjects.

[0025] FIG. 3 is a diagram showing the profiling results of MHC II.sup.+ dendritic cells in the subjects.

[0026] FIG. 4 is a diagram showing the profiling results of effector CD4 αβ T cells in the subjects.

[0027] FIG. 5 is a diagram showing the profiling results of effector CD8 αβ T cells in the subjects.

DETAILED DESCRIPTION OF EMBODIMENT

[0028] Different embodiments of the present disclosure are provided in the following description. These embodiments are meant to explain the technical content of the present disclosure, but not meant to limit the scope of the present disclosure. A feature described in an embodiment may be applied to other embodiments by suitable modification, substitution, combination, or separation.

Preparation of Antrodia cinnamomea Composition

[0029] The Antrodia cinnamomea composition of the present disclosure is consisted of: 50 wt % to 99 wt % of solid-state cultivated Antrodia cinnamomea mycelium; and 1 wt % to 50 wt % of an ethanol/water extract of a fruiting body of Antrodia cinnamomea. The Antrodia cinnamomea mycelium is solid cultivated with whole grains (the inoculation ratio is 5-15% (v/w)). After cultivating at 18˜30° C. for 3-5 months, the solid-state cultivated Antrodia cinnamomea mycelium are harvested and processed to obtain Antrodia cinnamomea mycelium powders. In addition, the extract of the fruiting body of Antrodia cinnamomea is prepared by the following procedures. The fruiting body of Antrodia cinnamomea is harvest from the camphorata segment, the harvested fruiting body is dried, the dried fruiting body is extracted by water (50˜100° C.) and ethanol (35˜50° C.) in a ratio of 1:8 to 1:12 (W/V) for 1˜10 hours, the extraction process is repeated for 1 to 2 times, and the obtained extract is concentrated for the following use.

[0030] The obtained powders of the solid-state cultivated Antrodia cinnamomea mycelium and the water/ethanol extract of the fruiting body of Antrodia cinnamomea are mixed in a ratio of 99:1 to 1:1 to obtain the Antrodia cinnamomea composition. One combination is: 80 g of the powders of the solid-state cultivated Antrodia cinnamomea mycelium is mixed with 20 g of the water/ethanol extract of the fruiting body of Antrodia cinnamomea. Another combination is: 90 g of the powders of the solid-state cultivated Antrodia cinnamomea mycelium is mixed with 10 g of the water/ethanol extract of the fruiting body of Antrodia cinnamomea.

[0031] The Antrodia cinnamomea composition named “LEAC102 sample” used in the following embodiments is manufactured and provided by Taiwan Leader Biotech Corporation, which is the first combination mentioned above. After analyzing the components in the Antrodia cinnamomea composition, the active ingredients comprised in each grain of the Antrodia cinnamomea composition include: 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), Safrole C (0.24 mg˜9 mg), Zhankuic acid B (0.09 mg˜5 mg), Zhankuic acid H (0.25 mg˜13 mg), Zhankuic acid K (0.15 mg˜8 mg), Zhankuic acid A (0.005 mg˜2 mg), Zhankuic acid C (0.03 mg˜2 mg), dehydrosulfurenic acid (0.14 mg˜11 mg) and dehydroeburicoic acid (0.15 mg˜15 mg).

Experimental Method

[0032] Healthy subjects were participated in the present embodiment. Flow cytometry was taken as an approach in evaluating immune regulatory responses for 4 times. In the present embodiment, all the subjects were orally up-taking LEAC102 sample twice daily for 3 months, and the intake amount was about 800˜1000 mg Antrodia cinnamomea composition each time. The associations of immune cells with indexes of immune modulatory function were analyzed by paired student t test analysis.

[0033] The reagents used in the present embodiment included: Ficoll-Paque PREMIUM (GE Healthcare cat#17-5442), Staining buffer (homemade reagent), eBioscience™ Foxp3/Transcription Factor Staining Buffer Set (eBioscience cat#00-5523-0), Pasteur Pipettes (Volac cat#D812), 1.5 ml eppendorf tubes (SSI cat#1260-00), 15 ml and 50 ml Plastic centrifuge tubes (Corning CentriStar cat#4307941/#430829), Thermo Scientific Nunc Serological Pipette (cat#170355/#170356/#170357), 96 well plate (V bottom) (for cell counting) (Basic Life cat#BL6001) and FACS™ Tubes (flow tube)(Corning cat#FLCON 352008).

[0034] Human PBMCs were isolated from buffy coats by Ficoll density gradient centrifugation technique (Ficoll-Paque™ PREMIUM; GE Healthcare, USA). After centrifugation, PBMCs were washed with 1×PBS and re-suspended with staining buffer (2% FBS+0.02% NaN.sub.3 in 1×PBS) for cell surface marker staining or intracellular staining.

[0035] PBMCs were prepared from ficolled peripheral blood and stained for cell surface markers including Fluorescein isothiocyanate (FITC)-labeled anti-human T-cell receptor alpha/beta (TCRα/β), CD33; Phycoerythrin (PE)-labeled anti-human CD4, CD14, CD25, CD39, PD-L-1, PD-1; Coupled Dye (ECD)-labeled CD45; APC-labeled anti-human TCR gamma/delta (TCRγ/δ), CD11c; and AlexaFluor® 488APC-labeled anti-human CD25, PD-1. Data acquisition was performed on Navios flow cytometer (Beckman Coulter) and analyzed by Kaluza software version 2.1 (Beckman Coulter).

[0036] Statistical analysis was performed in-depth by comprehensively screening phenotypes of immune subsets and comparing the difference at kinetic time points. The data at kinetic time points were qualitatively and statistically analyzed by paired Student's T test.

Results

[0037] Elucidation of Antrodia cinnamomea Composition on PD-L1.sup.+ Cell Subsets in PBMCs

[0038] PD-1/PD-L1 axis plays an essential role in regulating immune balance. PD-L1 is the protein that expresses on normal and cancerous cells and delivers the negative signal to PD-1 expressing T cell to suppress T cell activation. To further confirm the effect of the Antrodia cinnamomea composition of the present embodiment on the regulation of the PD-L1/PD-1 axis, PBMCs of the subjects were collected and determined by flow cytometer and antibodies against specific epitopes, and analyzed by the aforesaid software (*, P<0.05, **, P<0.01, ***, P<0.001). The results are shown in FIG. 1 and FIG. 2, wherein FIG. 1 is a diagram showing the profiling results of PD-L1.sup.+ B cells in the subjects, and FIG. 2 is a diagram showing the profiling results of PD-L1.sup.+ monocytes in the subjects.

[0039] As shown in FIG. 1, at the 12.sup.th and 16.sup.th week, the expression of PD-L1.sup.+ B cells are significantly lower than that of the 0 week. As shown in FIG. 2, at the 4.sup.th and 12.sup.th week, the expression of PD-L1.sup.+ monocytes are significantly lower than that of the 0 week. These results indicate that the expression level of PD-L1 on B cells and monocytes was decreased. These data revealed that orally up-taking of the Antrodia cinnamomea composition may help block the interaction of PD-L1 and PD-1.

Elucidation of Antrodia cinnamomea Composition on MHC-II.sup.+ DC in PBMCs

[0040] Dendritic cells (DCs) are potent antigen presenting and processing cells that help provide suitable antigens to both T cells and B cells. In Human DCs, all types of DCs demonstrate high levels of MHC class II related markers. To further confirm the effect of the Antrodia cinnamomea composition on the regulation of MHC-II positive cell subsets, PBMCs of the subjects were collected and determined by flow cytometer and antibodies against specific epitopes, and analyzed by the aforesaid software (*, P<0.05, **, P<0.01, ***, P<0.001). The results are shown in FIG. 3, which is a diagram showing the profiling results of MHC II.sup.+ dendritic cells in the subjects.

[0041] As shown in FIG. 3, at the 16.sup.th week, the expression of MHC-II.sup.+ DCs are significantly lower than that of the 0 week. These data revealed that orally up-taking of the Antrodia cinnamomea composition may help pay tribute to suppressing cross-presentation activities in the subjects.

Elucidation of Antrodia cinnamomea Composition on Effector CD4 αβ T Cells and Effector CD8 αβ T Cells in PBMCs

[0042] T cell is one of an essential immune subset against extracellular and intracellular pathogens. T cells also can distinguish into two major subgroups, CD4 and CD8 T cell, which indicated T helper cells and cytotoxic T cells, respectively. In addition, T cell becomes activated after encounters stimuli and up-regulates surface antigens expression. To confirm the effect of the Antrodia cinnamomea composition on the regulation of the effector CD4 αβ T cells and effector CD8 αβ T cells, PBMCs of the subjects were collected and determined by flow cytometer and antibodies against specific epitopes, and analyzed by the aforesaid software (*, P<0.05, **, P<0.01, ***, P<0.001). The results are shown in FIG. 4 and FIG. 5, which respectively are diagrams showing the profiling results of effector CD4 αβ T cells and effector CD8 αβ T cells in the subjects.

[0043] As shown in FIG. 4, at the 12.sup.th and 16.sup.th week, the expression of effector CD4αβ T cells in all the subjects are significantly higher than that of the 0 week. As shown in FIG. 5, at the 12.sup.th and 16.sup.th week, the expression of effector CD8αβ T cells in all the subjects are significantly higher than that of the 0 week. These results indicate the up-regulation of effector CD4 αβ T cells and effector CD8 αβ T cells expression. These data revealed that orally up-taking of the Antrodia cinnamomea composition have the potential to induce T cell activation.

[0044] The aforesaid results indicate that the Antrodia cinnamomea composition of the present disclosure has the effect of immune modulation by down-regulating the PD-L1.sup.+ B cells, the PD-L1.sup.+ monocytes and the MHC II.sup.+ dendritic cells and up-regulating the effector CD4 αβ T cells and the effector CD8 αβ T cells in healthy subjects. In particular, the Antrodia cinnamomea composition of the present disclosure may pay tribute to down-regulation of the PD-L1/PD-1 axis and up-regulation of the effector CD4 αβ T cells and the effector CD8 αβ T cells. Furthermore, the Antrodia cinnamomea composition of the present disclosure is effective in the modulation of the adaptive immunity. In the cell profiling results, not only the expressions of the MHC-II.sup.+ dendritic cells were significantly down-regulated, the expression levels of PD-L1.sup.+ monocytes were also decreased after 16 weeks of the administration of the Antrodia cinnamomea composition of the present disclosure. These discrete results showed that the Antrodia cinnamomea composition of the present disclosure may possess potential therapeutic effects in regulating the adaptive immune responses of the subjects.

[0045] Although the present disclosure has been explained in relation to its embodiment, it is to be understood that many other possible modifications and variations can be made without departing from the spirit and scope of the disclosure as hereinafter claimed.