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US20100227404A1 - Water extract of antrodia camphorata for immunostimulatory effect and preparation method thereof - Google Patents

Water extract of antrodia camphorata for immunostimulatory effect and preparation method thereof Download PDF

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US20100227404A1
US20100227404A1 US12/552,115 US55211509A US2010227404A1 US 20100227404 A1 US20100227404 A1 US 20100227404A1 US 55211509 A US55211509 A US 55211509A US 2010227404 A1 US2010227404 A1 US 2010227404A1
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water extract
acw
cell
extract
dcs
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Inventor
Yang-Chang Wu
Mei-Chin Lu
Fang-Rong Chang
Ying-Chi Du
Tung-Ying Wu
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Kaohsiung Medical University
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Kaohsiung Medical University
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Assigned to KAOHSIUNG MEDICAL UNIVERSITY reassignment KAOHSIUNG MEDICAL UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: CHANG, FANG-RONG, DU, YING-CHI, LU, MEI-CHIN, WU, TUNG-YING, WU, YANG-CHANG
Publication of US20100227404A1 publication Critical patent/US20100227404A1/en
Priority to US14/577,605 priority Critical patent/US9241962B2/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/06—Fungi, e.g. yeasts
    • A61K36/07—Basidiomycota, e.g. Cryptococcus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0639—Dendritic cells, e.g. Langherhans cells in the epidermis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/70—Undefined extracts
    • C12N2500/74—Undefined extracts from fungi, e.g. yeasts

Definitions

  • the present invention relates to an extract of a fruiting body of Antrodia camphorata and the preparing method thereof.
  • the present invention relates to a water extract of a fruiting body of A. camphorata (ACW) and the preparing method thereof, and the water extract has immunostimulatory effect on dendritic cells (DCs).
  • ACW A. camphorata
  • DCs dendritic cells
  • Antrodia camphorata is an endemic mushroom in Taiwan and grows in the internal heartwood (or the dark/humid wood surface) of the particular Cinnamomum kanehirai in 400 to 2000 meters altitude. Therefore, it is uneasily to find out the wide fruiting body of AC or identify the morphological appearance of this Aphyllophorales fungus. In addition, the price of AC is still high due to their biologically active components having potential pharmaceutical value.
  • the fruiting body of AC cannot be easily harvested and be artificially cultured.
  • mycelia products of AC are popular in the market and announce to own anticancer activity, reduced cancer therapy-related symptoms and other side effects, anti-oxidation, anti-hypersensitivity and immunostimulatory effects.
  • DCs dendritic cells
  • APCs antigen presenting cells
  • LPS lipopolysaccharide
  • polysaccharides produced from this medical mushroom can be new pharmaceuticals for inducing the maturation and activation of DCs. Therefore, when searching for new cancer immunotherapy agents, the edible and medicinal fungi being used for cancer treatment in folk medicine are promising candidates, wherein AC is the most popular one.
  • Taiwan Patent No. 1299665 discloses an extract of AC and the preparing method thereof, wherein polysaccharide is obtained from extraction of mycelia of Taiwanofungus camphoratus with ethanol so as to inhibit the activity of matrix metalloproteinases.
  • the extraction is not performed with the fruiting body of AC, and the product also cannot inhibit the growth of cancer cells.
  • Taiwan Patent No. I279439 discloses inoculate obtained from incubating mycelia of AC by adjusting the pH value at incubation, but there is not extraction method disclosed.
  • Taiwan Patent No. 591110 discloses that ⁇ -aminobutyric acid is extracted from the lyophilized mycelia of AC with water or organic solvents.
  • the above-mentioned inventions do not disclose any product of the wild fruiting body of AC extracted with water or organic solvent, and the effect of ACW on DCs is not identified in the immune response.
  • the water extract of the fruiting body of wild A. camphorata (abbreviated as ACW) is obtained by extracting the fruiting body of AC with water, and ACW can effectively induce immune response and stimulate DCs.
  • ACW is further sequentially extracted with ethyl acetate, ethanol and water to obtain an ethyl acetate (ACW-EA), an ethanol extract (ACW-E) and a second water extract (ACW-W) of the fruiting body of AC. It can be identified from the above three extracts that polysaccharide can induce maturation of DCs and activate T cells.
  • a fractionation extract of the fruiting body of Antrodia camphorata includes at least one characteristic of: (1) causing first signals of carbohydrate on a first 1 H nuclear magnetic resonance (NMR) spectrum; and (2) causing a plurality of hydrogen signals of anomeric carbons of the polysaccharides on a second 1 H NMR spectrum.
  • NMR nuclear magnetic resonance
  • the first signals have the first chemical shift ranged between 3.0 and 5.5, and the plurality of hydrogen signals have second chemical shifts at 4.44, 4.46, 4.91, 5.00 and 5.15 respectively.
  • the fractionation extract is a second water extract fractionized from a water extract of the fruiting body of the Antrodia camphorata.
  • a method for preparing a water extract of the fruiting body of AC includes steps of: (a) providing the fruiting body; and (b) boiling the fruiting body in the water to obtain the water extract.
  • the step (a) further includes a step (a1) of grinding the fruiting body as a powder
  • the step (b) further includes a step (b1) of refluxing the water after the boiling step.
  • the water extract has a precipitate therein, and the method further includes a step (c) of removing the precipitate from the water extract.
  • the step (c) is performed by at least one of treatments of filtering the water extract and centrifuging the water extract.
  • the step (c) further includes a step (d) of extracting the water extract with at least one organic solvent to obtain an organic solvent extract.
  • the at least one organic solvent has ethyl acetate and ethanol
  • the step (d) further includes steps of: (d1) extracting the water extract with ethyl acetate to obtain an ethyl acetate extract and a first residue; and (d2) extracting the first residue with ethanol to obtain an ethanol extract and a second residue.
  • the step (d2) further includes a step (d3) of extracting the second residue with water to obtain a second water extract.
  • a method for maturating a cell includes a step of administrating the cell with a water extract as mentioned above.
  • the cell is an immature dendritic cell (DC).
  • DC dendritic cell
  • the cell expresses at least one protein after the administration with the water extract, and the at least one protein is one selected from a group consisting of a CD86, a human leukocyte antigen (HLA)-DR and a combination thereof.
  • the at least one protein is one selected from a group consisting of a CD86, a human leukocyte antigen (HLA)-DR and a combination thereof.
  • the method further includes a step of providing a second cell, wherein the cell expresses a cytokine after the administration with the water extract, and the cytokine induces the second cell to express a second cytokine.
  • the cytokine includes interleukin-12 (IL-12), the second cell is helper T cell, and the second cytokine is interferon-gamma (TNF- ⁇ ).
  • IL-12 interleukin-12
  • TNF- ⁇ interferon-gamma
  • the cell progresses a cell migration induced by the water extract.
  • the cell expresses at least one protein after the administration with the water extract, and the at least one protein is one selected from a group consisting of a Bax, a Bcl-2, a nuclear factor kappa B (NF ⁇ B) p65, a phosphorylated p38 (p-p38), a phosphorylated c-Jun N-terminal kinase (p-JNK), a phosphorylated protein kinase B (p-Akt), a phosphorylated extracellular signal-regulated kinase (p-ERK) and a combination thereof.
  • a Bax a Bcl-2
  • NF ⁇ B nuclear factor kappa B
  • p-p38 phosphorylated p38
  • p-JNK phosphorylated c-Jun N-terminal kinase
  • p-Akt phosphorylated protein kinase B
  • p-ERK extracellular signal-regulated kinase
  • the cell is induced by the water extract to go into the htephosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) pathway and/or the mitogen-activated protein kinase (MAPK) pathway.
  • PI3K/Akt htephosphatidylinositol-3 kinase/protein kinase B
  • MAPK mitogen-activated protein kinase
  • FIG. 1 is a diagram showing expressions of CD86 and HLA-DR after immatured DCs (ImDCs) are stimulated with ACW, GLW and ABW respectively;
  • FIG. 2 is a diagram showing the carbohydrate contents of ACW, GLW and ABW;
  • FIG. 3 is a flowchart showing the preparing method of ACW and the subsequent organic solvent extracts and a second water extract in the first preferred embodiment of the present invention
  • FIG. 4 is a diagram showing expressions of CD86 and HLA-DR after ImDCs are stimulated with the respective extracts of AC;
  • FIG. 5 is a bar chart showing the carbohydrate content of the respective extracts of AC
  • FIGS. 6(A) to 6(D) are the diagrams showing 1 H NMR spectra of (A) ACW, (B) ACW-EA, (C) ACW-E and (D) ACW-W;
  • FIG. 6(E) is a diagram showing a locally amplified 1 H NMR spectrum of ACW-W;
  • FIG. 7 is a diagram showing expressions of CD86 and HLA-DR induced by treating DCs with polymyxin B in advance and then with LPS or ACW;
  • FIG. 8 is a 1 H NMR spectrum of LPS
  • FIG. 9 is a diagram showing the interleukin-12 (IL-12) produced from induction of DCs with different concentrations of ACW;
  • FIG. 10 is a diagram showing the migratory ability of ImDCs stimulated with different concentrations of ACW;
  • FIG. 11(A) is a diagram showing the relationship of T cell proliferation and different concentrations of ACW at different DCs/T cells ratios
  • FIG. 11(B) is a diagram showing IFN- ⁇ production by stimulating DCs and activating helper T cells at different concentrations of ACW;
  • FIG. 12(A) and FIG. 12(B) respectively are the diagrams showing protein expressions of signaling transduction pathway of the ACW-stimulated DCs, wherein FIG. 12(A) shows Bax, Bcl-2 and p65, and FIG. 12(B) shows p-ERK, ERK, p-p38, p38, p-JNK, JNK and p-Akt; and
  • FIGS. 13(A) and 13(B) respectively are the diagrams showing expressions of (A) CD86 and (B) IL-12 after DCs are inhibited by signaling transduction pathway protein inhibitors and then treated with ACW.
  • ACW (216.4 mg) was extracted with solvents (sequentially as ethyl acetate, ethanol and water) of increasing polarities to obtain three different extracts, 8.9% (19.3 mg) of ethyl acetate extract (ACW-EA), 38.5% (83.3 mg) of ethanol extract (ACW-E) and 52.6% (113.8 mg) of a second water extract (ACW-W). Percentage is referred to the weight percentage of ACW, and weight is referred to the dry material extracted. The amount of carbohydrate in each extract was determined using phenol-sulfuric acid method (Chen et al., 2007), and the spectrum characteristics therein were analyzed with NMR.
  • peripheral blood mononuclear cells were obtained from 12 healthy donors by centrifugation with the Ficoll-Hypaque (Amersham Bioscience, Uppsala, Sweden) density gradient centrifugation.
  • PBMCs peripheral blood mononuclear cells
  • PBMCs were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum, 1 mM L-glutamin, 100 ⁇ g/ml streptomycin and 100 U/ml penicillin for 2 hours.
  • the nonadherent cells were removed, and the T cells were purified by nylon wool separation, as the source of mononuclear T lymphocytes.
  • T cells were loaded into the column and the eluted nonadherent cell fraction was rich in T cells. As determined by FACS, the resulting cell population was 80% T cells with a high expression of CD3.
  • the adherent cells were cultured for 6 days in RPMI 1640 medium with 50 ng/ml of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). Half of the culture medium was replaced by fresh medium containing growth factors every 2 to 3 days. On day six of culture, cells were collected and incubated with anti-CD11C + microbeads in conjunction with the mini MACs system by following the manufacturer's instructions (Becton Dickinson, Mountain View, Calif.).
  • the CD11C-positive cells were above 90% pure to obtain ImDCs.
  • ImDCs were further treated with phosphate buffered saline (PBS), LPS (100 ng/ml) and different doses of ACW for an additional 2 days and then assessed by flow cytometry.
  • PBS phosphate buffered saline
  • LPS 100 ng/ml
  • the treated DCs were pretreated with 25 ng/ml of mitomycin C at 37° C. for 30 minutes and subsequently washed three times with culture medium before plating.
  • the DCs were mixed with T cells at a concentration of 1:10 and 1:20.
  • Cells were cultured for five days on 96-well flat-bottom plates and subsequently pulsed with 20 ⁇ l of WST-1 (2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, Roche, Germany) during the last 4 hours of culture.
  • the absorbance of measuring wavelength (450 nm) and the reference wavelength (650 nm) were measured for T cell proliferation. All samples were measured against a background control.
  • Cell culture supernatants were collected and analyzed for IL-4 and IFN- ⁇ by enzyme-linked immunosorbent assay (ELISA).
  • DCs were stained in PBS using fluorochrome-conjugated or isotype-matched control antibodies for 30 minutes at 37° C.
  • the cells were analyzed by flow cytometry using a FACScalibur flow cytometer and Cell Quest software (Becton Dickinson, Mountain View, Calif.).
  • IL-12, IL-10, IFN- ⁇ and IL-4 in the culture supernatants from DCs or T cells were evaluated by using highly sensitive human cytokine assay kits (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions.
  • Chemotaxis of DCs was measured by the migration through a polycarbonate filter with 8 ⁇ m pore size in transwell chambers.
  • DCs were treated for 48 hours with LPS, 25 and 50 ⁇ g/ml of ACW and assayed for migration.
  • the lower chambers of transwell plates were filled with 500 ⁇ l serum-free medium with or without 2 ⁇ 10 6 T lymphocytes/ml.
  • DCs (1 ⁇ 10 5 cells in 0.1 ml) resuspended in serum-free medium were deposited in the upper chambers of the transwell plates and allowed to migrate for 3 hours at 37° C. in 5% CO2.
  • the numbers of migrating DCs harvested from the lower chambers were counted by flow cytometry (60 second counts).
  • DCs were stimulated with or without LPS or indicated doses of ACW, then lysed with RIPA Reagent (140 mM NaCl, 10 mM EDTA (2-[2-(Bis(carboxymethyl)amino)ethyl-(carboxymethyl)amino]acetic acid), 10% glycerol, 1% Nonidet P-40, 20 mM Tris (pH 7.0), 1 ⁇ M pepstatin, 1 ⁇ g/ml aprotinin, 1 ⁇ g/ml leupeptin and 1 mM sodium orthovanadate) and protein concentrations of the extracts were measured by BCA assay (Pierce, Rockford, Ill.).
  • DCs were pretreated at 37° C. with ERK inhibitor PD98059 (25 ⁇ M), JNK inhibitor SP600125 (25 ⁇ M), p38 MAPK inhibitor SB203580 (25 ⁇ M) or PI3K/Akt inhibitor LY294002 (25 ⁇ M) for 2 hours before stimulation with ACW. After 2 days, phenotyping characteristics of DCs and IL-12 production in the supernatants of DCs were analyzed.
  • FIG. 1 is a diagram showing expressions of CD86 and HLA-DR after ImDCs are stimulated with ACW, GLW and ABW respectively.
  • Mean fluorescence intensity (MFI) for each treatment is expressed as a fold increase over PBS. It can be known from FIG. 1 that GLW- or ABW-treated DCs do not show a significant increase in phenotypic maturation as do by ACW-treated DCs.
  • the carbohydrate of these extracts are estimated by phenol-sulfuric acid method, and the carbohydrate content of ACW is found to be 82.6% more than those of GLW (52.3%) and ABW (28.3%) ( FIG. 2 ).
  • FIG. 3 is a flowchart showing the preparing method of ACW and the subsequent organic solvent extracts and a second water extract in the first preferred embodiment of the present invention.
  • the wild fruiting bodies of AC is extracted with water (step 102 ) to obtain a water extract of the fruiting body of A. camphorata (ACW) (step 103 ).
  • ACW is extracted with ethyl acetate to obtain ethyl acetate extract of the fruiting body of A.
  • camphorata (ACW-EA) (step 104 ) and a first residue (step 105 ), and then the first residue is extracted with ethanol to obtain an ethanol extract of the fruiting body of A. camphorata (ACW-E) (step 106 ) and a second residue (step 107 ). Finally, the second residue is extracted with water to obtain a second water extract of the fruiting body of A. camphorata (ACW-W) (step 108 ).
  • Yields of ACW-EA, ACW-E and ACW-W respectively are 8.9%, 38.5% and 52.6% of the weight of ACW.
  • ImDCs are stimulated with ACW and its fractionized extracts respectively.
  • DCs treated with ACW or ACW-W increase the expressions of CD86 (1.6- and 1.9-fold) and HLA-DR (1.5- and 1.7-fold) compared with the control (PBS).
  • the expressions of CD86 and HLA-DR of DCs induced by ACW-E and ACW-EA respectively are obviously less than those induced by ACW-W or ACW ( FIG. 4 ).
  • the carbohydrate content in ACW and the subsequent fractionized extracts are further analyzed with phenol-sulfuric acid method.
  • the carbohydrate content in ACW-W is the most abundant (88.2%), compared to 31.8% and 12.9% in ACW-E and ACW-EA, respectively ( FIG. 5 ).
  • FIGS. 6(A) to 6(D) are the diagrams showing 1 H NMR spectra of (A) ACW, (B) ACW-EA, (C) ACW-E and (D) ACW-W.
  • ACW-W has the highest ratio of carbohydrate signals ranged between 3.0 and 5.5, and the specific hydrogen signals of anomeric carbons of the polysaccharides at 4.44, 4.46, 4.91, 5.00 and 5.15 ppm ( FIG. 6(E) ).
  • polymyxin B test identifies that ACW indeed can induce the maturation of DCs, and this activity does not originate from LPS contamination.
  • Polymyxin B is an antibiotic used for inactivating potential LPS contamination when evaluating the activity of different agents on immune cells. Therefore, whether polymyxin B blocks the phenotypic maturation of ACW-induced DCs is determined. The results show that polymyxin B accelerates an increased expressions of CD86 and HLA-DR by ACW, but significantly inhibits the expressions of CD86 and HLA-DR on LPS-induced DCs ( FIG. 7 ). Accordingly, ACW is able to stimulate the maturation of DCs without LPS contamination.
  • ACW-W and LPS are compared at the identical conditions with 1 H NMR spectrum. It can be found that ACW-W almost only has signal characteristic of carbohydrate when merging and comparing FIG. 6(D) with FIG. 8 , and does not show long-chain signals of LPS at 0.70 to 2.40 ppm. Therefore, the second water extract (ACW-W) with characteristic 1 H NMR signals of polysaccharides has mainly contribution to the immunostimulatory activity of ACW in causing the maturation of dendritic cells.
  • FIG. 9 is a diagram showing IL-12 produced from induction of DCs with different concentrations of ACW.
  • the ability of ACW (25 ⁇ g/ml and 50 ⁇ g/ml) to stimulate DCs and produce IL-12 is stronger than that of LPS, and the determined concentration of IL-12 are 235.53 ⁇ 2.54 (PBS), 698.45 ⁇ 5.46 (LPS), 758.14 ⁇ 26.54 (ACW-25) and 855.7 ⁇ 56.21 (ACW-50) pg/ml.
  • FIG. 10 is a diagram showing the migratory ability of ImDCs stimulated with different concentrations of ACW.
  • the stimulation of different ACW (12.5, 25 and 50 ⁇ g/ml) on ImDCs is able to effectively induce the migration of DCs, wherein the maximal cell migration to ACW is generated at 25 ⁇ g/ml ACW and 50 ⁇ g/ml ACW produces a diminished migratory response.
  • FIG. 11(A) is a diagram showing the relationship of T cell proliferation and different concentrations of ACW at different DCs/T cells ratios.
  • FIG. 11(A) comparing with PBS and 100 ng/ml LPS, 25 and 50 ⁇ g/ml ACW can efficiently stimulate DCs and activate T cells.
  • FIG. 11(B) which is a diagram showing IFN- ⁇ produced by stimulating DCs and activating helper T cells at different concentrations of ACW.
  • FIG. 11(B) is a diagram showing IFN- ⁇ produced by stimulating DCs and activating helper T cells at different concentrations of ACW.
  • ACW can efficiently stimulate DCs, activate helper T cells and efficiently increase the production of IFN- ⁇ induced by helper T cells. It can be known from the above results that ACW is able to induce DCs to produce abundant IFN- ⁇ and ACW-induced DCs are able to induce na ⁇ ve T cells to Th 1 pathways.
  • Induction of apoptosis is one of the escape mechanism of tumor cells from the immune surveillance system, while apoptotic inhibition of DCs enhances the antigen-specific immune response. Therefore, it needs to determine the cytotoxicity of ACW to DCs. It is found that more than 90% of the cells exhibit the characteristic phenotypes of mature DCs after the stimulation with ACW on day eight. ACW is not cytotoxic to DCs after long-term incubation. In addition, ACW treatment on DCs can dramatically decrease Bax expression, whereas increase Bcl-2 and NF ⁇ B p65 expressions ( FIG. 12(A) ).
  • FIG. 12(B) is the diagram showing protein expressions of signaling transduction pathway of the ACW-stimulated DCs.
  • FIG. 12(B) comparing with PBS and LPS, higher levels of p-p38, p-JNK and p-Akt are activated in DCs treated with 25 and 50 ⁇ g/ml ACW.
  • higher levels of p-ERK is activated in DCs treated with 12.5 ⁇ g/ml ACW, but significantly declined with higher doses of ACW.
  • the levels of nonphosphorylated ERK remains stable in different concentrations of ACW.
  • DCs are pretreated with the inhibitors (SB203580, PD98059, SP600125 and LY294002, respectively) to inhibit the activation of p38, ERK1/2, JNK, PI3K, and then are stimulated with ACW.
  • the results show that p38 (SB203580), JNK (SP600125) and Akt (LY294002) are able to inhibit CD86 expression of the ACW-treated DCs ( FIG. 13(A) ).
  • inhibition of p38 and Akt significantly blocks the up-regulation of IL-12 production of DCs induced by ACW.
  • the ERK inhibitor (PD98059) upregulates the IL-12 production by DCs in response to ACW ( FIG. 13(B) ). Taken together, it is concluded that ACW induces the activation of Akt and p38 leading to the differentiation and functional maturation of DCs.
  • ACW has better ability for inducing the maturation of DCs.
  • ACW is able to induce the maturation of DCs via the activation of PI3K/Akt and p38/MAPKs signaling transduction pathways, enhances T cell proliferation and IFN- ⁇ production, and polarizes them toward the Th1 pathway.
  • ACW further enhances DC survival by inhibiting apoptotic pathways. Accordingly, ACW is able to applied in cancer immunotherapy.
  • the extracts obtained from ACW sequentially extracted with ethyl acetate, ethanol and water can be found that the carbohydrate contents therein influences the level of DC activation, and demonstrates that the inducing immune components of ACW are originated from polysaccharides.

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CN103733991A (zh) * 2013-12-18 2014-04-23 柳州市天姿园艺有限公司 牛樟芝扦插枝的培育方法
CN104359933A (zh) * 2011-01-26 2015-02-18 高雄医学大学 樟芝子实体三萜类组合物、制备与分析方法
US20150157673A1 (en) * 2013-12-05 2015-06-11 National Taiwan Normal University METHOD FOR CONTROLLING OBESITY USING Antrodia camphorata
US9241962B2 (en) 2009-03-04 2016-01-26 Kaohsiung Medical University Water extract of Antrodia camphorata for immunostimulatory effect and preparation method thereof
US9603882B2 (en) 2013-08-13 2017-03-28 Industrial Technology Research Institute Method for modulating Th17 cells and method for treating a disease related to modulation of Th17 cells
US9751956B2 (en) 2015-03-06 2017-09-05 Ultra-Microrigin Biomedical Technology Co., Ltd. Method of using supercritical fluid technology to separate and purify functional components of Antrodia camphorata

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI549614B (zh) * 2013-08-15 2016-09-21 zong-ming Ye Extraction of Antrodia extraction method and apparatus

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030113297A1 (en) * 2001-05-18 2003-06-19 Jinn-Chu Chen Liver-caring medicine containing antrodia camphorata
US20030148517A1 (en) * 2000-11-13 2003-08-07 Jinn-Chu Chen Preparation and compositions for antrodia camphorata mycelium biologically active material
US20060052337A1 (en) * 2004-08-17 2006-03-09 Masao Hattori Polysaccharide and composition from Antrodia camphorata and use thereof
US20080312334A1 (en) * 2007-06-12 2008-12-18 Golden Biotechnology Corporation Cyclohexenone compounds from antrodia camphorata to treat autoimmune diseases

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TW591110B (en) 2001-12-03 2004-06-11 Grape King Entpr Inc A method for producing gamma-aminobutyric acid with Antrodia camphorate mycelium and composition thereof
TWI279439B (en) 2002-03-29 2007-04-21 Council Of Agriculture Processes for producing an antrodia camphorata culture having pharmacological activity, processes for obtaining a pharmacologically active composition from a culture of a. camphorata, products produced thereby and pharmaceutical compositions for the...
TWI299665B (en) 2006-05-09 2008-08-11 Univ Chia Nan Pharm & Sciency A method for preparing the extracts from taiwanofungus camphoratus with a capacity for inhibiting the activity of matrix metalloproteinases
TWI426916B (zh) * 2006-10-17 2014-02-21 Simpson Biotech Co Ltd 具有護肝功效之樟芝多醣體
TWI448294B (zh) 2009-03-04 2014-08-11 高雄醫學大學 增強免疫活性之樟芝子實體水萃取物及其製備方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030148517A1 (en) * 2000-11-13 2003-08-07 Jinn-Chu Chen Preparation and compositions for antrodia camphorata mycelium biologically active material
US20030113297A1 (en) * 2001-05-18 2003-06-19 Jinn-Chu Chen Liver-caring medicine containing antrodia camphorata
US20060052337A1 (en) * 2004-08-17 2006-03-09 Masao Hattori Polysaccharide and composition from Antrodia camphorata and use thereof
US20080312334A1 (en) * 2007-06-12 2008-12-18 Golden Biotechnology Corporation Cyclohexenone compounds from antrodia camphorata to treat autoimmune diseases

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Jen-Leun et al. "Antioxidant Properties of Methanolic Extracts from two kinds of Antrodia Camphorata Mycelium" (Food Chemistry 86, (2004) pages 25-31). *
Jen-Leun et al., "Antioxidant properties of methanolic extracts from two kinds of antrodia camphorata mycelia" Food Chemistry, Vol 86, (2004), pages 25-31 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9241962B2 (en) 2009-03-04 2016-01-26 Kaohsiung Medical University Water extract of Antrodia camphorata for immunostimulatory effect and preparation method thereof
CN104359933A (zh) * 2011-01-26 2015-02-18 高雄医学大学 樟芝子实体三萜类组合物、制备与分析方法
US9603882B2 (en) 2013-08-13 2017-03-28 Industrial Technology Research Institute Method for modulating Th17 cells and method for treating a disease related to modulation of Th17 cells
US20150157673A1 (en) * 2013-12-05 2015-06-11 National Taiwan Normal University METHOD FOR CONTROLLING OBESITY USING Antrodia camphorata
CN103733991A (zh) * 2013-12-18 2014-04-23 柳州市天姿园艺有限公司 牛樟芝扦插枝的培育方法
US9751956B2 (en) 2015-03-06 2017-09-05 Ultra-Microrigin Biomedical Technology Co., Ltd. Method of using supercritical fluid technology to separate and purify functional components of Antrodia camphorata

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