Zhankuic Acid Research Articles

Naturally Occurring Plant-Based Anticancerous Candidates as Potential ERK2 Inhibitors: In-Silico Database Mining and Molecular Dynamics Simulations.

The evolutionarily conserved extracellular signal-regulated kinase 2 (ERK2) is involved in regulating cellular signaling in both normal and pathological conditions. ERK2 expression is critical for human development, while hyperactivation is a major factor in tumor progression. Up to now, there have been no approved inhibitors that target ERK2, and as such, here we report on screening of a naturally occurring plant-based anticancerous compound-activity-target (NPACT) database for prospective ERK2 inhibitors. More than 1,500 phytochemicals were screened using in-silico molecular docking and molecular dynamics (MD) approaches. NPACT compounds with a docking score lower than a co-crystallized LHZ inhibitor (calc.-10.5 kcal/mol) were subjected to MD simulations. Binding energies (ΔGbinding) of inhibitor-ERK2 complexes over the MD course were estimated using an MM-GBSA approach. Based on MM-GBSA//100 ns MD simulations, the steroid zhankuic acid C (NPACT01034) demonstrated greater binding affinity against ERK2 protein than LHZ, with ΔGbinding values of -50.0 and -47.7 kcal/mol, respectively. Structural and energetical analyses throughout the MD course demonstrated stabilization of zhankuic acid C complexed with ERK2 protein. The anticipated ADMET properties of zhankuic acid C indicated minimal toxicity. Moreover, in-silico evaluation of fourteen ERK2 inhibitors in clinical trials demonstrated the higher binding affinity of zhankuic acid C towards ERK2 protein.

Bioactive metabolites of edible mushrooms efficacious against androgenic alopecia: Targeting SRD5A2 using computational approach

IntroductionAndrogenic alopecia (AGA), scalp hair loss, occurs due to the hyperactivity of steroid 5α-reductase2 (SRD5A2), which metabolizes testosterone into 5α-dihydroxytestosterone. Finasteride, an FDA-approved drug producing many side effects, is a commonly used competitive inhibitor for SRD5A2 to treat AGA. As a number of mushroom species have been used to treat patients having hair loss problems since ancient times, in the present paper, the bioactive metabolites in mushrooms were computationally screened to assess SRD5A2 inhibitory properties and compared with Finasteride. MethodologyA virtual screening approach in conjunction with molecular dynamics simulation and MMPBSA methods has been applied to identify potential inhibitors against SRD5A2. SwissADME tool and AutoDock Vina were used to screen the library of 152 bioactive mushroom metabolites. All MD simulations were carried out using GROMACS 5.1.4 suite and GROMOS96 43a1 force field. The stability of SRD5A-ligand complexes was determined in terms of RMSD, RMSF, Rg, SASA, and hydrogen bonding. The binding energy of complexes was calculated using MMPBSA. Results and ConclusionVirtual screening and MD simulation studies revealed that out of the 152 metabolites, three bioactives, i.e., Zhankuic acid A (−11.5 kcal/mol), Sterenin M (−10.4 kcal/mol), Melleolide K (−10.2 kcal/mol), and drug Finasteride having the least binding energy are the potential inhibitors of SRD5A2. These molecules also exhibited stable interaction with SRD5A2 during MD simulation. This study will pave the way for the experimental evaluation and validation of the medicinal potentials of screened mushroom compounds to treat androgenic alopecia.

Chemoreversal Agents from Taiwanofungus Genus and Their More Potent Methyl Derivatives Targeting Signal Transducer and Activator of Transcription 3 (STAT3) Phosphorylation.

Multidrug resistance (MDR), for which the mechanisms are not yet fully clear, is one of the major obstacles to cancer treatment. In recent years, signal transducer and activator of transcription 3 (STAT3) were found to be one of the important MDR mechanism pathways. Based on the previous research, zhankuic acid A, B, and C were found to have collateral sensitivity effects on MDR cancer cells, and MDR inhibitory activity of zhankuic acid methyl ester was found to be better than that of its acid. Therefore, we executed a systematic examination of the structure–activity relationship of zhankuic acid methyl ester derivatives to collateral sensitivity in MDR cancer cells. The results showed that compound 12 is the best in terms of chemoreversal activity, where the reversal fold was 692, and the IC50 value of paclitaxel combined with 10 μM compound 12 treatment was 1.69 nM in MDR KBvin cells. Among all the derivatives, methyl ester compounds were found to be better than their acids, and a detailed discussion of the structure–activity relationships of all of the derivatives is provided in this work. In addition, compounds 8, 12, and 26 were shown to influence the activation of STAT3 in KBvin cells, accounting for part of their chemoreversal effects. Our results may provide a new combined therapy with paclitaxel to treat multidrug-resistant cancers and provide a new therapy option for patients.

Bioactive naphthoquinones and triterpenoids from the fruiting bodies of Taiwanofungus salmoneus

Drug resistance of cancer cells stands for the major problem of the treatment failure for chemotherapy or target therapy. Overexpression of efflux pumps leading to multidrug resistance (MDR) is still an important issue needed to be solved. In the present study, Taiwanofungus salmoneus was selected as the topic and eleven undescribed constituents including four naphthoquinones salmonones A-D (1–4) and seven triterpenoids salmoneatins A-G (5–11), along with one chromanone (12) and two benzenoids (13 and 14) reported from the natural sources for the first time, as well as twenty-one known compounds were characterized. The structures of undescribed compounds were established by the spectroscopic and spectrometric analyses. In addition, the plausible biosynthetic mechanism of purified naphthoquinones was proposed and these compounds may be the excellent chemotaxonomic markers. Moreover, the isolates were evaluated for their P-gp inhibitory effects and the results showed that most of the examined compounds were effective. Among the tested compounds, 5, 10, 2,3-dimethoxy-5-(2ʹ,5ʹ-dimethoxy-3ʹ,4ʹ-methylenedioxyphenyl)-7-methyl-[1,4]naphthoquinone, zhankuic acid A methyl ester, and camphoratin F can reverse the resistance of paclitaxel or vincristine with the reversal folds in the range of 51093.3 and 259.5. These experimental data would initiate the possible development of Taiwanofungus salmoneus for the cancer therapy in the future.

Molecular docking and molecular dynamic studies: screening of phytochemicals against EGFR, HER2, estrogen and NF-KB receptors for their potential use in breast cancer

Breast cancer (BC) is a second common malignancy in female globally. Hence, identification of novel therapeutic agents is extremely important. Molecular docking and MD simulation are the important tools in the process of drug discovery for searching the potential hits. The structure-based drug designing technique also reveals the information about ligands behavior in computational environment. Docking tools help in visualization and analysis of protein–ligand complex at atomic level. Molecular dynamics shows the stability of the molecules in the receptor cavity in the simulated environment. In this research work, we have screened potent phytochemicals against the BC. We docked the phytochemicals and examined the binding affinities of ligands towards the EGFR, HER2, estrogen and NF-κB receptors. Pristimerin, ixocarpalactone A, viscosalactone B and zhankuic acid A have shown higher binding affinities and energies towards targeted receptors among the screened phytochemicals. MD simulation study shows stability of docked complex for pristimerin and HER2 receptor. These phytochemicals can be repurposed for their anticancer activity. This in-silico work provides a strong ground for further investigation of their anticancer activity.

Zhankuic Acids A, B and C from Taiwanofungus Camphoratus Act as Cytotoxicity Enhancers by Regulating P-Glycoprotein in Multi-Drug Resistant Cancer Cells.

Since P-glycoprotein (P-gp)-related multidrug resistance (MDR) remains the most important unsolved problem in cancer treatment, scientists are attempting to find potential structures from natural resources. The aim of the present study was to elucidate whether the triterpenoids from Taiwanofungus camphoratus could reverse cancer MDR by influencing P-gp efflux pump. Substrates efflux assay and P-gp ATPase activity assay were conducted to reveal the molecular mechanisms of P-gp inhibition, while SRB assay, cell cycle analyses and apoptosis analyses were performed to confirm the cancer MDR modulating effects. The results indicated that Zhankuic acids A, B and C (ZA-A, ZA-B and ZA-C) impacted P-gp efflux function in competitive, noncompetitive and competitive manners, respectively. Furthermore, these triterpenoids all demonstrated inhibitory patterns on both basal P-gp ATPase activity and verapamil-stimulated ATPase activity. In terms of MDR reversal effects, ZA-A sensitized the P-gp over-expressing cell line (ABCB1/Flp-InTM-293) and MDR cancer cell line (KB/VIN) toward clinically used chemotherapeutic drugs, including doxorubicin, paclitaxel and vincristine, exhibiting the best cytotoxicity enhancing ability among investigated triterpenoids. The present study demonstrated that ZA-A, ZA-B and ZA-C, popular triterpenoids from T. camphoratus, effectively modulated the drug efflux transporter P-gp and reversed the cancer MDR issue.

Constituents and Anti-Multidrug Resistance Activity of Taiwanofungus camphoratus on Human Cervical Cancer Cells.

Resistance to anti-cancer drugs is one of the main factors of treatment failure resulting in high morbidity. Among the reasons of resistance, overexpression of efflux pumps leading to multidrug resistance is an important issue that needs to be solved. Taiwanofungus camphoratus has been used as a nutritional supplement to treat various cancers. However, its effects on the resistance to chemotherapeutic agents are still unknown. In this study, we report four new chemical constituents of T. camphoratus isolated from an ether extract: camphoratins K (1) and N (2) and benzocamphorins G (3) and I (4). Furthermore, we evaluated zhankuic acids A–C for their P-glycoprotein (P-gp) inhibitory effects. The results showed that zhankuic acid A was the most potent P-gp inhibitor compound and (at 20 μM) could reverse drug resistance in human cancer cells, restoring an IC50 of 78.5 nM for doxorubicin, of 48.5 nM for paclitaxel, and of 321.5 nM for vincristine, indicating a reversal fold of 48, 38, and 45 times, respectively. This study provides support for the use of T. camphoratus in the further development of cancer therapy.

The JAK inhibitor antcin H exhibits direct anticancer activity while enhancing chemotherapy against LMP1-expressed lymphoma

Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The latent membrane protein 1 (LMP-1) of EBV constitutively activates the JAK/STAT signaling pathway and contributes to the proliferation of EBV-infected primary human B lymphocytes. Thus, targeting LMP1-induced JAK/STAT signaling may prove effective in treating B-cell lymphomas. The extract of the fruiting body of Antrodia cinnamomea, has been reported to have cytotoxicity on blood cancer cells. Here, we report that the bioactivity of antcin H, an analog of the JAK2 inhibitor zhankuic acid A (ZAA), inhibits LMP1-induced JAK/STAT related signaling and induces lymphoma cell line apoptosis. Moreover, antcin H enhances low-dose methotrexate (MTX) cytotoxicity against lymphoma cells. Treatment of antcin H with low-dose MTX significantly suppressed tumor growth and prolonged the survival of tumor-bearing mice. Our findings indicate antcin H as a potential therapeutic agent for the treatment of EBV-infected cancer cells.

Suppression of Cell Growth, Migration and Drug Resistance by Ethanolic Extract of Antrodia cinnamomea in Human Lung Cancer A549 Cells and C57BL/6J Allograft Tumor Model.

The purpose of this study was to investigate the inhibitory activities of ethanolic extracts from Antrodia cinnamomea (EEAC) on lung cancer. Cell proliferation and cell cycle distribution were analyzed using (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay and flow cytometry, respectively. Wound-healing assay, Western blotting, and a murine tumor model were separately used to examine cell migration, protein expression, and tumor repression. Our results showed that EEAC induced cell cycle arrest at the G0/G1 phase resulting decreased cell viability in A549 cells. Moreover, EEAC up-regulated the growth-suppressing proteins, adenosine 5′-monophosphate-activated protein kinase (AMPK), p21 and p27, but down-regulated the growth-promoting proteins, protein kinase B (Akt), mammalian tarfet of rapamycin (mTOR), extracellular signal-regulating kinase 1/2 (ERK1/2), retinoblastoma protein (Rb), cyclin E, and cyclin D1. EEAC also inhibited A549 cell migration and reduced expression of gelatinases. In addition, our data showed that tumor growth was suppressed after treatment with EEAC in a murine allograft tumor model. Some bioactive compounds from EEAC, such as cordycepin and zhankuic acid A, were demonstrated to reduce the protein expressions of matrix metalloproteinase (MMP)-9 and cyclin D1 in A549 cells. Furthermore, EEAC enhanced chemosensitivity of A549 to paclitaxel by reducing the protein levels of caveolin-1. Our data suggests that EEAC has the potential to be an adjuvant medicine for the treatment of lung cancer.

Precursor-feeding strategy on the triterpenoid production and anti-inflammatory activity of Antrodia cinnamomea

Antrodia cinnamomea has commercially been used in the formulation of nutraceuticals and functional foods. A. cinnamomea was fed exogenous sterols including squalene, cholesterol, and stigmasterol to enhance its triterpenoid content. Four triterpenoids were identified in A. cinnamomea, namely dehydrosulphurenic acid (De-sul), zhankuic acid A (ZaA), 15α-acetyl-dehydrosulphurenic acid (15α) and dehydroeburicoic acid (De-eb), and one polyphenyl compound, 4,7-dimethoxy-5-methyl-1,3-benzodioxole (4,7-D). Maximum ZaA and 15α contents of 2.84 and 48.07μg/mg dry weight, respectively were achieved by 100μM squalene-feeding, and maximum De-sul and De-eb contents of 69.08 and 47.91μg/mg dry weight were achieved by 10 and 100μM stigmasterol-feeding, respectively. To study the anti-inflammatory potential of A. cinnamomea, lipopolysaccharide-induced nitric oxide (NO) production, NADPH oxidase (NOX), inducible NO synthetase (iNOS) and cyclooxygenase-2 (COX-2) expression in murine microglial cells, BV-2, were evaluated. ZaA and 50μM squalene-feeding of A. cinnamomea inhibited LPS-induced NO, iNOS and COX-2 expression in BV-2 cells.

Immunosuppressive effect of zhankuic acid C from Taiwanofungus camphoratus on dendritic cell activation and the contact hypersensitivity response.

Some ergostane triterpenoids from Taiwanofungus camphoratus have been shown to exhibit anti-inflammatory activity in vitro. However, the effect of ergostane triterpenoids on the immune response remains unknown. In this study, we elucidated that ergostane triterpenoids significantly decreased the cytokines and chemokine release by dendritic cells (DC) and that, in the case of zhankuic acid C (ZAC), the decrease was dose-dependent and inhibited DC maturation. ZAC inhibited the contact hypersensitivity response and infiltrative T cells in the ears of DNFB-stimulated mice. Thus, we demonstrate for the first time that ZAC exhibits an immunosuppressive effect on DC activation and the contact hypersensitivity response. It is suggested that ZAC can potentially be used for treating chronic inflammation and autoimmune diseases.

Antrodia cinnamomea Inhibits Migration in Human Hepatocellular Carcinoma Cells: The Role of ERp57 and PGK-1.

Evidences suggest that ERp57 and PGK-1 signaling lead to cancer cell proliferation and migration. We hypothesized that ERp57 and PGK-1 down-regulation may inactivate matrix metalloproteinase (MMP)-2, -9 expressions and inhibit hepatocellular carcinoma (HCC) migration. Antrodia cinnamomea is widely prescribed as an adjuvant to treat HCC in Taiwan. We aimed to investigate if ethanol extract of fruiting bodies of Antrodia cinnamomea (EEAC) and its active ingredients (i.e., zhankuic acid A, cordycepin, and adenosine) can modulate HCC cancer cells migration through ERp57 and PGK-1 and other molecular pathways such as PI3K/Akt and MAPK. ERp57 and PGK-1 siRNA were transfected into HCC to determine effects on MMP-2/-9 expressions and cell migration. We then examined the inhibitory effects of EEAC and its active ingredients on HCC migration and its related mechanisms including ERp57, PGK-1, PI3K/Akt, and MAPK signaling pathways. Down-regulation of ERp57 and PGK-1 by siRNA decreased MMP-2, -9 expressions and Transwell cell migration in HCC. Nontoxic EEAC markedly inhibited migration of HCC, and significantly inhibited activities and protein expressions of MMP-2 and -9, while the expression of the endogenous inhibitors (TIMP-1 and TIMP-2) of these proteins increased. Nontoxic EEAC and its active ingredients decreased ERp57, GLUD-1, GST-pi, and PGK-1 protein expressions. Finally, nontoxic EEAC inhibited the phosphorylated FAK, PI3K/Akt, and MAPK signaling. Our findings first indicate that EEAC and its ingredients effectively suppress HCC migration. Additionally, the molecular mechanisms appear to be mediated, in part, through the down-regulation of ERp57, PGK-1, MAPK, and PI3K/Akt.

Zhankuic acid A as a novel JAK2 inhibitor for the treatment of concanavalin A-induced hepatitis

Fruiting bodies of Taiwanofungus camphoratus have been widely used as an antidote for food poisoning and considered to be a precious folk medicine for anti-inflammation and hepatoprotection. Zhankuic acid A (ZAA) is its major pharmacologically active compound. Janus kinase 2 (JAK2), whose activation is involved in cytokine signaling, plays critical roles in the development and biology of the hematopoietic system. JAK2 has been implicated as a therapeutic target in inflammatory diseases. The HotLig modeling approach was used to generate the binding model for ZAA with JAK2, showing that ZAA could bind to the ATP-binding pocket of JAK2 exclusively via the H-bond. The interaction between ZAA and JAK2 was verified by antibody competition assay. Binding of ZAA to JAK2 reduced antibody recognition of native JAK2. The expressions of phosphorylated JAK2 and STATs were analyzed by immuno-blotting. ZAA reduced the phosphorylation and downstream signaling of JAK2, and inhibited the interferon (IFN)-γ/signal transducer and activator of transcription (STAT) 1/interferon regulatory factor (IRF)-1 pathway. The protective effect of ZAA on liver injury was evaluated in mice by Con-A-induced acute hepatitis. Pre-treatment with ZAA also significantly ameliorated acute liver injury in mice. Therefore, ZAA can inhibit JAK2 phosphorylation and protect against liver injury during acute hepatitis in mice. In this study, we present data that ZAA exerts anti-inflammatory effects through the JAK2 signaling pathway. As such, ZAA may be a potential therapeutic agent for the treatment of inflammatory diseases.

Zhankuic Acid A Isolated from Taiwanofungus camphoratus Is a Novel Selective TLR4/MD-2 Antagonist with Anti-Inflammatory Properties

TLR4, a membrane receptor that functions in complex with its accessory protein myeloid differentiation factor-2 (MD-2), is a therapeutic target for bacterial infections. Taiwanofungus camphoratus is highly valued as a medicinal mushroom for cancer, hypertension, and inflammation in traditional medicine. Zhankuic acid A (ZAA) is the major pharmacologically active compound of T. camphoratus. The mechanism of action of T. camphoratus or ZAA has not been fully elucidated. We analyzed the structure of human TLR4/MD-2 complex with ZAA by X-score and HotLig modeling approaches. Two Abs against MD-2 were used to verify the MD-2/ZAA interaction. The inflammation and survival of the mice pretreated with ZAA and injected with LPS were monitored. The modeling structure shows that ZAA binds the MD-2 hydrophobic pocket exclusively via specific molecular recognition; the contact interface is dominated by hydrophobic interactions. Binding of ZAA to MD-2 reduced Ab recognition to native MD-2, similar to the effect of LPS binding. Furthermore, ZAA significantly ameliorated LPS-induced endotoxemia and Salmonella-induced diarrhea in mice. Our results suggest that ZAA, which can compete with LPS for binding to MD-2 as a TLR4/MD-2 antagonist, may be a potential therapeutic agent for gram-negative bacterial infections.

Elucidating the inhibitory mechanisms of the ethanolic extract of the fruiting body of the mushroom Antrodia cinnamomea on the proliferation and migration of murine leukemia WEHI-3 cells and their tumorigenicity in a BALB/c allograft tumor model

The aim of this study was to explore whether the ethanolic extract of Antrodia cinnamomea (EEAC), a medical mushroom form Taiwan, could affect the proliferation and migration of WEHI-3 cells in vitro and to explore the antitumor effects of EEAC in BALB/c mice engrafted with WEHI-3 cells. The results showed that EEAC inhibited the proliferation of WEHI-3 cells, resulting in the accumulation of cell in G0/G1 and G2/M phases, as determined by flow cytometry. Moreover, EEAC markedly reduced the migration of WEHI-3 cells, as determined by a transwell assay. Treatment of WEHI-3 cells with EEAC also decreased MMP-9 protein expression and enzyme activity. The protein levels of p-Akt, p-ERK1/2 were also decreased, whereas the expression of p21 and p27 was increased. Furthermore, in an in vivo model, EEAC treatment reduced the infiltration of WEHI-3 cells into the liver and spleens and decreased tumor growth. Other bioactive compounds, such as cordycepin and zhankuic acid A, have been demonstrated to reduce the expression of MMP-9, cyclin E, cyclin D1 and to increase the expression of p21, p27. This is the first study to investigate that the mechanisms by which EEAC reduce the proliferation and migration of WEHI-3 cells in vitro, as well as the ability of EEAC to reduced infiltration of WEHI-3 cells into the liver and spleen in vivo. The results suggest that EEAC may prove to be useful in future antileukemic therapies.

Antileukemia component, dehydroeburicoic acid from Antrodia camphorata induces DNA damage and apoptosis in vitro and in vivo models

Antrodia camphorata (AC) is a native Taiwanese mushroom which is used in Asian folk medicine as a chemopreventive agent. The triterpenoid-rich fraction (FEA) was obtained from the ethanolic extract of AC and characterized by high performance liquid chromatography (HPLC). FEA caused DNA damage in leukemia HL 60 cells which was characterized by phosphorylation of H2A.X and Chk2. It also exhibited apoptotic effect which was correlated to the enhancement of PARP cleavage and to the activation of caspase 3. Five major triterpenoids, antcin K (1), antcin C (2), zhankuic acid C (3), zhankuic acid A (4), and dehydroeburicoic acid (5) were isolated from FEA. The cytotoxicity of FEA major components (1–5) was investigated showing that dehydroeburicoic acid (DeEA) was the most potent cytotoxic component. DeEA activated DNA damage and apoptosis biomarkers similar to FEA and also inhibited topoisomerase II. In HL 60 cells xenograft animal model, DeEA treatment resulted in a marked decrease of tumor weight and size without any significant decrease in mice body weights. Taken together, our results provided the first evidence that pure AC component inhibited tumor growth in vivo model backing the traditional anticancer use of AC in Asian countries.

Ethanol extracts of fruiting bodies of Antrodia cinnamomea exhibit anti-migration action in human adenocarcinoma CL1-0 cells through the MAPK and PI3K/AKT signaling pathways

Cancer metastasis is a primary cause of cancer death. Antrodia cinnamomea (A. cinnamomea), a medicinal mushroom in Taiwan, has been shown antioxidant and anticancer activities. In this study, we first observed that ethanol extract of fruiting bodies of A. cinnamomea (EEAC) exerted a concentration-dependent inhibitory effect on migration and motility of CL1-0 cells in the absence of cytotoxicity. The results of a gelatin zymography assay showed that A. cinnamomea suppressed the activity of matrix metalloproteinase (MMP)-2 and MMP-9 in a concentration-dependent manner. Western blot results demonstrated that treatment with A. cinnamomea decreased the expression of MMP-9 and MMP-2; while the expression of the endogenous inhibitors of these proteins, i.e., tissue inhibitors of MMP (TIMP-1 and TIMP-2) increased. Two major compounds from EEAC codycepin and zhankuic acid A alone and together inhibited MMP-9 and MMP-2 expressions. Further investigation revealed that A. cinnamomea suppressed the phosphorylation of p38, and JNK1/2. A. cinnamomea also suppressed the expressions of PI3K and phosphorylation of AKT. This is the first report confirming the anti-migration activity of this potentially beneficial mushroom against human lung adenocarcinoma CL1-0.

Chemical profiling of the cytotoxic triterpenoid-concentrating fraction and characterization of ergostane stereo-isomer ingredients from Antrodia camphorata

Antrodia camphorata (AC), also known as Antrodia cinnamomea, an endemic species in Taiwan, is one of the treasured medicinal mushrooms. AC is traditionally used for its chemopreventive biofunctions. In this investigation, we report a convenient method for concentrating the antiproliferative active triterpenoid-rich fraction (FEA), from ethanolic extract of AC (EEAC). A series of stereo-isomers of zhankuic acids ( 1– 8) from the FEA was purified by HPLC using an efficient acidic solvent system. The structures of compounds 1– 8 were elucidated based on spectroscopic data analysis, and the absolute configuration of α-chiral carboxylic acid at C-25 in the structures was assigned based on reaction with ( R)- and ( S)-1-(9-anthryl)-2,2,2-trifluoroethanol. Major ingredients of FEA (eight ergostanes 1– 8 and two lanostanes 9– 10) were further characterised by high-performance liquid chromatography-photodiode array detection/mass spectrometry (HPLC-PDA/MS). Compounds 1– 8 and their pair mixture forms (antcin K, antcin C, zhankuic acid C, and zhankuic acid A) were subjected to anti-proliferative assay against three human leukemia cell lines. Among them, the derivatives with carbonyl group at C-3 showed cytotoxicity with IC 50 values ranging from 16.44 to 77.04 μg/ml.

Differentiation of mycelia and basidiomes of Antrodia cinnamomea using certain chemical components.

Using 10 known components in Antrodia cinnamomea including 5 ergostanes (antcins C and K, and zhankuic acids A, B, and C), 4 lanostanes (sulphurenic acid, dehydrosulphurenic acid, eburicoic acid, and dehydroeburicoic acid), and 1 monophenyl (4,7-dimethoxy-5-methyl-1,3-benzodioxole) as standards, mycelia and basidiomes of A. cinnamomea were differentiated in this study. Natural basidiomes collected from wood of Cinnamomum kanehirai in natural forests and cultured basidiomes grown on potato dextrose agar medium contained all 10 test components. However, natural mycelia collected from the wood of C. kanehirai in a natural forest and liquid/solid cultured mycelia grown on potato dextrose broth/potato dextrose agar media contained the 4 lanostanes and 4,7-dimethoxy-5-methyl-1,3-benzodioxole but not the 5 ergostanes. These results indicate that the production of ergostanes is related to basidiomatal formation of A. cinnamomea, but is not related to the substrate on which the organism is grown.

Identification of triterpenoids from the fruiting body and mycelium of Antrodia camphorata with HPLC-ESI/MS

Antrodia camphorata Wu, Ryvarden & Chang (Polyporaceae) is an endemic parastic fungus in Taiwan. It grows slowly on the heartwood of Cinnamomum kanehirai and produced orange-red color fruiting body on the surface of wood. The fruiting body of A. camphorata has been used as antidote for food or drug intoxication and treatment for hepatoma. The extract or compounds isolated from fruiting body or mycelium of A. campharata showed immuno-enhance, neuroprotective, hepatoprotective, cytotoxicity on tumor cells, anti-inflammatory, and antioxidative, activities. The bioactive components of A. camphorata were identified as polysaccharides, triterpenoids, steroids, bezenoids, and maleic acd/succinic acid derivatives. High-performance liquid chromatography-photodiode array detector-electrospray ionization mass spectrometry (HPLC-PAD-ESI/MS) was developed to characterize the triterpenoids in A. camphorata mycelium and fruiting body. Zhankuic acid D, dehydroeburicoic acid (or antcin B), dehydroesulfurenic acid, zhankuic acid, antcin C, and antcin A were identified with selected ion monitoring (SIM) of 481, 467, 483, 485, 469, and 453 amu ions from ethanol extract of A. camphorata fruiting body. Antcin C, antcin A, zhankuic acid C, dehydresulfurinic acid, and dehydroeburicoic acid (or antcin B) were identified with SIM of 469, 453, 485, 483, and 467 amu ions from ethanol extract of A. camphorata mycelium. This HPLC-PAD-ESI/MS is suitable to identify the triterpenoids in the commercial products of A. camphorata mycelium or fruiting body.