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Inhibitory effects of antrodins A-E from Antrodia cinnamomea and their metabolites on hepatitis C virus protease
Abstract
Antrodia cinnamomea is a highly valued folk medicine used for liver cancer, a disease often caused by the long term infection of hepatitis C virus (HCV). In the present study, the maleic and succinic acid constituents (antrodins A-E) of this medicinal fungus, the in vivo metabolites of antrodin C and the analogue of one of the metabolites were tested for their inhibitory activity on HCV protease. Most of the compounds showed potent inhibitory activity, with antrodin A being the most potent (IC(50) = 0.9 microg/mL). Antrodin A was isolated as one of the constituents of A. cinnamomea and was also detected as an in vivo metabolite of the major constituent antrodin C. The mode of inhibition for antrodin A on HCV protease was revealed by a Lineweaver-Burk plot as competitive inhibition. These results strongly support the use of this folk medicine for liver cancer and HCV infection which is a global problem.
... Antrodin C is the most widely studied antrodin compound, with studies mainly focusing on its biological activity and pharmacology. Antrodin C has been shown to substantially inhibit hepatitis C virus and Lewis lung carcinoma tumour cells, and it exhibits better inhibition of TGF-β1-induced epithelial-mesenchymal transition and breast cancer cell metastasis [8,9]. ...
... wrbA may respond to environmental stress when multiple electron transfer chains are compromised or when the environment is highly oxidised, which may favour antrodin C synthesis. Antrodin C is a triquinane-type sesquiterpene with potent inhibitory activity against the hepatitis C virus [9,30]. The significant downregulation of inositol-and MAC-related ubiquinone genes may be attributed to the fact that the addition of inositol and cis-butenedioic acid leads to the activation of the antrodin C biosynthesis pathway. ...
Antrodin C, a bioactive component of Taiwanofungus camphoratus, exhibits good immunophysiological and antitumour activities, including a broad spectrum of anticancer effects. Exogenous additives can bind to metabolites during the submerged culture of T. camphoratus and affect secondary metabolite yields. However, the lack of molecular genetic studies on T. camphoratus has hindered the study of the antrodin C biosynthetic pathway. In this study, we conducted a ribonucleic acid-sequencing-based transcriptional analysis to identify the differentially expressed genes involved in the synthesis of antrodin C by T. camphoratus, using inositol and maleic acid (MAC) as exogenous additives. The addition of inositol significantly upregulated carbohydrate and sugar metabolism pathway genes (E3.2.1.14, UGDH, and IVD). When MAC was used, amino and nucleotide sugar metabolism and starch and sucrose metabolism pathways were significantly inhibited, and the associated genes (E3.2.1.14 and E3.2.1.58) were also significantly downregulated. The biosynthesis pathway genes for ubiquinone and other terpene quinones (COQ2, ARO8, and wrbA), which may play an important role in antrodin C synthesis, were significantly downregulated. This study advances our understanding of how the additives inositol and MAC affect metabolite biosynthesis in T. camphorates. This could be beneficial in proposing potential strategies for improving antrodin C production using a genetic approach.
... Taiwanofungus camphorates (M.ZangC.H.Su) Sheng H. Wu et al. is a treasured Taiwanese mushroom which only parasitizes in the inner cavity of the endemic species Cinnamomum kanehirae Hayata, Lauraceae or the bull camphor tree [15,16]. Taiwanofungus camphoratus is known as the ruby in Taiwan's forest as a result of its excellent biological activities, which include antihepatotoxic, anticancer, anti-inflammatory, antihypertensive, neuroprotective, and antioxidant properties [17][18][19]. In 2016, its anticancer effect was useful for locating antroquinonol, a ubiquinone derivative isolated from the fruiting body of T. camphoratus, and was successfully entered into Phase II clinical trials for treating NSCLC due to its excellent anti-lung cancer effect [20]. ...
... According to reports, more than 80% of all bioactive mushroom compounds are isolated from their fruiting bodies. However, compounds from mycelial are considered to have great future potential due to their low cost and a vast market demand [18]. Our preliminary experiments have also shown an anti-tumor effect of ADC on lung cells which was better than for other malignant cells and is similar to the anti-tumor activity of antroquinonol. ...
The current study aims to explore the possible anti-lung carcinoma activity of ADC as well as the underlying mechanisms by which ADC exerts its actions in NSCLC. Findings showed that ADC potently inhibited the viability of SPCA-1, induced apoptosis triggered by ROS, and arrested the cell cycle at the G2/M phase via a P53 signaling pathway. Interestingly, phenomena such as autophagosomes accumulation, conversion of the LC3-I to LC3-II, etc., indicated that autophagy could be activated by ADC. The blockage of autophagy-augmented ADC induced inhibition of cell proliferation, while autophagy activation restored cell death, indicating that autophagy had a protective effect against cell death which was induced by ADC treatment. Meanwhile, ADC treatment suppressed both the Akt/mTOR and AMPK signaling pathways. The joint action of both ADC and the autophagy inhibitor significantly increased the death of SPCA-1. An in vitro phase I metabolic stability assay showed that ADC was highly metabolized in SD rat liver microsomes and moderately metabolized in human liver microsomes, which will assist in predicting the outcomes of clinical pharmacokinetics and toxicity studies. These findings imply that blocking the Akt/mTOR signaling pathway, which was independent of AMPK inhibition, could activate ADC-induced protective autophagy in non-small-cell lung cancer cells.
... It is well-known as a traditional Chinese medicine (TCM) and the original inhabitants of Taiwan used this species for the prevention or treatment of numerous diseases including liver diseases, food poisoning and drug intoxication, diarrhoea, abdominal pain, hypertension, tumourigenic diseases and itchy skin [12,13]. It has been reported that an ethanol extract from the mycelia of A. camphorata displayed anti-HBV and anti-HCV activity in a dosedependent manner without cytotoxicity and some compounds have been isolated from the ethanol extract [14][15][16][17]. ...
... The earlier reports indicated that the fruiting body and mycelia of A. camphorata contain several secondary metabolites like terpenoids, diterpenic lactones, triterpene, benzenoids, lignans, benzoquinone derivatives, succinic and maleic derivatives, with anti-cancer, anti-inflammatory, anti-oxidant, hepatoprotective and anti-hepatitis B and C replication activity [23]. Antrodin A-E belong to succinic and maleic derivatives, showing anti-cancer, anti-inflammatory and anti-hepatitis B/C effects [14,15,17,24,25]. In conclusion, antrodin A and extracts from A. caphorata have antiviral effects, presumably a new antiviral mechanism that is different to acyclovir and it could be an alternative and complementary agent. ...
Antrodia camphorata, known as a Traditional Chinese Medicine (TCM), are widely used in treatment liver diseases and cancers. Besides those important activities, other biological activities, such as anti-inflammatory have been described. Herpes simplex virus infections represent one of the most serious public health concerns globally because of their devastating impact. Searching for new antiviral agents, especially those with different mechanisms of action, is a crucial goal and an unmet need for alternative and complementary therapy against HSV infection. In this study, an antiherpes screening was performed to obtained extracts from A. camphorata mycelia.
MTT method and FIC theorem and median-effect principle were used to evaluate anti-viral activity and calculate drug combination effect.
The crude ethanol extracts and isolated constituents showed inhibition of HSV replication at very low concentration. Fraction A and antrodin A showed viral inhibitory effect with reduction of viral cell-to-cell spread. In addition, neither fraction A nor antrodin A showed interaction in combination with acyclovir.
A.camphorata mycelia and antrodin A might be potential to use as anti-HSV agents and promising for future antiviral drug design.
... Among its diverse pharmacological activities, the evidence for hepatic protection is the most recognized and the strongest. Studies have shown that AC can inhibit hepatic tumor growth and retard the progression of hepatitis [3,4]. Although AC is currently used as a food supplement, the Food and Drug Administration (FDA) has not approved any AC extracts or purified compounds for clinical applications. ...
... Inhibition of viral proteases, which are crucial for viral replication, can interrupt viral protein maturation. Antrodins (A to E) and their metabolic analogues selectively inhibited HCV NS3/4A protease activity [3]. Among these, antrodin A (the major in vivo metabolite of antrodin C) showed the highest potency with an IC 50 of 0.9 μg/mL, while antrodin B showed lower potency with an IC 50 of > 100 μg/mL. ...
Antrodia cinnamomea (AC) is an endemic mushroom species of Taiwan, and has been demonstrated to possess diverse biological and pharmacological activities, such as anti-hypertension, anti-hyperlipidemia, anti-inflammation, anti-oxidation, anti-tumor, and immunomodulation. This review focuses on the inhibitory effects of AC on hepatitis, hepatocarcinoma, and alcohol-induced liver diseases (e.g., fatty liver, fibrosis). The relevant biochemical and molecular mechanisms are addressed. Overall, this review summarizes the hepatoprotective activities in vitro and in vivo. However, there is no doubt that human and clinical trials are still limited, and further studies are required for the development of AC-related products.
... Malic acid and succinic acid derivatives are the primary bioactive compounds in the liquid fermentation of A. camphorata. Among these compounds, antrodin A exhibits excellent inhibitory activity against the protease of the hepatitis C virus, while antrodin B and C demonstrate significant cytotoxicity against lung cancer cell lines [41]. Liquid fermentation allows for better control of the cultivation environment. ...
Antimicrobial resistance is a major threat to human health globally. Antrodia camphorata was grown in a malt/yeast extract broth liquid medium for 15 days. Then, 4-L fermentation broth was harvested, yielding 7.13 g of the ethyl acetate extract. By tracing the antimicrobial activity, 12.22 mg of the antimicrobial compound was isolated. The structure of 5-methyl-benzo [1,3]-dioxole-4,7-diol (MBBD) was elucidated using NMR and MS data analyses. The antibacterial activity of MBBD was detected through the microbroth dilution method. MBBD exhibited broad-spectrum antibacterial activity. The minimum inhibitory concentration (MIC) range of MBBD for drug-resistant pathogenic bacteria was 64–256 μg/mL, with the lowest MIC observed for Acinetobacter baumannii (64 μg/mL), followed by Pseudomonas aeruginosa (MIC = 128 μg/mL). Klebsiella pneumoniae, Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli were also sensitive, with an MIC of 256 μg/mL. The MIC range of MBBD against 10 foodborne pathogens was 12.5–100 μg/mL. Based on the results of this study, MBBD exhibits broad-spectrum antibacterial activity, particularly demonstrating excellent inhibitory effects against A. baumannii. MBBD will be good candidates for new antimicrobial drugs.
... Later studies have confirmed five maleic and succinic acid derivatives, which were named antrodins A-E [76]. Researchers have optimized their separation procedures due to their significant bioactivity. ...
Antrodia cinnamomea is a precious and popular edible and medicinal mushroom. It has attracted increasing attention due to its various and excellent bioactivities, such as hepatoprotection, hypoglycemic, antioxidant, antitumor, anticancer, anti-inflammatory, immunomodulation, and gut microbiota regulation properties. To elucidate its bioactivities and develop novel functional foods or medicines, numerous studies have focused on the isolation and identification of the bioactive compounds of A. cinnamomea. In this review, the recent advances in bioactivity, isolation, purification, and identification methods of active compounds from A. cinnamomea were summarized. The present work is beneficial to the further isolation and discovery of new active compounds from A. cinnamomea.
... Antrodia cinnamomea, a medicinal mushroom, produced antrodins A-E. Antrodins A showed potent inhibitory capabilities of HCV protease activity [97]. Five products that were obtained from the endophytic fungus Emericella nidulans, namely cordycepin, emericellin, ergosterol peroxide, myristic acid, and sterigmatocystin, reported having HCV NS3/4A protease inhibitory properties [98][99][100][101][102]. ...
The inhibition of viral protease is an important target in antiviral drug discovery and development. To date, protease inhibitor drugs, especially HIV-1 protease inhibitors, have been available for human clinical use in the treatment of coronaviruses. However, these drugs can have adverse side effects and they can become ineffective due to eventual drug resistance. Thus, the search for natural bioactive compounds that were obtained from bio-resources that exert inhibitory capabilities against HIV-1 protease activity is of great interest. Fungi are a source of natural bioactive compounds that offer therapeutic potential in the prevention of viral diseases and for the improvement of human immunomodulation. Here, we made a brief review of the current findings on fungi as producers of protease inhibitors and studies on the relevant candidate fungal bioactive compounds that can offer immunomodulatory activities as potential therapeutic agents of coronaviruses in the future.
... AC is a succinic/maleic acid derivative and is potentially one of the most biologically active components in A. camphorata. The effect of AC on antiinflammatory (Wu et al. 2008), antitumour (Nakamura et al. 2004), and antiviral (Phuong et al. 2009;Liu et al. 2010) activities has garnered particular attention. However, the difficulty of mass-producing AQ and AC has hampered their possible clinical application. ...
This study describes the application of in situ extractive fermentation (ISEF) to increase the yields of antroquinonol (AQ) and antrodin C (AC) from Antrodia camphorata S-29. In initial screening experiments, nine solvents were tested to identify the most suitable extractant for the in situ extraction of AQ and AC. These solvents included n-tetradecane, n-dodecane, n-decane, heavy paraffin, light paraffin, oleyl alcohol, oleic acid, butyl oleate, and isopropyl myristate. Of these, oleic acid was the most suitable solvent for the in situ extraction of AQ and AC. The use of oleic acid as an in situ extractant significantly improved AQ and AC productions, which were approximately 5-fold and 8-fold that of the control, respectively. The recovered oleic acid was treated with a silica gel solid-phase extraction column, which was able to rapidly adsorb the bioactive metabolites. The separated solvent hardly contained fermentation products and could be directly reused in ISEF. AQ and AC were obtained with purities of over 75% by silica gel column chromatography. The recoveries of AQ and AC reached 70.7 ± 0.8% and 81.5 ± 1.2%, respectively.
... There are five novel maleic acid and succinic acid derivatives isolated from the mycelia of A. cinnamomea (Antrodin A-E), which have shown potent cytotoxic effects on LLC tumor cell lines [86]. It was found that Antrodins could inhibit the activity of the HCV virus, with Antrodin B and Antrodin C having the strongest cytotoxic effects [86][87][88]. In a previous study, Antrodin B was shown to serve as a potential candidate for treatment of liver fibrosis [89]. ...
Edible and medicinal mushrooms have usually been considered as a sustainable source of unique bioactive metabolites, which are valued as promising provisions for human health. Antrodia cinnamomea is a unique edible and medicinal fungus widespread in Taiwan, which has attracted much attention in recent years for its high value in both scientific research and commercial applications owing to its potent therapeutic effects, especially for its hepatic protection and anticancer activity. Due to the scarcity of the fruiting bodies, the cultivation of A. cinnamomea by submerged fermentation appears to be a promising substitute which possesses some unique advantages, such as short culture time period and its high feasibility for scale-up production. However, the amount of fungal bioactive metabolites derived from the cultured mycelia of A. cinnamomea grown by submerged fermentation is much less than those obtained from the wild fruiting bodies. Hence, there is an urgent need to bridge such a discrepancy on bioactive metabolites between the wild fruiting bodies and the cultured mycelia. The objective of this article is to review recent advances and the future development of the mycelial submerged fermentation of A. cinnamomea in terms of enhancement for the production of fungal bioactive components by the optimization of culture conditions and the regulation of fungal metabolism. This review provides valuable information for further biotechnological applications of A. cinnamomea as well as other mushrooms being the source of bioactive ingredients by submerged fermentation.
... Cytotoxic, anti-inflammatory and hepatoprotective properties of the active components of A. cinnamomea extracts have been reported previously [23][24][25] . In addition, camphorataimide B, an active component of A. cinnamomea, exhibited potent cytotoxic activities in lung cancer cells (A549), leukemia cells (HL-60) and breast cancer cells (MCF-7) and (MDA-MB-231) 26 . ...
Abstract Hepatocellular carcinoma (HCC) has been recognized worldwide as one of the major causes of cancer death. The medicinal fungus Antrodia cinnamomea (A. cinnamomea) has been served as a functional food for liver protection. The aim of the present study was to investigate the potential activity of A. cinnamomea extracts as a safe booster for the anticancer activity of sorafenib, a multi-kinase inhibitor approved for the treatment of HCC. The biologically active triterpenoids in the ethanolic extracts of A. cinnamomea (EAC) were initially identified by HPLC/LC/MS then the different extracts and sorafenib were assessed in vitro and in vivo. EAC could effectively sensitize HCC cells to low doses of sorafenib, which was perceived via the ability of the combination to repress cell viability and to induce cell cycle arrest and apoptosis in HCC cells. The ability of EAC to enhance sorafenib activity was mediated through targeting mitogen-activated protein (MAP) kinases, modulating cyclin proteins expression and inhibiting cancer cell invasion. Moreover, the proposed combination significantly suppressed ectopic tumor growth in mice with high safety margins compared to single-agent treatment. Thus, this study highlights the advantage of combining EAC with sorafenib as a potential adjuvant therapeutic strategy against HCC.
... Considering the cytotoxic activity, it was reported that camphorataimide B displayed a potent anticancer activity in human breast cancer, leukemia cells, and human lung cancer cells (Lin et al., 2012). For hepatoprotective activity, maleic and succinic acid derivatives from the AC mycelia were involved in inhibition of HCV protease (Phuong do et al., 2009). In addition, some of the A. cinnamomea extract components such as methyl antcinate A, antcin B, and antcin K were able to induce apoptotic cell death in HCC (Hsieh et al., 2010(Hsieh et al., , 2011Huang et al., 2015;Lai et al., 2016). ...
Antrodia cinnamomea (AC) is a medicinal fungal species that has been widely used traditionally in Taiwan for the treatment of diverse health-related conditions including cancer. It possesses potent anti-inflammatory and antioxidant properties in addition to its ability to promote cancer cell death in several human tumors. Our aim was to improve the anticancer activity of AC in hepatocellular carcinoma (HCC) through its cocultivation with ginger aiming at tuning the active ingredients. HCC cell lines, Huh-7 and HepG2 were used to study the in vitro anticancer activity of the ethanolic extracts of AC (EAC) alone or after the cocultivation in presence of ginger (EACG). The results indicated that the cocultivation of AC with ginger significantly induced the production of important triterpenoids and EACG was significantly more potent than EAC in targeting HCC cell lines. EACG effectively inhibited cancer cells growth via the induction of cell cycle arrest at G2/M phase and induction of apoptosis in Huh-7 and HepG2 cells as indicated by MTT assay, cell cycle analysis, Annexin V assay, and the activation of caspase-3. In addition, EACG modulated cyclin proteins expression and mitogen-activated protein kinase (MAPK) signaling pathways in favor of the inhibition of cancer cell survival. Taken together, the current study highlights an evidence that EACG is superior to EAC in targeting cancer cell survival and inducing apoptotic cell death in HCC. These findings support that EACG formula can serve as a potential candidate for HCC adjuvant therapy.
... We previously reported that Antrodin C, a maleimide derivative isolated from the mycelia of A. cinnamomea inhibits TGF-βinduced breast cancer cell metastasis via inhibition of epithelial-to-mesenchymal transition in vitro [29]. Another study shows that ADC inhibits hepatitis C virus (HCV) protease activity in vitro [42]. However, other biological effects of this potentially beneficial compound are not well elucidated. ...
In the present study, we investigated the effects of antrodin C (ADC), a maleimide derivative isolated from mycelia of Antrodia cinnamomea, on high glucose (HG, 30 mM)-accelerated endothelial dysfunction in vitro. HG-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs) was significantly ameliorated by ADC. In addition, treatment with ADC significantly prevented HG-induced senescence, growth arrest at the G 1-S transition phase and apoptosis in HUVECs. Moreover, the increased level of intracellular reactive oxygen species (ROS) under HG condition was significantly ameliorated by ADC. Further analysis revealed that ADC-mediated anti-oxidant effects were due to up-regulation of cellular anti-oxidant genes, such as HO-1 and NQO-1 via promotion of the transcriptional activity of Nrf2, which was further confirmed by the failure of ADC to protect HUVECs from HG-induced dysfunction under HO-1 inhibition or Nrf2 silencing. Furthermore, hyperosmotic glucose (HOG, 60 mM)-induced uncontrolled production of ROS, rapid apoptotic cell death and HUVEC injury were significantly prevented by ADC, whereas these preventive effects were barely observed in HO-1 inhibited or Nrf2 silenced cells. Taken together, these results suggest that ADC may represent a promising intervention in diabetic-associated cardiovascular diseases by activating the Nrf2-dependent cellular anti-oxidant defense system.
... a: R= 2,4-dimethoxy 5-pyrimidyl, R 1 = 3,4,5-trimethoxy; b: R= 2,4-dimethoxy 5-pyrimidyl, R 1 = 4-hydroxy-3-methoxy; c: R= 2,4-dimethoxy 5-pyrimidyl, R 1 = 3-hydroxy-4-methoxy; d: R= 2,4-dimethoxy 5-pyrimidyl, R 1 = 4-fluoro-3methoxy; e: R= 5-indyl, R 1 = 3,4,5-trimethoxy; f: R= 5-indyl, R 1 = 4-hydroxy-3-methoxy; g: R= 5-indyl, R 1 = 3-hydroxy-4methoxy; h: R= 5-indyl, R 1 = 4-fluoro-3-methoxy; i: R= 5-indyl, R 1 = 3-nitro-4-methoxy; j: R= 2-methyl-5-indyl, R1= 3,4,5-trimethoxy; k: R= 2-methyl-5-indyl, R1= 4-fluoro-3-methoxy; l: R= 2-methyl-5-indyl, R 1 = 2-fluoro-4-methoxy; m: R= 5-indazolyl, R 1 = 4-hydroxy-3-methoxy; n: R= 5-indazolyl, R 1 = 3-hydroxy-4-methoxy; o: R= 5-indazolyl, R 1 = 4fluoro-3-methoxy; p: R= 6-benzthiazolyl, R 1 = 3,4,5-trimethoxy; q: R= 2-methyl-6-benzthiazolyl, R 1 = 3-nitro-4methoxy; r: R= 2-mercapto-5-imidazolyl, R 1 = 3-hydroxy-4-methoxy; s: R= 2,4-dimethoxy 5-pyrimidyl, R 1 = 3,4,5trimethoxy; t: R= 5-triazolyl, R 1 = 2-fluoro-4-methoxy; u: R= 3-(4-methoxyaryl)-5-isoxazolyl, R 1 = 3,4,5-trimethoxy; v: R= 3-(4-methoxyaryl)-5-isoxazolyl, R 1 = 3-hydroxy-4-methoxy; w: R= 2,3,4-trimethoxyaryl, R 1 = 4-hydroxy-3-methoxy. mg/mL) [62]. Compound 53 has been shown to inhibit the production of pro-inflammatory mediators in macrophages (e.g., NO and IL-6) [63,64], although its limited availability has prevented more comprehensive screening [25,65]. ...
This work highlights the literature of one of the most valuable moieties in the field of organic chemistry. In this review, the chemistry of tetronic acid as a simple precursor to privileged heterocyclic motifs is described. The synthetic procedures of different fused heterocycles incorporating a furan moiety are described. Fused heterocycles are classified as bicyclic, tricyclic, tetracyclic and spiro-fused pyran derivatives.
... Taiwanese have a long history to use AC as a medicine for several body disorders, such as diarrhea, intoxication, hypertension, stomachache, inflammation, etc [23]. Recent studies further revealed that AC has some hepatoprotective effects, for example, inhibition of viral antigen activity of hepatitis viruses [24], reduction of oxidative stress of alcohol-induced liver diseases, and counteraction of liver fibrosis of the TAA-induced liver injury [25]. AC was also reported able to protect liver cells from free radical-induced oxidative stresses through the Nrf-2 activation and up-regulation of the MAP kinase-mediated antioxidant genes [26, 27]. ...
Whereas cisplatin (cis-diamminedichloroplatinum II) is a first-line medicine to treat solid cancerous tumors, it often causes serious side effects. New medicines that have an equivalent or even better therapeutic effect but with free or less side effects than cisplatin are highly anticipated in cancer therapy. Recent reports revealed that Antrodia cinnamomea (AC) possesses hepatoprotective activity in addition to anticancer. In this study, we wanted to know whether AC enhances chemo-sensitivity of cisplatin and/or alleviates cisplatin-induced hepatotoxicity, as well as the underlying mechanisms thereof. Our results indicated that AC inhibited proliferation of line-1 lung carcinoma cells and rescued hepatic HepG2 cells from cisplatin-induced cell death in vitro. The fact is that AC and cisplatin synergized to constrain growth of line-1 lung carcinoma cells in BALB/cByJ mice. Quantitative real-time PCR further revealed that AC promoted expression of apoptosis-related genes, while it decreased expression of NF-κB and VEGF in tumor tissues. In liver, AC reduced cisplatin-induced liver dysfunctions, liver inflammation and hepatic apoptosis in addition to body weight restoration. In summary, AC is able to increase cisplatin efficacy by triggering expression of apoptosis-related genes in line-1 lung cancer cells as well as to protect liver from tissue damage by avoiding cisplatin-induced hepatic inflammation and cell death.
... To determine the 50% inhibitory concentration (IC 50 ) of policresulen, different concentrations of policresulen (0, 0.001, 0.01, 0.1, 0.5, 1, 10, and 20 μg/mL) were prepared. The IC 50 value was calculated using Origin 5, and the inhibitor type was determined using the Lineweaver-Burk plot [32] . ...
Dengue is a severe epidemic disease caused by dengue virus (DENV) infection, for which no effective treatment is available. The protease complex, consisting of nonstructural protein 3 (NS3) and its cofactor NS2B, plays a pivotal role in the replication of DENV, thus may be a potential target for anti-DENV drugs. Here, we report a novel inhibitor of DENV2 NS2B/NS3 protease and its antiviral action.
An enzymatic inhibition assay was used for screening DENV2 NS2B/NS3 inhibitors. Cytotoxicity to BHK-21 cells was assessed with MTT assay. Antiviral activity was evaluated in BHK-21 cells transfected with Rlu-DENV-Rep. The molecular mechanisms of the antiviral action was analyzed using surface plasmon resonance, ultraviolet-visible spectral analysis and differential scanning calorimetry assays, as well as molecular docking analysis combined with site-directed mutagenesis.
In our in-house library of old drugs (∼1000 compounds), a topical hemostatic and antiseptic 2-hydroxy-3,5-bis[(4-hydroxy-2-methyl-5-sulfophenyl)methyl]-4-methyl-benzene-sulfonic acid (policresulen) was found to be a potent inhibitor of DENV2 NS2B/NS3 protease with IC50 of 0.48 μg/mL. Furthermore, policresulen inhibited DENV2 replication in BHK-21 cells with IC50 of 4.99 μg/mL, whereas its IC50 for cytotoxicity to BHK-21 cells was 459.45 μg/mL. Policresulen acted as a competitive inhibitor of the protease, and slightly affected the protease stability. Using biophysical technology-based assays and molecular docking analysis combined with site-directed mutagenesis, we demonstrated that the residues Gln106 and Arg133 of DENV2 NS2B/NS3 protease directly interacted with policresulen via hydrogen bonding.
Policresulen is a potent inhibitor of DENV2 NS2B/NS3 protease that inhibits DENV2 replication in BHK-21 cells. The binding mode of the protease and policresulen provides useful hints for designing new type of inhibitors against the protease.
... It is most well-known for its use as a traditional Chinese medicine that was first discovered by aboriginal Taiwanese. Over the past few years, A. cinnamomea has been reported to possess a wide range of biological functions, such as anti-oxidative, [6] vasorelaxatory, [7] anti-inflammatory, [8] anti-angiogenic, [9] anti-tumor, [10] anti-hepatic, [11] hepatoprotective, [12] and immunomodulative [13,14] activities. However, only one study has shown that the twig essential oil of C. kanehirae has strong antibacterial activity against nine pathogenic bacteria. ...
Cinnamomum kanehirae Hayata is an endemic tree species in Taiwan. Leaf essential oils from 26 sources of C. kanehirae were obtained by hydrodistillation and their chemical constituents were analyzed by gas chromatography-mass spectrometry (GC-MS). Fifty-eight compounds were identified from C. kanehirae leaf essential oils. The main constituents in leaf essential oils were linalool, 1,8-cineole, β-selinene, 1-hexadecyne, and α-cadinol. According to the chemical compositions of leaf essential oils and their relative contents obtained from GC-MS analyses, cluster analysis, and principal component analysis, the 26 sources examined were classified into five chemotypes: linalool type, linalool/1,8-cineole type, 1,8-cineole type, linalool/α-cadinol type, and mixed type. The biochemical correlations between the major constituents of C. kanehirae were examined and their relationship is discussed.
... Antrodia cinnamomea, a Taiwan-specific mushroom, has been reported to have numerous biological activities including hepatoprotection, anti-inflammation, 1 antihepatitis C virus activity, 2 and anticancer activity. 3 Liver cancer ranks fourth in cancerrelated mortality around the world (WHO 2008) and is the second leading cause of cancer deaths in Taiwan. ...
Liver cancer is the second leading cause of cancer deaths in Taiwan as per the 2011 statistics and ranks fourth in cancer-related mortality in the world. Recent researches have shown that Antrodia cinnamomea, a Taiwan-specific medicinal mushroom, has biological activities, including hepatoprotection, anti-inflammation, antihepatitis B virus activity, and anticancer activity. In the present study, the antiproliferative activity and molecular mechanisms of antcin K, the most abundant ergostane triterpenoid from the fruiting bodies of basswood cultivated A. cinnamomea, were investigated using human hepatoma Hep 3B cells. The results showed that antcin K effectively reduced Hep 3B cells viability within 48 hours. Antcin K induced phosphatidylserine exposure, chromatin condensation, and DNA damage, but did not significantly increase autophagosome content or cause cell expansion and cell lysis. Thus, the principal mode of Hep 3B cells death induced by antcin K was apoptosis, rather than autophagy or necrosis. In-depth investigation of the molecular mechanisms revealed that antcin K first promoted reactive oxygen species generation and adenosine triphosphate depletion, leading to endoplasmic reticulum stress and resulting in mitochondrial membrane permeability changes. After losing the mitochondrial membrane potential, caspase-independent and caspase-dependent apoptosis-related proteins were released, including HtrA2, apoptotic-induced factor, endonuclease G, and cytochrome c. Cytochrome c activated caspase-9 and caspase-3, and cut downstream protein PARP, ultimately leading to cell apoptosis. These results suggested that antcin K induced mitochondrial and endoplasmic reticulum stress-mediated apoptosis in human hepatoma cells. Coupled with these findings, antcin K has a potential to be a complementary agent in liver cancer therapy.
... Hepatitis C virus is one of leading cause of liver cancer in developed countries. It is estimated that 3% of the total world population is infected with this virus (Stauber & Stadlbauer, 2006).The effect of different maleic and succinic acid derivatives isolated from the AC mycelia (ACM) on HCV protease, a crucial enzyme for viral maturation through the conversion of the viral nonfunctional proteins to the functional form, was examined using SensoLyteTM 520 HCV protease assay kit (Do Thi et al., 2009). It was found that antrodin A (4) (Fig. 3) exhibited the most potent activity with IC 50 0.9 μg/mL. ...
... Fluorescence was measured by TECAN GENios plate reader at excitation/emission 485/530 nm. The assay was carried out on BD Falcon™ Microtest™ 384-well 120 µl black assay plates (lot#05391155) following the procedure described in a previous paper with DMSO as solvent for all compounds [10]. ...
To evaluate oleanolic acid derivatives on liver disease related bioactivities, 29 oleanolic acid derivatives of several series were tested for their inhibitory activity on hepatitis C viral protease and for their cytotoxic effects on Hep G2 cells.
The amino derivatives showed potent cytotoxicity, among which, the beta-amino isomer exhibited more distinct cytotoxicity than the alpha-isomer. The cytotoxicity of hemiesters and hemiamides varied as the chain lengths varied. The oxalic and malonic hemiesters showed weaker cytototoxicity than oleanolic acid, while those with longer chain lengths showed higher cytotoxicity. Contrary to the cytotoxic activity, the free amino derivatives showed little inhibitory activity on HCV protease. All the hemiesters and hemiamides showed high activity against HCV protease.
The findings that addition of amino-group enhanced the cytotoxicity and that introduction of acidic group increased the inhibition on HCV protease may be useful for further design and synthesis of triterpene derivatives as drug candidates for liver diseases.
... Antrodia cinnamomea is an alternative medicine that has been administered for treatment of HCVinduced hepatocellular carcinomas, and specific components of this fungus displayed functionalities that were explored for anti-HCV activity. Phuong et al. [66] report an IC 50 of 0.9 μg/mL in a cell-free assay, presenting encouraging preliminary data from which to design more advanced study. ...
Hepatitis C virus (HCV) causes significant morbidity and mortality worldwide with nearly 3% of the world population infected by this virus. Fortunately, this virus does not establish latency, and hence it may be possible to eradicate it. HCV is strongly associated with liver cirrhosis and hepatocellular carcinoma and is currently treated with pegylated interferon-alpha (peg-IFN-alpha) and ribavirin. Unfortunately, these limited treatment options often produce significant side effects, and currently, complete eradication of virus with combined drug modalities has not yet been achieved for the majority of chronically HCV-infected individuals. Restricted treatment options, lack of a universal cure for HCV and the link between chronic infection, liver cirrhosis and hepatocellular carcinoma necessitate design of novel drugs and treatment options. Understanding the relationship between the immune response, viral clearance and inhibition of viral replication with pharmacology-based design can ultimately allow for complete eradication of HCV. This review focuses upon significant novel preclinical and clinical specifically targeted antiviral therapy (STAT-C) drugs under development, highlights their mechanism of action, and discusses their impact on systemic viral loads and permanent clearance of infection.
... Isobutyl-4-[4-(3-methyl-2butenyloxy)phenyl]-1H-pyrrole-2,5dione (Antrodin B or Camphorataimide B) M, BIn vitro anti-inflammatory, anti-HBV and anti-HCV[26,29,69] ...
Antrodia camphorata is a unique mushroom of Taiwan, which has been used as a traditional medicine for protection of diverse health-related conditions. In an effort to translate this Eastern medicine into Western-accepted therapy, a great deal of work has been carried out on A. camphorata. This review discusses the biological activities of the crude extracts and the main bioactive compounds of A. camphorata. The list of bioactivities of crude extracts is huge, ranging from anti-cancer to vasorelaxation and others. Over 78 compounds consisting of terpenoids, benzenoids, lignans, benzoquinone derivatives, succinic and maleic derivatives, in addition to polysaccharides have been identified. Many of these compounds were evaluated for biological activity. Many activities of crude extracts and pure compounds of A. camphorata against some major diseases of our time, and thus, a current review is of great importance. It is concluded that A. camphorata can be considered as an efficient alternative phytotherapeutic agent or a synergizer in the treatment of cancer and other immune-related diseases. However, clinical trails of human on A. camphorata extracts are limited and those of pure compounds are absent. The next step is to produce some medicines from A. camphorata, however, the production may be hampered by problems related to mass production.
Medicinal mushrooms, such as Taiwanofungus camphoratus, Inonotus obliquus, and Tropicoporus linteus, have been used in traditional medicine for therapeutic purposes and promotion of overall health in China and many East Asian countries for centuries. Modern pharmacological studies have demonstrated the large amounts of bioactive constituents (such as polysaccharides, triterpenoids, and phenolic compounds) available in these medicinal mushrooms and their potential therapeutic properties. Due to the rising demand for the health-promoting medicinal mushrooms, various cultivation methods have been explored to combat over-harvesting of the fungi. Evidence of the robust pharmacological properties, including their anticancer, hypoglycemic, hypolipidemic, antioxidant, and antiviral activities, have been provided in various studies, where the health-benefiting properties of the medicinal fungi have been further proven through numerous clinical trials. In this review, the cultivation methods, available bioactive constituents, therapeutic properties, and potential uses of T. camphoratus, I. obliquus and T. linteus are explored.
This study aimed to investigate the protective effect of Antrodin A (AdA) from Antrodia camphorata (A. camphorata) mycelium on alcohol-induced gut microbiota and liver metabolomic disorders. In acute alcoholic liver injury mice, AdA ameliorated alcoholic exposure-induced hepatic lipid deposition (TC and TG), oxidative stress (MDA), inflammation (TNF-α, IL-1β, IL-6, IL-17 and IFN-γ), and liver damage via modulating microbiome and metabolomic responses. AdA restored the composition of intestinal flora with an increase in the relative abundance of Lactobacillus and Dubosiella and a decrease in Clostridium_sensu_stricto_1, Lachnospiraceae_NK4A136_group, Prevotellaceae_NK3B31_group, and Prevotellaceae_UCG-001. Besides, AdA favorably regulated alcohol-induced metabolic disorders, including glutathione metabolism (S-(2-hydroxyethyl)glutathione and glutathione oxidized), ascorbate and aldarate metabolism (l-ascorbic acid), and taurine and hypotaurine metabolism (taurine). In conclusion, AdA in A. camphorata is a beneficial active ingredient to treat the microbiomic and metabolic disturbance induced by alcohol intake.
BACKGROUND
In situ extractive fermentation (ISEF) is an important technique for improving metabolite productivity. The different extractants can induce the synthesis of different bioactive metabolites of Antrodia camphorata during ISEF. However, a lack of research on the molecular genetics of A. camphorata during ISEF currently hinders such studies on metabolite biosynthetic mechanisms.
RESULTS
To clarify the differentially expressed genes during ISEF, the gene transcriptional expression features of A. camphorata S‐29 were analysed. The addition of n‐tetradecane as an extractant during ISEF showed more pronounced up‐regulation of ubiquinone and other terpenoid‐quinone biosynthesis pathway genes (CoQ2, wrbA and ARO8). When oleic acid was used as an extractant, the terpenoid backbone biosynthesis and ubiquinone and other terpenoid‐quinone biosynthesis pathways were significantly enriched, and genes (IDI, E2.3.3.10, HMGCR atoB, and CoQ2) related to these two pathways were also significantly up‐regulated. The CoQ2 genes encode puru‐hydroxybenzoate:polyprenyltransferase, playing an important role in antroquinonol synthesis. The IDI, E2.3.3.10, HMGCR and atoB genes of the terpenoid backbone biosynthesis pathway might play an important role in the synthesis of the triquine‐type sesquiterpene antrodin C.
CONCLUSION
This investigation advances our understanding of how two different extractants of n‐tetradecane and oleic acid affect the biosynthesis of metabolites in A. camphorata. It is beneficial to provide potential strategies for improving antrodin C and antroquinonol production by genetic means. © 2020 Society of Chemical Industry
Antioxidant metabolites contribute to alleviating oxidative stress caused by reactive oxygen species (ROS) in microorganisms. We utilized oxidative stressors such as hydrogen peroxide supplementation to increase the yield of the bioactive secondary metabolite antioxidant antrodin C in submerged fermentations of the medicinal mushroom Antrodia cinnamomea. Changes in the superoxide dismutase and catalase activities of the cells indicate that ROS are critical to promote antrodin C biosynthesis, while the ROS production inhibitor diphenyleneiodonium cancels the productivity-enhancing effects of H2O2. Transcriptomic analysis suggests that key enzymes in the mitochondrial electron transport chain are repressed during oxidative stress, leading to ROS accumulation and triggering the biosynthesis of antioxidants such as antrodin C. Accordingly, rotenone, an inhibitor of the electron transport chain complex I, mimics the antrodin C productivity-enhancing effects of H2O2. Delineating the steps connecting oxidative stress with increased antrodin C biosynthesis will facilitate the fine-tuning of strategies for rational fermentation process improvement.
In recent years, Antrodia cinnamomea has attracted great attention around the world as an extremely precious edible and medicinal mushroom. Ubiquinone derivatives, which are characteristic metabolites of A. cinnamomea, have shown great bioactivities. Some of them have been regarded as promising therapeutic agents and approved into clinical trial by US FDA. Although some excellent reviews have been published covering different aspects of A. cinnamomea, this review brings, for the first time, complete information about the structure, bioactivity, chemical synthesis, biosynthesis and metabolic regulation of ubiquinone derivatives in A. cinnamomea. It not only advances our knowledge on the bioactive metabolites especially the ubiquinone derivatives in A. cinnamomea, but also provides valuable information for the investigation on other edible and medicinal mushrooms.
Antrodia cinnamomea (A. cinnamomea), a popular medicinal mushroom in Taiwan, is widely used to prevent or treat liver diseases. Systematic studies on the anti-inflammatory effect of A. cinnamomea and its molecular mechanisms have not yet been fully investigated. HPLC fingerprint analysis identified seven ergostane-type triterpenoids from A. cinnamomea water extract (ACW), including high amounts of Antcin K (AC), Antcin C, Antcin H, Dehydrosulphurenic acid, Antcin B, Antcin A and Dehydroeburicoic acid. Here, we explored the effects and mechanisms of ACW and the highest content AC on N-nitrosodiethylamine (DEN) induced liver inflammation, fibrosis and carcinogenesis in rats. In the in vitro study, we measured how ACW and AC dose-dependently scavenged O[Formula: see text], H2O2 and HOCl by a chemiluminescence analyzer. In the in vivo experiment, oral intake ACW and AC significantly inhibited DEN-enhanced hepatocellular inflammation, fibrosis and carcinoma by pathologic observation, the elevated bile and liver reactive oxygen species (ROS) amounts, plasma [Formula: see text]-glutamyl transpeptidase, and oxidative stress including 3-nitrotyrosine, 4-hydroxynonenal and Kuppfer cell infiltration (ED-1 stains) in the inflammatory livers. DEN enhanced nuclear factor-[Formula: see text]B (NF-[Formula: see text]B) translocation, whereas ACW and AC suppressed DEN-enhanced NF-[Formula: see text]B translocation through the inhibition of its upstream signaling of p85/phosphoinositide-3-kinase, mitogen activated protein kinase and CYP2E1 expression. In conclusion, DEN can induce hepatocellular inflammation, fibrosis and carcinoma by increasing NF-[Formula: see text]B translocation to the nucleus, and oxidative injury. ACW and its active component, Antcin K, counteract DEN-induced hepatic injury and inflammation by the protective and therapeutic mechanisms of a direct scavenging ROS activity and an upregulation of anti-oxidant defense mechanisms.
This work highlights the literature of one of the most valuable moieties in the field of organic chemistry. In this review, the chemistry of tetronic acid as a simple precursor to privileged heterocyclic motifs is described. The synthetic procedures of different fused heterocycles incorporating a furan moiety are described. Fused heterocycles are classified as bicyclic, tricyclic, tetracyclic and spiro-fused pyran derivatives.
Embelia ribes Burm of Myrsinaceae family has been widely used as an herb in the traditional medicine of India. Embelin is an active component extracted from the fruits of Embelia ribes. It has a wide spectrum of biological activities and is not toxic at low dose. This review focuses on the physical?chemical properties and bioactivities of Embelin, as well as its effects on chronic diseases such as tumors, autoimmune inflammatory diseases, parasitic infections, microbial infections, diabetes, obesity, and cardio-cerebral vascular diseases. The underlying mechanisms of the effects are also discussed. As a multiple-targeted therapeutic agent, Embelin has the potential to be used widely for the treatment of a variety of chronic diseases, including malignant tumors.
Aim:
Antrodia Camphorate is a mushroom which is widely used in Asian countries to prevent and treat various diseases including liver diseases. However, the active ingredients that contribute to the biological functions remain elusive. The purpose of the present study is to test the hepatoprotective effect of Antcin H, a major triterpenoid chemical isolated from Antrodia Camphorate, in murine models of acute liver injury.
Results:
We found that Antcin H pretreatment protected against liver injury in both acetaminophen and galactosamine/TNFα models. More importantly, Antcin H also offered a significant protection against acetaminophen-induced liver injury when it was given one hour after acetaminophen. The protection was verified in primary mouse hepatocytes. Antcin H prevented sustained JNK activation in both models. We excluded an effect of Antcin H on acetaminophen metabolism and TNF receptor signaling and excluded a direct effect as a free radical scavenger or JNK inhibitor. Since the sustained JNK activation through its interaction with mitochondrial Sab, leading to increased mitochondrial ROS is pivotal in both models, we examined the effect of Antcin H on p-JNK binding to mitochondria and impairment of mitochondrial respiration. Antcin H inhibited the direct effect of p-JNK on isolated mitochondrial function and binding to isolated mitochondria.
Innovation and conclusion:
Our study has identified Antcin H as a novel active ingredient that contributes to the hepatoprotective effect of Antrodia Camphorate and Antcin H protects against liver injury through disruption of the binding of p-JNK to Sab which interferes the ROS-dependent self-sustaining activation of MAPK cascade.
Large-scale submerged fermentation (SmF) of Antrodia camphorata (A. camphorata) usually encounters challenges including tedious preparation of mycelial inoculum, long fermentation period (10-14d), and poor repeatability. Here we developed an asexual reproduction-based repeated batch fermentation (RBF) process for bioactive metabolites production by A. camphorata ATCC 200183. Compared with traditional batch fermentation, production time was shortened to 58d from 80d (overall time for eight cycles) using the RBF process established in this study, and accordingly, the productivities of bioactive metabolites (including antrodins) were improved by 40-60%. Kinetic parameters (α is 2.1-18.7 times as β) indicated that the cell growth was the major contribution for bioactive metabolites production. The RBF shows excellent batch-repeatability (Pearson correlation coefficient of 0.998±0.001), together with advantages of energy-efficient, low cost, and labor-saving, RBF process can be implemented to SmF by other filamentous fungi.
Copyright © 2015 Elsevier Ltd. All rights reserved.
Antrodin C is one of the most potent bioactive components produced by the medicinal mushroom Antrodia camphorata. However, almost all studies in this field have focused on the biological activity of Antrodin C and relatively rare information has been reported regarding the biosynthetic process of Antrodin C. In this study, the strategies of pH-shift and glucose feeding for enhanced production of Antrodin C in submerged fermentation of A. camphorata were successfully applied in stirred bioreactors. The critical parameters for pH-shift and glucose feeding were systematically investigated. On one hand, the optimal culture pH for cell growth was distinct with Antrodin C biosynthesis and the maximum Antrodin C production was obtained by maintaining the first-stage culture at initial pH 4.5 and adjusted to 6.0 at day 8. On the other hand, it was beneficial for the Antrodin C accumulation with the initial glucose concentration of 40 g/L and feeding glucose to keep the residual sugar above 10 g/L. The maximum Antrodin C production (1,549.06 mg/L) was about 2.1-fold higher than that of control in 15-L stirred bioreactors by taking advantage of the integrated strategy of pH-shift and glucose feeding. These results would be helpful for the design of a highly efficient Antrodin C biosynthesis process.
The short and biomimetic synthesis of the potent HCV protease inhibitor antrodin A, (I), in three steps and 73% overall yield represents a significant improvement over previous routes.
Antrodia camphorata is a medicinal fungus and antrodin C is one of the main bioactive components of A. camphorata in the submerged fermentation. To explore the good culture method, the factors influenced the production of antrodin C by A. camphorata in solid state fermentation was investigated in this study. Different solid substrates and external nitrogen sources were tested for their efficiency in producing antrodin C. The response surface methodology (RSM) was applied to evaluate the influence of several variables that includes the concentration of soy bean meal, initial moisture content and inoculum density on antrodin C production in solid state fermentation. The experimental results show that the optimum fermentation medium for antrodin C production by A. camphorata was composed of 0.578 g soy bean meal, 0.05 g Na2 HPO4 , 0.05 g MgSO4 for 100 g rice, with 51.83% initial moisture content, 22 days culture time, 28 °C culture temperature and 35.54% inoculum density. At optimized conditions, 6617.36 ± 92.71 mg kg(-1) yield of antrodin C was achieved. The solid state fermentation is one of the good cultural methods to improve the production of antrodin C by A. camphorata. This article is protected by copyright. All rights reserved.
Abstract In this study, a biphasic fermentation system coupling use of surfactant and in situ extractant was established to enhance Antrodin C production in submerged fermentation of Antrodia camphorata. Effects of several crucial factors such as the selection, concentration and addition time of surfactants and in situ extractants on the mycelial growth and production of Antrodin C were systematically studied. Results showed that the optimal surfactant and extractant were Tween-80 and soybean oil, respectively. The maximum production of Antrodin C (247.09 ± 0.97 mg/L) was obtained when 0.1% (w/v) Tween-80 (as surfactant) and 10% (v/v) soybean oil (as in situ extractant) both supplied at the logarithmic growth phase of fermentation (the 72th h and the 96th h, respectively), corresponding to 3.6 times significant increase compared with that of the control (53.23 ± 0.86 mg/L).
The first synthesis of antrocinnamomin D and a new synthesis of antrodins A and B have been achieved in 6–8 steps and high overall efficiency (51, 46, and 43%, respectively) from commercially available methyl 4-hydroxyphenylacetate. Key steps include Fürstner–Kochi iron-catalyzed sp2–sp3 cross-coupling and 2-silyloxyfuran oxyfunctionalization.
Medicinal mushroom Antrodia cinnamomea is a higher Basidiomycetes endemic to Taiwan, where it is commonly used as a traditional folk medicine. It is well known for its multiple biologic activities and its potential for commercial development. Here, ten full lengths of cytochrome P450 (CYP) genes (ac-1 to ac-10) from A. cinnamomea were cloned and identified. With the exception of ac-3 and ac-8, which will probably be assigned as new CYP families, these genes had more than 40% amino acid identity and close evolutionary relationships to known CYPs. Among the ten genes, only Ac-7 did not possess a transmembrane domain but had an N-terminal signal peptide, so it was considered a novel extracellular CYP. The ten A. cinnamomea CYPs had different expression profiles in different growth conditions. In general, they were strongly expressed during the formation of fruiting bodies, especially in natural basidiomycetes. The expression of six CYPs of A. cinnamomea (ac-1 to ac-3 and ac-5 to ac-7) were strictly inhibited in the mycelia cell type. It was therefore concluded that these CYPs are most active in the fruiting bodies of A. cinnamomea.
In an attempt to identify potential HCV NS3 protease inhibitors lead compounds, a series of novel indoles (10a-g) was designed. Molecular modeling study, including fitting to a 3D-pharmacophore model of the designed molecules (10a-g), with HCV NS3 protease hypothesis using catalyst program was fulfilled. Also, the molecular docking into the NS3 active site was examined using Discovery Studio 2.5 software. Several compounds showed significant high simulation docking score and fit values. The designed compounds with high docking score and fit values were synthesized and biologically evaluated in vitro using an NS3 protease binding assay. It appears that most of the tested compounds reveal promising inhibitory activity against NS3 protease. Of these, compounds 10a and 10b demonstrated potent HCV NS3 protease inhibitors with IC(50) values of 9 and 12μg/mL, respectively. The experimental serine protease inhibitor activities of compounds 10a-g were consistent with their molecular modeling results. Inhibitors from this class have promising characteristics for further development as anti-HCV agents.
A novel lysophospholipase (LysoPL) from the basidiomycetous fungi Antrodia cinnamomea named ACLysoPL was cloned, heteroexpressed in Escherichia coli and characterized.
The gene encoding ACLysoPL was obtained from expressed sequence tags from A. cinnamomea. The full length of this gene has a 945 -bp open reading frame encoding 314 amino acids with a molecular weight of 35.5 kDa. ACLysoPL contains a lipase consensus sequence (GXSXG) motif and a Ser-His-Asp catalytic triad. A putative peroxisomal targeting signal type 1 was found in the C-terminal. Heterologous expression of ACLysoPL in E. coli showed that the enzyme preferentially hydrolyses long-chain acyl esterases at pH 7 and 30 degrees C. ACLysoPL is a psychrophilic enzyme about 40% of whose maximum activity remained at 4 degrees C. The LysoPL activities with lysophospholipids as substrate were analysed by gas chromatography/mass spectrometry.
We have identified and characterized a gene named ACLysoPL encoding a protein performing LysoPL and esterase activities.
This is the first LysoPL of A. cinnamomea identified and characterized at the molecular level.
A major obstacle in the treatment of chronic hepatitis C virus (HCV) infection has been the lack of effective, well-tolerated therapeutics. Notably, the recent development of the HCV cell culture infection system now allows not only for the study of the entire viral life cycle, but also for the screening of inhibitors against all aspects of HCV infection. However, in order to screen libraries of potential antiviral compounds, it is necessary to develop a highly reproducible, accurate assay for HCV infection adaptable for high-throughput screening (HTS) automation. Using an internally quenched 5-FAM/QXL 520 fluorescence resonance energy transfer (FRET) substrate containing the HCV NS3 peptide cleavage sequence, we report the development of a simple, mix-and-measure, homogenous, cell-based HCV infection assay amendable for HTS. This assay makes use of synchronized, nondividing human hepatoma-derived Huh7 cells, which support more-reproducible long-term HCV infection and can be readily scaled down to a 96-well-plate format. We demonstrate that this stable cell culture method eliminates common problems associated with standard cell-based HTS, such as cell culture variability, poor reproducibility, and low signal intensity. Importantly, this HCV FRET assay not only can identify inhibitors that act throughout the viral life cycle as effectively as more-standard HCV assays, such as real-time quantitative PCR and Western blot analysis, but also exhibits a high degree of accuracy with limited signal variation (i.e., Z' > or = 0.6), providing the basis for a robust HTS campaign for screening compound libraries and identifying novel HCV antivirals.
樟芝菌糸体中の新規マレイン酸及び琥珀酸誘導体(antrodin A-E)の定量分析法を確立する目的で,高速液体クロマトグラフィー(HPLC)による分析条件を検討した。Antrodin A-Eは,移動相:1%AcOH/H_2O-CH_3CN (0 min, 60: 40; 40min, 30:70);流速:1.0ml/min;測定波長:235nm;カラム温度:室温の条件下,ODSカラムを用い定量を行った。この方法による日内と日間の相対的な標準偏差は4.6%及び4.1%(n=5)であり,0.1 (or 0.2)μgから1.2μgまでの濃度範囲で良好な直線性が得られた。Antrodin A-Eの回収率はそれぞれ,99.8, 102.9, 101.0, 100.5及び99.9%であった。本測定法は迅速且つ正確であり,樟芝菌糸体中の新規マレイン酸及び琥珀酸誘導体の定量に適していると考えられる。 For the purpose of quantitative determination of novel maleic acid and succinic acid derivatives, antrodins A-E, in the mycelium of Antrodia cinnamomea, a high-performance liquid chromatography (HPLC) method was applied to separation of these compounds under the conditions, where the mobile phase was a linear gradient of 1% AcOH/H_2O-CH_3CN, the flow rate was 1.0 ml/min and the detecting wavelength was 235 nm, using an ODS column. The relative standard deviations of this method were less than 4.6% and 4.1% (n=5) for interday and intraday assays, respectively. A good linear correlation was obtained in a range of 0.1 (or 0.2) μg-1.2μg. The recoveries of antrodins A, B, C, D and E were 99.8, 102.9, 101.0, 100.5 and 99.9%, respectively. This method was rapid, accurate and suitable for the quantitative determination of maleic acid and succinic acid derivatives in the mycelium of Antrodia cinnamomea.
Polysaccharides were extracted from fruiting bodies and cultured mycelia from five Antrodia camphorata strains. Polysaccharide profiles of the five strains, as determined by high-performance anion-exchange chromatography, showed varying yields and composition of neutral sugars. A. camphorata fruiting bodies also had different polysaccharide patterns compared to the cultured mycelium. Analysis of 26-day-old mycelia showed that the neutral sugars galactose, glucose, mannose, and galactosamine were predominant. All mycelia polysaccharide preparations exhibited anti-hepatitis B virus activity. Polysaccharides from strain B86 at a concentration of 50 mug ml(-1) showed the highest level of anti-hepatitis B surface antigen effect, which was higher than a-interferon at a dosage of 1000 U ml(-1). Only strains B86 and 35398 had substantial anti-hepatitis B e antigen activities. None of the polysaccharides exhibited cytotoxic effects.
A simple assay was developed based on intramolecular fluorescence resonance energy transfer for detection of the activity of hepatitis C virus (HCV) serine proteinase. Two quenched-fluorogenic substrates, (7-methoxycoumarin-4-yl)acetyl (Mca) Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-(2,4-dinitrophenyl, Dnp) Lys (Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Ser-Lys[Dnp], QF-1) and Mca-Asp-Asp-Ile-Val-Pro-Cys-Ser-Met-Lys(Dnp)-Arg-Arg (QF-2), which derived from the NS5A/5B junction of the HCV polyprotein, were designed. Kinetic studies revealed that QF-1 and QF-2 had high affinity for a recombinant enzyme which is a fusion protein of maltose binding protein and almost entire nonstructural protein (MBP–NS3), with Km values comparable to that of longer substrate based on the same cleavage site. QF-1 and QF-2 were cleaved by MBP-NS3 efficiently with kcat values of 7.5 and 4.2 min−1, respectively. QF-2 was also found to be a good substrate of ΔNS3 which contained serine proteinase part of NS3 with kcat value of 4.3 min−1. The cleavage reaction is detected continuously by the elevation of the fluorescence due to release from quenching. The fluorescence of the substrates increases in proportion to progress of the cleavage reaction under the standard conditions. This method was applied for screening of HCV serine protease inhibitors using a fluorescence multiwell plate reader. A group of natural occurring products, flavonoids, was subjected to be screened. Two flavonoids out of 25 were found to inhibit the enzyme moderately at a concentration of 100 μM. The data agreed with those obtained by high-performance liquid chromatography (HPLC). This method is suited to sensitive quantitation of the enzyme reaction as well as the high throughput analysis of the inhibitors.
Variants of chronic hepatitis delta (CHD) course were studied in 94 patients. Slow progress (for decades) occurred most frequently, while rapid progress to hepatic cirrhosis (for 1-2 years) or benign course presenting as greater than 3-year remission were rare findings. In addition to routine therapeutic modalities, except corticosteroids, 43 CHD patients received recombinant alpha 2-interferon (reaferon). Follow-up results evaluated separately for hepatic cirrhosis and free of it CHD patients support clinical promise of Soviet recombinant alpha 2-interferon against CHD.
One hundred fifty-two methanol and water extracts of different parts of 71 plants commonly used in Sudanese traditional medicine were screened for their inhibitory effects on hepatitis C virus (HCV) protease (PR) using in vitro assay methods. Thirty-four extracts showed significant inhibitory activity (>/=60% inhibition at 100 microg/mL). Of these, eight extracts, methanol extracts of Acacia nilotica, Boswellia carterii, Embelia schimperi, Quercus infectoria, Trachyspermum ammi and water extracts of Piper cubeba, Q. infectoria and Syzygium aromaticum, were the most active (>/=90% inhibition at 100 microg/mL). From the E. schimperi extract, two benzoquinones, embelin (I) and 5-O-methylembelin (II), were isolated and found as potent HCV-PR inhibitors with IC(50) values of 21 and 46 microM, respectively. Inhibitory activities of derivatives of I against HCV-PR as well as their effects on other serine proteases were also investigated.
Polysaccharides were extracted from fruiting bodies and cultured mycelia from five Antrodia camphorata strains. Polysaccharide profiles of the five strains, as determined by high-performance anion-exchange chromatography, showed varying yields and composition of neutral sugars. A. camphorata fruiting bodies also had different polysaccharide patterns compared to the cultured mycelium. Analysis of 26-day-old mycelia showed that the neutral sugars galactose, glucose, mannose, and galactosamine were predominant. All mycelia polysaccharide preparations exhibited anti-hepatitis B virus activity. Polysaccharides from strain B86 at a concentration of 50 microg ml(-1) showed the highest level of anti-hepatitis B surface antigen effect, which was higher than alpha-interferon at a dosage of 1000 U ml(-1). Only strains B86 and 35398 had substantial anti-hepatitis B e antigen activities. None of the polysaccharides exhibited cytotoxic effects.
Five new maleic and succinic acid derivatives were isolated from the mycelium of Antrodia camphorata. Their structures were determined by various spectroscopic means. Maleimide derivatives 2 and 3 showed appreciable cytotoxic activity against LLC cells.
Antrodia camphorata is a popular folk medicine that has attracted great attention due to its fame for antitumor activity against cancer. However, there is little information available about its action. In the present study, we purified a unique polysaccharide component from A. camphorata mycelia (AC-PS) and found that it has pronounced anti-tumor effects on both in vitro and in vivo model. Our results showed that AC-PS alone did not show any direct cytotoxic effect to human leukemic U937 cells, even at high concentration (200 microg/ml). However, it could inhibit the proliferation of U937 cells via activation of mononuclear cells (MNCs). Treatment of U937 cells with AC-PS-stimulated-MNC-CM could significantly inhibit its proliferation with 55.3% growth inhibition rate. The in vitro antitumor activity was substantiated by the in vivo therapeutical study of AC-PS in sarcoma 180-bearing mice. Intraperitoneal and oral administration of AC-PS, 100 and 200 mg/kg significantly suppressed the tumor growth with the inhibition rate of 69.1% and 58.8%, respectively. In vivo studies also showed that several immunoparameters, such as the spontaneous proliferation of spleen cells, after AC-PS administration, were two-fold higher than in control mice. Furthermore, the cytolytic activity of spleen cells also increased from 9.8 +/- 1.1% in control mice to 34.2 +/- 5.5% and 48.2 +/- 2.5%, after oral and intraperitoneal treatment, respectively. Besides, the mice serum interleukin-12 levels increased significantly by AC-PS treatment. Considering all these results, it is suggested that AC-PS elicit its anti-tumor effect by promoting a Th1-dominant state and killer activities.
Currently available anti-HCV therapy is effective in only half of the patients and limited by side effects that often necessitate discontinuation. Therefore, new treatment strategies are being developed including (i) the optimization of current regimens, (ii) the use of additional agents working via novel mechanisms, and (iii) anti-fibrotic strategies. Many new antiviral compounds are now being studied in preclinical and clinical trials. This review will focus on drugs that have already entered the stage of phase 2 or phase 3 studies.
Mycelia of Antrodia cinnamomea were extracted with chloroform and hot water. A neutral polysaccharide named ACN2a separated from the water extract was purified using 10% CCl3COOH, and repeated column chromatography on HW-65 and DE-52 cellulose. Its structure was determined by chemical and spectroscopic analyses. ACN2a was composed of Gal, Glc, Fuc, Man and GalN (in the ratio 1:0.24:0.07:0.026:faint), in which an alpha-D-(1-->6)-Gal linkage accounted for 73% of all linkages. The ratio of branch points was about 16% of the total residual numbers, and branches were attached to C-2 of galactosyl residues of the main chain. ACN2a had an average molecular weight of 12.9x10(5) Daltons, [alpha]D25=+115 degrees (c=0.44, H2O); [eta]=0.0417dl.g-1, Cp=0.2663 cal/(g. degrees C). The hepatoprotective effect of ACN2a was evaluated using a mouse model of hepatic injury that was induced by Propionibacterium acnes (P. acnes) and lipopolysaccharide (LPS). The administration of ACN2a (0.4, 0.8 g/kg/d, p.o.), significantly prevented increases in serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) enzyme activities in mice treated with P. acnes-LPS, indicating hepatoprotective activity in vivo.
Submerged cultures were used to identify growth-limiting nutrients by Antrodia cinnamomea strains. The mycelial biomass and EPS production by A. cinnamomea BCRC 35396 were markedly higher than other A. cinnamomea strains. A relatively high C/N ratio was favorable for both the mycelial growth (5.41 g/l) and EPS production (0.55 g/l); the optimum ratio was 40. The glucose was available utilized preferentially for mycelial growth, rather than for EPS production. Flushing the culture medium with nitrogen had a stimulating effect on both mycelial growth and EPS production. In addition, peptone, yeast extract and malt extract appeared to be important and significant component for EPS production. Phosphate ion, magnesium ion and thiamine were probably not essential for mycelial growth. By optimizing the effects of additional nutrition, the results showed that 5% (w/v) glucose, 0.8% (w/v) peptone, 0.8% (w/v) yeast extract, 0.8% (w/v) malt extract, 0.03% (w/v) KH2PO4, 0.1% (w/v) MgSO4 .7H2O and 0.1% (w/v) thiamine could lead to the maximum production of EPS (1.36 g/l).
Investigation of the effect of Antrodia camphorata mycelium on fulminant hepatitis mode animals. The 21st annual Meeting of Medi-cal and Pharmaceutical Society for Wakan-Yaku. Abstract paper
- A Hirakawa
- T Yokozawa
- Gao Jj
- Hattori
Hirakawa A, Yokozawa T, Gao JJ, Hattori M. 2004. Investigation of the effect of Antrodia camphorata mycelium on fulminant hepatitis mode animals. The 21st annual Meeting of Medi-cal and Pharmaceutical Society for Wakan-Yaku. Abstract paper. Medical and Pharmaceutical Society for WAKAN-YAKU, Toyama, Japan, 79–79.
The Use and the Effect of Ganoderma
- Tsai Zt
- Liaw
- Sl
Tsai ZT, Liaw SL. 1982. The Use and the Effect of Ganoderma. Sheng-Yun Publisher Inc.: Taichung, Taiwan, 116 –117.