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An acidic mucilage was isolated from the outer-layer seed coat of shaddock (Citrus grandis Osbeck) by water extraction at room temperature, and purified by DE-52 anion-exchange chromatography using 0.26 – 0.37 M NaCl. This purified mucilage was almost entirely composed of galacturonic acid residues. Glycosyl-linkage analysis showed that the backbone was 1→4 linked. Fourier Transform Infrared Spectroscopic analysis and determination of the degree of methylation further revealed that the mucilage was a low-degree (11.94%) esterified homogalacturonan. Size exclusion chromatography showed that the major molecular weight distribution of 610.9 kDa.

The purpose of this research was to physiochemically characterize the temporal expression profiles of polysaccharides from cold-stressed rice. Rice seedlings (at the three-leaf stage) were subjected to cold treatment, and polysaccharides were characterized by size-exclusion chromatography (SEC) and high-performance anion-exchange chromatography (HPAEC). In general, the molecular weight distribution of polysaccharides was similar during the 6-day exposure to cold stress. We observed that very-low-molecular-weight (

Poria cocos has recently been commercially used to formulate nutraceuticals and functional foods in Taiwan. In this study, P. cocos, was cultured and polysaccharides were extracted, followed by fractionation with size-exclusion column chromatography to give a water-soluble 1,6-branched 1,3-α- d -galactan (PC-II). The predominant monosaccharide was galactose after complete hydrolysis of the polysaccharide. The chemical structure was characterised by a monosugar analysis, along with 1H, 13C, and 2D nuclear magnetic resonance spectroscopy, including NOESY and HMBC experiments for linkage and sequence analysis. This polysaccharide is composed of a high-galactose-type undecasaccharide repeating unit with the following structure: Download : Download full-size image PC-II showed no toxicity to endothelial cells (ECs) and dose-dependently suppressed production of the interferon (IFN)-γ-induced inflammation marker, IP-10. However, PC-II showed no effect on cancer cell cytotoxicity or antiangiogenic activity. These results suggest that PC-II may play roles in regulating the anti-inflammatory process.

Sulfated polysaccharides (SPSs) of Antrodia cinnamomea were extracted by an exhaustive papain digestion and characterized, and their biological functions were evaluated. In this study, we demonstrated the existence of SPSs in the medicinal fungus, A. cinnamomea . Since no SPSs had previously been identified in any fungal organism, we attempted to characterize those from A. cinnamomea . SPSs from A. cinnamomea inhibited in vitro Matrigel tube formation, in an angiogenesis model, in a dose-dependent manner. Furthermore, using serum deprivation-induced apoptosis in neuronal-like PC12 cells as a stress model, the SPSs of A. cinnamomea were effective in preventing serum-deprived apoptosis. Compositional analysis revealed that myo-inositol, fucose, galactose, and glucose were the neutral sugars in SPSs of A. cinnamomea , and these SPSs had a high sulfate content. The sulfation degree paralleled their antiangiogenic and neuroprotective activities. In this work, we report novel data on the structure, antiangiogenic, and neuroprotective effects of these fungal SPSs.

Fomitopsis pinicola is used as a medicinal fungus in Asia. Polysaccharides extracted from 35-day-cultured fungus and its ethanolic extract showed no toxicity to endothelial cells (ECs). Extracted polysaccharides had a strong inhibitory effect on vascular endothelial growth factor (VEGF)-induced tube formation in ECs in a dose-dependent manner. Furthermore, the ethanolic extract dose-dependently suppressed production of the interferon (IFN)-γ-induced inflammation marker, IP-10. High-molecular-weight of 5367 and 1056 kDa accounted for 19.6% and 19.3% of the total polysaccharides, and a low-molecular-weight one of 14.7 kDa accounted for 53.6% of the total polysaccharides. Chemical analyses of the polysaccharides revealed that myo-inositol, fucose, galactose, glucose, mannose, and fructose were neutral sugars in the polysaccharide. Six nucleoside-type compounds including cytidine, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine, inosine, and thymidine were identified in the ethanolic extract. These results suggest that different extracts from F. pinicola play different roles in regulating the angiogenic process and inflammation.

Antrodia cinnamomea (formerly A. camphorata) has recently and commercially been used in the formulation of nutraceuticals and functional foods in Taiwan. Because of its diverse properties, the neuroprotective effect was investigated using a fermented A. cinnamomea extract in this study. Serum deprivation-induced apoptosis in neuronal-like pheochromocytoma (PC12) cells was used as a cell stress model, and it was found that A. cinnamomea was effective in preventing serum-deprived apoptosis according to results of an MTT assay and Hoechst staining. Serum deprivation resulted in decreased phosphorylation of extracellular signal-regulated kinase (ERK) and increased phosphorylations of c-Jun NH2-terminal kinase (JNK) and p38, of the family of mitogen-activated protein kinases (MAPKs); however, A. cinnamomea reversed these phenomena, supporting the antagonistic effects between ERK and JNK-p38 in regulating cell survival. The previously identified active component of A. cinnamomea, adenosine (ADO), also exerted the same effects as A. cinnamomea in preventing apoptosis and regulating phosphorylations of MAPKs. Although an inhibitor of the ERK upstream activator blocked A. cinnamomea-induced ERK phosphorylations, it failed to block the protection of A. cinnamomea and ADO. A protein kinase A (PKA) inhibitor blocked the protection by both A. cinnamomea and ADO. Both JNK and p38 inhibitors were effective in preventing the phosphorylations of JNK and p38 and serum deprivation-induced apoptosis. Collectively, A. cinnamomea prevented serum deprivation-induced PC12 cell apoptosis through a PKA-dependent pathway and by suppression of JNK and p38 activities.

Solanum lyratum , a medicinal plant, has been used to treat cancers, tumors, and warts for many years. Undifferentiated cell cultures were mainly used to study the precursor-feeding strategy for the production of secondary metabolites of α-solanine, solanidine, and solasodine for pharmaceutical usage. In this study, S. lyratum cells were fed with exogenous plant sterols including cholesterol, stigmasterol, and mixed sterols (β-sitosterol, campesterol, and dihydrobrassicasterol). The results showed that none of the plant sterols exhibited an effect on cell growth as compared to that of the control. Cellular concentrations of solanidine and solasodine were relatively higher than α-solanine levels in all the treatments. The maximal solasodine level in cells was 11.19 mg/g dry weight (DW) after 0.05–1 mg/l stigmasterol feeding, which was about 10-fold higher than the control. With regard to solanidine levels, the maximal level in cells was 5.84 mg/g DW after feeding with 20 mg/l cholesterol. This is the first report on the in vitro enhancement of solanidine and solasodine, steroidal alkaloids with medicinal value, from S. lyratum .

Extracted polysaccharides from medicinal fungi, including Antrodia cinnamomea, Antrodia malicola, Antrodia xantha, Antrodiella liebmannii, Agaricus murrill, and Rigidoporus ulmarius, were investigated for their effects on vascular endothelial growth factor (VEGF)-induced tube formation in endothelial cells (ECs). Chemical analysis revealed that myo-inositol, sorbitol, fucose, galactosamine, glucosamine, galactose, glucose, and mannose were the neutral sugars in these polysaccharides. These fungal polysaccharides showed no toxicity to ECs. For the inhibition of endothelial tube formation, extracted polysaccharides from A. xantha and R. ulmarius were shown to produce greater inhibition compared to those from other fungi. Fucose, glucose and mannose were the predominant monosaccharides from these two fungi. These results suggest that monosaccharides may play a role in the inhibitory effect of these fungi on endothelial tube formation. In contrast to the inhibition on tube formation from polysaccharides of A. cinnamomea and A. malicola, polysaccharides from A. xantha and R. ulmarius, with molecular weight between 2693-2876 and 304-325 kDa, were critical for this inhibitory activity. Our results show that polysaccharides isolated from A. xantha and R. ulmarius provide greater antiangiogenesis than those from commercialized A. murrill (Brazilian mushroom) and A. cinnamomea. These studies provide a basis for the potential development of these polysaccharides for antiangiogenesis usage.

Antrodia camphorata (A. camphorata) is a rare medicinal fungus with antioxidative, vasorelaxtative, anti-inflammatory and anti-hepatitive effects. However, the neuroprotective effect has not been studied. By using serum deprivation-induced apoptosis in neuronal-like PC12 cells as a cell stress model, we found that A. camphorata is effective in preventing serum-deprived apoptosis. Inhibitors of both a serine/threonine kinase and a specific protein kinase A (PKA) inhibited the protective effect of A. camphorata, indicating that A. camphorata prevents serum-deprived PC12 cell apoptosis through a PKA-dependent mechanism. A transcription inhibitor, actinomycin D, and a protein synthesis inhibitor, cyclohexamide, both attenuated the protective effect of A. camphorata, indicating a requirement for gene expression for protection by A. camphorata. On the other hand, A. camphorata also increased phosphorylated CREB, a transcription factor, which is H-89-inhibitable in this study, suggesting the possibility that A. camphorata prevents serum deprivation-induced PC12 cell apoptosis through a PKA/CREB-dependent pathway.

Sulfated polysaccharides (SPSs) from two edible fungal species, including two strains of Antrodia cinnamomea and Poria cocos , were isolated. Fucose, glucosamine, galactose, glucose, and mannose were the major sugars in the SPSs, and these SPSs had a high sulfate content. The area percentage of low-molecular-weight SPSs (1–100 kDa) covered almost half of the SPS mixture of the A. cinnamomea strains. In contrast, high-molecular-weight SPSs (>1000 kDa) of P. cocos covered a large proportion of the area at 30.06%. SPSs from A. cinnamomea B86 showed stronger inhibition of endothelial cell (EC) tube formation in an in vitro assay of angiogenesis, than did A. cinnamomea 35396 or P. cocos . The degree of sulfation paralleled their antiangiogenic activity. When tumor cells were concurrently exposed to doxorubicin (DOX) and fungal SPSs, SPSs synergistically increased the cytotoxicity of DOX to different degree up to 50-fold. Fungal SPSs may offer new applications for combinational-therapy drugs.

Antrodia cinnamomea (formerly named Antrodia camphorata) is a rare medicinal fungus. We previously reported that it exhibits antioxidative, vasorelaxative, anti-inflammatory, and anti-angiogenic effects. When serum deprivation-induced apoptosis in neuronal-like PC12 cells was used as a stress model, the extract of A. cinnamomea displayed effectiveness in preventing serum-deprived apoptosis. Since our previous data show that the extract of A. cinnamomea contains adenosine (ADO), we attempt to investigate if the active component is ADO and to identify its targeting site in this study. After pre-incubation with ADO deaminase, neither ADO nor the extract of A. cinnamomea exerted any protection, demonstrating that the active component of A. cinnamomea is ADO. Furthermore, an ADO A2A receptor (A2A-R) antagonist was used and was able to block the protective effects of ADO and the extract of A. cinnamomea, demonstrating that the ADO targeting site in this model is A2A-R. Taken together, the protective effect of A. cinnamomea is owed to its active component, ADO, which acts through activation of A2A-R to prevent serum deprivation-induced PC12 cell apoptosis.

Lipopolysaccharide (LPS) is a highly proinflammatory molecule isolated from bacteria. This study demonstrated the existence of LPS in a medicinal fungus, Antrodia camphorata. Because no LPS had been identified in any fungus organism, the purification of LPS from A. camphorata was attempted. LPSs from six strains of A. camphorata (35396, 35398, 35716, B71, B85, and B86) were isolated. Chemical and functional properties were investigated on the fungus LPS. Compositional analysis revealed that sorbitol, fucose, galactose, and glucose were the neutral sugars in LPS of A. camphorata. Galactosamine, glucosamine, galactose, and glucose were the predominant monosaccharide species in E. coli O129 LPS molecules, whereas galactosamine and glucosamine were absent in A. camphorata LPS. Because these properties are different from those of bacterial LPS, the functions between fungus and bacterial LPS are also discussed. The vascular endothelial lining of blood vessels, which controls leucocyte traffic and activation, may be one of the primary targets of LPS action during sepsis. Assays for biological activity were performed on endothelial cells with anti-inflammatory effects associated with sepsis. A. camphorata LPS apparently showed a lesser extent of cytotoxicity than bacterial LPS. In contrary to the proinflammatory property of bacterial LPS, LPS from A. camphorata differentially reversed bacterial LPS-induced intercellular adhersion molecule-1 and monocyte adhesion; both were indicators during inflammatory process. In conclusion, basic chemical properties categorized A. camphorata extracts into lipopolysaccharide. However, the detailed functional structures and bioactivities of A. camphorata LPS were totally different from those of bacterial LPS. The investigation of the existence and anti-inflammatory effect of fungus LPS is at present a truly novel and important finding. These results show that LPS isolated from A. camphorata offers a novel therapeutic target for anti-inflammation against E. coli infection.

The aim of this research was to investigate the hyphal growth-promoting factors (HGFs) of Antrodia camphorata from the host-related species, Cinnamomum camphora (CC) and the underlying chemical produced. The HGF was identified in the polysaccharide fraction of CC at levels ranging from 80 to 320 mg L(-1), and it maximally stimulated growth to 5.50 g L(-1) during a 14-day culture period compared to that of the control of 2.88 g L(-1). We also investigated the nature and chemical composition of the CC polysaccharide. Herein, size-exclusion column chromatography followed by high-performance anion-exchange chromatography after complete hydrolysis of the CC polysaccharide was performed to derive its molecular weight and sugar composition. The Mw values of the CC polysaccharide were determined to be 728.2, 187.5, 28.7, 7.5, and 1.9 kDa. Compositional analysis of the CC polysaccharide showed that galactosamine, mannose, and glucose were the major monosaccharides. Time-course studies of mycelial extracts of cultures revealed that prolonged incubation with the water-soluble extracts of CC resulted in an increase in the relative amounts of two lanostane-type compounds, i.e., dehydrosulphurenic acid and 15alpha-acetyl-dehydrosulphurenic acid, which are found in the fruiting bodies of A. camphorata. This finding offers the possibility of the reliable production of this medicinal fungus under laboratory conditions compared to its limited slow growth in nature.

The main purposes of this study were to investigate the regulation of polysaccharides isolated from A. cinnamomea on vascular endothelial growth factor (VEGF)-induced cyclin D1 expression and down stream signaling pathway that may correlate with their anti-angiogenc effects in endothelial cells (ECs). Crude and fractionated polysaccharides (Fra-1 to Fra-4) of A. cinnamomea showed slightly toxicity to ECs as compared with their inhibition concentration on angiogenic-related gene expression. The crude extract and fractionated fractions, except for Fra-2, of A. cinnamomea polysaccharides significantly decreased VEGFR2 phosphorylation on tyrosine 1054/1059, cyclin D1 promotor activity, and protein expression induced by VEGF. Crude extract of A. cinnamomea polysaccharides inhibited the binding of VEGF to KDR/flk-1 in a dose-dependent manner. These results indicated that inhibition of VEGF interaction with VEGF receptor 2 is the mechanism serves A. cinnamomea as a protective mechanism composing the anti-angiogenesis function. Furthermore, A. cinnamomea polysaccharides also blocked VEGF-induced migration and capillary-like tube formation of ECs on Matrigel. Taken together, these results indicate that A. cinnamomea polysaccharides inhibit cyclin D1 expression through inhibition of VEGF receptor signaling, leading to the suppression of angiogenesis.

We have previously reported that polysaccharides extracted from fruiting bodies or cultured mycelia of Antrodia camphorata exhibit an anti-hepatitis B virus effect. In this study, we intended to elucidate the anti-inflammatory potency of six mycelial extracts, namely PDB-ext, CK-ext, CM-ext, CO-ext, CC-ext, and CKO-ext, isolated from mycelia of A. camphorata cultured with six different media including potato dextrose broth (PDB) and five water-soluble fractions from the wood of different Cinnamomum species, i.e. C. kanehirae (CK), C. micranthum (CM), C. osmophloeum (CO), C. camphora (CC), and C. kotoense (CKO), against reactive oxygen species (ROS) production induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA) in peripheral human neutrophils (PMN) or mononuclear cells (MNC). ROS produced by PMN or MNC act as inflammatory mediators and also signal immune responses. Pretreatment with these mycelial extracts (1-50 microg ml(-1)) concentration-dependently diminished fMLP- or PMA-induced ROS production in PMN or MNC, as measured by lucigenin-amplified chemiluminescence, with 50% inhibition concentrations (IC(50)) ranging from 2 to 20 microg ml(-1). Among these extracts evaluated, CM-ext, CO-ext, or CKO-ext exhibited higher potency than the others. Using high performance liquid chromatography, we identified two lanostane-type compounds, i.e. dehydrosulfurenic acid and 15alpha-acetyl-dehydrosulfurenic acid, which could be involved in the anti-inflammatory actions of these extracts. The anti-inflammatory actions of these extracts were not due to cytotoxic effects. In summary, these data suggest that extracts from cultured mycelia of A. camphorata display anti-inflammatory effects by inhibiting ROS production in human leukocytes at a pharmacologically applicable concentration. The biological activities of these extracts were further promoted when the culture medium was replaced with water-soluble fractions isolated from the wood of CM, CO or CKO.

Antrodia camphorata, a medicinal fungus, has been used to treat cardiovascular diseases such as hypertension for many years. The purpose of this study was to examine the effects of mycelia extracts, from five Antrodia camphorata strains, on vascular tension and underlying mechanisms were explored. In isolated rat aortic rings, accession B86 caused concentration-dependent vasorelaxation with maximal relaxation of 40.34 ± 7.53% whereas accessions 35398, 35396 and B71 had mild vasorelaxing effects. Strain B85 evoked potent vasorelaxation, partly through an endothelium-dependent mechanism that was inhibited by Nω-nitro-L-arginine and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one (ODQ) but not by antagonist of K+ channels, tetraethylammonium. In cultured endothelial cells, B85 stimulated nitric oxide (NO) release and augmented the level of the intracellular Ca2+ concentration. HPLC and LC-MS-MS analysis revealed the presence of adenosine. Our results suggest that B85 produced strongest vasorelaxation in aortic preparations among five test strains. B85 acts in part on endothelial cells by activating the Ca2+-NO-cGMP pathway to reduce smooth muscle tone. However, K+ channels had no apparent roles. Adenosine could possibly be involved in the endothelium-dependent pathway of B85-induced vasorelaxation.

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.