Comprehensive Transcriptomic Analysis of Cordyceps militaris Cultivated on Germinated Soybeans.

The ascomycete fungus Cordyceps militaris infects lepidopteran larvae and pupae and forms characteristic fruiting bodies. Owing to its immune-enhancing effects, the fungus has been used as a medicine. For industrial application, this fungus can be grown on geminated soybeans as an alternative protein source. In our study, we performed a comprehensive transcriptomic analysis to identify core gene sets during C. militaris cultivation on germinated soybeans. RNA-Seq technology was applied to the fungal cultures at seven-time points (2, 4, and 7-day and 2, 3, 5, 7-week old cultures) to investigate the global transcriptomic change. We conducted a time-series analysis using a two-step regression strategy and chose 1460 significant genes and assigned them into five clusters. Characterization of each cluster based on Gene Ontology and Kyoto Encyclopedia of Genes and Genomes databases revealed that transcription profiles changed after two weeks of incubation. Gene mapping of cordycepin biosynthesis and isoflavone modification pathways also confirmed that gene expression in the early stage of GSC cultivation is important for these metabolic pathways. Our transcriptomic analysis and selected genes provided a comprehensive molecular basis for the cultivation of C. militaris on germinated soybeans.

Fermented Cordyceps militaris Extract Ameliorates Hepatosteatosis via Activation of Fatty Acid Oxidation.

Nonalcoholic fatty liver disease is a progressive disease involving the accumulation of lipid droplets in the liver. In this study, we investigated the anti-hepatosteatosis effects of fermented Cordyceps militaris extract (CME) in AML-12 hepatocytes. Although the levels of adenosine and cordycepin were reduced in the extracts of CM grown on germinated soybean (GSCE) and fermented CM grown on germinated soybean (GSC) by Pediococcus pentosaceus ON188 (ON188E), the expression of fatty acid oxidation (FAO) genes were upregulated only by GSC-ON188E treatment in a dose-dependent manner. In contrast, a lipogenic gene, stearoyl Coenzyme A desaturase 1, was downregulated by ON188E. Formation of intracellular lipid droplets by the addition of oleic acid was reduced by ON188E to levels observed in WY14643-treated cells. When cells were treated with ON188E, sphingosine kinase 2 mainly responsible for hepatic sphingosine 1-phosphate (S1P) synthesis was upregulated and S1P was elevated. Collectively, the fermented GSC extract activates FAO through elevation of S1P synthesis and has potential as a therapeutic for hepatosteatosis.

Identification of an anticancer compound against HT-29 cells from Phellinus linteus grown on germinated brown rice.

OBJECTIVE: To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate (EtOAc) extract of Phellinus linteus grown on germinated brown rice (PB). METHODS: EtOAc extract of PB was partitioned with n-hexane, EtOAc, and water-saturated n-butanol. Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR, respectively. Cytotoxicity against HT-29 cells was tested by SRB assay. RESULTS: The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line. Atractylenolide I, a eudesmane-type sesquiterpene lactone, a major anticancer substance of PB, was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC. This structure was elucidated by one- and two-dimensional NMR spectroscopic data. Atractylenolide I has not been reported in mushrooms or rice as of yet. The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells. CONCLUSIONS: Atractylenolide I might contribute to the anticancer effect of PB.

Antrodia camphorata grown on germinated brown rice inhibits HT-29 human colon carcinoma proliferation through inducing G0/G1 phase arrest and apoptosis by targeting the ß-catenin signaling.

Antrodia camphorata (AC) has been used as a traditional medicine to treat food and drug intoxication, diarrhea, abdominal pain, hypertension, pruritis (skin itch), and liver cancer in East Asia. In this study, we investigated anticancer activities of AC grown on germinated brown rice (CBR) in HT-29 human colon cancer cells. We found that the inhibitory efficacy of CBR 80% ethanol (EtOH) extract on HT-29 and CT-26 cell proliferation was more effective than ordinary AC EtOH 80% extract. Next, 80% EtOH extract of CBR was further separated into four fractions; hexane, ethyl acetate (EtOAc), butanol (BuOH), and water. Among them, CBR EtOAc fraction showed the strongest inhibitory activity against HT-29 cell proliferation. Therefore, CBR EtOAc fraction was chosen for further studies. Annexin V-fluorescein isothiocyanate staining data indicated that CBR EtOAc fraction induced apoptosis. Induction of G0/G1 cell cycle arrest on human colon carcinoma cell was observed in CBR EtOAc fraction-treated cells. We found that CBR decreased the level of proteins involved in G0/G1 cell cycle arrest and apoptosis. CBR EtOAc fraction inhibited the ß-catenin signaling pathway, supporting its suppressive activity on the level of cyclin D1. High performance liquid chromatography analysis data indicated that CBR EtOAc fraction contained adenosine. This is the first investigation that CBR has a greater potential as a novel chemopreventive agent than AC against colon cancer. These data suggest that CBR might be useful as a chemopreventive agent against colorectal cancer.

Ethanol extract of Cordyceps militaris grown on germinated soybeans attenuates dextran-sodium-sulfate- (DSS-) induced colitis by suppressing the expression of matrix metalloproteinases and inflammatory mediators.

The effect of Cordyceps militaris (CM) grown on germinated soybeans (GSC) in the inflammatory bowel disease (IBD) model was studied. To demonstrate the preventive effect of GSC extract in a dextran-sodium-sulfate- (DSS-) induced acute colitis mouse model, GSC was administered 2 days before DSS coadministration. GSC significantly suppressed DSS-induced disease activity index (DAI) as well as histopathological scores, compared to control or CM-treated group. To elucidate the anti-IBD activity of GSC, we checked the level of matrix metalloproteinases (MMPs) and inflammatory mediators. GSC extract decreased the level of MMP-3 and -9 mRNAs and p53 proteins. The level and activity of LPS-induced MMP-9 were reduced in GSC-treated RAW264.7 cells. It also attenuated the level of inducible nitric oxide synthase (iNOS) and tumor necrosis factor- (TNF-) α mRNAs both in colon tissue and in macrophage cells. These results suggest that GSC can be applied as a protective agent against IBDs.

Ethanol Extract of Antrodia camphorata Grown on Germinated Brown Rice Suppresses Inflammatory Responses in Mice with Acute DSS-Induced Colitis.

The anti-inflammatory activity of Antrodia camphorata (AC) grown on germinated brown rice (CBR) extract was evaluated in vitro and in vivo. CBR suppressed the release of nitric oxide (NO) and prostaglandin (PG) E2 from lipopolysaccharide-(LPS-)stimulated RAW264.7 cells. CBR inhibited the level of inducible nitric oxide synthase (iNOS) and cyclooxygenase-(COX-)2 proteins, and it activated p38-MAPK, extracellular signal-related kinases (ERK), and NF- κ B in LPS-stimulated RAW264.7 macrophages. LPS-induced tumor necrosis factor- α (TNF- α ) and interleukin-6 (IL-6) mRNA expression was reduced in CBR-treated RAW264.7 cells. In concert with in vitro data, CBR suppressed the levels of dextran-sulfate-sodium-(DSS-)induced iNOS and COX-2 proteins in the colon tissue. CBR treatment inhibited activated p38-MAPK, ERK, and NF- κ B proteins in the colon tissue of DSS-induced mice. TNF- α and IL-6 mRNA expression was reduced in DSS+CBR-treated mice. The disease activity index and histological scores were significantly lower in CBR-treated mice (500 mg/kg/day) than in DSS-treated mice (P < 0.05 versus DSS). This is the first report of anti-inflammatory activity of CBR in DSS-induced acute colitis. These results suggest that CBR is a promising, potential agent for preventing acute colitis through the inhibition of NF- κ B signaling and its upstream signaling molecules, including MAPKs.

Extract of Rhus verniciflua Bark Suppresses 2,4-Dinitrofluorobenzene-Induced Allergic Contact Dermatitis.

Rhus verniciflua Stokes (RV) has traditionally been used as a food supplement and a traditional herbal medicine for centuries in Korea. Recent studies suggest that RV has potent antioxidative, antitumor, and anti-inflammatory properties. In this study, the anti-inflammatory effects of RV from mice sensitized with 2,4-dinitrofluorobenzene (DNFB) and activated macrophages were investigated. The results showed that RV reduced ear swelling and hyperplasia of ear tissue as well as an increase in vascular permeability, which are characteristics of allergic contact dermatitis (ACD) with evident histomorphological changes in epidermis and dermis. Decreased numbers of infiltrated mast cells were seen in RV extract treated group, using toluidine blue staining. RV extract significantly regulates the expression of inducible nitric oxide synthase (iNOS) at the translational level in activated macrophages. Furthermore, RV extract and its active compound, fisetin, attenuated the level of tumor necrosis factor- α (TNF- α ) and interleukin 6 (IL-6) mRNA in LPS-stimulated macrophages. Anti-ACD effect of RV extract may be due to the suppression of iNOS and proinflammatory cytokines which might be mediated via the NF κ B signaling pathways. Collectively, RV extract has potential for alleviating ACD-like symptoms induced by DNFB in the mouse.

Antrodia camphorata Grown on Germinated Brown Rice Suppresses Melanoma Cell Proliferation by Inducing Apoptosis and Cell Differentiation and Tumor Growth.

Antrodia camphorata grown on germinated brown rice (CBR) was prepared to suppress melanoma development. CBR extracts were divided into hexane, EtOAc, BuOH, and water fractions. Among all the fractions, EtOAc fraction showed the best suppressive effect on B16F10 melanoma cell proliferation by CCK-8 assay. It also showed the increased cell death and the changed cellular morphology after CBR treatment. Annexin V-FITC/PI, flow cytometry, and western blotting were performed to elucidate anticancer activity of CBR. The results showed that CBR induced p53-mediated apoptotic cell death of B16F10. CBR EtOAc treatment increased melanin content and melanogenesis-related proteins of MITF and TRP-1 expressions, which supports its anticancer activity. Its potential as an anticancer agent was further investigated in tumor-xenografted mouse model. In melanoma-xenografted mouse model, melanoma tumor growth was significantly suppressed under CBR EtOAc fraction treatment. HPLC analysis of CBR extract showed peak of adenosine. In conclusion, CBR extracts notably inhibited B16F10 melanoma cell proliferation through the p53-mediated apoptosis induction and increased melanogenesis. These findings suggest that CBR EtOAc fraction can act as an effective anticancer agent to treat melanoma.

Hair growth-promoting activity of hot water extract of Thuja orientalis.

BACKGROUND: Thuja orientalis has been traditionally used to treat patients who suffer from baldness and hair loss in East Asia. The present study sought to investigate the hair growth-promoting activity of T. orientalis hot water extract and the underlying mechanism of action. METHODS: After T. orientalis extract was topically applied to the shaved dorsal skin of telogenic C57BL/6 N mice, the histomorphometric analysis was employed to study induction of the hair follicle cycle. To determine the effect of T. orientalis extract on the telogen to anagen transition, the protein expression levels of ß-catenin and Sonic hedgehog (Shh) in hair follicles were determined by immunohistochemistry. RESULTS: We observed that T. orientalis extract promoted hair growth by inducing the anagen phase in telogenic C57BL/6 N mice. Specifically, the histomorphometric analysis data indicates that topical application of T. orientalis extract induced an earlier anagen phase and prolonged the mature anagen phase, in contrast to either the control or 1% minoxidil-treated group. We also observed increases in both the number and size of hair follicles of the T. orientalis extract-treated group. Moreover, the immunohistochemical analysis reveals earlier induction of ß-catenin and Shh proteins in hair follicles of the T. orientalis extract-treated group, compared to the control or 1% minoxidil-treated group. CONCLUSION: These results suggest that T. orientalis extract promotes hair growth by inducing the anagen phase in resting hair follicles and might therefore be a potential hair growth-promoting agent.

The inhibitory activity of atractylenolide Ш, a sesquiterpenoid, on IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA).

ETHNOPHARMACOLOGICAL RELEVANCE: AT Ш, a sesquiterpenoid, is the major component of Atractylodes japonica Koidz that has been used as a traditional oriental medicine. AIM OF THE STUDY: We investigated the anti-allergic activity of AT Ш and its mechanism of action. MATERIALS AND METHODS: The released amount of ß-hexosaminidase in mast cells, a key parameter of degranulation, was measured. Anti-allergic potential of AT Ш was evaluated using passive cutaneous anaphylaxis in vivo. The anti-allergic mechanism of AT Ш was investigated by immunoblotting analysis, RT-PCR and measurement of [Ca(2+)]i in mast cells. RESULTS: AT Ш significantly inhibited IgE/Ag-mediated degranulation with an IC(50) value (36 ± 4 µM) in RBL-2H3 cells without affecting cell viability. It also suppressed IgE/Ag-mediated passive cutaneous anaphylaxis (PCA) response with an ED(50) value (65 ± 41 mg/kg) in vivo. AT Ш suppressed the production of interleukin (IL-4) and tumor necrosis factor (TNF)-alpha mRNAs more potent than the Src-family kinase inhibitor PP2 in RBL-2H3 cells at all concentrations. In order to elucidate the anti-allergic mechanisms of AT Ш in mast cells, we examined the activated levels of signaling molecules. AT Ш inhibited the phosphorylation of Lyn, Fyn, Syk, LAT, PLCγ, Gab2, Akt, p38, and JNK kinases expression. IgE/Ag-mediated [Ca(2+)]i elevation was significantly inhibited by AT Ш. CONCLUSIONS: Our study suggests that AT Ш might be used as a therapeutic agent for allergic diseases.

Cordyceps militaris Grown on Germinated Soybean Induces G2/M Cell Cycle Arrest through Downregulation of Cyclin B1 and Cdc25c in Human Colon Cancer HT-29 Cells.

Cordyceps militaris (CM) is an insect-borne fungus that has been used in traditional Chinese medicine because of its wide range of pharmacological activities. In this paper, we studied CM grown on germinated soybean (GSC) and investigated the possible mechanisms underlying antiproliferative effect of GSC on HT-29 human colon cancer cells. In comparison with CM extracts and germinated soybean (GS) BuOH extracts, BuOH extracts of GSC showed remarkable inhibitory and antiproliferative effects on HT-29 colon cancer cells. After GSC treatment, HT-29 cells became smaller and irregular in shape. High G2/M phase cell populations were observed in the GSC-treated group. The levels of cyclin B1 and Cdc25 in the GSC-treated group were lower than those in the control group. These findings suggest that GSC BuOH extracts might act as an effective anti-proliferative agent by inducing G2/M cell cycle arrest in colon cancer cells.

A new isoflavone glycitein 7-O-beta-D-glucoside 4''-O-methylate, isolated from Cordyceps militaris grown on germinated soybeans extract, inhibits EGF-induced mucus hypersecretion in the human lung mucoepidermoid cells.

A new isoflavone glycitein 7-O-beta-d-glucoside 4''-O-methylate (CGLM) has been isolated recently from Cordyceps militaris grown on germinated soybean extract and has antioxidant activity. In the present study, CGLM was investigated for its suppression of airway mucous hyper-secretion in epidermal growth factor (EGF)-treated human lung mucoepidermoid cells. NCI-H292 cells were treated with CGLM for 1 h, followed by EGF treatment for 24 h. The decrease in cyclooxygenase-2 (COX-2) production was correlated with reduced levels of protein and mRNA of inducible matrix metalloproteinase 9 (MMP-9) and also MUC5AC gene expression. CGLM directly inhibited down-regulated NF-κB activity, and significantly inhibited the phosphorylation of p38 and ERK1/2 (p42/p44) in NCI-H292 cells. These results suggest that CGLM protects NCI-H292 cells from EGF-induced damage by down-regulation of COX-2, MMP-9 and MUC5AC gene expression, mediated via blocking the NF-kappa-B and p38/ERK MAPK pathways.

Antiallergic activity of novel isoflavone methyl-glycosides from Cordyceps militaris grown on germinated soybeans in antigen-stimulated mast cells.

Isoflavones are known to possess immunomodulating and antiallergic activities. Previously we identified novel isoflavone methyl-glycosides (daidzein 7-O-ß-d-glucoside 4″-O-methylate (CDGM), glycitein 7-O-ß-D-glucoside 4″-O-methylate (CGLM), genistein 7-O-ß-D-glucoside 4″-O-methylate (CGNMI) and genistein 4'-O-ß-D-glucoside 4″-O-methylate (CGNMII)) from Cordyceps militaris grown on germinated soybeans (GSC). The biological activity of novel isoflavone methyl-glycosides, however, remains unknown. In this study, CGNMII showed the strongest inhibition of degranulation. Additionally, the release of interleukin (IL)-4 and tumor necrosis factor (TNF)-α was decreased by CGNMII in antigen-stimulated RBL-2H3 cells. To elucidate the antiallergic mechanism of CGNMII, we examined whether it affected levels of signaling molecules responsible for degranulation. The levels of activated Lyn, Syk, PLCγ1 and LAT proteins were reduced in CGNMII treated RBL-2H3 cells. CGNMII also inhibited the activation of AKT and ERK1/2 proteins. These results suggest that CGNMII might be used as a therapeutic agent for allergic diseases.

The Src family kinase Fgr is critical for activation of mast cells and IgE-mediated anaphylaxis in mice.

Mast cells are critical for various allergic disorders. Mast cells express Src family kinases, which relay positive and negative regulatory signals by Ag. Lyn, for example, initiates activating signaling events, but it also induces inhibitory signals. Fyn and Hck are reported to be positive regulators, but little is known about the roles of other Src kinases, including Fgr, in mast cells. In this study, we define the role of Fgr. Endogenous Fgr associates with FcεRI and promotes phosphorylation of Syk, Syk substrates, which include linkers for activation of T cells, SLP76, and Gab2, and downstream targets such as Akt and the MAPKs in Ag-stimulated mast cells. As a consequence, Fgr positively regulates degranulation, production of eicosanoids, and cytokines. Fgr and Fyn appeared to act in concert, as phosphorylation of Syk and degranulation are enhanced by overexpression of Fgr and further augmented by overexpression of Fyn but are suppressed by overexpression of Lyn. Moreover, knockdown of Fgr by small interfering RNAs (siRNAs) further suppressed degranulation in Fyn-deficient bone marrow-derived mast cells. Overexpression of Fyn or Fgr restored phosphorylation of Syk and partially restored degranulation in Fyn-deficient cells. Additionally, knockdown of Fgr by siRNAs inhibited association of Syk with FcεRIγ as well as the tyrosine phosphorylation of FcεRIγ. Of note, the injection of Fgr siRNAs diminished the protein level of Fgr in mice and simultaneously inhibited IgE-mediated anaphylaxis. In conclusion, Fgr positively regulates mast cell through activation of Syk. These findings help clarify the interplay among Src family kinases and identify Fgr as a potential therapeutic target for allergic diseases.

A novel imidazo[1,5-b]isoquinolinone derivative, U63A05, inhibits Syk activation in mast cells to suppress IgE-mediated anaphylaxis in mice.

Mast cells play a pivotal role in IgE-mediated allergic responses. Development of specific inhibitors against FcεRI-associated proximal signaling molecules in mast cells may represent a promising therapeutic strategy for allergic diseases. We examined whether a novel synthetic compound, 3-butyl-1-chloro-8-(2-methoxycarbonyl)phenyl-5H-imidazo[1,5-b]isoquinolin-10-one (U63A05), could suppress antigen-stimulated degranulation and cytokine secretion in mast cells and IgE-mediated passive cutaneous anaphylaxis (PCA) in mice. U63A05 reversibly and dose-dependently inhibited degranulation of rat basophilic leukemia (RBL)-2H3 mast cells and bone marrow-derived mast cells (BMMCs) stimulated by antigen (IC(50) values for RBL-2H3 and BMMCs were 4.1 and 4.8 µM, respectively). The secretion of inflammatory cytokines was also suppressed in antigen-stimulated mast cells. However, degranulation by thapsigargin, a typical calcium inducer, was not inhibited by U63A05. U63A05 exerts its inhibitory effect, to the same extent as in degranulation, on the activating phosphorylation of Syk and downstream signaling molecules, including LAT and SLP-76. Further downstream, the activating phosphorylations of Akt, Erk1/2, p38, and JNK were also inhibited. Finally, antigen-stimulated PCA was dose-dependently suppressed in mice (ED(50), 26.3 mg/kg). Taken together, the results suggest that U63A05 suppresses the activation of mast cells and the mast cell-mediated allergic response through the inhibition of Syk activation in mast cells.

Topical application of Polygonum multiflorum extract induces hair growth of resting hair follicles through upregulating Shh and ß-catenin expression in C57BL/6 mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonum multiflorum has traditionally been used for treating patients suffering from baldness and hair loss in East Asia. AIM OF THE STUDY: The present study sought to investigate the hair growth promoting activities of Polygonum multiflorum and its mechanism of action. MATERIALS AND METHODS: The Polygonum multiflorum extract was topically applied to the shaved dorsal skin of telogenic C57BL6/N mice. To determine the effect of Polygonum multiflorum extract in telogen to anagen transition, the expression of ß-catenin and Sonic hedgehog (Shh) was determined by immunohistochemistry analysis. RESULTS: Polygonum multiflorum extract promoted hair growth by inducing anagen phase in telogenic C57BL6/N mice. In Polygonum multiflorum extract treated group, we observed increase in the number and the size of hair follicles that are considered as evidence for anagen phase induction. Immunohistochemical analysis revealed that earlier induction of ß-catenin and Shh were observed in Polygonum multiflorum extract treated group compared to that in control group. CONCLUSION: These results suggest that Polygonum multiflorum extract promotes hair growth by inducing anagen phase in resting hair follicles.

Morus bombycis Koidzumi extract suppresses collagen-induced arthritis by inhibiting the activation of nuclear factor-κB and activator protein-1 in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Morus bombycis Koidzumi is widely distributed in Asia. In Korea, it has been used in traditional medicine because of its apparent anti-inflammatory, antioxidant, and hepatoprotective properties. AIM OF THE STUDY: Although the extract of Morus bombycis Koidzumi (MB) has long since been used as a traditional anti-inflammatory medicine in Korea, its effect on arthritis remains unknown. We aimed to investigate the anti-arthritis activity of MB and the mechanism underlying it. MATERIALS AND METHODS: The anti-arthritis activity of MB was assessed by using mouse models of type II collagen-induced arthritis (CIA). The clinical arthritis index and histopathological changes were evaluated in mice. Reverse transcriptase-polymerase chain reaction (RT-PCR), electrophoretic mobility shift assay (EMSA), and other biologic approaches were used for measuring the effect of MB on arthritis and understanding the underlying mechanism. RESULTS: MB significantly decreased the clinical arthritis index in CIA mice; this was confirmed by examining histological changes in joints. Infiltration of immune cells, synovial hyperplasia, cartilage destruction, and bone erosion in the hind paw were largely suppressed by MB. The mRNA levels of matrix metalloproteinase (MMP)-1/MMP-3, inflammatory cytokines (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6), and chemokines (macrophage inflammatory protein (MIP)-1, monocyte chemoattractant protein (MCP)-1, RANTES) were significantly suppressed by MB in a dose-dependent manner. The number of osteoclasts in the hind tibia was also significantly decreased. With regard to the mechanism, MB suppressed the activation of nuclear factor (NF)-κB and activator protein (AP)-1 in CIA mice. CONCLUSIONS: MB produced an anti-arthritis effect in CIA mice by inhibiting the production of critical inflammatory mediators and osteoclasts through the downregulation of NF-κB and AP-1.

An extract of Phellinus linteus grown on germinated brown rice inhibits inflammation markers in RAW264.7 macrophages by suppressing inflammatory cytokines, chemokines, and mediators and up-regulating antioxidant activity.

The immunomodulatory activity of an organic extract of Phellinus linteus grown on slightly germinated brown rice (PBR) was previously demonstrated. Here, we investigated the possible anti-inflammatory activity of the PBR extract by analyzing its effect on the expression of macrophage-derived cytokines, chemokines, and mediator genes that participate in immune and inflammatory responses and diseases. The extract profoundly inhibited the induction of cytokines and chemokines, including tumor necrosis factor-α, chemokine (C-X-C motif) ligand-10, granulocyte-macrophage colony-stimulating factor, and interleukin-6, in lipopolysaccharide (LPS)-stimulated RAW264.7 mouse macrophage cells. It also greatly inhibited LPS-stimulated production of nitric oxide (NO) and prostaglandin E(2) in RAW264.7 cells by suppressing the expression of inducible NO synthase and cyclooxygenase-2. PBR extract inhibited NO production with a twofold lower half-maximal inhibitory concentration value than P. linteus extract. To elucidate the underlying mechanism of action, we examined the effect of the PBR extract on the LPS-induced phosphorylation of mitogen-activated protein kinases (MAPKs) in RAW264.7 cells. PBR extract greatly inhibited extracellular signal-regulated kinase and c-Jun N-terminal kinase phosphorylation and slightly inhibited p38 MAPK phosphorylation. It also significantly increased intracellular glutathione peroxidase activity and heme oxygenase-1 protein expression. Thus, the PBR extract has anti-inflammatory activity in LPS-stimulated RAW264.7 cells by virtue of its ability to suppress the production of inflammatory cytokines and chemokines via inhibition of MAPK activation and up-regulation of antioxidant activities.

The ethyl acetate extract of PGP (Phellinus linteus grown on Panax ginseng) suppresses B16F10 melanoma cell proliferation through inducing cellular differentiation and apoptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Phellinus linteus and Panax ginseng have been widely used as traditional herbal medicines to treat various diseases including cancer in East Asia. AIM OF THE STUDY: The present study sought to investigate the possible mechanism in anti-proliferative effect of Phellinus linteus that was grown on Panax ginseng (PGP) on B16F10 melanoma cells. MATERIALS AND METHODS: The anti-proliferative effect of PGP on B16F10 was evaluated by CCK-8 assays. Apoptotic cells were detected by flow cytometry analysis. The proteins involved in apoptosis and cellular differentiation were assessed by immunoblot analysis. Ginsenosides contents of PG or PGP were analyzed using HPLC. RESULTS: The ethyl acetate fraction (EtOAc) of PGP exhibited the strongest anti-proliferative activity among PGP fractions (butanol or water) on B16F10 cells. PGP EtOAc extract showed stronger inhibitory effect than Panax ginseng (PG) or Phellinus linteus (PL) EtOAc extract on B16F10 melanoma cell proliferation. PGP EtOAc extract induced the dendrite-like structures and the melanin production in B16F10 cells. PGP EtOAc extract increased a sub-G1 cell population through inducing p53/p21 and activated caspase-8 protein expression in B16F10 cells. Notably, PGP EtOAc extract contained ginsenosides Rd, Rg3, Rb2, Rg1 and Rb1 more than PG EtOAc extract. Rd and Rg3 significantly inhibited B16F10 cell proliferation. CONCLUSION: Our data suggest that PGP EtOAc extract inhibits B16F10 cell proliferation through inducing apoptosis and cellular differentiation.

Metabolomics revealed novel isoflavones and optimal cultivation time of Cordyceps militaris fermentation.

Germinated soybean (GS) cultivated with Cordyceps militaris (GSC) might be a promising efficacious source of novel bioactive compounds. In this study, the metabolome changes between GS and GSC were investigated by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) analysis coupled with a multivariate data set. Principal component analysis (PCA) and orthogonal projection to latent structures discriminate analysis (OPLS-DA) of GSC clearly showed higher levels of soyasaponin Bd, soyasaponin Bc(II), daidzein, genistein, four isoflavones (compounds 1-4), glycerol, proline, glutamine, pentitol, fructose, inositol, octadecanoic acid, and sucrose together with lower levels of pyroglutamic acid, citric acid, histidine, and palmitic acid in GSC than in GS. The structures of compounds 1-4 were analyzed by mass and NMR spectroscopy and were determined to be novel isoflavone methyl-glycosides (daidzein 7-O-beta-d-glucoside 4''-O-methylate (1), glycitein 7-O-beta-d-glucoside 4''-O-methylate (2), genistein 7-O-beta-d-glucoside 4''-O-methylate (3), and genistein 4'-O-beta-d-glucoside 4''-O-methylate (4)). Multivariate statistical models showed that metabolic changes of GSC were maximal within 1 week after the C. militaris inoculation, consistent with the strongest antioxidant activity of GSC cultivated for 1 week. This metabolomics study provides valuable information in regard to optimizing the cultivation process for bioactive compound production and describes an efficient way to screen for novel bioactive compounds from GSC.