Immunomodulatory Effects of the Stout Camphor Medicinal Mushroom, Taiwanofungus camphoratus (Agaricomycetes)-Based Health Food Product in Mice.

Taiwanofungus camphoratus is a unique medicinal mushroom endemic to Taiwan, and it is used as a folk medicine in East Asian countries. The aim of the present study was to investigate the immunomodulatory effects of "leader Antrodia cinnamomea capsule" (LAC), a health food product containing solid-state cultivated mycelial powder of T. camphoratus. For the in vivo studies, mice were orally administered LAC (76, 250, and 760 mg/kg b.w.) for 30 days, and its effects on cell-mediated humoral immune function were examined. The results of the concanavalin A-induced splenic lymphocyte proliferation test showed that LAC significantly increased splenic lymphocyte proliferation compared with the control. In addition, serum hemolysin analysis showed that LAC treatment significantly increased the half value of serum hemolysin (HC50) in mice compared with the control. Moreover, treatment with LAC significantly increased the phagocytic index as measured by carbon clearance and natural killer cell activity. Taken together, these findings provide strong evidence that LAC can modulate immune function.

Effects of Taiwanofungus camphoratus on non-specific and specific immune activities in mice.

Taiwanofungus camphoratus is a precious medicinal fungus endemic to Taiwan and has been used as traditional medicine for a long time. Many pharmacological studies have revealed that T. camphoratus possessed various biological activities, such as immunomodulatory effects, anticancer activity and liver protective function. The aim of this study is to investigate the non-specific and antigen (ovalbumin [OVA])-specific immunomodulation effects of solid-state cultivated powder of T. camphoratus (Leader Antrodia cinnamomea [LAC]) in BABL/c male mice. In non-specific and antigen-specific immune function studies, 8-week-old mice were orally administered with LAC for 6 and 8 weeks, respectively. The results have shown that the proliferation of splenic immune cells, phagocytic activity of macrophages and cytolytic activity of natural killer cells were enhanced by LAC. Additionally, LAC increased the levels of IL-2, TNF-α, INF-γ, GM-CSF and serum OVA-IgG and OVA-IgM. These findings provided evidences that LAC had the immunomodulation effects on both antigen-specific and non-specific immune responses in mice.

Taiwanin A inhibits MCF-7 cancer cell activity through induction of oxidative stress, upregulation of DNA damage checkpoint kinases, and activation of p53 and FasL/Fas signaling pathways.

This study investigates the anti-MCF-7 breast cancer cell effects and the underlying pharmacological activity and mechanism of taiwanin A, a major lignan isolated from Taiwania cryptomerioides. Our results show that taiwanin A time-dependently induced reactive oxygen species level and DNA damage in MCF-7 cells, which were likely activated kinases ataxia telangiectasia mutated (ATM) and checkpoint kinase (Chk). Taiwanin A could also up-regulate p53, phosphorylated p53, p21(Cip1), and p27(Kip1) and down-regulate the G(2)/M checkpoint cyclin-dependent kinase1 (Cdk1)-cyclin A/B, leading to induction of G(2)/M cell-cycle arrest in MCF-7 cells. Blockade of p53 gene expression by siRNA further demonstrated that the cell-cycle arrest induced by taiwanin A was p53-dependent. The FasL/Fas-mediated apoptotic signaling cascade was involved in taiwanin A-induced apoptosis via activation of caspases-10 and -7 (but not caspase-8), and proteolytic cleavage of poly(ADP-ribose) polymerase (PARP). In contrast, mitochondria-initiated apoptotic pathway was not involved. This is the first report to delineate novel mechanism of the action of taiwanin A against MCF-7 cells, suggesting this lignan may have value for development as an anti-breast cancer agent.

Deoxyelephantopin, a novel multifunctional agent, suppresses mammary tumour growth and lung metastasis and doubles survival time in mice.

BACKGROUND AND PURPOSE: Elephantopus scaber L. (Asteraceae) is a traditional herbal medicine with anti-cancer effects. We evaluated the in vitro and in vivo efficacy of a major sesquiterpene lactone constituent of E. scaber, deoxyelephantopin (DET), against mammary adenocarcinoma and the underlying molecular mechanism. EXPERIMENTAL APPROACH: A variety of cellular assays, immunoblotting and immunohistochemistry, as well as both orthotopic and metastatic TS/A tumour models in BALB/c mice, were used. Test mice were pretreated and post-treated with DET or paclitaxel and mammary tumour growth evaluated. KEY RESULTS: DET (< or =2 microg x mL(-1)) significantly inhibited colony formation, cell proliferation, migration and invasion of TS/A cells and induced G(2)/M arrest and apoptosis in TS/A cells. c-Jun N-terminal kinase-mediated p21(Waf1/Cip1) expression and caspase activation cascades were up-regulated by DET, effects suppressed by N-acetyl-L-cysteine. Moreover, tumour necrosis factor alpha-induced matrix metalloproteinase-9 enzyme activity and expression and nuclear factor-kappa B activation were abolished by DET. Pretreatment with DET was more effective than paclitaxel, for profound suppression of orthotopic tumour growth (99% vs. 68% reduction in tumour size) and lung metastasis of TS/A cells (82% vs. 63% reduction in metastatic pulmonary foci) and prolonged median survival time (56 vs. 37 days, P < 0.01) in mice. The levels of cyclooxygenase-2 and vascular endothelial growth factor in metastatic lung tissues of TS/A-bearing mice were attenuated by DET. CONCLUSIONS AND IMPLICATIONS: Our data provide evidence for the suppression of mammary adenocarcinoma by DET with several mechanisms and suggest that DET has potential as a chemopreventive agent for breast cancer.

Comparative metabolomics approach coupled with cell- and gene-based assays for species classification and anti-inflammatory bioactivity validation of Echinacea plants.

Echinacea preparations were the top-selling herbal supplements or medicines in the past decade; however, there is still frequent misidentification or substitution of the Echinacea plant species in the commercial Echinacea products with not well chemically defined compositions in a specific preparation. In this report, a comparative metabolomics study, integrating supercritical fluid extraction, gas chromatography/mass spectrometry and data mining, demonstrates that the three most used medicinal Echinacea species, Echinacea purpurea, E. pallida, and E. angustifolia, can be easily classified by the distribution and relative content of metabolites. A mitogen-induced murine skin inflammation study suggested that alkamides were the active anti-inflammatory components present in Echinacea plants. Mixed alkamides and the major component, dodeca-2E,4E,8Z,10Z(E)-tetraenoic acid isobutylamides, were then isolated from E. purpurea root extracts for further bioactivity elucidation. In macrophages, the alkamides significantly inhibited cyclooxygenase 2 (COX-2) activity and the lipopolysaccharide-induced expression of COX-2, inducible nitric oxide synthase and specific cytokines or chemokines [i.e., TNF-α, interleukin (IL)-1α, IL-6, MCP-1, MIP-1ß] but elevated heme oxygenase-1 protein expression. Cichoric acid, however, exhibited little or no effect. The results of high-performance liquid chromatography/electron spray ionization/mass spectrometry metabolite profiling of alkamides and phenolic compounds in E. purpurea roots showed that specific phytocompound (i.e., alkamides, cichoric acid and rutin) contents were subject to change under certain post-harvest or abiotic treatment. This study provides new insight in using the emerging metabolomics approach coupled with bioactivity assays for medicinal/nutritional plant species classification, quality control and the identification of novel botanical agents for inflammatory disorders.

Hepatoprotective phytocompounds from Cryptomeria japonica are potent modulators of inflammatory mediators.

Cryptomeria japonica is an important plantation conifer tree in Asia. This study aimed to characterize the anti-inflammatory and hepatoprotective activities of the phytocompounds from C. japonica wood on LPS- or TPA-induced activation of proinflammatory mediators and CCl(4)-induced acute liver injury in mice. A CJH7-2 fraction was purified from C. japonica extracts following bioactivity-guided fractionation, and it exhibited significant activities on inhibition of NO production and iNOS expression as well as up-regulating HO-1 expression in LPS-stimulated macrophages. CJH7-2 also potently inhibits COX-2 enzymatic activity (IC(50)=5 microg/mL) and TPA-induced COX-2 protein expression in mouse skin (1mg/200 microL/site). CJH7-2 (10 mg/kg BW) can prevent CCl(4)-induced liver injury and aminotransferases activities in mice. Chemical fingerprinting analysis showed that terpenes are the major bioactive compounds in the CJH7-2 fraction. This is the first study to demonstrate that chemical constituents from the wood extract of C. japonica possess anti-inflammatory activities in vitro and in vivo that may play a role in hepatoprotection.

A novel polyacetylene significantly inhibits angiogenesis and promotes apoptosis in human endothelial cells through activation of the CDK inhibitors and caspase-7.

A novel bioactive polyacetylene compound, 1,2-dihydroxy-5(E)-tridecene-7,9,11-triyne (compound 1), was identified from the Bidens pilosa extract using an ex vivo primary human umbilical vein endothelium cell (HUVEC) bioassay-guided fractionation protocol. Our results demonstrate that compound 1 (at 2.5 microg/mL) possessed significant anti-angiogenic effects, as manifested by an inhibition of HUVEC proliferation, migration, and the formation of tube-like structures in collagen gel. Moreover, compound 1 induced HUVECs to undergo cell death in a concentration- and time-dependent manner. The mechanisms underlying these pharmacological effects include reduced expression of cell cycle mediators such as CDK4, cyclins D1 and A, retinoblastoma (Rb) and vascular endothelial growth factor receptor 1 (VEGFR-1), and promotion of caspase-mediated activation of CDK inhibitors p21(Cip1) and p27(Kip). Moreover, apoptotic induction in HUVECs mediated by compound 1 was found to be in part through overexpression of FasL protein, down-regulation of anti-apoptotic Bcl-2, and activation of caspase-7 and poly(ADP-ribose) polymerase. This study demonstrates the potent anti-angiogenic and apoptotic activities of compound 1, suggesting that phytocompounds such as polyacetylenes deserve more attention regarding their potential as candidates for anti-angiogenic therapeutics.

Ethyl caffeate suppresses NF-kappaB activation and its downstream inflammatory mediators, iNOS, COX-2, and PGE2 in vitro or in mouse skin.

Ethyl caffeate, a natural phenolic compound, was isolated from Bidens pilosa, a medicinal plant popularly used for treating certain inflammatory syndromes. The purpose of this study was to investigate the structural activity, and the anti-inflammatory functions and mechanism(s) of ethyl caffeate. Ethyl caffeate was found to markedly suppress the lipopolysaccharide (LPS)-induced nitric oxide (NO) production (IC(50) = 5.5 microg ml(-1)), mRNA and protein expressions of inducible nitric oxide synthase (iNOS), and prostaglandin E(2) (PGE(2)) production in RAW 264.7 macrophages. Transient gene expression assays using human cox-2 promoter construct revealed that ethyl caffeate exerted an inhibitory effect on cox-2 transcriptional activity in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells. Immunohistochemical studies of mouse skin demonstrated that TPA-induced COX-2 expression was significantly inhibited by ethyl caffeate with a superior effect to that of celecoxib, a nonsteroidal anti-inflammatory drug. The phosphorylation and degradation of inhibitor kappaB (IkappaB) and the translocation of nuclear transcription factor-kappaB (NF-kappaB) into the nucleus, as well as the activation of mitogen-activated protein kinases (MAPKs) induced by LPS in macrophages, were not affected by ethyl caffeate. Ethyl caffeate, however, could inhibit NF-kappaB activation by impairing the binding of NF-kappaB to its cis-acting element. These results suggest that ethyl caffeate suppresses iNOS and COX-2 expressions partly through the inhibition of the NF-kappaB.DNA complex formation. Structure-activity relationship analyses suggested that the catechol moiety and alpha,beta-unsaturated ester group in ethyl caffeate are important and essential structural features for preventing NF-kappaB.DNA complex formation. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory and therapeutic properties of ethyl caffeate.

Induction of apoptosis in MCF-7 human breast cancer cells by phytochemicals from Anoectochilus formosanus.

The antitumor activity of Anoectochilus formosanus Hayata (Orchidaceae), a popularly used folk medicine in the treatment of cancers in Asia, was investigated in MCF-7 human mammary carcinoma cells. Plant extracts of A. formosanus were observed to induce apoptosis of MCF-7 cells as evidenced by cell-morphological changes, an early redistribution of plasma membrane phosphatidylserine, and DNA content distribution studies. Bioactivity-guided fractionation of A. formosanus extracts produced a specific ethyl acetate (EA)-partitioned fraction in which apoptotic activity was enriched. The chemical profile and candidate index compounds of the active EA fraction were obtained using HPLC and various spectral analyses. Western blot analysis showed that upon treatment of MCF-7 cells with the EA fraction, cleavage of pro-caspases-8, -9, and -7, and poly(ADP-ribose) polymerase as well as significant release of mitochondrial cytochrome c into the cytosol were readily observed. Flow cytometry showed that the Fas ligand protein was overexpressed in EA-treated MCF-7 cells. Functional genomic studies indicated that specific genes related to cytoskeleton rearrangement, apoptotic signal transduction, and various transcription factors were differentially regulated in EA-treated MCF-7 cells. Putative apoptotic signaling pathways of MCF-7 cells in response to the EA extract of A. formosanus are proposed.

Antioxidant properties and phytochemical characteristics of extracts from Lactuca indica.

Lactuca indica (Compositae) is an edible wild vegetable, used as a folk medicine in anti-inflammatory, antibacterial, and other medications in Asia. This is the first scientific evaluation of the chemopreventive therapeutic properties of L. indica using five antioxidation assay systems. An extract from L. indica was found to possess significant free radical scavenging activity, effectively protecting phix174 supercoiled DNA against strand cleavage and reducing oxidative stress in human promyelocytic leukemia HL-60 cells. Moreover, extracts of L. indica almost totally inhibited nitric oxide production and the mRNA expression of inducible nitric oxide synthase, at a dosage of 100 microg/mL, in LPS-stimulated macrophage RAW264.7 cells. Bioactivity-guided chromatographic fractionation and metabolite profiling coupled with spectroscopic analyses revealed that the six phenolic compounds, that is, protocatechulic acid (1), methyl p-hydroxybenzoate (2), caffeic acid (3), 3,5-dicaffeoylquinic acid (4), luteolin 7-O-beta-glucopyranoside (5), and quercetin 3-O-beta-glucopyranoside (6), are the major antioxidative constituents in the L. indica extract.