1 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. 2 Ethyl caffeate was found to markedly suppress the lipopolysaccharide (LPS)-induced nitric oxide (NO) production (IC 50 ¼ 5.5 mg ml À1 ), mRNA and protein expressions of inducible nitric oxide synthase (iNOS), and prostaglandin E 2 (PGE 2 ) production in RAW 264.7 macrophages. 3 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. 4 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. 5 The phosphorylation and degradation of inhibitor kB (IkB) and the translocation of nuclear transcription factor-kB (NF-kB) 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-kB activation by impairing the binding of NF-kB to its cis-acting element. These results suggest that ethyl caffeate suppresses iNOS and COX-2 expressions partly through the inhibition of the NF-kB Á DNA complex formation. 6 Structure-activity relationship analyses suggested that the catechol moiety and a,b-unsaturated ester group in ethyl caffeate are important and essential structural features for preventing NFkB Á DNA complex formation. This study provides an insight into the probable mechanism(s) underlying the anti-inflammatory and therapeutic properties of ethyl caffeate.

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. Waf1/Cip1 expression and caspase activation cascades were up-regulated by DET, effects suppressed by N-acetyl-L-cysteine. Moreover, tumour necrosis factor a-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.

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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.

Calocedrus formosana Florin (Cupressaceae) is an endemic tree to Taiwan. To evaluate the antioxidant activity of the plant extracts from heartwood, bark, and leaf of C. formosana, assays for 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and superoxide anion scavenging activities, as well as prevention of DNA strand cleavage were performed in this study. Similar IC 50 values against the DPPH radical were found for the heartwood and bark extracts at approximately 23 µg/ml. Moreover, the heartwood extract exhibited the highest inhibitory activity against superoxide radicals among the test samples; a 2.3-fold lower value of IC 50 for superoxide radical inhibition was found in the heartwood extract relative to that of (ϩ)-catechin. Much less effect on inhibition of DPPH and superoxide radicals was found from the leaf extract of C. formosana. More than 70% of superoxide radicals were inhibited in the presence of 10 µg/ml heartwood extract, whereas only 15% inhibition was obtained from the leaf extract. The heartwood extract, at a dose of approximately 0.5 mg/ml, apparently completely prevented the FX174 supercoiled DNA cleavage induced by ultraviolet photolysis of H 2 O 2 , as judged by agarose gel electrophoresis. This report also suggests that the antioxidant activities of the plant extracts of C. formosana are in good correlation with their phenolic contents.

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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.

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.