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Background: Cytokines such as TNF-alpha and IL-1 beta are known for their contribution to inflammatory processes in liver. In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1 beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization., Results: The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately up-regulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1 beta proposed an "IL-1 beta-like effect" of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase activity of the up-regulated Zc3h12a have to be considered., Conclusions: Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signaling pathway. Gene expression profile suggests IL-17 alone and in concert with TNF-alpha a role in sustaining liver inflammatory processes. IL-17 might exceed this function by RNA stabilization.

The anti-inflammatory activity of hispidol A 25-methyl ether (hispidol A 25-Me ether), a triterpenoid isolated from Ponciri Immaturus Fructus, was studied in lipopolysaccharide (LPS)-stimulated RAW264.7 murine macrophages. It was revealed that hispidol A 25-Me ether dose-dependently inhibits nitric oxide (NO) production by down-regulating inducible nitric oxide synthase (iNOS). It also reduces prostaglandin E(2) (PGE(2)) production by inhibiting cyclooxygenase-2 (COX-2) expression proven on both mRNA as well as on protein levels. In addition, hispidol A 25-Me ether inhibits mRNA expressions of major pro-inflammatory cytokines including the tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interleukin-6 (IL-6). Interestingly, hispidol A 25-Me ether probably exhibits a glucocorticoid-like activity, exerting functional inhibition of NF-kappaB without inhibition of DNA binging as de novo synthesis of IkappaB-alpha was induced and thereby NF-kappaB activity was reduced. Furthermore, administrations of hispidol A 25-Me ether (1 and 10mg/kg, i.p., v/w.) were tested in two animal experiments involving acute inflammation, namely, the carrageenan-induced paw edema swelling test and the acetic acid-induced vascular permeability assay, and showed concentration-related inhibitory activities. The anti-inflammatory property of hispidol A 25-Me ether seems to resemble the effects of the class of naturally occurring anti-inflammatory agents, glucocorticoids, which inhibit transcriptions of important inflammatory mediators., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Cucurbitacin R is known to exhibit an anti-inflammatory effect in different experimental models of inflammation. In this article, we outline the effect of cucurbitacin R on T lymphocyte proliferation, cytokine production, and nuclear factor activation, as well as its influence on various experimental models of delayed-type hypersensitivity (DTH) in mice. Cucurbitacin R reduced the proliferation of phytohemagglutinin A-stimulated human T lymphocytes (IC(50), 18 microM), modifying the cell cycle, as well as the production of cytokines [interleukin (IL)-2, IL-4, IL-10, and especially interferon-gamma] and the induction of the principal cyclins implicated in the cell cycle (A(1), B(1), D(2), and E). These effects are brought on by a novel, selective inhibition of nuclear factor AT (NFAT) by cucurbitacin R, with no concomitant effect on other transcription factors such as activator protein-1. In addition, we tested the in vivo effects of cucurbitacin R in three experimental models of DTH, as well as its effects on T lymphocyte proliferation, the cell cycle, cytokines, and cyclins. Although cucurbitacin R was found to reduce the inflammatory response brought on by both oxazolone and dinitrofluorobenzene, its activity was even more pronounced against sheep red blood cell-induced edema in mouse paws, with a clear reduction in the production of IL-1beta, IL-4, and tumor necrosis factor alpha in the inflamed paw. In conclusion, cucurbitacin R has the potential to be a new immunosuppressive agent with antiproliferative effects through the inhibition of the NFAT with anti-inflammatory activity in DTH reactions.

Apoptosis is regulated by several signaling pathways which are extensively linked by crosstalks. Boolean or logical modeling has become a promising approach to capture the qualitative behavior of such complex networks. Here we built a large-scale literature-based Boolean model of the central intrinsic and extrinsic apoptosis pathways as well as pathways connected with them. The model responds to several external stimuli such as Fas ligand, TNF-alpha, UV-B irradiation, interleukin-1beta and insulin. Timescales and multi-value node logic were used and turned out to be indispensable to reproduce the behavior of the apoptotic network. The coherence of the model was experimentally validated. Thereby an UV-B dose-effect is shown for the first time in mouse hepatocytes. Analysis of the model revealed a tight regulation emerging from high connectivity and spanning crosstalks and a particular importance of feedback loops. An unexpected feedback from Smac release to RIP could further increase complex II formation. The introduced Boolean model provides a comprehensive and coherent description of the apoptosis network behavior. It gives new insights into the complex interplay of pro- and antiapoptotic factors and can be easily expanded to other signaling pathways.

Herein we report a feasibility study for a new concept to detect DNA binding protein NF-kappaB based on a DNA triple helix formation in combination with a fluorescence resonance energy transfer (FRET). The new principle avoids expensive antibodies and radioactivity and might have implications for assays of other DNA binding proteins.

We report on the structure elucidation of arnamial, a new Delta(2,4)-protoilludane everninate ester from the fungus Armillaria mellea, and on the apoptotic activity of arnamial as well as the cytotoxic activity of structurally related compounds on selected human cancer cells. Arnamial showed cytotoxicity against Jurkat T cells, MCF-7 breast adenocarcinoma, CCRF-CEM lymphoblastic leukemia, and HCT-116 colorectal carcinoma cells at IC50 = 3.9, 15.4, 8.9, and 10.7 microM, respectively, and the related aryl ester melledonal C showed cytotoxic activity against CCRF-CEM cells (IC50 = 14.75 microM). [1,2-13C2]Acetate feeding supports a polyketide origin of the orsellinic acid moiety of arnamial.

A new GC-FID method, which allows rapid and reliable quantitation of nicotine in tobacco leaf extracts, was developed and validated. To avoid nicotine adsorption on the column, an amine-deactivated capillary column was used. The method developed was applied to study the degradation of nicotine in a fermented aqueous extract, and a loss of nearly 20% of nicotine over 12 months was observed. Careful inspection of GC-MS runs from concentrated samples of the same extract revealed the presence of nicotine metabolites such as nornicotine, anatabine, myosmine, 2,3'-bipyridyl, and 2-pyrrolidinone.

BaP1, a zinc-dependent endopeptidase belonging to the P-I class of snake venom metalloproteinases, exerts multiple tissue-damaging activities, leading to hemorrhage, myonecrosis, dermonecrosis, blistering, and edema. Interestingly, this metalloproteinase shows a high degree of structural homology with the catalytic domain of human adamalysins and matrix metalloproteinases, especially at the strictly conserved zinc binding motif and the so-called Met turn. This highlights BaP1 as an interesting model concerning inhibitor design for several medicinally important metalloproteinases, such as tumor necrosis factor alpha converting enzyme. Here, we report the first crystal structure of BaP1 complexed with a peptidomimetic inhibitor. Suitable crystals were obtained at four different pH values (4.6, 6.5, 7.5, and 8.0), and four high-resolution structures (1.46, 1.14, 1.08, and 1.05 A) were established. These structures and the detailed analysis of the structure-activity relationship of the bound inhibitor form a basis for the design of potent BaP1 inhibitors. The latter can be used for the treatment of local pathological effects caused by snake bites, mainly due to metalloproteinases such as BaP1. Besides, the high-resolution structure is an excellent starting point for the rational development of inhibitors for human metalloproteinases. The finding of a flexible loop region may have a great impact on further studies as to date little is known about the structural dependencies of the hemorrhagic activity of snake venom metalloproteinases.

To investigate possible congruencies between DNA sequence data and secondary chemistry, we compared nuclear ribosomal DNA (nrDNA) sequence data, sesquiterpene lactone (STL) contents, and cytometric data from 35 accessions of 16 Arnica (Asteraceae) species and two outgroup taxa (Layia hieracioides and Madia sativa), using phylogenetic inference and principal component analysis (PCA). Several groups supporting multiple accessions of the same species (of A. montana, A. longifolia, A. gracilis, and A. chamissonis) are congruent between the phylogenetic trees based on nrDNA and STL data. Sesquiterpene lactone profiles were found to be highly consistent within multiple samples of A. montana and A. longifolia respectively. Moreover, sesquiterpene lactone data support subspecies classifications of A. chamissonis and A. parryi, with additional support from DNA sequence data and cytometric data. Morphology, STL data (PCA), cytometric data and DNA sequence data suggest a hybrid origin of one accession (A. gracilis × longifolia). In A. gracilis, A. latifolia, and Layia hieracioides, previously not investigated for STLs, we found large amounts of xanthalongin derivatives. This is the first time STLs have been reported from subtribe Madiinae. © The Willi Hennig Society 2009., (© The Willi Hennig Society 2009.)

Preparations of Harpagophytum procumbens and of Salix species are successfully used for the treatment of degenerative rheumatism and painful arthrosis. For the quality control of both drugs, rapid methods of quantification are desirable. Here we report the development of two HPTLC methods enabling the determination of harpagoside in Harpagophyti radix and of salicin in Salicis cortex. We focused on a standardized methodology and thorough validation including two laboratories. The methods allow the analyses of up to 16 samples in parallel demonstrating the proposed methods as very rapid and cost efficient.

The anti-inflammatory effects of glycyrol, a benzofuran coumarin isolated from Glycyrrhizae Radix, were studied. Glycyrol of 5, 25 and 50 microM dose-dependently inhibited nitric oxide (NO) production by down-regulating inducible nitric oxide synthase (iNOS), and alleviated cyclooxygenase-2 (COX-2) expression in LPS-stimulated RAW264.7 macrophages, in both the mRNA and the protein. Furthermore, glycyrol dose-dependently decreased the mRNA of the pro-inflammatory cytokines IL-1beta and IL-6. LPS-induced NF-kappaB activation was prevented in RAW264.7 macrophages by inhibition of I-kappaBalpha phosphorylation. In addition, administration of glycyrol (30 and 100 mg/kg, i.p) reduced the thickness of carrageenan-induced mouse-paw edema swelling. Taken together, our results indicate that glycyrol is an important anti-inflammatory constituent of Glycyrrhizae Radix, and that its anti-inflammatory effect is attributed to the inhibition I-kappaBalpha phosphorylation.

Luteolin is a flavone which occurs in medicinal plants as well as in some vegetables and spices. It is a natural anti-oxidant with less pro-oxidant potential than the flavonol quercetin, the best studied flavonoid, but apparently with a better safety profile. It displays excellent radical scavenging and cytoprotective properties, especially when tested in complex biological systems where it can interact with other anti-oxidants like vitamins. Luteolin displays specific anti-inflammatory effects at micromolar concentrations which are only partly explained by its anti-oxidant capacities. The anti-inflammatory activity includes activation of anti-oxidative enzymes, suppression of the NFkappaB pathway and inhibition of pro-inflammatory substances. In vivo, luteolin reduced increased vascular permeability and was effective in animal models of inflammation after parenteral and oral application. Although luteolin is only a minor component in our nutrition (less than 1 mg/day) epidemiological studies indicate that it has the potential to protect from diseases associated with inflammatory processes such as cardiovascular disease. Luteolin often occurs in the form of glycosides in plants, but these are cleaved and the aglycones are conjugated and metabolized after nutritional uptake which has to be considered when evaluating in vitro studies. Some data for oral and topical bioavailability exist, but more quantitative research in this field is needed to evaluate the physiological and therapeutical potential of luteolin.

Luteolin is a flavonoid which is part of our daily nutrition in relatively low amounts (less than 1 mg/day). Nevertheless, some epidemiological studies suggest an inverse correlation between luteolin intake and the risk of some cancer types. Luteolin displays specific anti-inflammatory and anti-carcinogenic effects, which can only partly be explained by its anti-oxidant and free radical scavenging capacities. Luteolin can delay or block the development of cancer cells in vitro and in vivo by protection from carcinogenic stimuli, by inhibition of tumor cell proliferation, by induction of cell cycle arrest and by induction of apoptosis via intrinsic and extrinsic signaling pathways. When compared to other flavonoids, luteolin was usually among the most effective ones, inhibiting tumor cell proliferation with IC(50) values between 3 and 50 microM in vitro and in vivo by 5 to 10 mg/kg i.p., intragastric application of 0.1-0.3 mg/kg/d, or as food additive in concentrations of 50 to 200 ppm. Luteolin has been shown to penetrate into human skin, making it also a candidate for the prevention and treatment of skin cancer.

Sesquiterpene lactones (SL), secondary plant metabolites from flowerheads of Arnica, exert anti-inflammatory effects mainly by preventing nuclear factor (NF)-kappaB activation because of alkylation of the p65 subunit. Despite its known immunosuppressive action, Arnica has been classified as a plant with strong potency to induce allergic contact dermatitis. Here we examined the dual role of SL as anti-inflammatory compounds and contact allergens in vitro and in vivo. We tested the anti-inflammatory and allergenic potential of SL in the mouse contact hypersensitivity model. We also used dendritic cells to study the activation of NF-kappaB and the secretion of interleukin (IL)-12 in the presence of different doses of SL in vitro. Arnica tinctures and SL potently suppressed NF-kappaB activation and IL-12 production in dendritic cells at high concentrations, but had immunostimulatory effects at low concentrations. Contact hypersensitivity could not be induced in the mouse model, even when Arnica tinctures or SL were applied undiluted to inflamed skin. In contrast, Arnica tinctures suppressed contact hypersensitivity to the strong contact sensitizer trinitrochlorobenzene and activation of dendritic cells. However, contact hypersensitivity to Arnica tincture could be induced in acutely CD4-depleted MHC II knockout mice. These results suggest that induction of contact hypersensitivity by Arnica is prevented by its anti-inflammatory effect and immunosuppression as a result of immune regulation in immunocompetent mice.

In addition to known heliangolides, a new eudesmanolide was isolated from the leaf rinse extract of Viguiera robusta (Asteraceae). Structural elucidation was based on spectral analysis. It is the first report on eudesmanolides in members of the subgenus Calanticaria of Viguiera. In this work, the main isolated compound, the furanoheliangolide budlein A, besides its previously reported in vitro and in vivo anti-inflammatory activities, inhibited human neutrophil elastase release. The inhibition was at the concentration of (16.83 +/- 1.96) microM for formylated bacterial tripeptide (fMLP) stimulation and (11.84 +/- 1.62) microM for platelet aggregation factor (PAF) stimulation, being slightly less active than the reference drug parthenolide. The results are important to demonstrate the potential anti-inflammatory activities of sesquiterpene lactones and corroborate the previous studies using other targets.

Glycogen synthase kinase-3 (GSK-3) is known to modulate cell survival and apoptosis through multiple intracellular signaling pathways. However, its hepatoprotective function and its role in activation of NF-kappaB and anti-apoptotic factors are poorly understood and remain controversial. Here we investigated whether inhibition of GSK-3 could induce apoptosis in the presence of TNF-alpha in primary mouse hepatocytes. We show that pharmacological inhibition of GSK-3 in primary mouse hepatocytes does not lead to TNF-alpha-induced apoptosis despite reduced NF-kappaB activity. Enhanced stability of IkappaB-alpha appears to be responsible for lower levels of nuclear NF-kappaB and hence reduced transactivation. Additionally, inhibition of GSK-3 was accompanied by marked upregulation of beta-catenin, AP-1, and CREB transcription factors. Stimulation of canonical Wnt signaling and CREB activity led to elevated levels of anti-apoptotic factors. Hence, survival of primary mouse hepatocytes may be caused by the activation and/or upregulation of other key regulators of liver homeostasis and regeneration. These signaling molecules may compensate for the compromised anti-apoptotic function of NF-kappaB and allow survival of hepatocytes in the presence of TNF-alpha and GSK-3 inhibition.

Sesquiterpene lactones are the active components of a variety of medicinal plants from the Asteraceae family. They possess biological activities such as the inhibition of NF-kappaB and the release inhibition of the vasoactive serotonin. On the basis of a data set of 54 SLs, we report the development of a quantitative model for the prediction of serotonin release inhibition. Comparing this model with a previous investigation of the target NF-kappaB, structural features necessary for specific compounds could be acquired. Atomic properties encoded by radial distribution function and molecular surface potentials encoded by autocorrelation were used as descriptors. Whereas some descriptors describe the structural requirements for both activities, other descriptors can be used to decide whether an SL is more active to NF-kappaB or to serotonin release. Again, counter propagation neural networks proved to be a valuable tool to establish structure-activity relationships that are necessary for the search for and optimization of lead structures.

Human neutrophil elastase (HNE), a serine protease, is involved in the regulation of inflammatory processes and controlled by endogenous proteinase inhibitors. Abnormally high levels of HNE can cause degradation of healthy tissues contributing to inflammatory diseases such as rheumatoid arthritis, and also psoriasis and delayed wound healing. In continuation of our research on HNE inhibitors we have used the recently developed binding mode model for a group of cinnamic acid derivative elastase inhibitors and created bornyl (3,4,5-trihydroxy)-cinnamate. This ligand exhibited improved binding affinity predicted by means of free energy calculations. An organic synthesis scheme for the ligand was developed and its inhibitory activity was tested toward the isolated enzyme. Its IC(50) value was found to be three times lower than that of similar compounds, which is in line with the computational result showing the high potential of free energy calculations as a tool in drug development.

Background: Bisphosphonates have shown direct antitumoral activity in vitro, in vivo and even in clinical studies, but the exact mechanism for this has not yet been elucidated. In this study the antiangiogenic potency of zoledronic acid and clodronate were evaluated., Materials and Methods: The effects of zoledronic acid and clodronate on the proliferation of endothelial cells and different tumor cells, and on the activity of protein kinases were investigated. Furthermore in vitro experiments were performed to evaluate the underlying antiangiogenic mechanism of action. Both bisphosphonates were examined in vivo at different doses and in daily subcutaneous application in a murine renal cell carcinoma model (RENCA). The antiangiogenic activity was evaluated by immunohistochemical staining (CD31) and by determination of mouse vascular endothelial growth factor (VEGF) serum concentration., Results: Zoledronic acid and clodronate inhibited proliferation of endothelial cells at lower concentrations than the different tumor cell lines did. This effect was more pronounced for zoledronic acid. The activity of almost all tested kinases was inhibited by zoledronic acid, whereas clodronate showed no effect. In the RENCA model, a significant effect of zoledronic acid on the primary tumor in a bell-shaped dose response curve with the highest efficacy between 100 Bg/kg 2xd and 200 Bg/kg 1xd, was observed. The mean vessel density (MVD) was significantly reduced by both bisphosphonates at different concentrations. This is the first report on increased mouse VEGF serum concentrations in the RENCA model., Conclusion: The results indicate that these bisphosphonates, particularly zoledronic acid, possess antitumoral and antiangiogenic activity.

Background: Tormentil extracts (TE) have antioxidative properties and are used as a complementary therapy for chronic inflammatory bowel disease. In individual patients with ulcerative colitis (UC) positive effects have been observed., Goals: To assess the safety, pharmacology, and clinical effects of different doses of TE in patients with active UC., Study: Sixteen patients with active UC [clinical activity index (CAI) >/=5] received TE in escalating doses of 1200, 1800, 2400 and 3000 mg/d for 3 weeks each. Each treatment phase was followed by a 4-week washout phase. The outcome parameters were side effects, CAI, C-reactive protein, and tannin levels in patient sera., Results: Mild upper abdominal discomfort was experienced by 6 patients (38%), but did not require discontinuation of the medication. During therapy with 2400 mg TE per day, median CAI and C-reactive protein improved from 8 (6 to 10.75) and 8 (3 to 17.75) mg/L at baseline to 4.5 (1.75 to 6) and 3 (3 to 6) mg/L, respectively. During therapy, the CAI decreased in all patients, whereas it increased during the washout phase. Neither undegraded nor metabolized tannins could be detected by liquid-mass spectrometry (LC-MS) in patient sera., Conclusions: TE appeared safe up to 3000 mg/d. Tannins from TE are not systemically absorbed. The efficacy in patients with UC should be further evaluated.

The imbalance between human neutrophil elastase (HNE) and endogenous serine proteinase inhibitors is considered to cause a variety of HNE-mediated inflammatory disorders. Thus, HNE has been the object of intensive research to find potent inhibitors that target its destructive and pro-inflammatory action. This review focuses on natural compounds which have been demonstrated to inhibit the enzyme itself or its release from neutrophils. Some of the natural compounds discussed here may serve as lead structures suitable to be used for the development of semi-synthetic inhibitors, but up to now none has been found active enough to be directly used in therapy.

Preparations of Arnica montana L. are widely used for the topical treatment of inflammatory diseases. The anti-inflammatory activity is mainly attributed to their sesquiterpene lactones (SLs) from the helenalin and 11alpha,13-dihydrohelenalin type. To study the penetration kinetics of SLs in Arnica preparations, a stripping method with adhesive tape and pig skin as a model was used. For the determination of SLs in the stripped layers of the stratum corneum (SC), a gas chromatography/mass spectrometry method was developed and validated. Thereby the amount of helenalin derivatives was calculated as helenalin isobutyrate, and 11alpha,13-dihydrohelenalin derivatives as 11alpha,13-dihydrohelenalin methacrylate. This GC-MSD method is suitable also to determine low amounts of SLs in Arnica preparations. The penetration behaviour of one gel preparation and two ointment preparations was investigated. The SLs of all preparations show a comparable penetration in and a permeation through the stratum corneum, the uppermost part of the skin. Interestingly, the gel preparation showed a decrease of the penetration rate over 4h, whereas the penetration rate of ointments kept constant over time. Moreover, we could demonstrate that the totally penetrated amount of SLs only depends on the kind of the formulation and of the SLs-content in the formulation but not on the SLs composition or on the used extraction agent.

Sesquiterpene lactones (SQTLs) are shown to possess anti-inflammatory as well as cytotoxic activity. No study, however, links both activities. We, therefore, hypothesized that SQTL-treated, dying cells might induce an anti-inflammatory response in cocultured THP-1 macrophages. Here we show that SQTLs bearing either an alpha,beta-unsaturated cyclopentenone or an alpha-methylene-gamma-lactone induce different forms of cell death. Whereas the cyclopentenone SQTL induced typical apoptosis, the alpha-methylene-gamma-lactone SQTLs-induced cell death lacked partly classical signs of apoptosis, such as DNA fragmentation. All SQTLs, however, activated caspases and the nuclear morphology of cell death was dependent on caspase activation. Most interestingly, alpha-methylene-gamma-lactone SQTLs induced a more pronounced phosphatidylserine (PS) exposure than the cyclopentenone SQTL. Especially, 7-hydroxycostunolide (HC), with an alpha-methylene-gamma-lactone substituted with a hydroxyl group, showed a striking fast and pronounced PS translocation. This result was in agreement with a strong activation of phagocytosis in cocultured THP-1 macrophages. Interestingly, HC-treated Jurkat cells led to an early (3.5 h) but transient increase in TNF-alpha levels in macrophage coculture. Release of TGF-beta remained unaffected after 18 h. We propose that this type of SQTL may influence local inflammation by transiently activating the immune system and help to clear cells by inducing a form of cell death that promotes phagocytosis.

Complex cellular networks regulate regeneration, detoxification and differentiation of hepatocytes. By combining experimental data with mathematical modelling, systems biology holds great promises to elucidate the key regulatory mechanisms involved and predict targets for efficient intervention. For the generation of high-quality quantitative data suitable for mathematical modelling a standardised in vitro system is essential. Therefore the authors developed standard operating procedures for the preparation and cultivation of primary mouse hepatocytes. To reliably monitor the dynamic induction of signalling pathways, the authors established starvation conditions and evaluated the extent of starvation-associated stress by quantifying several metabolic functions of cultured primary hepatocytes, namely activities of glutathione-S-transferase, glutamine synthetase, CYP3A as well as secretion of lactate and urea into the culture medium. Establishment of constant metabolic activities after an initial decrease compared with freshly isolated hepatocytes showed that the cultured hepatocytes achieve a new equilibrium state that was not affected by our starving conditions. To verify the highly reproducible dynamic activation of signalling pathways in the in vitro system, the authors examined the JAK-STAT, SMAD, PI3 kinase, MAP kinase, NF-kappaB and Wnt/beta-catenin signalling pathways. For the induction of gp130, JAK1 and STAT3 phosphorylation IL6 was used, whereas TGFbeta was applied to activate the phosphorylation of SMAD1, SMAD2 and SMAD3. Both Akt/PKB and ERK1/2 phosphorylation were stimulated by the addition of hepatocyte growth factor. The time-dependent induction of a pool of signalling competent beta-catenin was monitored in response to the inhibition of GSK3beta. To analyse whether phosphorylation is actually leading to transcriptional responses, luciferase reporter gene constructs driven by multiple copies of TGFbeta-responsive motives were applied, demonstrating a dose-dependent increase in luciferase activity. Moreover, the induction of apoptosis by the TNF-like cytokine Fas ligand was studied in the in vitro system. Thus, the mouse hepatocyte in vitro system provides an important basis for the generation of high-quality quantitative data under standardised cell culture conditions that is essential to elucidate critical hepatocellular functions by the systems biology approach.

Human neutrophil elastase (HNE) is a serine protease, which is present in its active form in inflamed tissue as well as in psoriatic lesions. In extension of our research on natural compounds as inhibitors of HNE or of its release, several phenolics of different size were tested. The ellagitannins agrimoniin and pedunculagin were the most potent direct HNE inhibitors (IC (50) = 0.9 and 2.8 microM, respectively). Ligand docking calculations provided evidence that inhibition may occur in an unspecific manner. Agrimoniin also showed anti-proliferative effects in the ATP assay (IC (50) = 3.2 microM), suggesting that this type of tannin could have beneficial effects in the treatment of diseases such as psoriasis. Tests with other phenolics combined with ligand docking experiments revealed that, besides the presence of ORTHO-dihydroxy groups, a specific lipophilic shape is necessary for an inhibitory activity. The phenolic genistein deserves special interest as an inhibitor of elastase release because its effect was remarkably potent (IC (50) = 0.6 microM).

Two Brazilian species of Dimerostemma (Asteraceae) were chemically investigated. Two known sesquiterpene lactones (STLs), a germacrolide and an eudesmanolide, were isolated from D. episcopale while D. brasilianum afforded the new germacranolide 1beta,5beta/10alpha-trihy-droxy-8alpha-angeloyloxy-germacra-3,11(13)-dien-6alpha,12-olide in addition to a known one. Structure identification based on NMR and MS analyses. 1beta,10alpha,4alpha,5beta-Diepoxy-8alpha-angeloyloxy-costunolide isolated from D. brasilianum was studied for its anti-inflammatory activity. This STL completely inhibited DNA binding of the transcription factor NF-kappaB at a concentration of 5 microm and 10 microM in Jurkat T and Raw 264.7 cells, respectively. Elastase release from human neutrophils was reduced to 50% at a concentrations of 23 microM after stimulation with PAF and of 27 microM after stimulation with fMLP without showing cytotoxic effects. Additionally, elastase was also directly inhibited.

Nuclear factor-kappaB (NF-kappaB) is a central transcriptional factor and a pleiotropic regulator of many genes involved in immunological responses. Dysregulation of NF-kappaB can lead to a wide variety of diseases, including those related to inflammation and cancer. Therefore, NF-kappaB and the signalling pathways that regulate its activity have become a focal point for intense drug discovery and development by a number of drug companies and academic groups. Small-molecule inhibitors of regulatory enzymes or of NF-kappaB itself, gene therapy, decoy oligonucleotides or various biologicals have been reported and patented as potential candidates for drug development. This article reviews patents claiming NF-kappaB inhibitors during the period 2002 - 2005. Some of these inhibitors may be developed to therapeutics and used for the treatment of inflammatory diseases and tumour chemoresistance.

Twenty-four structurally different SLs were studied for their inhibition on IL-8 production in HeLa229 cells and different IC50-values were obtained. QSAR analyses revealed that the alpha-methylene-gamma-lactone and the presence and reactivity of a second reaction center, expressed by LUMO2, are the most important descriptors for IL-8. Using two SLs as examples, we demonstrated that SLs prevent DNA binding of AP-1, which has binding sites in the IL-8 promoter together with NF-kappaB and C/EBP, and that this is probably due to directly targeting AP-1. p38 MAPK, which plays a role in AP-1 activation as well as in IL-8 regulation, was not influenced by SLs. These data show that NF-kappaB and AP-1, and consequently IL-8 may be interesting targets in antiinflammation research and that the small molecules of SLs may be powerful candidates with promising properties for therapeutic modulation of the inflammatory response.

A variety of sesquiterpene lactones (SLs) possess considerable anti-inflammatory activity. Several studies have shown that they exert this effect in part by inhibiting the activation of the transcription factor NF-kappaB. In the present study we elaborated on the investigation of a data set of 103 structurally diverse SLs for which we had previously developed several different QSAR equations dependent on the skeletal type. Use of 3D structure descriptors resulted in a single model for the entire data set. In particular, local radial distribution functions (L-RDF) were used that centered on the methylene-carbonyl substructure believed to be the site of attack of cysteine-38 of the p65/NF-kappaB subunit. The model was developed by using a counterpropagation neural network (CPGNN), attesting to the power of this method for establishing structure-activity-relationships. The investigations shed more light onto the influence of the chemical structure on NF-kappaB inhibitory activity.

Chemicals and metal ions often induce allergic contact dermatitis. We review here recent advances in the development of in vitro assays for prediction of skin sensitizing potency based on chemical and biological reactivity as well as in the identification of physiological binding partners and immunological pathomechanisms of chemical and metal ion induced disease.

The altitudinal variation on the contents of secondary metabolites in flowering heads of Arnica montana was assessed. Plants of A. montana cultivar ARBO were grown in nine experimental plots at altitudes between 590 and 2230m at Mount Patscherkofel near Innsbruck/Austria. The total contents of sesquiterpene lactones and flavonoids were not positively correlated with the altitude of the growing site. However, the proportion of flavonoids with vicinal free hydroxy groups in ring B to flavonoids lacking this feature significantly increased with elevation. Additionally, the level of caffeic acid derivatives also positively correlated with the altitude of the growing site. In particular amounts of 1-methoxyoxaloyl-3,5-dicaffeoylquinic acid significantly increased in higher sites and samples from the summit region contained 85% more of this compound than samples from valley sites. These results are discussed with regards to chemosystematic studies comparing samples collected in different altitudes as well as in the light of a UV-B protective and radical scavenging function of phenolics and their significance for plant life in environments with elevated UV-B radiation.

Investigation of the aerial parts of Vernonia triflosculosa afforded three hirsutinolides of which 8alpha-(4alpha-hydroxymethacryloyloxy)-10alpha-hydroxy-1,13-dimethoxy-hirsutinolide is new, three ent-kaurane diterpenes, among which the 19-[alpha-L-arabinopyranosyl-(1-->2)-beta-D-glucopyranosyl] esters of 16beta-hydroxy-ent-kauran-19-oic acid and of 16beta,17-hydroxy-ent-kauran-19-oic acid are also new. Diterpenes are reported here for the first time in the large genus Vernonia. Their structures were elucidated using 1D and 2D NMR measurement as well as ESI, CIMS, and HRMS analysis. Two hirsutinolides were studied for their NF-kappaB DNA binding activity in HaCaT cells (a human cell line similar to keratinocytes) and for their inhibition on IL-8 production in HeLa cells.

Enteric Yersinia bacteria trigger transcription and secretion of the proinflammatory chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in epithelial cells. Both chemokines are controlled by NF-kappaB. The NF-kappaB-binding site in the IL-8 promoter preferentially binds Rel p65/p65 homodimers and p50/p65 heterodimers while the NF-kappaB-binding motifs of the MCP-1 promoter preferably bind p50/p65 heterodimers and p50/p50 homodimers. Sesquiterpene lactones inhibit the transcription factor NF-kappaB by alkylating the p65 subunit. In this study we investigated the inhibitory effects of sesquiterpene lactones and the NF-kappaB inhibitor SN50 on NF-kappaB p50 and p65 subunits in Yersinia-triggered IL-8 and MCP-1 production. The sesquiterpene lactones blocked Yersinia-triggered IL-8 and MCP-1 production in a dose-dependent manner. In contrast, SN50 inhibited IL-8 production at high concentrations whereas it diminished the amount of secreted MCP-1 significantly already at low concentrations. By means of electrophoretic mobility shift assays we demonstrate that sesquiterpene lactones inhibit Yersinia-triggered activation of NF-kappaB by inhibiting Rel p65, but not Rel p50. Our results also demonstrate that SN50 is useful for inhibition of nuclear translocation of the NF-kappaB p50 subunit but cannot be considered a general NF-kappaB inhibitor.

Investigation of an ethanolic extract prepared from fresh Arnica montana flowers afforded three new 1,5- trans-guaianolides, of which 11alpha,13-dihydro-2-O-tigloylflorilenalin and the respective 2-O-isovaleryl derivative are reported for the first time. Additionally, three new and one known 2beta-ethoxy-2,3-dihydrohelenalin esters were isolated. GC/MS studies of the extract after a two year storage at 4 degrees C demonstrated that the latter were artefacts that had been formed by addition of ethanol to the cyclopentenone structure of helenalin. Formation of these adducts gave compounds possessing an inhibitory activity comparable to that of 11alpha,13-dihydrohelenalin derivatives in the NF-kappaB EMSA and the IL-8 ELISA in vitro assays as well as in the in vivo croton oil-induced mouse ear edema test for one adduct, namely 2beta-ethoxy-6-O-acetyl-2,3-dihydrohelenalin. As expected, 6-O-(2-methylbutyryl)- and 6-O-methacryloyl-helenalin exhibited a stronger activity in the NF-kappaB EMSA and IL-8 ELISA. Sesquiterpene lactones seem to be the most important NF-kappaB inhibiting compounds in the Arnica extract. Bioguided fractionation using the luciferase reporter gene assay resulted in the isolation of only moderately active compounds, such as 6-acetoxy-2,2-dimethylchroman-4-one and 10-acetoxy-8,9-epoxythymol isobutyrate.

Neutrophil granulocytes are effector cells in innate and humoral immunity. They are involved in inflammatory processes by releasing pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). We here report an optimisation of an HNE release assay using microplates. Special attention has been directed to overcome the often observed activation of neutrophils during isolation from fresh blood. This so-called basal stimulation can take place without addition of external stimulants and can cause severe problems during the assay. We demonstrated that bovine serum albumin (BSA), lipopolysaccharide (LPS), use of different blood donors, heparin and ethylenediaminetetraacetic acid (EDTA) do not cause basal stimulation, but may be due to mechanical stress and the immune system of the blood donor. Here, the number of eosinophils may play a role in the induction of activation. Basal stimulation was overcome when a hypertonic solution, such as sucrose- with N-(2-hydroxyethyl)-piperazine-N'-2-ethanesulfonic acid (HEPES) buffer, was used during centrifugation and the isolated granulocytes were left in phosphate buffered saline (PBS) for 30 min before stimulation. Sesquiterpene lactones (SLs), known for their anti-inflammatory activity were used for evaluation of the assay and were observed to inhibit HNE release at micromolar concentrations. Whereas N-formyl-methionyl-leucylphenylalanine (fMLP), platelet-activating factor (PAF) and basal stimulation resulted in similar IC50 values, phorbol-12-myristate-13-acetate (PMA) as a stimulant needed higher concentrations of SLs. The molecular inhibitory mechanism of SLs is discussed.

Phospholipases A(2) (PLA(2)) are important constituents of snake venoms, being responsible for several of their toxic actions. Extracts from plants used in folk medicine were screened for inhibition of the enzymatic activity of myotoxin I, a PLA(2) from Bothrops asper. Piper umbellatum and Piper peltatum extracts tested positive, and their fractionation resulted in the isolation of 4-nerolidylcatechol. Its inhibitory effects towards toxic activities of two Bothrops myotoxins, representing catalytically active (Asp49) and catalytically inactive (Lys49) types of group II PLA(2)s, respectively, were characterized. The enzyme activity of B. asper myotoxin I was completely inhibited by 4-nerolidylcatechol at an inhibitor:toxin ratio of 10:1 (wt/wt) with an IC50 of approximately 1mM. In addition, 4-nerolidylcatechol inhibited representatives of groups I and III of PLA(2)s. Its preincubation with Bothrops myotoxins significantly reduced their myotoxic and edema-inducing activities in animal experiments. However, when 4-nerolidylcatechol was administered in situ, immediately after toxin injection, its inhibitory ability was substantially lower or negligible. This might be explained by the rapid action of these toxins in vivo, together with the slow inactivation of PLA(2) activity observed in vitro. Electrophoretic and chromatographic analyses of myotoxins ruled out major changes in protein charge, hydrophobicity, or gross molecular mass being involved in the inhibition mechanism. Mass spectrometry determinations are consistent with the covalent modification of myotoxin by one molecule of 4-nerolidylcatechol. Finally, a novel compound was isolated from both Piper species, sharing the nerolidyl skeleton, but nevertheless not being inhibitory towards the PLA(2)s studied.

Four new hopane-type saponins, glinusides F, G, H, and I (1-4), and the known succulentoside B (5), as well as the two known flavones 5,7,4'-trihydroxyflavone-6,8-di-C-glucoside (vicenin-2) and 5,7,4'-trihydroxyflavone-8-C-sophoroside (vitexin-2' '-O-glucoside), were isolated from the seeds of Glinus lotoides growing in Ethiopia. On the basis of the spectroscopic data analysis, including 2D NMR and HRESIMS, the new structures were characterized as 3beta-O-beta-D-xylopyranosyl-6alpha-O-beta-D-xylopyranosyl-16beta-O-beta-D-xylopyranosyl-22-hydroxyhopane (1), 3beta-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyl-6alpha,16beta-dihydroxy-22-O-alpha-L-rhamnopyranosylhopane (2), 3beta-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-D-xylopyranosyl-6alpha-O-beta-D-xylopyranosyl-16beta-hydroxy-22-O-alpha-L-rhamnopyranosylhopane (3), and 3beta-O-alpha-L-rhamnopyranosyl-(1-->2)-beta-d-xylopyranosyl-6alpha-O-beta-D-xylopyranosyl-16beta-O-beta-D-xylopyranosyl-22-hopane (4).

Sesquiterpene lactones (SLs) are the active compounds of a variety of traditionally used medicinal plants from the Asteraceae family. They are known to possess a considerable antiinflammatory activity in different inflammation models. They inhibit the transcription factor NF-kappaB probably by alkylating cysteine38 in the DNA binding domain of the p65 subunit. Here we investigate a set of 103 different sesquiterpene lactones representing 6 structural groups (44 germacranolides, 16 heliangolides, 22 guaianolides, 9 pseudoguaianolides, 2 hypocretenolides, 10 eudesmanolides) for their NF-kappaB inhibiting properties and the resulting IC(100)-values were submitted to a QSAR study. Properties important for the inhibition potency are discussed for the whole data set and for subsets of the different structural classes.

Alcoholic preparations of Arnica montana are widely used for the topical treatment of various inflammatory diseases. Sesquiterpene lactones (SLs) are mainly responsible for their anti-inflammatory activity. Here we have studied the penetration kinetics of Arnica tinctures prepared from dried Arnica flowers originating from different chemotypes as well as of their respective dominating SLs, helenalin isobutyrate and 11alpha,13-dihydrohelenalin acetate. Some alcoholic preparations of fresh Arnica flowers and an Arnica fresh plant gel were also included in the study. We used the stripping method with adhesive tape and pig skin as a model and determined the quantity of SLs in the stripped layers of the stratum corneum (SC). Thus, we observed the penetration into and permeation through this uppermost part of the skin. Whereas isolated SLs permeate through the SC only in a very small amount, permeation of SLs was much higher when they were present in the tinctures. Furthermore, differences of permeation were observed between helenalin and dihydrohelenalin derivatives. Permeation through the SC could be determined for the tested Arnica preparations of fresh Arnica flowers with two preparations showing the best penetration behaviour of all the tested substances. Moreover, the effects of incubation time as well as of repeated applications were investigated with one preparation. Altogether, this study shows that a sufficient amount of SLs might permeate the skin barrier by using Arnica preparations to exert anti-inflammatory effects and that the topical use of plant preparations may be advantageous compared to the isolated compounds.

Sesquiterpene lactones (SLs) are potent anti-inflammatory substances. It was previously shown that the anti-inflammatory effect could be partly explained by the inhibition of the transcription factor NF-kappaB. Whether they inhibit the DNA binding of NF-kappaB, the activation of the IkappaB-kinase, or both is still a matter of debate. The data supporting these hypotheses were obtained using different cell systems. In this contribution we analyzed the mechanism of the sesquiterpene lactone-mediated inhibition using different cell systems, and showed that in all the cell lines analyzed, SLs inhibited both NF-kappaB binding and the IkappaB-kinase, but that the former played a more preponderant role in the inhibition. These results again confirm the importance of cysteine 38 in the inhibition and regulation of NF-kappaB's function. Moreover, we compared the selectivity of the SL parthenolide with that of N-ethyl maleimide (NEM). We showed that NEM directly alkylated p65 as well as p50 of NF-kappaB, whereas SLs possess a selectivity towards p65. Finally, we studied the transactivating properties of various p65 mutants, to analyze the effect of exchanged cysteine residues in the DNA binding domain of NF-kappaB/p65 on its function and demonstrated that the transactivating potential of the mutants did not correlate with their DNA binding strenght., (Copyright 2004 Elsevier Inc.)

Reporter enzymes such as firefly luciferase or beta-galactosidase of Escherichia coli are frequently used to study transcriptional activity of genes and to investigate the effects of novel compounds on gene or transcription factor activity. It is generally assumed that the activity of these enzymes is unaffected by the treatment conditions. Therefore, this factor is not considered when interpreting the data obtained. Biologically active compounds such as sesquiterpene lactones (SLs) have also been tested in reporter gene assays for their influence on gene expression. Here we show in in vitro and ex vivo experiments that SLs inhibit firefly luciferase activity probably by direct targeting of the enzyme while beta-galactosidase remains almost completely unaffected. The loss of luciferase activity after SL treatment could be an effect of their sulfhydryl-modifying potency and the subsequent alteration of the enzyme's tertiary structure. These results demonstrate that the effect of the test substance on the reporter enzyme used should be taken into consideration when the transcriptional effect of novel compounds is investigated.

The reinvestigation of the aerial parts of Montanoa hibiscifolia afforded four new eudesmanolides (1-4), three of them with a rare endoperoxide structural element and the fourth with a rare carbonyl function. It also afforded three unusual montabibisciolides (5-7), two (5 and 7) of which are new natural compounds. Additionally, seven germacrolides (8-10 and 12-15) and one melampolide (11) could be isolated, including two new germacrolides (8 and 9). Their structures were elucidated using 1D and 2D NMR measurement as well as ESI, CIMS, and HRMS analyses. Low-energy conformations were obtained by molecular mechanics calculations. The (13)C NMR data of five compounds are reported for the first time. Six sesquiterpene lactones (4, 6, 10, 11, 12, and 14) were investigated for their inhibitory activity on DNA binding of the transcription factor NF-kappa B using Jurkat T cells as well as RAW 264.7 cells. Besides the alpha-methylene-gamma-butyrolactone moiety the epoxy group in the acyl residue might take part in the NF-kappa B inhibitory activity of sesquiterpene lactones.

The interactions of the three sesquiterpene lactones (SLs) dihydrohelenalin acetate, dihydrohelenalin methacrylate and helenalin isobutyrate from Arnica montana and of parthenolide from Tanacetum parthenium as well as of three ethanolic Arnica preparations with human blood proteins using different matrices, human serum albumin (HSA), plasma and whole blood, were investigated. The extent of protein binding in human plasma or to human albumin differed significantly between individual SLs (dihydrohelenalin methacrylate < dihydrohelenalin acetate < helenalin isobutyrate << parthenolide), e. g., 30 % to 50 % of the SLs were bound to plasma. On the whole, SLs in the ethanolic preparations showed a lower degree of protein binding. Interestingly, although HSA has a reactive thiol group at its cysteine-34 position which is prone to react with the alpha,beta-unsaturated carbonyl of SLs, our studies showed that less than 6 % of SLs are bound to this position within 60 minutes. Thus, a reaction with other amino acids as well as non-covalent interactions with plasma proteins have to be considered. Considering the biological activity of SLs in whole blood, our studies demonstrate that knowledge of their plasma protein binding is important for interpreting the reported data of in vitro and ex vivo assays.

Oral administration of the chloroform extract from Tanacetum larvatum (Griseb. ex Pant.) Kanitz caused a dose-dependent anti-inflammatory effect in the carrageenan-induced rat paw oedema test. The obtained anti-inflammatory effect was 8.6, 32.8, 37.0 and 49.5% for the extract doses of 25, 50, 100 and 200mg/kg, respectively, being statistically significant at a dose of 50mg/kg. Indomethacin had a strong anti-inflammatory effect of 73.4% at a dose of 8mg/kg, but large gastric lesions were detected. When the plant extract in the highest tested dose (200mg/kg) was concomitantly given with indomethacin, the anti-inflammatory effect was slightly enhanced, but the gastric lesions were significantly reduced. The anti-inflammatory and anti-ulcer activity may be mainly due to the inhibition of DNA binding of the transcription factor NF-kappaB by components of the plant extract. This was proven in an electrophoretic mobility shift assay at a concentration of 50microg/ml. Due to its anti-inflammatory as well as anti-ulcer effects, Tanacetum larvatum should especially be used combined with those drugs that are known both for their strong anti-inflammatory activities and the ulcerogenic side effects such as NSAIDs.

From the aerial parts of Oyedaea verbesinoides nine ent-kauranes and a sesquiterpene were isolated. ent-9alpha-Hydroxy-kaur-16-en-19-oic acid, ent-15beta-tigloyloxy-9alpha-hydroxy-kaur-16-en-19-oic acid, ent-15beta-angeloyloxy-9alpha-hydroxy-kaur-16-en-19-oic acid, ent-16alpha-hydroxykaurane and 1alpha-angeloyloxy-carotol are new for the genus or the species and ent-15beta-angeloyloxy-7alpha,9alpha-dihydroxy-kaur-16-en-19-oic acid is reported for the first time. Structure elucidation was based on one and two dimensional NMR as well as ESI and CI-MS analysis. Some diterpenes were proven to exhibit inhibitory effects on smooth muscle contraction on rat aorta.

Structure-activity relationship of cinnamic acid derivatives as inhibitors of the human neutrophil elastase is reported. Comparison of the inhibitory concentrations (IC50 values) with the results of the ligand docking calculations revealed that the structure element of the aromatic ortho-dihydroxy groups combined with a lipophilic residue seems to be a prerequisite for an optimal binding within the active site.

We used flow cytometry to investigate the inhibitory effect of sesquiterpene lactones (SLs) on T-cell activation measured by the expression of its early marker CD69, and on interleukin (IL)-2, a mediator of activation, in whole blood. SLs are biologically active compounds found especially in plants from the Asteraceae family. Overnight treatment of blood with these substances led to the inhibition of CD69 and IL-2 expression. Interestingly, bifunctional SLs showed a weaker activity than monofunctional substances, which is in contradiction with the data obtained so far, using other biological test systems. Additionally, SLs did not completely inhibit CD69 or IL-2 expression. We also determined their toxicity and observed only a low effect. Up to now, studies on cytotoxicity have only been performed using cultured cell lines. From these results it may be supposed that these natural compounds preferentially show toxic effects towards transformed cell lines. Altogether, the results demonstrated that SLs effectively inhibit the activation of the T-lymphocyte response in whole blood and proved the utility of a whole blood system in studying their biological effects.

Preparations from Arnica flowers have been used in traditional medicine since a long time for the treatment of inflammatory diseases. Sesquiterpene lactones are considered as their main active compounds. Previously, it was shown that these natural products attack inflammatory processes at a very central point by inhibiting the transcription factors NF-kappa B and NF-AT at micromolar concentrations. Both transcription factors regulate the transcription of genes encoding for many inflammatory mediators. Thus, these new insights on their molecular mode of action are an important contribution for a better understanding of the antiinflammatory activity of preparations from Arnica. First clinical studies show that they can support the treatment of rheumatic diseases. The agreed use is important to avoid undesirable side effects., (Copyright 2003 S. Karger GmbH, Freiburg)

Sesquiterpene lactones (SLs) are natural products responsible for the anti-inflammatory activity of a variety of medicinal plants, mainly from the Asteraceae family. Here, we investigated whether they also influence the process of exocytosis of pro-inflammatory enzymes, such as the human neutrophil elastase (HNE). Altogether, eight structurally different SLs from the eudesmanolide, guaianolide, pseudoguaianolide, and germacranolide type were studied. Neutrophils were isolated from fresh human blood. After pre-incubation with different concentrations of the respective SL and cytochalasin B, the exocytosis of elastase was initiated either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Inhibition of HNE release was measured by p-nitroaniline formation. The SLs exhibited an inhibitory effect on elastase release from neutrophils challenged either by platelet activating factor or N-formyl-methionyl-leucyl-phenylalanine. Concentration-response curves were recorded and the IC(50) values ranged from 2 to 30 microM. Studies on isolated HNE showed that a selective direct inhibition on HNE can be excluded. Interestingly, the inhibitory activity did not correlate with the number of alpha,beta-unsaturated carbonyl functions. The structure-activity relationship and the molecular mechanism are discussed.