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Chinese herbal medicinal (CHM) extracts from fourteen plants were investigated in cell-based in vitro assays for their effect on nuclear factor κB (NF-κB), a key regulator of inflammation, as well as on peroxisome proliferator-activated receptors (PPARs) being key regulators of genes involved in lipid and glucose metabolism. 43% of the investigated CHMs showed NF-κB inhibitory and 50% PPARα and PPARγ activating effects. Apolar extracts from cortex and flos of Albizia julibrissin Durazz. and processed rhizomes of Arisaema sp. and Pinellia ternata (Thunb.) Breit. that effectively inhibited TNF-α-induced NF-κB activation and dosedependently activated PPARα and PPARγ were further investigated. Bioassay-guided fractionation and analysis by GC- MS led to the identification of fatty acids as PPAR agonists, including linoleic and palmitic acid. [ABSTRACT FROM AUTHOR]

Inflammation is a hallmark of some of today's most life-threatening diseases such as arteriosclerosis, cancer, diabetes and Alzheimer's disease. Herbal medicines (HMs) are re-emerging resources in the fight against these conditions and for many of them, anti-inflammatory activity has been demonstrated. However, several aspects of HMs such as their multi-component character, natural variability and pharmacodynamic interactions (e.g. synergism) hamper identification of their bioactive constituents and thus the development of appropriate quality control (QC) workflows. In this study, we investigated the potential use of Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy as a tool to rapidly and non-destructively assess different anti-inflammatory properties of ethanolic extracts from various species of the Genus Lonicera (Caprifoliaceae). Reference measurements for multivariate calibration comprised in vitro bioactivity of crude extracts towards four key players of inflammation: Nitric oxide (NO), interleukin 8 (IL-8), peroxisome proliferator-activated receptor β/δ (PPAR β/δ), and nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB). Multivariate analysis of variance (MANOVA) revealed a statistically significant, quantitative pattern-activity relationship between the extracts' ATR-FTIR spectra and their ability to modulate these targets in the corresponding cell models. Ensemble orthogonal partial least squares (OPLS) discriminant models were established for the identification of extracts exhibiting high and low activity with respect to their potential to suppress NO and IL-8 production. Predictions made on an independent test set revealed good generalizability of the models with overall sensitivity and specificity of 80% and 100%, respectively. Partial least squares (PLS) regression models were successfully established to predict the extracts' ability to suppress NO production and NF-κB activity with root mean squared errors of cross-validation (RMSECV) of 8.7% and 0.05-fold activity, respectively., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Melampyrum pratense L. (Koch) is used in traditional Austrian medicine for the treatment of different inflammation-related conditions. In this work, we show that the extracts of M. pratense stimulated peroxisome proliferator-activated receptors- (PPARs-) α and - γ that are well recognized for their anti-inflammatory activities. Furthermore, the extract inhibited the activation of the proinflammatory transcription factor NF- κ B and induction of its target genes interleukin-8 (IL-8) and E-selectin in vitro. Bioassay-guided fractionation identified several active flavonoids and iridoids including melampyroside and mussaenoside and the phenolic compound lunularin that were identified in this species for the first time. The flavonoids apigenin and luteolin were distinguished as the main components accountable for the anti-inflammatory properties. Apigenin and luteolin effectively inhibited tumor necrosis factor α (TNF- α )-induced NF- κ B-mediated transactivation of a luciferase reporter gene. Furthermore, the two compounds dose-dependently reduced IL-8 and E-selectin protein expression after stimulation with lipopolysaccharide (LPS) or TNF- α in endothelial cells (ECs). The iridoids melampyroside and mussaenoside prevented the elevation of E-selectin in LPS-stimulated ECs. Lunularin was found to reduce the protein levels of the proinflammatory mediators E-selectin and IL-8 in ECs in response to LPS. These data validate the ethnomedical use of M. pratense for the treatment of inflammatory conditions and point to the constituents accountable for its anti-inflammatory activity.

The organosulfur compound ajoene, a constitutent of garlic, has been shown to induce apoptosis in a leukemic cell line as well as in blood cells of a leukemic patient. The mechanisms of action of ajoene, however, are unknown. The present study aims to characterize the molecular events leading to ajoene-triggered apoptosis. We show here that ajoene (20 microM) leads to a time-dependent activation of caspase-3-like activity as well as to the proteolytic processing of procaspase-3 and -8. Activation of caspases was necessary for ajoene-induced apoptosis since the broad-range caspase inhibitor zVAD-fmk completely abrogated ajoene-mediated DNA fragmentation. Although the initiator caspase-8 was activated, the CD95 death receptor was not involved in death signaling since the HL-60 clone used was shown to express a functionally inactive CD95 receptor. Furthermore, ajoene induced the release of cytochrome c, which was not inhibited by zVAD-fmk indicating that cytochrome c release precedes caspase activation. Ajoene also led to a dissipation of the mitochondrial transmembrane potential. Overexpression of Bcl-x(L) clearly diminished ajoene-induced caspase activation as well as apoptosis. These results indicate that apoptosis in leukemia cells triggered by ajoene is based on the activation of a mitochondria-dependent caspase cascade which includes also the activation of the initiator caspase-8.

4,5-Diaminofluorescein (DAF-2) and its membrane-permeable derivate DAF-2 diacetate are fluorescent probes that have been developed to perform real-time biological detection of nitric oxide (NO). Their use for intracellular imaging, however, has recently been seriously questioned and data using DAF-2 for extracellular NO detection at low levels, as for example released from endothelial cells, are rare. Here we show that a reliable detection of low levels of NO in biological systems by DAF-2 is possible (a) by using low DAF-2 concentrations (0.1 microM) and (b) by subtracting the DAF-2 auto-fluorescence from the measured total fluorescence. The described method allows easy real-time detection of endothelial NO formation.

Apoptosis is required for proper tissue homeostasis. Defects in apoptosis signaling pathways, thus, contribute to carcinogenesis and chemoresistance. A major goal in chemotherapy is, therefore, to find cytotoxic agents that restore the ability of tumor cells to undergo apoptosis. We show here that the sesquiterpene lactone helenalin (10-50 microM) induces apoptosis in leukemia Jurkat T cells even if they lack the CD95 death receptor or overexpress the antiapoptotic proteins Bcl-x(L) or Bcl-2. Activated peripheral blood mononuclear cells, however, are not affected (10-50 microM helenalin). Helenalin led to a time-dependent (0-24 h) cleavage of the specific caspase-3-like substrate Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin as well as to the proteolytic processing of procaspase-3 and -8. Caspase activation was a necessary requirement for apoptosis because the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD-fmk, 50 microM) completely abrogated helenalin-induced DNA fragmentation as well as phosphatidylserin translocation. Although the initiator caspase-8 was activated, the helenalin-induced signaling pathway did not require the CD95 death receptor as shown using cells without or with an antibody (ZB4)-blocked CD95 receptor. Helenalin also did not induce CD95 or CD95-ligand expression. On the other hand, helenalin was found to induce the release of cytochrome c from mitochondria that was not inhibited by the caspase inhibitor zVAD-fmk, which indicated that cytochrome c release precedes caspase activation. Cytochrome c release was accompanied by dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), which was partly inhibited by zVAD-fmk, which suggests that caspases are involved in loss of DeltaPsi(m). Most importantly, overexpression of the mitochondria protecting proteins Bcl-x(L) or Bcl-2 failed to confer resistance to helenalin-induced apoptosis, although the data presented here suggest that helenalin induces a mitochondria-dependent pathway. Thus, helenalin is a promising experimental cytotoxic agent that possibly points to new strategies to overcome apoptosis resistance attributable to overexpression of antiapoptotic Bcl-2 proteins.

Hypocretenolides, a small group of sesquiterpene lactones with an unusual ring structure, are constituents of a small number of species from the Lactuceae tribe (Asteraceae). Three biogenetically closely related 14-hypocretenolides from Leontodon hispidus L. were investigated for a putative anti-inflammatory activity. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate significantly exhibited in vivo anti-inflammatory activity in the croton oil-induced mouse ear edema. To obtain first information regarding the molecular targets which might be affected by this constituent, two in vitro bioassays were performed: (i) DNA binding activity of the transcription factor NF-kappa B was evaluated by electrophoretic mobility shift assay (EMSA) using TNF-alpha-activated Jurkat T cells and (ii) nitrite accumulation in cell culture supernatants of LPS-activated RAW 264.7 macrophages was determined as a parameter for inducible nitric oxide synthase (iNOS)-dependent nitric oxide release. In order to gain information about structure-activity relationships, additionally the aglycone 14-hydroxyhypocretenolide and its D-glycoside were investigated in these in vitro systems. 14-Hydroxyhypocretenolide-beta-D-glucoside-4'-14"-hydroxyhypocr etenoate as well as its aglycone exhibited activity in both test systems, whereas the D-glucoside was not or only weakly active.

In the field of inflammation research the inducible nitric oxide synthase (iNOS) became an important pharmacological target, since overproduction of nitric oxide (NO) after induction of this enzyme seems to be associated with numerous pathological conditions. NO released from cells can be detected and quantified photometrically as its stable product nitrite by a simple colorimetric reaction (Griess reaction). The aim of our study was to investigate whether this method might be suitable for the bio-guided fractionation of anti-inflammatory plant extracts. For this purpose we assayed extracts as well as fractions of the roots of Curcuma zanthorrhiza Roxb, which contain the known iNOS inhibitor curcumin, and compared the obtained activity with their curcumin content. Furthermore, leaf extracts of Betula pendula Roth, to which defined amounts of curcumin were added, were examined to clarify the question whether chlorophyll might interfere with the test system. The presented results suggest that the Griess assay is indeed suitable to guide fractionation of plant extracts in order to isolate highly active compounds. Factors, however, which might restrict the broad application of this assay are the limited selection of solvents which do not interfere with the system and high contents of chlorophyll in plant extracts.

The pharmacological role of garlic in prevention and treatment of cancer has received increasing attention, but thorough investigations into the molecular mechanisms of action of garlic compounds are rare. The present study demonstrates that ajoene, a major compound of garlic induces apoptosis in human leukemic cells, but not in peripheral mononuclear blood cells of healthy donors. The effect was dose and time dependent. Apoptosis was judged by three criteria, morphology of cells, quantification of subdiploid DNA content by flow cytometry, and detection of DNA fragmentation by gel electrophoresis. Ajoene increased the production of intracellular peroxide in a dose- and time-dependent fashion, which could be partially blocked by preincubation of the human leukemic cells with the antioxidant N-acetylcysteine. Interestingly, N-acetylcysteine-treated cells showed a 50% loss of ajoene-induced apoptosis. Moreover, ajoene was demonstrated to activate nuclear translocation of the transcription factor nuclear factor kappaB, an effect that was abrogated in N-acetylcysteine-loaded cells. These results suggested that ajoene might induce apoptosis in human leukemic cells via stimulation of peroxide production and activation of nuclear factor kappaB. This is a novel aspect in the biological profile of this garlic compound and an important step in elucidating the underlying molecular mechanisms of its antitumor action.

Inducible nitric oxide synthase dependent production of nitric oxide (NO) plays an important role in inflammation. We investigated whether pristimerin ((20alpha)-3-hydroxy-2-oxo-24-nor-friedela-1(10),3,5,7-te traen-carboxylic acid-(29)-methylester), an antitumoral, antimicrobial as well as anti-inflammatory plant compound, has an effect on the inducible NO synthase system in lipopolysaccharide-activated RAW 264.7 macrophages. Pristimerin dose dependently (IC50: 0.2-0.3 microM) reduces nitrite accumulation, a parameter for NO synthesis, in supernatants of lipopolysaccharide-stimulated (1 microg/ml, 20 h) macrophages. This effect correlates with a reduced inducible NO synthase enzyme activity measured by conversion of [3H]L-arginine to [3H]L-citrulline and significantly lower levels of enzyme protein (Western blotting) in homogenates of cells cotreated with lipopolysaccharide and pristimerin (12 h). Northern blot analysis and polymerase chain reaction (PCR) showed decreased inducible NO synthase mRNA levels in activated macrophages exposed to pristimerin (4 h). Electrophoretic mobility shift assay (EMSA) demonstrated a markedly reduced binding activity of nuclear factor-kappa B (NFkappaB) in nuclear extracts of pristimerin-treated cells. These results suggest that pristimerin inhibits the induction of inducible NO synthase by a mechanism which involves inhibition of NFkappaB activation. This feature of pristimerin is likely to contribute to its anti-inflammatory activity.