Inhibition of NF-κB-dependent cytokine and inducible nitric oxide synthesis by the macrocyclic ellagitannin oenothein B in TLR-stimulated RAW 264.7 macrophages.

Immunomodulatory effects of oenothein B (1), a macrocyclic ellagitannin from various Onagraceae species, have been described previously. However, the mechanisms underlying the anti-inflammatory activity of 1 have not been fully clarified. The effects of 1 were investigated on inducible nitric oxide synthase, TLR-dependent and TLR-independent signal transduction cascades, and cytokine expression using murine macrophages (RAW 264.7). Compound 1 (10-60 µg/mL) reduced NO production, iNOS mRNA, and iNOS protein levels in a dose-dependent manner, without inhibition of iNOS enzymatic activity. It reduced the binding of the NF-κB p50 subunit to the biotinylated-consensus sequence and decreased nuclear p65 translocation. Gallic acid as a subunit of the macrocyclic ellagitannin 1 showed a far lower inhibitory activity. Nitric oxide production was reduced by 1 after stimulation using TLR2 (Pam2CSK4) and TLR4 (Kdo2) agonists, but this compound did not inhibit inducible nitric oxide synthesis after stimulation using interferon-gamma. IL-1beta, IL-6, and TNF-alpha mRNA synthesis was clearly reduced by the addition of 1. Oenothein B (1) inhibits iNOS after stimulation with LPS, TLR2, and TLR4 agonists via inhibition of TLR/NF-κB-dependent inducible nitric oxide and cytokine synthesis independent from IFN-gamma/JAK/STAT pathways. The full molecular structure of this macrocyclic ellagitannin seems to be required for its immunomodulatory actions.

Aqueous extracts of Cimicifuga racemosa and phenolcarboxylic constituents inhibit production of proinflammatory cytokines in LPS-stimulated human whole blood.

Cimicifuga racemosa (black cohosh) is commonly used in traditional medicines as treatment for menopausal symptoms and as an antiinflammatory remedy. To clarify the mechanism of action and active principle for the antiinflammatory action, the effects of aqueous C. racemosa root extracts (CRE) and its major constituents on the release of the proinflammatory cytokines IL-6, TNF-alpha, IFN-gamma, and the chemokine IL-8 were investigated in lipopolysaccharide (LPS)-stimulated whole blood of healthy volunteers. CRE (3 microg/microL and 6 microg/microL) reduced LPS-induced release of IL-6 and TNF-alpha in a concentration- and time-dependent manner and almost completely blocked release of IFN-gamma into the plasma supernatant. Except for IFN-gamma, these effects were attenuated at longer incubation periods. IL-8 secretion was stimulated by CRE. As shown by quantitative real-time RT-PCR, effects on cytokines were based on preceding changes in mRNA levels except for IL-8. According to their content in CRE, the phenolcarboxylic compounds caffeic acid, ferulic acid, and isoferulic acid, as well as the triterpene glycosides 23-epi-26-deoxyactein and cimigenol-3-O-xyloside, were tested at representative concentrations. Among these, isoferulic acid was the prominent active principle in CRE, responsible for the observed inhibition of IL-6, TNF-alpha, and IFN-gamma, but not for IL-8 stimulation. The effect of this compound may explain the antiinflammatory activities of CRE and its beneficial actions in rheumatism and other inflammatory diseases.

Inhibition of inducible nitric oxide synthesis by Cimicifuga racemosa (Actaea racemosa, black cohosh) extracts in LPS-stimulated RAW 264.7 macrophages.

OBJECTIVES: Cimicifuga racemosa (Actaea racemosa, black cohosh) is used as an anti-inflammatory, antipyretic and analgesic remedy in traditional medicines. The present study focuses on the effects of C. racemosa root extracts on inducible nitric oxide synthase (iNOS) in lipopolysaccharide-stimulated murine macrophages (RAW 264.7). METHODS: C. racemosa rhizome and phosphate-buffered saline extracts were analysed for phenolcarboxylic acids and triterpene glycosides using an HPLC photodiode array/evaporative light-scattering detector system. iNOS was characterised by measurement of iNOS protein (immunoblotting), iNOS mRNA (semiquantitative competitive RT-PCR), nitric oxide production (nitrite levels) and nuclear translocation of nuclear factor-kappaB (p65 subunit) protein. KEY FINDINGS: Incubation of lipopolysaccharide-stimulated macrophages with aqueous C. racemosa extracts (0-6 mg/ml) inhibited nitrite accumulation in a concentration-dependent manner. C. racemosa extracts also reduced iNOS protein expression and iNOS mRNA levels in a dose-dependent manner. C. racemosa extracts did not significantly inhibit iNOS activity and did not affect nuclear translocation of nuclear factor-kappaB (p65 subunit) protein. Incubation with the extract was associated with a concentration-dependent reduction of interferon beta and interferon regulatory factor 1 mRNA. Among the triterpene glycosides, 23-epi-26-deoxyactein was identified as an active principle in C. racemosa extracts. CONCLUSIONS: Extracts from the roots of C. racemosa inhibit nitric oxide production by reducing iNOS expression without affecting activity of the enzyme. This might contribute to the anti-inflammatory activities of C. racemosa.