Apigenin and its structural analogues chrysin and luteolin were used to evaluate their capacity to inhibit the production of pro-inflammatory cytokines by lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC). Furthermore, flowcytometric analysis was performed to compare the effects of apigenin, chrysin, luteolin, quercetin and naringenin on the different cell types present in PBMC. LPS-stimulated PBMC were cultured in the presence of the flavonoids and TNFalpha, IL-1beta and IL-6 were measured in the supernatants. In parallel, metabolic activity of the PBMC was determined by measuring succinate dehydrogenase activity. Apigenin, chrysin and luteolin dose-dependently inhibited both pro-inflammatory cytokine production and metabolic activity of LPS-stimulated PBMC. With increasing concentration of apigenin, chrysin or luteolin the monocytes/macrophages disappeared as measured by flowcytometry. This also appeared to occur in the non-LPS-stimulated PBMC. At the same time there was an increase in dead cells. T- and B-lymphocytes were not affected. Quercetin and naringenin had virtually no effects on cytokines, metabolic activity or on the number of cells in the studied cell populations. In conclusion, monocytes were specifically eliminated in PBMC by apigenin, chrysin or luteolin treatment in vitro at low concentrations (around 8 microM), in which apigenin appeared to be the most potent.

Oral antigen application induces tolerance, leading to suppression of a subsequent systemic challenge with this antigen. The suppression is mediated by mucosal regulatory T (Tr) cells that may differentiate from naive peripheral T cells in the gut-draining lymphoid tissue. However, little is known about the initial steps of this differentiation process. In this study we show that 48 h after oral OVA treatment, antigen-specific T cells in mesenteric lymph nodes (MLN) and Peyer's Patches (PP) were activated and had divided up to four times. The first division was already seen in PP after 24 h. Analysis of surface marker expression and cytokine secretion of the dividing antigen-specific T cells revealed that they sequentially obtained an activation-and memory-like phenotype. These cells secreted IL-2 in most stages of division but only transiently IFN-+ whereas no IL-4 or IL-10 secretion was detected. Remarkably, 48 h after antigen application, isolated dividing cells were suppressive, as they transferred tolerance to naive mice. Even though CD25 was expressed heterogeneously, both CD25+ and CD25 -OVA-specific T cells from MLN could transfer tolerance. Together these findings show that differentiation of functional Tr cells occurs in the MLN and PP within 2 days after antigen ingestion and involves the generation of CD25 + and CD25 -antigenspecific T cells.

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In this study, an extract of Pterocarpus marsupium Roxb. containing pterostilbene has been evaluated for its PGE2-inhibitory activity in LPS-stimulated PBMC. In addition, the COX-1/2 selective inhibitory activity of P. marsupium (PM) extract was investigated. Biological activity, as well as safety of PM extract was evaluated in healthy human volunteers. PM extract, pterostilbene and resveratrol inhibited PGE2 production from LPS-stimulated human peripheral blood mononuclear cells (PBMC) with IC50 values of 3.2 +/- 1.3 microg/mL, 1.0 +/- 0.6 microM and 3.2 +/- 1.4 microM, respectively. When pterostilbene content of PM extract is calculated, PGE2 production inhibition of PM extract is comparable to PGE2 production inhibition of purified pterostilbene. Furthermore, in a COX-1 whole blood assay (WBA) PM extract was not effective while in a COX-2 WBA, PM extract decreased PGE2 production indicating COX-2 specific inhibition. In healthy human volunteers, the oral use of 450 mg PM extract did not decrease PGE2 production ex vivo in a WBA. Pterostilbene levels in serum were increased, but were 5-fold lower than the observed IC50 for PGE2 inhibition in LPS-stimulated PBMC. No changes from base-line of the safety parameters were observed and no extract-related adverse events occurred during the study. In conclusion, this is the first study to describe the selective COX-2 inhibitory activity of a Pterocarpus marsupium extract. Moreover, the PGE2 inhibitory activity of PM extract was related to its pterostilbene content. In humans, 450 mg PM extract resulted in elevated pterostilbene levels in serum, which were below the active concentration observed in vitro. In addition, short-term supplementation of 450 mg PM extract is considered to be a safe dose based on the long history of use, the absence of abnormal blood cell counts and blood chemistry values and the absence of extract-related adverse events. This strongly argues for a dose-finding study of PM extract in humans to corroborate the in vitro observed inhibitory activity on PGE2 production in order to resolve the potential use of PM extract in inflammatory disorders and/or inflammatory pain.

A standardized CO(2) extract from Humulus lupulus L. (hop extract) was investigated for its selective COX-1/2 inhibitory properties. An in vitro model of inflammation using lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMC) was used as a model to investigate the effect of hop extract on PGE(2) production. COX-1/2 selective inhibition by the hop extract was investigated in a COX-1 whole blood assay (WBA) and a COX-2 WBA. To evaluate the in vivo activity of hop extract, it was administered orally to C57BL/6 mice in which inflammation of the right joint was induced by injecting zymosan intra-articularly. Ex vivo PGE(2) production of LPS-stimulated blood cells was determined. Also, the effect of hop extract on healthy and arthritic cartilage was investigated as well as effects on inflammatory joint swelling. Hop extract inhibited PGE(2) production by LPS-stimulated PBMC without compromising the metabolic activity of these cells. Furthermore, hop extract showed a decline in PGE(2) production in the COX-2 whole blood assay (WBA) with an IC(50) of 20.4 microg/mL, while in the COX-1 WBA no inhibition of PGE(2) production was observed. This indicates a COX-2 selective inhibition. The COX-1 inhibitor SC-560 inhibited PGE(2) production in the COX-1 WBA but not in the COX-2 WBA. At 2 microM, celecoxib inhibited PGE(2) production in the COX-2 WBA by 92 % and in the COX-1 WBA by 50 %. When hop extract was administered orally to C57BL/6 mice in which joint inflammation was induced with zymosan, PGE(2) production in ex vivo LPS-stimulated whole blood was significantly decreased by 24 %, suggesting that hop extract becomes bioavailable. Furthermore, oral administration of hop extract showed no negative or positive effects on healthy cartilage proteoglycan synthesis, or on zymosan-induced arthritic cartilage proteoglycan synthesis. However, no effect of oral administration of 1.25 mg hop extract daily was observed on joint swelling. In conclusion, this standardized CO(2) extract of Humulus lupulus could be a useful agent for intervention strategies targeting inflammatory disorders and/or inflammatory pain.