OBJECTIVE: In the present study, BBP was investigated for it's in vivo innate and adaptive immune responses mediated by different humoral and cellular immune factors.
METHODS: Male Balb/c mice were orally fed with BBP (5, 10 and 20 mg/kg) for a period of 14 days and immunized with sheep red blood cells (sRBC) on day 0 for the determination of adaptive responses. The effects of BBP on phagocytosis process of neutrophils isolated from blood of treated/untreated animals were determined. The ceruloplasmin and lysozyme serum levels and myeloperoxidase (MPO) plasma level were also monitored. The mechanism was further explored by assessing its effects on the proliferation of T and B lymphocytes, T-lymphocytes subsets CD4+ and CD8+ and on the secretion of Th1/Th2 cytokines as well as serum immunoglobulins (IgG, IgM) and delayed type hypersensitivity (DTH) reaction.
RESULTS: BBP showed a significant dose-dependent reduction on the migration of neutrophils, Mac-1 expression, phagocytic activity and reactive oxygen species (ROS) production. In comparison to the sensitized control group, a dose-dependent inhibition was observed on lymphocyte proliferation along with the downregulation of effector cells expression and release of cytokines. Moreover, a statistically significant decrease was perceived in serum levels of ceruloplasmin, lysozyme and immunoglobulins and MPO plasma level of BBP-treated mice. BBP also dose-dependently inhibited sheep red blood cells (sRBC)-induced swelling rate of mice paw in DTH.
CONCLUSION: These findings suggest the potential of BBP as a potent immunosuppressive agent.
METHODS: Isolation of compounds from G. segetum leaves was conducted using vacuum liquid chromatography (VLC) and column chromatography (CC). Two new compounds, namely 4,5,4'-trihydroxychalcone and 8,8'-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol, together with stigmasterol and β-sitosterol were isolated from G. segetum methanol extract and their structures were determined spectroscopically. The presence of gallic acid and rutin in the extract was determined quantitatively by a validated HPLC method. G. segetum methanol extract and its constituents were investigated for their effects on chemotaxis, phagocytosis, β2 integrin (CD18) expression, and reactive oxygen species (ROS) of polymorphonuclear leukocytes (PMNs), lymphocytes proliferation, cytokine release and nitric oxide (NO) production of phagocytes.
RESULTS: All the samples significantly inhibited all the innate immune responses tested except CD 18 expression on surface of leukocytes. Among the samples, 8,8'-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol exhibited the strongest inhibitory on chemotaxis, phagocytosis, ROS and NO production. The compound exhibited exceptionally strong inhibitions on ROS and chemotaxis activities with IC50 values lower than the positive controls, aspirin and ibuprofen, respectively. 4,5,4'-Trihydroxychalcone revealed the strongest immunosuppressive activity on proliferation of lymphocytes (IC50 value of 1.52 μM) and on release of IL-1β (IC50 value of 6.69 μM). Meanwhile rutin was the most potent sample against release of TNF-α from monocytes (IC50, 16.96 μM).
CONCLUSION: The extract showed strong immunosuppressive effects on various components of the immune system and these activities were possibly contributed mainly by 4,5,4'-trihydroxychalcone, 8,8'-(ethene-1,2-diyl)-dinaphtalene-1,4,5-triol and rutin.
METHODS: The release of prostaglandin E2 (PGE2) and pro-inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β in a culture supernatant was determined by ELISA. Determination of cyclooxygenase-2 (COX-2) protein and the activation of MAPKs molecules (JNK, ERK and p38 MAPK), NF-κB and Akt in LPS-induced U937 human macrophages were investigated by immunoblot technique. The relative gene expression levels of COX-2 and pro-inflammatory cytokines were measured by using qRT-PCR. The major metabolites of P. amarus were qualitatively and quantitatively analyzed in the extract by using validated reversed-phase high performance liquid chromatography (HPLC) methods.
RESULTS: P. amarus extract significantly inhibited the production of pro-inflammatory mediators (TNF-α, IL-1β, PGE2) and COX-2 protein expression in LPS-induced U937 human macrophages. P. amarus-pretreatment also significantly downregulated the increased mRNA transcription of pro-inflammatory markers (TNF-α, IL-1β, and COX-2) in respective LPS-induced U937 macrophages. It downregulated the phosphorylation of NF-κB (p65), IκBα, and IKKα/β and restored the degradation of IκBα, and attenuated the expression of Akt, JNK, ERK, and p38 MAPKs phosphorylation in a dose-dependent manner. P. amarus extract also downregulated the expression of upstream signaling molecules, TLR4 and MyD88, which play major role in activation of NF-κB, MAPK and PI3K-Akt signaling pathways. The quantitative amounts of lignans, phyllanthin, hypophyllahtin and niranthin, and polyphenols, gallic acid, geraniin, corilagin, and ellagic acid in the extract were determined by HPLC analysis.
CONCLUSION: The study revealed that P. amarus targeted the NF-κB, MAPK and PI3K-Akt signaling pathways to exert its anti- inflammatory effects by downregulating the prospective inflammatory signaling mediators.