A new prenylated benzoquinone from Cyathocalyx pruniferus abrogates LPS-induced inflammatory responses associated with PGE2, COX-2 and cytokines biosynthesis in human plasma.

In our previous laboratory findings, Cyathocalyx pruniferus extracts exhibited platelet-activating factor inhibition, suggesting their anti-inflammatory potential. Hence, this study was designed with the aim to isolate phyto-constituents from C. pruniferus with potent anti-inflammatory activities. Column and volume liquid chromatography were used for isolation of phyto-constituents. The structure elucidation was carried out using spectroscopic analysis (HRESI-MS, 1H and 13C-NMR) and compared with published literature. For cytotoxicity analysis, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide assay was performed on peripheral blood mononuclear cells. Anti-inflammatory activities were evaluated against the levels of inflammatory cytokines (IL-1ß and IL-6), prostaglandin-E2 (PGE2) and cyclooxegenase-2 (COX-2), in lipopolysaccharide (LPS)-induced human plasma using ELISA and radioimmunoassay (RIA). The chromatographic purification of methanol leaves extract afforded 13 (1-13) secondary metabolites. Additionally, cytotoxicity analysis suggested that isolates were non-cytotoxic at 100 µM. In anti-inflammatory evaluation, 2-octaprenyl-1, 4-benzoquinone (5) produced strong (≥ 70%) inhibition of PGE2, COX-2, IL-1ß and IL-6 at 50 µM. Moreover, 2-octaprenyl-1,4-benzoquinone (5) exhibited concentration-dependent inhibition with IC50 values (µM) of 11.21, 6.61, 2.20 and 3.56 as compared to controls; indomethacin for PGE2 (11.84) and dexamethasone in COX-2 (5.19), IL-1ß (1.83) and IL-6 (3.76) analysis, respectively. In conclusion, two new compounds including 2-octaprenyl-1, 4-benzoquinone (5) and 14-methyloctadec-1-ene (6) are reported for the first time from plant species. Additionally, 2-octaprenyl-1, 4-benzoquinone (5) dose-dependently suppressed the production of pro-inflammatory mediators involved in acute and chronic inflammation at non-cytotoxic concentrations.

Phyllanthus amarus prevents LPS-mediated BV2 microglial activation via MyD88 and NF-κB signaling pathways.

BACKGROUND: Phyllanthus amarus has been shown to attenuate lipopolysaccharide (LPS)-induced peripheral inflammation but similar studies in the central nervous system are scarce. The aim of the present study was to investigate the neuroprotective effects of 80% ethanol extract of P. amarus (EPA) in LPS-activated BV2 microglial cells. METHODS: BV2 microglial cells c for 24 h, pre-treated with EPA for 24 h prior to LPS induction for another 24 h. Surface expression of CD11b and CD40 on BV2 cells was analyzed by flow cytometry. ELISA was employed to measure the production of pro-inflammatory mediators i.e. nitric oxide (NO) and tumor necrosis factor (TNF)-α. Western blotting technique was used to determine the expression of inducible nitric oxide synthase (iNOS), myeloid differentiation protein 88 (MYD88), nuclear factor kappa B (NF-κB), caspase-1, and mitogen activated protein kinase (MAPK). RESULTS: Qualitative and quantitative analyses of the EPA using a validated ultra-high pressure liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method indicated the presence of phyllanthin, hypophyllanthin, niranthin, ellagic acid, corilagin, gallic acid, phyltetralin, isolintetralin and geraniin. EPA suppressed the production of NO and TNFα in LPS-activated BV2 microglial cells. Moreover, EPA attenuated the expression of MyD88, NF-κB and MAPK (p-P38, p-JNK and p-ERK1/2). It also inhibited the expression of CD11b and CD40. EPA protected against LPS-induced microglial activation via MyD88 and NF-κB signaling in BV2 microglial cells. CONCLUSIONS: EPA demonstrated neuroprotective effects against LPS-induced microglial cells activation through the inhibition of TNFα secretion, iNOS protein expression and subsequent NO production, inhibition of NF-κB and MAPKs mediated by adapter protein MyD88 and inhibition of microglial activation markers CD11b and CD40.

Expression of recombinant human epidermal growth factor in Escherichia coli and characterization of its biological activity.

Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.