METHODS: Cells were pre-incubated with 32µM of 15dPGJ2 and stimulated with 1ng/mL of IL-1β as an in vitro model of inflammation. Western immunoblotting was used to detect phosphorylated p-65 and phosphorylated c-Jun as markers of NF-κB and AP-1 activation, respectively. mRNA expression of the pro-inflammatory cytokines IL-6, IL-8, and TNF-α was examined, and protein expression of COX-2 and PGE2 were detected by western immunoblotting and ELISA respectively. Myometrial contractility was examined ex-vivo using a myograph.

RESULTS: 15dPGJ2 inhibited IL-1β-induced activation of NF-κB and AP-1, and expression of IL-6, IL-8, TNF-α, COX-2 and PGE2 in myocytes, with no effect on myometrial contractility or cell viability. Despite inhibiting IL-1β-induced activation of NF-κB, expression of IL-6, TNF-α, and COX-2, 15dPGJ2 led to activation of AP-1, increased production of PGE2 and increased cell death in VECs and AECs.

Methods: NQC was synthesised and characterised using spectroscopic techniques. The compound was tested for its anti-inflammatory effect using Lipopolysaccharide from Escherichia coli (LPSEc) induced inflammation in mouse macrophages (RAW 264.7 cells). The effect of NQC on inflammatory cytokines was measured using enzyme-linked immune sorbent assay (ELISA). The Nrf2 activity of the compound NQC was determined using 'Keap1:Nrf2 Inhibitor Screening Assay Kit'. To obtain the insights on NQC's activity on Nrf2, molecular docking studies were performed using Schrödinger suite. The metabolic stability of NQC was determined using mouse, rat and human microsomes.

Results: NQC was found to be non-toxic at the dose of 50 µM on RAW 264.7 cells. NQC showed potent anti-inflammatory effect in an in vitro model of LPSEc stimulated murine macrophages (RAW 264.7 cells) with an IC50 value 26.13 ± 1.17 µM. NQC dose-dependently down-regulated the pro-inflammatory cytokines [interleukin (IL)-1β (13.27 ± 2.37 μM), IL-6 (10.13 ± 0.58 μM) and tumor necrosis factor (TNF)-α] (14.41 ± 1.83 μM); and inflammatory mediator, prostaglandin E2 (PGE2) with IC50 values, 15.23 ± 0.91 µM. Molecular docking studies confirmed the favourable binding of NQC at Kelch domain of Keap-1. It disrupts the Nrf2 interaction with kelch domain of keap 1 and its IC50 value was 4.21 ± 0.89 µM. The metabolic stability studies of NQC in human, rat and mouse liver microsomes revealed that it is quite stable with half-life values; 63.30 ± 1.73, 52.23 ± 0.81, 24.55 ± 1.13 min; microsomal intrinsic clearance values; 1.14 ± 0.31, 1.39 ± 0.87 and 2.96 ± 0.34 µL/min/g liver; respectively. It is observed that rat has comparable metabolic profile with human, thus, rat could be used as an in vivo model for prediction of pharmacokinetics and metabolism profiles of NQC in human.

MATERIALS AND METHODS: The cows with mastitis were divided into two groups. In antibiotic control group, the cows were given tetraneomycin ointment. In conditioned-DPBS of AMSCs treatment group, amniotic membrane was collected for AMSCs after delivery. With expression of surface antigen and potential of tri-linage differentiation, AMSCs were injected into mammary glands. Then, milk was sampled every three days to monitor the effect of both treatments. The quality of milk was measured with pH, titratable acidity, free calcium ions and somatic cell count.

RESULTS: Our results demonstrated the Bovine AMSCs expressed CD44, low levels of CD4 and no CD105. Bovine AMSCs demonstrated the differentiation capability in the tri-cell lineages. Mastitis treatment with conditioned-DPBS from AMSCs (experimental group) and conventional antibiotics (control group) showed insignificant difference in pH value and titratable acidity. The level of ionic calcium concentration in the conditioned-DPBS group decreased from 3rd day to 12th day, while the level in the antibiotic group decreased from 0 day to 12th day. The somatic cell number was similar in both groups, which meet the standard of Taiwan milk collection.

AIM OF THE STUDY: However, so far there is no literature available on the anti-inflammatory activity of this species. Henceforth, based on the above background and our previous laboratory findings, we hypothesize that phytoconstituents of A. elliptica could possess anti-inflammatory potential against inflammatory mediators including prostaglandin-E2 (PGE2), cyclooxegenase-2 (COX-2) and cytokines (IL-1β and IL-6).

MATERIALS AND METHODS: Vacuum and column chromatography techniques were employed for the isolation of phytoconstituents. The structure elucidation was carried out using HRESI-MS, 1H and 13C-NMR analysis and compared with the published literature. For cytotoxicity analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed on peripheral blood mononuclear cells. In-vitro anti-inflammatory activities were evaluated against the levels of PGE2, COX-2, IL-1β and IL-6 in lipopolysaccharide (LPS)-induced human plasma using enzyme-linked immunosorbent assay and radioimmunoassay.