Physalin pool from Physalis angulata L. leaves and physalin D inhibit P2X7 receptor function in vitro and acute lung injury in vivo.

P2X7 receptor promotes inflammatory response and neuropathic pain. New drugs capable of impairing inflammation and pain-reducing adverse effects extracted from plant extracts have been studied. Physalis angulate L. possesses traditional uses and exhibits antiparasitic, anti-inflammatory, antimicrobial, antinociceptive, antimalarial, antileishmanial, immunosuppressive, antiasthmatic. diuretic, and antitumor activities. The most representative phytochemical constituents identified with medicinal importance are the physalins and withanolides. However, the mechanism of anti-inflammatory action is scarce. Although some physalins and withanolides subtypes have anti-inflammatory activity, only four physalins subtypes (B, D, F, and G) have further studies. Therefore, we evaluated the crude ethanolic extract enriched with physalins B, D, F, and G from P. angulata leaves, a pool containing the physalins B, D, F, G, and the physalins individually, as P2X7 receptor antagonists. For this purpose, we evaluated ATP-induced dye uptake, macroscopic currents, and interleukin 1-ß (IL-1ß) in vitro. The crude extract and pool dose-dependently inhibited P2X7 receptor function. Thus, physalin B, D, F, and G individually evaluated for 5'-triphosphate (ATP)-induced dye uptake assay, whole-cell patch-clamp, and cytokine release showed distinct antagonist levels. Physalin D displayed higher potency and efficacy than physalin B, F, and G for all these parameters. In vivo mice model as ATP-induced paw edema was potently inhibited for physalin D, in contrast to physalin B, F, and G. ATP and lipopolysaccharide (LPS)-induced pleurisy in mice were reversed for physalin D treatment. Molecular modeling and computational simulation predicted the intermolecular interactions between the P2X7 receptor and physalin derivatives. In silico results indicated physalin D and F as a potent allosteric P2X7 receptor antagonist. These data confirm physalin D as a promisor source for developing a new P2X7 receptor antagonist with anti-inflammatory action.

Evaluation and comparison of three different separation techniques for analysis of retroamide enantiomers and their biological evaluation against h-P2X7 receptor.

The P2X receptors are seven-transmembrane domain G protein-coupled receptors and the 7 subtypes of P2X receptors identified in humans, and named P2X1 to P2X7, are channel receptors whose endogenous ligand is ATP. New antagonists of the P2X7 receptor were developed, since this purinergic receptor was highlighted to be involved in many diseases such as different types of pain, cancer, ischemia, neurodegenerative diseases (including Parkinson's and Alzheimer's diseases) characterized by inflammatory processes. With the aim of evaluate the impact of chirality on the pharmacological activity of a new P2X7R antagonist, a semi-preparative method was developed in supercritical fluid chromatography (SFC). Among four polysaccharide based chiral stationary phases: Chiralcel OD-H and OJ-H and Chiralpak AS-H and AD-H, the last one namely amylose tris (3,5-dimethylphenylcarbamate) with a mobile phase consisted of carbon dioxide-ethanol (80:20, v/v), led to the successful separation of the enantiomers in short run time and with good resolution. Limits of detection and quantification were calculated and were found equal for compound 1, to 1.37 µM and 4.57 µM respectively, for peak 1 and were equal to 1.60 µM and 5.30 µM respectively, for peak 2 at λ=210 nm. Before carrying out the pharmacological evaluation of each enantiomer, two complementary methodologies, e.g. liquid chromatography and capillary electrophoresis were performed in parallel to improve the limits of detection and quantification to assess the enantiomeric purity. HPLC using a Chiralpak AD stationary phase led to four times lower limits of detection and quantification with regard to SFC. In the same time, capillary electrophoresis involving dual cyclodextrins system constituted of a SBE-ß-CD and a MM-ß-CD mixture enhanced the signal-to-noise ratio and led to similar limits of detection and quantification with regard to SFC. No trace of the other enantiomer was found in the isolated one. Biological activities of individual enantiomers were then evaluated and revealed no cytotoxicity against cell lines and a significant difference in terms of their IC50 values with respect to the investigated racemate (6.43 µM): 3.49 µM for the (R)-enantiomer and >10(-4)µM for the (S)-enantiomer, for compound 1, showing that, this antagonist activity is stereospecific.

Emodin inhibits ATP-induced IL-1ß secretion, ROS production and phagocytosis attenuation in rat peritoneal macrophages via antagonizing P2X7 receptor.

CONTEXT: Previous in vitro studies have demonstrated that emodin (1,3,8-trihydroxy-6-methyl-anthraquinone), an anthraquinone derivative from the rhizome of Rheum palmatum L., can inhibit the activation of P2X7 receptors (P2X7R) as a potential antagonist. However, the effects of emodin on P2X7R-related inflammatory processes remain unclear. OBJECTIVE: This study aimed to investigate the effects of emodin on different inflammation responses of macrophages induced by ATP, the natural ligand of P2X7R. MATERIALS AND METHODS: Rat peritoneal macrophages were treated with millimolar ATP and emodin (0.1, 0.3, 1, 3, 10 µM) or brilliant blue G (BBG, 0.1, 1, 10 µM). Cytosolic Ca²âº concentration ([Ca²âº]c) was detected by fluorescent Ca²âº imaging. Interleukin-1ß (IL-1ß) release was measured by rat IL-1ß ELISA kits. Reactive oxygen species (ROS) generation was examined by dihydroethidium (DHE) fluorescent staining. Phagocytic activity was tested by neutral red uptake assay. RESULTS: We found that the [Ca²âº](c) increase evoked by ATP (5 mM) was inhibited by emodin, in a dose-dependent manner with IC50 of 0.5 µM. Furthermore, emodin reduced the IL-1ß release induced by ATP (2 mM) in lipopolysaccharide (LPS)-activated macrophages, with an IC50 of 1.6 µM. Emodin also strongly suppressed the ROS production and phagocytosis attenuation triggered by ATP (1 mM), with IC50 values of 1 µM and 0.7 µM, respectively. Besides, BBG, a specific antagonist of P2X7R, exhibited similar suppressive effects on these inflammation responses. CONCLUSION: These results showed the inhibitory effects of emodin on ATP-induced [Ca²âº](c) increase, IL-1ß release, ROS production and phagocytosis attenuation in rat peritoneal macrophages, by inhibiting the activation of P2X7R.

Effect of Rheedia longifolia leaf extract and fractions on the P2X7 receptor in vitro: novel antagonists?

Recently, the P2X(7) receptor has been reported to be associated with chronic inflammatory and neuropathic pain. Because Rheedia longifolia extract has analgesic and anti-inflammatory activity, we evaluated the in vitro inhibitory potential of methanol extract and fractions from its leaves on the P2X(7) purinergic receptor. The activity of P2X(7) was studied with a dye uptake assay and with the whole-cell patch clamp technique in mouse peritoneal macrophages treated with methanol extract of R. longifolia leaves and fractions. The dye uptake was evaluated by flow cytometry and fluorescence microscopy. The R. longifolia extract and some fractions showed an inhibitory effect on the P2X(7) purinergic receptor in a dose-dependent manner. The ethyl acetate fraction exhibited the most potent inhibitory effects. The methanol extract and the butanol fraction showed the same inhibitory effects, despite their lower potency compared with the other fractions. The R. longifolia extract and some of its fractions may be anti-inflammatory because of their inhibitory effect on the P2X(7) receptor. Further investigation is needed to determine the pattern of inhibition and selectivity. Chromatographic analysis indicated the presence of bisflavonoids in the methanol extract fractions. A member of this chemical family is the most probable active compound responsible for the P2X(7) inhibitory effects present in the R. Longifolia extract and fractions.