Fig 1: Effects of 8-C-rhamnosyl apigenin injected 15 min after the injection of sPLA2 from Crotalus durissus terrificus. In (A) we observe the anti-inflammatory effect of 8CR that neutralizes the edema induced by sPLA2; In (B), we see the protective effect of the flavonoid on the myotoxic activity induced by sPLA2; (C–F) show the hind paw tissue homogenate quantification of cPLA2, PGE-2, MDA, and COX-2. All biochemical determinations were established by swollen hind paw tissue homogenate analysis of five animals. In (C–E), each column of biochemical determination represents the mean ± SEM of five animals and * statistically significant differences (n = 5, p < 0.05). All animals were treated with 200 μg (8 mg/kg) 8CR prior to injection of sPLA2, and after the experiments swollen hind paw tissue homogenate from each animal was prepared for specific analysis following the procedures described in each protocol supplied by the manufacturers. AV (crotalic and bothropic commercial veterinary propose antivenom, Lema-Injex, São Paulo, Brasil). Indo (Indomethacin, Sigma-Aldrich, St. Louis, MO, USA).
Fig 2: (A) Chemical structure of 8-C-rhamnosyl apigenin (8CR) isolated from Peperomia obtusifolia and the botanical illustration of the plant; (B) Comparative effects of 8CR, DuP-697, and SC-560 on cyclooxygenase-1 (COX-1) and COX-2 enzymatic activities. Each point represents the mean ± SEM of six animals in the percentage of remaining enzymatic activity of COX-1 or COX-2 without inhibitors; (C) Effects of 8CR, aristolochic acid (Aa), quercetin (Q), and quercitrin (Qn) on the enzymatic activity of sPLA2 isolated from Crotalus durissus terrificus; (D) The enzymatic effect of COX-2 in the presence of oleyloxyethyl phosphorylcholine (OP) and 8CR. All of the compounds used in this section were dissolved according to the supplier’s information by adjusting the concentrations for 128, 64, 32, and 16 μM, and all assays were done with the material provided by the manufacturer. * indicates significant differences relative to a standard. All analyses were performed using analysis of variance (ANOVA, p < 0.05), and each bar represents n = 5.
Fig 3: Rats given lethal doses of M. fulvius venom subcutaneously (SC) were treated with a single dose of Varespladib 8 mg/kg intravenously (IV) or excipient (control). (a) Plot showing survival benefit (Excipient n = 6, Varespladib n = 12). Dose of venom indicated inside the red dots indicate 4 mg/kg and 8 mg/kg venom doses for which there was no apparent difference in time to death (287 ± 110 min vs. 240 ± 68, respectively; p = 0.32); (b) snake venom-induced rise in sPLA2 activity is suppressed by varespladib (N = 3 each group; * p < 0.001); time of varespladib addition post venom-addition is noted by “T =” for each group; (c) treatment with varespladib prevented intravascular hemolysis in the same 12 animals. Tubes shown are blood drawn at the 4 h time point.
Fig 4: Comparison of the native secretory phospholipase A2 (sPLA2) and sPLA2 treated with 8CR in relation to: (A) chromatographic retention times; (B) circular dichroism curves and (C) fluorescence of the protein.
Fig 5: In vitro dose-response curves for varespladib and its orally bioavailable prodrug methyl-varespladib tested against 20 medically important snake venoms. While demonstrating high degrees of potency against snake venoms, neither varespladib nor methyl-varespladib showed high degrees of potency against bee venom sPLA2 (positive control). N = 1 run unless otherwise specified number of replicates. Error bars signify s.d. a. Agkistrodon brevicaudus blomhoffi, b. Bungarus caeruleus, c. B. fasciatus, d. Crotalus adamanteus, e. Crotalus atrox, f. Crotalus scutulatus, g. Dendroaspis polylepis, h. Echis carinatus, i. Laticauda semifasciata, j. Micrurus fulvius, k. Naja naja atra, l. Naja naja kaouthia, m. Naja naja naja, n. Notechis scutatus scutatus, o. Ophiophagus hannah, p. Oxyuranus scutellatus, q. Pseudechis australis, r. Vipera berus, s. Daboia russelli, t. Crotalus durissus terrificus, u. Bee venom (Apis mellifera) purified sPLA2 positive (+) control.
Supplier Page from Abcam for Phospholipase A2 Activity Assay Kit (Colorimetric)