The leaves of Annona purpurea have yielded several alkaloids with anti-aggregation activities against rabbit platelets. This is promising in the search for agents that might act against platelets and reduce the incidence of cardiovascular diseases. Since significant differences in platelet function have been reported between human and animal platelets, a study focusing on the effect of A. purpurea extracts against human platelet activation is necessary. The compounds in an A. purpurea ethanolic extract underwent bio-guided fractionation and were used for in vitro human platelet aggregation assays to isolate the compounds with anti-platelet activity. The bioactive compounds were identified by spectroscopic analysis. Additional platelet studies were performed to characterize their action as inhibitors of human platelet activation. The benzylisoquinoline alkaloid norpurpureine was identified as the major anti-platelet compound. The IC50 for norpurpureine was 80 μM against platelets when stimulated with adenosine 5′-diphosphate (ADP), collagen and thrombin. It was pharmacologically effective from 20 to 220 μM. Norpurpureine (220 μM) exhibited its in vitro effectiveness in samples from 30 healthy human donors who did not take any drugs during the 2 weeks prior to the collection. Norpurpureine also gradually inhibited granule secretion and adhesion of activated platelets to immobilized fibrinogen. At the intra-platelet level, norpurpureine prevented agonist-stimulated calcium mobilization and cAMP reduction. Structure–activity relationship analysis indicates that the lack of a methyl group at the nitrogen seems to be key in the ability of the compound to interact with its molecular target. Norpurpureine displays a promising in vitro pharmacological profile as an inhibitor of human platelet activation. Its molecular target could be a common effector between Ca2+ and cAMP signaling, such as the PLC-PKC-Ca2+ pathway and PDEs. This needs further evaluation at the protein isoform level.

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y₁ receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y₁receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y₁-NTPDase1 fusion protein. Stable expression of the P2Y₁-NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca²⁺response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADPβS to stimulate inositol phosphate accumulation was similar, and the EC₅₀ of ADPβS was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y₁ and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y₁ receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y₁ receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y₁ receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.

This study explores the synthesis of nanohydroxyapatite (nHA) using high-frequency ultrasonic irradiation (UI), a chemically friendly technique aligned with green chemistry principles. Synthesis was achieved by varying the UI time (15, 30, 45, and 60 minutes) and the reaction medium. Water-based mixtures were used: water/acetone (W/ACET), water/tetrahydrofuran (W/THF), and water/ethanol (W/ETOH). The resulting nHA was characterized structurally using FTIR, XRD, TGA, and SEM. These analyses revealed the formation of carbonated nHA similar to biological apatite, with distinct morphologies and particle sizes dependent on the solvent used. All synthesized materials exhibited high thermal stability and yields exceeding 80%. Hemocompatibility studies showed that nHA samples obtained in the W/THF mixture presented low hemotoxicity (1-2 % hemolysis), and did not affect platelet ADP aggregation processes. Microbiological assays showed a significant reduction in P. aeruginosa biomass production (p<0.05) after 24 hours, particularly for nHA synthesized with the W/ETOH mixture. There was also a less bacterial colonization, cellular aggregates or formation of specialized structures when was treated with this bioceramic, compared to the commercial hydroxyapatite, suggesting potential antibiofilm properties. This research presents an original and efficient method for producing nHA as a promising biomaterial for tissue engineering and bone replacement applications.

This study explores the synthesis of nanohydroxyapatite (nHA) using high-frequency ultrasonic irradiation (UI), a chemically friendly technique aligned with green chemistry principles. Synthesis was achieved by varying the UI time (15, 30, 45, and 60 min) and the reaction medium. Water-based mixtures were used, that is, water/acetone (W/ACET), water/tetrahydrofuran (W/THF), and water/ethanol (W/ETOH). The resulting nHA was characterized structurally using FTIR, XRD, TGA, FESEM, and specific surface area determination. These analyses revealed the formation of carbonated nHA similar to biological apatite, with distinct morphologies and particle sizes dependent on the solvent system used. All synthesized materials exhibited high thermal stability and yields exceeding 80%. Hemocompatibility studies showed that nHA samples obtained in the W/THF mixture presented very low hemotoxicity (hemolysis < 2%), and did not affect platelet ADP aggregation processes. Microbiological assays showed a significant reduction in Pseudomonas aeruginosa biomass production ( p < 0.05) after 24 h, particularly for nHA synthesized with the W/ETOH mixture. There was also a less bacterial colonization, cellular aggregates, or formation of specialized structures after treatment with this bioceramic, compared to the commercial hydroxyapatite, suggesting potential antibiofilm properties. This research presents an original and efficient method for producing nHA as a promising biomaterial for tissue engineering.