Novel Insights into the Biological Activity of Croton lechleri Twigs Extracts and Advancements in Their Sustainable Recovery.

Sangre de drago, the sap of Croton lechleri Müll. Arg. tree, has been used for centuries in traditional medicine owing to its diverse biological activities. Extracts derived from different parts of the species contain a multitude of phytochemicals with varied applications. Twigs, however, are among the least studied parts of the plant. This study unveils new biological activities of Croton lechleri twig extracts recovered by applying Soxhlet and advanced green techniques. For all extracts, total phenolic content and antioxidant activity were determined. Subsequently, four were selected, and their cytotoxic effects were assessed on both normal (HaCat) and malignant melanoma (A375) skin cell lines using the MTT assay and trypan blue exclusion assay. All showed dose-dependent cytotoxicity, with the Soxhlet ethanol extract demonstrating the highest selectivity towards A375 cells over HaCat cells. The extracts induced apoptosis and necrosis, as confirmed by Annexin V/PI dual-labeling and flow cytometry, highlighting their ability to trigger programmed cell death in cancer cells. The selective inhibition of cell cycle progression in A375 compared to HaCat observed both for Soxhlet ethanol and pressurized ethanol extracts induces cell cycle arrest at multiple points, primarily in the G1 and G2/M phases, and significantly reduces DNA synthesis as evidenced by the decrease in the S-phase population, confirmed by the EdU assay. Consequently, the Soxhlet extract composition was analyzed using LC-MS, which revealed their richness in polyphenolic compounds, particularly flavonoids from the flavonol subclass.

Quantitative assessment of the contribution of high resolution mass spectrometric analysis to the reliability of compound confirmation.

Applications of high resolution mass spectrometry (HRMS) in food safety and residue analysis have increased remarkably over the last few years. The high resolution detection of ions reportedly enhances the assay selectivity but quantitative assessment of HRMS contribution to the assay selectivity has not yet been undertaken. We devised a method to assess the impact of instrument resolution on the probability that a spectral assignment to a given compound was made in error. The method allows for evaluating the quality of a spectral assignment based on resolution and the number of fragmentation stages. It thus provides a firm basis for comparing analytical methods performed on very different mass spectrometric instrumental platforms as well as in the context of the current regulatory framework.

Identification of the related substances of tilmicosin by liquid chromatography/ion trap mass spectrometry.

Structures of seven impurities of the veterinary drug tilmicosin have been elucidated by multiple fragmentation with ion trap tandem mass spectrometry. All related compounds possess the main lactone ring of tilmicosin. The differences in their structures are due to the hydroxyl, mycaminose, 3,5-dimethylpiperidine and mycinose groups connected to C(3), C(5), C(6), C(14) of the lactone ring, respectively. The following compounds of the impurity profile of tilmicosin were identified: B - tilmicosin with a hydroxyl group at C(3); C - tilmicosin without a methyl group at the N-atom connected to C(3) of the mycaminose ring; D - tilmicosin with a hydroxyl group at C(6) of the mycaminose ring; E - tilmicosin with a methoxy group at C(3), F - desmicosin; G - 20-dihydrodesmicosin; and H - tilmicosin without a mycaminose ring. Isomers of the compounds B, C, D, E and H were identified by their mass chromatograms and retention times. The concentrations of the impurities varied in the range of 0.1% to 2.9%.

Ligand-based design, synthesis and primary in vivo screening of pyrrole derivatives as potential tricyclic anti-inflammatory agents.

Twelve new compounds were designed as 5-aryl-1H-pyrrole analogs of celecoxib (CAS 169590-42-5) and were synthesized by Paal-Knorr cyclization in three series according to 1H-substitution: derivatives with salicylic acid, pyrazolone or isonicotinamide residues. The average physico-chemical and steric similarity between the prototype and the new analogs (completed with two previously synthesized related products) was assessed to be 82 % and therefore considered as a reliable prerequisite for spatial compatibility and effective binding to the cyclooxygenase (COX) enzymes. The anti-inflammatory effects were determined in acute inflammation model using the carrageenan-induced rat paw edema assay on male Wistar rats (180-200 g) at doses of 10, 20 and 40 mg/kg, i.p. Six of the new products showed higher percent of inhibition (up to 100 %) compared to the highly selective COX-2 inhibitor celecoxib and the nonselective indometacin (CAS 53-86-1) used as reference compounds. Ethyl 1-(1,5-di-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-4-pyrazolyl)-2-methyl-5-phenyl-1H-3-pyrrolecarboxylate (2b), ethyl 5-(4-chlor-ophenyl)-2-methyl-1-[(4-pyridylcarbonyl) amino]-1H-3-pyrrolecarboxylate (3c) and 5-[3-acetyl-2-methyl-5-(4-methylphenyl)-1H-1-pyrrolyl] -2-hydroxybenzoic acid (4b) were pointed out as the most active representatives of each of the three tested sub-series.