The present work describes validated high-performance liquid chromatography (HPLC) and high-performance thin-layer chromatography (HPTLC) methods for the simultaneous determination of colchicine and khellin. The isocratic reversed-phase HPLC separation was performed on a 5 µm C18 column (Luna, Phenomenex, Torrance, CA). Good resolution between colchicine and khellin was achieved using a mixture of acetonitrile–10 mM NaH₂PO₄ (pH 3.0, 35:65 v/v) as a mobile phase. Quantitation was achieved with ultraviolet detection at 245 nm based on peak area. The HPTLC separation was conducted on Merck HPTLC aluminum sheets of silica gel 60 F254 as stationary phase using methylene chloride–methanol (95:5 v/v) as a mobile phase. Quantification was also achieved using densitometric measurements at 245 nm. Both methods revealed reasonable validation parameters concerning selectivity, linearity, accuracy, precision and limits of detection and quantitation.

Background/Objectives: A promising anticancer strategy is the simultaneous inhibition of the receptor tyrosine kinases VEGFR-2 and c-Met, which are essential for tumor angiogenesis, growth, and metastasis. In this study, a novel series of piperidinyl-based benzoxazole derivatives was designed and synthesized as potential dual VEGFR-2/c-Met inhibitors. Methods: The kinase inhibitory potential of the derivatives was evaluated in comparison to reference inhibitors, Sorafenib (VEGFR-2 inhibitor) and Staurosporine (c-Met inhibitor). Cytotoxicity was assessed across breast, prostate (PC-3), and lung (A549) cancer cell lines. Mechanistic studies included cell-cycle analysis, apoptosis assays, gene expression profiling of apoptosis-related markers, and molecular docking within the ATP-binding pockets of both kinases. Results: Compounds 5a, 5g, 5h, 11a, and 11b showed strong inhibition of both kinases (IC₅₀ = 0.145–0.970 μM for VEGFR-2 and 0.181–1.885 μM for c-Met). Selective cytotoxicity was observed against breast cancer cells, with compound 11b (p-fluorophenyl derivative) exhibiting high selectivity toward MCF-7 over normal breast cells (MCF-10A) and potency comparable to or exceeding Sorafenib. Mechanistically, 11b induced G₂/M cell-cycle arrest and apoptosis (total apoptosis = 48.34%), accompanied by upregulation of p53, BAX, and caspase-9 and downregulation of Bcl-2. Molecular docking confirmed stable binding within the ATP-binding sites of both kinases. Conclusions: Compound 11b was established as a novel, selective, dual VEGFR-2/c-Met inhibitor with strong potential for targeted breast cancer therapy.

ShK from Stichodactyla helianthus has established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in actinarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family in Actinia tenebrosa and Telmatactis stephensoni , characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom of T . stephensoni . We identified ten SA8 genes in two clusters and six SA8 genes in five clusters for T. stephensoni and A. tenebrosa , respectively. Nine SA8 T. stephensoni genes were found in a single cluster and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity. Our results provide the first demonstration that SA8 is a unique gene family in actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom of T . stephensoni .