Evaluation of the Physicochemical Properties and Potential Immunomodulatory Effects of Bovine Thymic Peptide-Based Preparations

Among the bioactive molecules of animal origin, thymic peptides are particularly important because of their immunomodulatory properties and potential application in pharmaceutical and nutraceutical formulations. This study aimed to assess two formulations of thymic peptides obtained via two new isolation protocols from calf thymus, focusing on their physicochemical and biological characteristics, compared with the active pharmaceutical ingredient Thymus Factor X® (API TFX), which contains thymic peptides with proven clinical utility. The physicochemical characteristics included basic chemical composition, solubility, sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS‒PAGE) reversed-phase high-performance liquid chromatography (RP‒HPLC), size exclusion high-performance liquid chromatography (SEC‒HPLC) profiles, amino acid composition, and thermal properties, whereas the biological activity was assessed via a nuclear factor kappa-light-chain-enhancer (NF‒κB) reporter assay and cell viability analysis. The results showed that the preparations differed significantly in terms of their properties. API TFX and thymic preparation b (Pb) are soluble in water, whereas thymic preparation a (Pa) has low solubility. Pa displayed the broadest molecular weight distribution (approximately 3–25 kDa), with prominent bands at ~ 25 kDa and ~ 15 kDa, alongside numerous lower-mass fractions below 10 kDa. Amino acid analysis revealed that Pa is rich in glutamic acid, proline, and aspartic acid. In contrast, Pb and API TFXs were predominantly composed of low-molecular-weight peptides (< 10 kDa) and enriched in glutamic acid, lysine, and proline. Pa significantly inhibited the secretion of secreted active embryonic alkaline phosphatase (SEAP) by a human monocytic cell line derived from acute monocytic leukemia patient (THP-1) cells (inhibitors ranging from 12 to 33%). The level of inhibition of the NF-κB pathway in human monocytic THP‑1 cells was dose dependent. The Pb and API TFX used at a dose of 10 µg/ml also inhibited the activation of this pathway. The levels of SEAP in the presence of Pb and API TFX (0.1 µg/ml) were the same as those of lipopolysaccharide (LPS). These findings add to our understanding of the mechanisms of the immunomodulatory action of thymic peptides. These preparations have been shown to modulate the NF-κB pathway in human monocytic THP‑1 cells. These findings confirm the pharmaceutical profile of API TFX and identify a new preparation, Pa, as a promising candidate for further development as a functional immunomodulatory ingredient. The results underscore the value of comprehensive structural and functional profiling in evaluating thymic peptide preparations.