Hanin V. Development of a method for obtaining and evaluating the hepatotropic activity of chlorogenic acid

The processes of development and aging of biological systems remain among the most challenging unresolved problems in biology and medicine. Aging is driven by complex, nonlinear interactions between the organism and environmental factors collectively referred to as the exposome. These interactions result in systemic metabolic remodeling aimed at maintaining organismal integrity and viability, a phenomenon referred to as dynamic adaptation. However, prolonged adaptation may lead to the gradual loss of biological responsiveness to environmental stimuli, resulting in chronic pathological states, most of which are age-related. A representative model of such temporal interactions is the disturbance of copper homeostasis leading to Cu-induced liver fibrosis. Previous studies conducted in our laboratory demonstrated that repeated exposure of experimental animals to copper ions induced resistance to copper toxicity and hormetic effects, while simultaneously promoting liver fibrosis. These findings raise a fundamental question: why does an adaptive response that initially enhances survival ultimately result in irreversible pathological conditions? The aim of this work was to develop a concept explaining the mechanisms underlying chronic state formation using the Cu-induced liver fibrosis model and to investigate the preventive potential of natural polyphenolic compounds enriched with chlorogenic acid. The objectives were to: formulate and substantiate the concept of "exposome-induced metabolic resetting"; investigate the role of copper imbalance in biological regulation using mammals, yeast, and bacteria as model systems; evaluate the effects of sunflower meal polyphenols on circulating immunocompetent cells in animals with Cu-induced liver fibrosis; develop and characterize supramolecular complexes of colostrum casein with polyphenolic compounds for chlorogenic acid stabilization; and assess their effects on immunological and biochemical parameters in experimental animals. Sunflower meal polyphenols were selected because currently available hepatoprotective drugs show limited efficacy against advanced liver fibrosis, whereas natural polyphenols possess broad biological activity, are readily available from agricultural by-products, and represent promising therapeutic candidates. Their practical application is limited by oxidative instability; therefore, complexation with colostrum casein was proposed as an approach to improve chlorogenic acid stability. The study employed physicochemical, biophysical, biochemical, cytological, microbiological, biotechnological, and physiological methods, including quantum dot synthesis, UV and fluorescence spectroscopy, electrical conductivity measurements, enzyme activity assays, blood cell analysis, microbial growth evaluation, production of yeast and microalgal autolysates, and experimental modeling of Cu-induced liver fibrosis. A new concept termed "exposome-induced metabolic resetting" was developed. According to this concept, chronic pathology formation proceeds through sequential stages involving adaptation, establishment of metabolic and epigenetic memory, and stabilization of adaptive metabolic patterns into persistent metabolic states referred to as resetting. These metabolic programs remain self-sustaining even after elimination of the initiating environmental factor. It is proposed that restoration of normal metabolism requires induction of a reverse metabolic resetting mediated by natural bioactive compounds. A simple and efficient method for extracting polyphenolic compounds from sunflower meal was developed. Phenylephrine-based quantum dots were synthesized, and a sensitive fluorescence assay for chlorogenic acid determination was established. The extracted polyphenolic fraction contained at least 15% chlorogenic acid. For the first time, supramolecular complexes of casein and sunflower meal polyphenols were obtained and characterized. Their formation was shown to depend on hydrogen bonding, while molecular docking identified nine potential chlorogenic acid binding sites within β-casein exhibiting different binding affinities. Oral administration of sunflower meal polyphenols restored immunocompetent cell numbers to control levels during the early stages of Cu-induced liver fibrosis. This anti-inflammatory effect was more pronounced after oral than intraperitoneal administration, suggesting the possible involvement of the gut microbiome in mediating polyphenol activity. Furthermore, the developed casein–polyphenol supramolecular complexes provided prolonged stabilization of chlorogenic acid, normalized immunocompetent cell counts, and restored alanine aminotransferase activity to control values. These findings demonstrate that chlorogenic acid-enriched casein–polyphenol supramolecular complexes represent promising functional food ingredients for the prevention of Cu-induced liver fibrosis during the early stages of disease development.