There is limited knowledge regarding α-lactalbumin amyloid aggregation and its mechanism. We examined the formation of α-lactalbumin amyloid fibrils (α-LAF) in the presence of cations (Mg²⁺, Ca²⁺, Na⁺, K⁺, NH₄⁺, and Cs⁺) in the form of chloride salts at two concentrations. We have shown that studied cations affect the conformation of α-lactalbumin, the kinetics of its amyloid formation, morphology, and secondary structure of α-LAF in a different manner. The higher salts concentration significantly accelerated the aggregation process. Both salt concentrations stabilized α-lactalbumin's secondary structure. However, the presence of divalent cations resulted in shorter fibrils with less β-sheet content. Moreover, strongly hydrated Mg²⁺ significantly altered α-lactalbumin's tertiary structure, followed by Na⁺, NH₄⁺, K⁺, and weakly hydrated Cs⁺. On the other hand, Ca²⁺, despite being also strongly hydrated, stabilized the tertiary structure, supposedly due to its high affinity towards α-lactalbumin. Yet, Ca²⁺ was not able to inhibit α-lactalbumin amyloid aggregation. Graphic abstract

Human insulin may undergo fibrillization under specific conditions, impairing its function and promoting its accumulation in amyloid deposits. Oregano (Origanum vulgare L.) leaves are rich in biologically active compounds such as polyphenols. Thus, we investigated their ability to inhibit insulin amyloid aggregation. The oregano aqueous extract phytochemical analysis (LC-MS/MS-DAD) revealed the presence of four major compounds: lithospermic acid (LA), rosmarinic acid (RA), oreganol A (OA), and luteolin-7-O-diglucuronide (L7dG), respectively. Compounds and their mixtures were subsequently screened for anti-amyloid activity and evaluated against oregano lyophilizate (LYO) utilizing ThT assay, AFM and ATR-FTIR analyses. LYO inhibited insulin fibrillization more effectively than its main constituent RA, prolonging the lag phase approximately two-fold. L7dG has been the most effective of the tested individual compounds, prolonging the lag phase by roughly 20 %, followed by LA, whereas OA was ineffective. Subsequently, we measured the anti-amyloid activity of two kinds of equimolar mixtures: either containing individually active compounds or analogous mixtures to which inactive OA was added. Surprisingly, LA:OA mixture proved to be the most effective. However, adding L7dG to the OA mixtures led to activity loss. The interactions of oregano polyphenols with the amyloidogenic regions of insulin were elucidated using molecular docking, explaining observed changes in their anti-amyloid activity. We conclude that when investigating the anti-amyloid activity of samples of natural origin and determining the activity of the extracts and their individual main components, it is necessary to consider their mutual interactions, which can significantly affect the final effect of the analyzed mixture.

Alzheimer’s disease (AD) is one of the most common causes of dementia, accounting for more than 60% of all cases. It is a neurodegenerative disease in which symptoms such as a decline in memory, thinking, learning, and organizing skills develop gradually over many years and eventually become more severe. To date, there is no effective treatment for the cause of Alzheimer’s disease, and the existing pharmacological options primarily help manage symptoms. Treatment is mainly based on acetylcholinesterase (AChE) inhibitors such as donepezil, rivastigmine, and galantamine, which exhibit numerous adverse cardiovascular and gastrointestinal effects due to excessive stimulation of peripheral cholinergic activity involving muscarinic receptors. Therefore, in addition to the obvious drugs that act on the cause of the disease, new drugs based on AChE inhibition that show the fewest side effects are needed. One potential drug could be a new compound under study, tetrahydroacridine derivative (CHDA), which showed significant potential to inhibit the AChE enzyme in previous in vitro studies. The present study shows that while having very potent AChE inhibitory properties, CHDA is a compound with low toxicity to nerve cell culture and living organisms. In addition, it exhibits dissociative activity against amyloid β fibrils, which is extremely important for applications in Alzheimer’s disease therapy.