Access of Group

At present time, only the use of cholinesterases inhibitors and NMDA receptor antagonists in the treatment of AD is justified from EBM point of view. Despite the efficacy of these drugs  AD therapy has partial character. Using low molecular drugs (small molecules) can affect only some of the pathophysiological processes from the whole complex of factors that play a role in the patophysiology of the disease.

Our approach as a contribution to the research of potential drugs for Alzheimer’s disease of natural origin consists of the determination of effects of selected tertiary alkaloids (isoquinoline, indole and steroid) to certain enzyme systems in the human brain and involves the following steps: 

1. Screening for alkaloids in morphological parts of plants and fruiting bodies of fungi belonging to the taxonomic units:

Magnoliideae (orders Magnoliales, Laurales),

Ranunculideae (orders Ranunculales, Hydrastidales, Berberidales, Papaverales),

Rosidae (order Rutales),

Lamiidae (order Gentianales)

Liliideae (orders Amaryllidales, Liliales)

Ascomycota

Basidiomycota

The procedure involves the following steps:

• preparation of summary (S) and alkaloidal (A) extracts from vegetabile (mushroom) material originating from different geographical areas,
• TLC, and detection with reagents for alkaloids according to the alkaloid types,
• bioautographic method on TLC for potential inhibitors of AChE and BuChE with semiquantitative evaluation (HPTLC, using galanthamine and rivastigmine as reference substances); non-alkaloidal taxa are excluded. 

2. Determination of inhibitory activity against human AChE (erythrocytic) and human BuChE (serum) of the extracts S and A using Ellman´s method. Only active extracts, more precisely  A‑extract, if IC₅₀ < 100 µg of dry weight / mL, are processed further.
     
3. Determination of antioxidative activity of both types of extracts (DPPH, ORAC, deoxyribose, TBA-MDA, FRAP).
     
4. Determination of qualitative and relative quantitative profile of active alkaloid taxa using LC/MS and GC/MS, and comparison with the database predicting the structure of alkaloids for development of isolation strategy from the biological material.
     
5. Obtaining plant (fungal) materials, isolation of alkaloids and determination of their structure (MS, NMR, optical rotation), preparation of salts (halogenides) and bases determination in the salts. Salts are used for biological tests, which require solubility in water, or rather  in buffer).
     
6. Determination of biological activity on the selected enzyme systems in vitro

• inhibitors of AChE and BuChE represent the first step of therapeutic intervention in the therapy of AD as effective cognitives confirmed by EBM. Current therapeutic range of these drugs is narrow (donepezil, galanthamine, rivastigmine), and therefore new potential drugs are sought. Therefore, the whole study of the biological activity of alkaloids by ADINACO Research Group is based primarily on the detection of inhibitory effects against AChE and BuChE and subsequently on other effects,
• inhibition of BACE1,
• inhibition  of Aβ formation and aggregation,
• inhibition  of prolyl endopeptidase and prolyl dipeptidase,
• antioxidative activity (see above). 

7. Further study of prospective compounds

For substances for which IC₅₀ (AChE, BuChE) ≤ 40 μM:

• study of acute toxicity (apoptosis, Ames test, Tetrahymena, neuroglia cell lines),
• preparation of semisynthetic derivatives (and subsequent finding of biological activity by cascade tests),
• methods of molecular docking,
• BACE1 inhibition,
• Aβ formation retardation, and inhibition of its aggregation,
• prolyl endopeptidase and prolyl dipeptidase inhibition,
• antioxidative activity.

Substances for which IC₅₀ (AchE, BuChE) > 40 μM are further monitored only for:

• BACE1 inhibition,
• Aβ formation retardation, and inhibition of its aggregation,
• prolyl endopeptidase and prolyl dipeptidase inhibition,
• antioxidative activity.