Understanding demulcent activity

Ahmad, Basir

Abstract

A demulcent is defined in the oxford dictionary as a soothing agent that protects mucous membranes and relieves irritation by forming a protective film (Ireland, 2020). There is no scientific or clinical evidence supporting this mode of action. We therefore aimed to develop in vitro models to investigate, for the first time, the mechanism of action of demulcents, pharmacologically characterise them and identify potential target proteins to ultimately select the most efficacious preparations for cough.
We chose glycerol (3.26mM to 1.09M), sucrose (880μM to 292mM), glycyrrhizin (35.7mM), dextran (4μM to 1.3mM) and carrageenan (30μg/ml to 1mg/ml) as our test demulcents with our main focus on dextran. We validated an old method from 1973 for labelling dextran with FITC to enable future labelling of polysaccharides of interest that are not available commercially. We used flow cytometry, fluorescence microscopy and confocal microscopy to show dextran interactions with the cells. We used LDH assay to test glycerol, sucrose, carrageenan and dextran for cytotoxicity. We used FlexStation 3 for a plate-based calcium signalling assay to test the agonist and antagonist effect of glycerol, sucrose, dextran and carrageenan on HEK293 cells transfected with TRPA1, TRPV1, TRPV4 and TRPM8. Using the same setup further, we tested the effect of increasing salt concentration on dextran inhibition of TRPA1, TRPV1, TRPV4 and TRPM8. We also used ATP determination assay to test the effect of dextran on the release of ATP from HEK293 cells transfected with TRPV4.
We successfully labelled dextran with FITC. Our fluorescence and confocal microscopy results show that the FITC dextran and texas-red dextran were taken up by A549 cells. FITC dextrans were present mainly at the cell junctions and some were found at the cell surface. Dextrans were also present throughout the cells with clusters around the cell surface membrane. Calcium signalling assay results showed that dextran significantly suppressed TRPA1 (IC50 677μM, up to 73.1%, P<0.0001), TRPV1 (co-expressed with TRPA1, IC50 3.76μM, up to 70.1%, P<0.0001), TRPV4 (IC50 112μM, up to 73.3%, P=0.0005) and TRPM8 (IC50 39μM, up to 49.4%, P=0.01) receptor responses to cinnamaldehyde, capsaicin, GSK1016790A and WS5 respectively compared to calcium working buffer vehicle. Dextran also significantly reduced the release of ATP (IC50 115μM, up to 24.6%, P=0.01). Likewise, glycerol significantly suppressed TRPA1 (IC50 722mM, up to 64%, P<0.0001), TRPV4 (IC50 110mM, up to 49.3%, P=0.0018), TRPM8 (IC50 167mM, up to 53.5%, P=0.003) and TRPV1 (co-expressed with TRPA1, IC50 1.06M, up to 55.6%, P<0.0001) response to their respective specific agonists compared to calcium working buffer vehicle. Similarly, sucrose suppressed TRPA1 (IC50 28mM, up to 36.8%, P<0.0001) and TRPV4 (IC50 110mM, up to 37%, P=0.0063) while carrageenan suppressed only TRPV4 (IC50 100μg/ml, up to 16.2%, P=0.0475) response to their specific agonists compared to calcium working buffer vehicle.
The definition of demulcent might not be as straightforward as mentioned in the oxford dictionary. Demulcents may have multiple mechanisms of action including macropinocytosis. We conclude that dextran might be the best demulcent among our test demulcents and that its mechanism of action is most likely a surface phenomenon as its effects are reversible. Dextran interacts with the cell surface membrane and is an antagonist for TRPA1, TRPV1 (co-expressed with TRPA1), TRPV4 and TRPM8.

Citation

Ahmad, B. Understanding demulcent activity. (Thesis). Hull York Medical School. https://hull-repository.worktribe.com/output/4790538

• Thesis Type: Thesis
• Deposit Date: Aug 22, 2024
• Keywords: Medicine
• Public URL: https://hull-repository.worktribe.com/output/4790538
• Additional Information: Hull York Medical School; University of Hull and University of York
• Award Date: Jun 1, 2024