Abstract
The widely accepted strategy to justify the use of medicinal plant extracts in diseases with inflammatory background is their examination on in vitro models using immune cells. It is also a key initial step of research for active principles, which could be then isolated and tested on more advanced models, becoming new pharmacologically active lead molecules. The crucial aspect which has not been so far addressed in this context, is the presence of pyrogens in plant preparations. The aim of this study was the examination of pyrogens interference with in vitro evaluation of anti-inflammatory activity of plant extracts using human primary neutrophils model together with introduction of effective method of interfering factors elimination. The obtained results showed that chosen plant extracts contained pyrogens, which were responsible for concentration-dependent stimulation of pro-inflammatory cytokines production by human neutrophils in vitro in the same extent as LPS did. The ultrafiltration method was successfully applied for pyrogens elimination, which effectiveness was confirmed using LAL test. The determined interference of pyrogens implies the necessity of their consideration and removal when in vitro studies include direct addition of plant extracts to the cell culture, what can be obtained by ultrafiltration, which does not affect extract composition.
Highlights
The widely accepted strategy to justify the use of medicinal plant extracts in diseases with inflammatory background is their examination on in vitro models using immune cells
Extracts after ultrafiltration 1n...9n Non-filtered extracts CDK Cyclin-dependent kinase DAMPS Damage-associated molecular patterns FBS Fetal bovine serum LAL Limulus amebocyte lysate LPS Lipopolysaccharide lipopolysaccharide from R. sphaeroides (LPS-RS) Ultrapure lipopolysaccharide from Rhodobacter sphaeroides NETs Neutrophil extracellular traps PAMPs Pathogen-associated molecular patterns propidium iodide (PI) Propidium iodide rsc Roscovitine TLR Toll-like receptor uroA Urolithin A
The commonly raised limitation of this strategy is the question regarding the bioavailability of plant extract constituents. This drawback can be addressed if we consider in vitro examination of plant extracts as an initial step before conducting in vivo studies or as a screening strategy which leads to a selection of the most effective extraction procedure and active composition followed by bio-guided fractionation and subsequent isolation of active principles which will be subjected for further pharmacological evaluations[5]
Summary
The widely accepted strategy to justify the use of medicinal plant extracts in diseases with inflammatory background is their examination on in vitro models using immune cells It is a key initial step of research for active principles, which could be isolated and tested on more advanced models, becoming new pharmacologically active lead molecules. The crucial aspect which so far has not been addressed in the in vitro studies of plant extracts is the possible presence of pyrogenic factors in tested preparations deriving from bacteria residing on raw plant materials Their interference with in vitro assays, especially targeted on the modulation of the inflammatory response can occur detrimental by leading to false-positive or false-negative results. Bacterial endotoxins, such as lipopolysaccharide, are the most common pyrogens, which if present even at a very low concentration in tested extracts can potentially interfere with examined inflammation processes leading to disrupted results
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