Nigella sativa itself and as a part of a balm inhibits activity of puried 20S proteasome

Ubiquitin-dependent proteasomal proteolysis is the perspective target for the therapy of the high amount of different diseases. the special attention is paid to the therapy of inammatory processes of both origins: infection and aseptic. Besides proteolytic processing of the inhibitor of NFkappaB proteasome is important for activation of polymorphonuclear neutrophils (PMN) and neutrophil extrcellular traps (NETs) formation. Basing on the mentioned above information we have evaluated some plant extracts included in the medical compound «Healthy Tonsils» that is used for the treatment of the chronic tonsillitis for their ability to inhibit activity of 20S proteasome.


Methods.
For in vitro experiments the puri ed 20S proteasome was used. 20S proteasome was incubated for 30 minutes at 36 C degrees in a mixture with phytobalm components. After the next 30 minutes of incubation with 6 microM solution of corresponded uorogenic substrates the measurement of uorescence products was performed (exciting/emission wave length was 360/440) on the spectro uorimeter Hitachi-4000 with the use of free 7-amido-4-methylcoumarine for a standard curve.

Results.
In the same concentrations the compounds investigated has demonstrated different ability to inhibit activity of puri ed 20S proteasome. The most effective compound was the extract of Nigella sativa that inhibits chemotrypsin-like and caspase-like activities of puri ed 20S proteasome on 69 % and 85 % correspondingly.

Conclusions.
Thus, our results allow to explaine the anti-in ammatory activity of phytobalm particularly by ability of some of its compounds to inhibit proteasomal proteolysis.

Background
Ubiquitin-dependent proteasomal proteolysis is the perspective target for the therapy of the high amount of different diseases. The clinical study in this direction has started from the usage of speci c proteasomal inhibitors in the therapy of multiple myeloma and some other cancers [1]. The range of pathologies that possibly can be treated with proteasomal inhibitors was expanded during lust years. In particular, the special attention is paid to the therapy of in ammatory processes of both origins: infection and aseptic [2,3]. This is completely justi ed by the mechanism of activation of NFkappaB -the transcription factor under the control of which is the expression of the majority of proin ammatory genes starting from the cytokines, cell adhesion molecules, and nishing with the components of in ammasome [4]. Besides proteolytic processing of the inhibitor of NFkappaB proteasome is important for activation of polymorphonuclear neutrophils (PMN) and neutrophil extrcellular traps (NETs) formation. Proteasomal inhibitors effectively counteract this important mechanism of alteration. The ability of proteasomal inhibitors to induce apoptotic and autophagic cell death mechanisms of in ammatory cells should be also mentioned. Disturbances of these mechanisms may lead to the transition of an in ammation to a chronic state.
The search of proteasomal inhibitors of natural origin has started at the end of XX century and is still going on [5]. Researchers are interested in such a molecules due to their low toxicity (debugging of utilization processes in the organism), their high availability, low price, and wide range of action points comparing to the target inhibitors of synthetic origin [6,7].
Basing on the mentioned above information we have evaluated some plant extracts for their ability to inhibit activity of 20S proteasome. Anti-in ammatory properties of these extracts are already well known in phytotherapy, and this was the main selection criterion for including them in medical compound «Healthy Tonsils» that is used for the treatment of the chronic tonsillitis. Measurement of the proteasomal activities For in vitro experiments the puri ed 20S proteasome (ICN, USA) was used. 20S proteasome (2.5 microg) was incubated for 30 minutes at 36 C degrees in a mixture with phytobalm components. After the next 30 minutes of incubation with 6 microM solution of corresponded uorogenic substrates the measurement of uorescence products was performed (exciting/emission wave length was 360/440) on the spectro uorimeter Hitachi-4000 with the use of free 7-amido-4-methylcoumarine for a standard curve. In the each measurement the percentage of the inhibition of correspondent substrate cleavage was estimated under the in uence of speci c proteasomal inhibitor clasto-lactacystin beta-lactone in probes with the same concentration of DMSO or ethanol.

Methods
Statistical analysis. The data analysis was performed using the R statistical environment (version 3.5). All of the quantitative factors were checked for normality of the data distribution when using the Kolmogorov-Smirnov test. One-Way Analysis of Variance (ANOVA) was used to determine the differences between the group averages. The results were considered statistically signi cant at p < 0.05.

Results
Results of the experiments are noted in Table 1. In the same concentrations the compounds investigated has demonstrated different ability to inhibit activity of puri ed 20S proteasome. The most effective compound was the extract of Nigella sativa and Peru balsam derived from a tree Myroxylon balsamum var. pereirae. But only the rst one of the mentioned compounds demonstrated dose dependent effect (Fig. 1).

Discussion
Thus, as a result of our experiments the ability of Nigella sativa extract to in uence on the activity of puri ed 20S proteasome was demonstrated. The study of this substance is very actual due to in particular thymoquinone, the active component of the Nigella sativa oil has a very pronounced antiproliferative property demonstrated on different cell lines [9]. Further research in this direction has demonstrated that antiproliferative effects of thymoquinone are in a part caused by the ability to inhibit proteasomal proteolysis [8]. These results support our data due to thymoquinone is one of the compounds of Nigella sativa extract. But it is also not excluded that other active components of this extract [10,11,12] also have ability to inhibit activity of proteasome. Our results indirectly indicate this suggestion due to the mixture of all components of the balm salved in DMSO also inhibits puri ed 20S proteasome activity in a dose dependent manner ( Fig. 1. C,D). It is not excluded of course that this effect can be explained by the in uence of some other components of the balm that have not demonstrated such a property in a single experiment. Clinical observations of the usage of the phytobalm in a children and adolescence suffering from tonsillitis indicate signi cant decrease of in ammation that was supported also with ultrasound study results.

Conclusion
Thus, our results allow to explaine the anti-in ammatory activity of phytobalm particularly by ability of some of its compounds to inhibit proteasomal proteolysis.

Declarations
Ethics approval and consent to participate: The work is performed only in vitro without using of cell lines, isolated cells, laboratory animals and patients materials.

Consent for publication: Not applicable
Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no known competing nancial interests or personal relationships that could have appeared to in uence the work reported in this paper.
Funding: This research did not receive any speci c grant from funding agencies in the public, commercial, or not-for-pro t sectors.