An in vitro test, carried out in a private laboratory in Spain in 2015, researched the molecular mechanisms of the immunostimulant effect of a natural product rich in pronutrients.

Pronutrients are molecules of botanical origin that produce the activation of a target cell, which in this case are macrophages, and induce an increase in the translation of RNA to proteins.

In order to calculate the effect of pronutrients on the mRNA-protein translation rate, a technique called SUnSET (Surface Sensing of Translation) was used.

SUnSET technique is a non-radioactive method for monitoring protein synthesis in cell populations, based on the use of puromycin. Puromycin is a tyrosyl-tRNA analogue that can join newly synthesized proteins by a peptide bond, so the detection of puromycin by an anti-puromycin antibody (a technique called Western Blot-ELISA) can be correlated with the translation rate mRNA-protein.

Picture 1: Protein synthesis and puromycin incorporated to a protein.

METHODOLOGY

• In vitro assays were performed in 12 wells, where 1.2 × 10⁵ cells/ml were added in a final volume of 2 ml per well.
• After 4 days of culture, confluence was observed, and the medium was replaced with different supplements, following the scheme described below:
  • ASSAY A (Control): Only puromycin is added.
  • ASSAY B (Samples): Puromycin and the natural immunostimulant are added.
  • ASSAY C (Control). Puromycin and cycloheximide are added. Cycloheximide is an inhibitor of protein synthesis.

RESULTS (Picture 2)

• Assay A indicates the basic protein synthesis, visible thanks to the use of puromycin marker.
• Assay B shows an increase in protein synthesis using immunostimulant pronutrients (Nº 2 and 3 in the picture, labelled with stars), visible thanks to the puromycin marker.
• Assay C is used as an evidence of the inhibition of protein synthesis caused by the administration of cycloheximide, revealed thanks to the use of puromycin as a marker.

Picture 2: Western Blot results

CONCLUSIONS

In conclusion, this natural immunostimulant, based on pronutrients, increases mRNA-protein translation rate.

A higher rate of mRNA-protein translation correlates with an increase in the production of tissue-specific proteins and, therefore, a better performance of the target organ.

This improvement at the physiological level leads to an increased resistance to infectious diseases and lower mortalities in farm.

This natural immunostimulant based on pronutrients, manufactured by Biovet S.A., is commercially available under the name Alquernat Inmuplus.