A method of obtaining biologically active components from Saccharomyces cerevisiae yeast cells and a therapeutic agent based on them

摘要

FIELD: medicine; pharmaceuticals.SUBSTANCE: method is proposed to produce total RNA and zymosan polysaccharide from yeast cells Saccharomyces cerevisiae, strain of VKPM Y-448 and therapeutic agent based thereon. Method is characterized by that it involves destruction of yeast cells in buffer with pH 7.4, containing 10 mM Tris, 20 mM EDTA and 0.5 M NaCl, treatment with sodium dodecylsulphate (SDS) in concentration of 1.0 % at 20–30 °C for 20–50 min and chloroform in concentration of up to 25 % for 20–30 minutes at 20–30 °C. Obtained mixture is centrifuged for 20 minutes, followed by separation of the supernatant containing the aqueous extract of total RNA from the residue—a clot of proteins and polysaccharides containing zymosan, washed from the bulk of the lipids. SDS solution is added to the obtained aqueous cell extract containing total RNA to a final concentration of 2 % and held for 60–90 minutes at 0 °C until precipitation of the SDS complex with denatured proteins, which is removed by centrifugation at 8000 g, 20 min, 0 °C, and total RNA is obtained by deposition from supernatant liquid lithium chloride solution with salt concentration of 3.5 M with removal of main pool of impurity proteins together with remaining solution during washing. Obtained residue is washed twice with 3.5 M lithium chloride salt solution and dissolved in water. Solution is clarified by centrifugation at 8000 g, 20 min, 4 °C and passed through a filter with pore diameter of 0.22 mcm, after which RNA is precipitated by adding ethanol to concentration of 50–55 %, and the formed precipitate is separated by centrifugation at 8000 g, 20 min, 4 °C, washed with 60 % ethanol and dissolved in isotonic solution with output of total RNA preparation 200–240 mg of 100 g of yeast biomass. Produced protein-polysaccharide clot containing zymosan is suspended in 1 % sodium dodecyl sulphate solution, suspension is stirred at 50–60 °C for 20–30 minutes and zymozan polysaccharide is separated from protein solution by centrifugation at 8000 g, 15 min, 20 °C. Obtained precipitate is washed, the purified zymosan polysaccharide is dried at a temperature of +40–45 °C for one day and transferred into a fine powder state. What is also presented is an antiviral and antibacterial agent containing total RNA and zymosan polysaccharide obtained by the above method and having ratio of 1:2.5 to 1:10, as well as solvent or ointment base with target additives at the following quantitative content of components, wt %: total RNA—0.2–3.0, zymosan polysaccharide—0.5–30.0, solvent or ointment base with target additives—balance up to 100 %.EFFECT: invention enables to obtain total RNA and zymosan polysaccharide in one technological process, as well as obtaining an agent exhibiting high interferon-inducing and phagocytosis-stimulating activities.4 cl, 2 dwg, 4 tbl, 8 ex