Antioxidant agent of nanostructured selenium with hepatoprotective effect and methods for producing and using it

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry, namely to selenium nanocomposites of natural hepatotrophic galactose-containing polysaccharide matrixes, representing water-soluble orange-red powders containing zerovalent selenium (Se0) nanoparticles sized 1-100 nm in the quantitative content of 0.5 - 60 wt %, possessing antioxidant activity for treating and preventing redox-related pathologies, particularly for treating toxic liver damage, to a method for producing and to an antioxidant agent containing the above nanocomposites.

EFFECT: invention provides the targeted agent delivery to liver cells, as well as higher agent accessibility and lower toxic action of selenium.

7 cl, 11 ex, 4 tbl

 

The invention relates to pharmaceutics, in particular, can be used for the prevention and treatment of redox-dependent diseases, as well as for replenishment of selenium in animals and humans, which provides a target hepatotropic antioxidant protection and restores the body's essential trace mineral selenium, and also decrease its toxicity in therapeutic use.

Known complex multivitamin preparations regulating metabolic processes and reducing the frequency of manifestation of selenium deficiency in animals, including birds, containing selenium and oil solvent, while it contains selenium as sodium Selenite, retinol acetate as vitamin A, an oil solvent is corn oil germ.

[The patent. RU №2250099, CL A61K 9/08, A61K 33/04, A61K 31/07, A61K 35/78, publ. 20.04.2005.].

Known preparation containing sodium Selenite and vitamin E, which has been successfully used for prevention of postpartum diseases in cattle. Thus, the complex application of sodium Selenite and vitamin E helped reduce mastitis in herds of 30% or more [Smith, A. M. and M. F. Picciano Evidence for increased selenium requirement for the rat during pregnancy and lactation. - J. of Nutrition, 1986, V. H6, N6, p.1068-1079.; Smith I., Hogan, J. Vitamin E and selenium can help lover incident of mastitis. - Zarge anim. Veter., 1988, 43, 6, 20-24].

The disadvantages of these drug�are in their poor digestibility and relatively high toxicity.

Known agent-based Nanomolecular selenium-enzyme, which has a pronounced immune and metabolic effects. Its production method includes the extraction of protein from protein-containing vegetable raw materials with organic solvents followed by the addition of selenium, treating the mixture in a high-frequency sinusoidal electromagnetic field, then the trypsin by adding to the formed nanoparticles of selenium-enzyme molecule of selenium ions and re-incubation in high-frequency electromagnetic field 20-30 GHz. The resulting tool is a Nanomolecular selenoproteins matrix containing selenium, obtained by thermolysis of Selenomethionine, wherein the molar ratio of protein and selenium is 1:8.

[The patent. RU №2242234, CL A61K 33/04, A61K 35/78, A61P 37/02, publ. 20.12.2004.].

This tool has only immune and metabolic effect, and nanoscale is selenium-enzyme a macromolecule, and the dimension of the particles of selenium is not defined.

Also known immunomodulatory composition for animals which comprises as the active substance milk whey protein - lactoferrin, as solvent - distilled water. Active substance includes, in addition to lactoferrin whey proteins of milk lacteal�min and lactoglobulin, this active substance is an aqueous solution of a mixture of serum proteins of milk with a total protein concentration of 10 g/l, containing 10-20% lactoferrin, 20-30% of lactoalbumin, 60-70% lactoglobulin, furthermore, the said active substance further comprises nanoparticles of selenium, which are directly formed in the process of formation of the composition when added to an aqueous solution of a mixture of three proteins of a 1 M solution of hydrazine hydrochloric acid and 1 M solution of sodium Selenite Na2SeO3. Then the resulting mixture was adjusted with distilled water to 100 wt.%. Stop the reaction by bringing the pH of the solution to 7.2 with 1 M sodium hydroxide solution and freed from low molecular weight compounds by dialysis against distilled water. The selenium nanoparticles included in the composition of the active substance, which give the songs additional antioxidant effect.

[Patent RU No. 2485964, CL A61K 35/20 ETF; A61K 33/04; A61P 37/02; B82B 1/00;, publ. 27.06.2013.].

The disadvantage of this composition is that as the active active substance use animal proteins (from cow's milk, which provides the potential allergologist, and also greatly affects the increase in the cost of the drug. In addition, to stop the reaction of formation of nanoscale� selenium is necessary to control the pH of the environment, as well as an additional stage of dialysis for release from low molecular weight compounds.

Patented as a preparation for injection, which increases the immune resistance of the organism of animals, which can be used to regulate metabolic processes and reduce the incidence of selenium deficiency in animals, in which selenium is contained in the nanosized state. The drug is produced by reduction of inorganic derivative of selenium in a high oxidation state (H2SeO3) with the formation of selenium atoms in the zero oxidation state or nolvadent, which then condense with the formation of selenium nanoparticles (up to 100 nm and stabilized by polyelectrolytes (macromolecular natural or synthetic nitrogen-containing polymer capable of stabilizing the nanoparticles of selenium). The drug includes water for injection, nitrogen-containing high molecular polymer as a stabilizer, selenium in nanoscale condition and the zero valence (Se0).

[The patent. RU №2392944, CL A61K 31/758, AL 33/04, A61P 3/02 published 27.06.2010 a.].

The disadvantages of this drug are the use as a stabilizer nitrogen-containing synthetic or natural polymer of protein nature, which determines their potential allergologist, in addition, these polymers do not �bladud directed hepatotropic effect.

The closest in technical essence and achieved the positive effect in relation to the proposed invention is the selenium nonprepared on the basis of the polysaccharides of the fungus Pleurotus tuber-regium with antitumor activity. Synthesis of nanocomposite involves two stages: first, add to polysaccharide ascorbic acid (reductant precursor selenium), and then dropwise the aqueous solution of selenium dioxide or metal Selenite. The appearance of the red color of the reaction mixture indicates the formation of nanoparticles of elemental selenium. The dropping finish in a time when the colour of the solution ceases to increase. The use of specific polysaccharides of the fungus Pleurotus tuber-regium as necessary to control the size of the resultant particles of selenium in the target nanocomposite, and the existence of joint antitumor action of these polysaccharides nanoparticles and selenium.

[US Patent 2013029931 "Pleurotus tuber-regium polysaccharide handling nano-selenium hydrosol with anti-tumor activity and preparation method thereof.", CL A61K 31/715; A61P 35/00, published on 31.01.2013].

The disadvantages of this drug are a narrow range of tested biological action (only the antitumor activity), as well as the lack of information about the target hepatotropic action derived nanocomposite (and polysaccharides that are part of this nano�of opposite).

The object of the invention is to develop a new highly effective antioxidant selenium preparation on the basis of hepatotropic galactosaemia polysaccharide matrices, in which selenium is contained in the nanosized state.

The technical result, which is achieved by the invention, is reduced to the targeted delivery of the drug to the liver cells through the implementation of specific receptor binding of hepatocytes with galaktoznoj fragments of polysaccharides, as well as to improve absorption of the drug and reduce the toxic effect of selenium due to the fact that this element is used in nanoscale condition.

Means on the basis of this drug proposed for the treatment and prevention of redox-related pathologies (in particular, toxic liver damage), and is a water-soluble powder red-orange color, containing selenium zero valence (Se0) in an amount of from 0.5 to 60% in the form of a nanocomposite with particle sizes of selenium in the range of 1-100 nm. The nanocomposite prepared from various selenium-containing raw material directly in an aqueous solution of natural galactosidase hepatotropic polysaccharide (arabinogalactan, galactomannan, carrageenan, etc.), which, moreover, is the stabilizer of the nanoparticles are then cleaned� his planting in 96% ethanol or other alcohol, or acetone and dried. In order to receive the funds antioxidant dry polysaccharide nanocomposite of selenium dissolved in distilled water, with the following ratio of components in wt.%:

selenium-containing nanocompositeIs 0.0001 to 1.0
distilled waterelse

The ratio of the nanocomposite and water set so that in the prepared antioxidant tool, the selenium concentration was close to the effective dose of selenium in sodium Selenite used for the prevention and treatment caused by selenium deficiency in the body white muscle disease (0.1-0.2 mg Na2SeO31 kg weight of the animal, or 0.0457-0.0913 mg of pure selenium per 1 kg of animal weight, or 3.0 mg once daily dose).

Affinity galactosaemia polysaccharide matrix to asialoglycoprotein receptors hepatocyte membrane allows this therapeutic agent is directed to selenium associate with membranes of hepatocytes, as well as potentially implement it into these cells by receptor-induced endocytosis. It is important to emphasize that when using galactosaemia polysaccharides as a matrix carrier proishoditb rational delivery of substances to the bodies (depot) and not observed its redistribution in favour of RES [Medvedev S. A. The larch arabinogalactan is a promising nutrient matrix for nutrient metals / S. Medvedev et al. / / Chemistry and computational simulation. Butlerov communications. - 2002. - No. 7. - P. 45-50., Krivtsov, G. G., Zhdanov R. I. // Questions of medical chemistry. - 2000. - No. 3. S. - 25-30]. That is, the combination has with polysaccharides, which have affinity to asialoglycoprotein receptors of the liver and spleen, helps to ensure directed receptora-mediated delivery of selenium to the cells of organs depot, and specifically the liver (and possible subsequent endocytosis intracellular delivery Se) depending on the needs of this element that can be defined as "intelligent" mechanism.

Nanocomposite with selenium galactosaemia polysaccharide matrix is proposed to obtain three different ways from a variety of selenium-containing starting compounds in the presence of various galactosaemia polysaccharides, for example, or arabinogalactan, galactomannan, or carrageenan, etc.:

1. The recovery of selenium dioxide in water - se acid (H2SeO3or its salts using sodium borohydride in aqueous solution galactosidase polysaccharide at room temperature with the formation of selenium atoms in the zero oxidation state in accordance with the scheme:

8H2SeO3+4NaBH4=8Se0+Na 2B4O7+2NaOH+15H2O

The recovery of selenium to the zero-valence state and the subsequent formation of selenium nanoparticles occurs in the matrix galactosidase polysaccharide causing hepatotropic properties of the resulting nanocomposite, as well as acting as a stabilizer of the nanoparticles at the time of formation of the nanocomposite. Isolation and purification from impurities prepared nanocomposite is produced by planting in a fourfold excess water-miscible organic solvent (acetone, ethanol, etc.) followed by filtration and drying.

2. The recovery of commercial elemental selenium (in the form of a powder, powder, granules or blocks) to polysilane - and selenide anions hydrazine hydrate in an alkaline environment. When this occurs the dissolution of elemental selenium and education vysokoreaktsionnosposobnyh selenide anions according to the equation:

2Se+4NaOH+N2H4·H2O=2Na2Se+N2+5H2O

[E. N. Derjaguin, N. In. Resavska, L. K. Paperna, E. P. Levanova, E. N. Sukhomazova, N. And. Carcavi. "Synthesis halogenorganics compounds in the primary and recovery systems // Izvestiya an. Chemical Bulletin, 2005, No. 11. S. 2395-2405.]

Generated selenide anions are characterized by limited long-term stability, which determines the necessity of their obtaining �neposredstvenno before the synthesis of selenium nanoparticles. Then to the resulting solution containing the selenide anions, was added an aqueous solution galactosidase polysaccharide and oxidized by blowing oxygen at room temperature, which ensures the formation of selenium atoms in the zero oxidation state, and the polysaccharide acts as a stabilizing ligand for forming nanoparticles Se0. Isolation and purification from impurities prepared nanocomposite also occurs by planting in a fourfold excess water-miscible organic solvent (acetone, ethanol, etc.) followed by filtration and drying.

3. Oxidation of organiltsinkgalogenidov ammonium, alkali and alkaline earth metals hydrogen peroxide, which takes place in accordance with the scheme:

M[Se2PR2]n+2H2O2=2Se0+M[O2PR2]n+2H2O

M = a cation of ammonium or alkali or alkaline earth metal, n=1, 2.

R = alkyl-, aryl-, hetaryl-, arylalkyl, getariako-

The interaction occurs in aqueous solutions of polysaccharides, followed by the formation of selenium nanoparticles and their simultaneous stabilization of polysaccharide matrix at a temperature of 35-40°C. isolation and purification from impurities prepared nanocomposite is also by planting in a fourfold excess water-miscible orga�quarter of solvent (acetone, ethanol, etc.) followed by filtration and drying.

Depending on the production method has, the size of the generated nanoparticles is 1-100 nm, which was determined by transmission electron microscopy, as well as regenerative analysis.

Recomposit selenium-based natural galactosidase hepatotropic polysaccharide is a hybrid organic-inorganic materials as inorganic phases which are nanoscale particles of elemental selenium in the amount of 0.5-60% and a size of 1-100 nm, and the organic phase is natural galactosidase polysaccharides with hepatotropic activity: arabinogalactan, galactomannan, carrageenan and others.

Regardless of the method of producing nanoparticles of selenium, Se allocation0-containing nanocomposite is carried out by placing the reaction mixture in a fourfold excess water-miscible organic solvent (acetone, ethanol, etc.). When this occurs the formation of precipitates selenium-containing nanocomposite due to the insolubility in an organic solvent of high molecular weight galactosidase polysaccharide (arabinogalactan, galactomannan, carrageenan, etc.), which is the main phase of the nanocomposite. All other organic and inorganic precursors, reducing agents and products �x transformations are of low molecular weight and therefore dissolve in water-organic mixtures and are easily separated from the nanocomposite in the process of further filtration and washed on the filter. Completes the process of obtaining their nanocomposites by simple air drying. The obtained nanocomposites are water-soluble powder orange-red color, with selenium from 0.5 to 60%.

Antioxidant-based nanoparticles and selenium galactosidase polysaccharide is prepared by dissolving the obtained selenium-containing nanocomposites in distilled water. The ratio of components in the received tool varies in the following ranges (wt.%)

selenium-containing nanocompositeIs 0.0001 to 1.0
distilled waterelse
the content of selenium0.0457-0.0913 mg / kg of animal weight, or 3.0 mg once daily dose human

The following examples of the preparation of a drug for the treatment and prevention of redox-associated pathologies (in particular, toxic liver damage), and study of their antioxidant activity, illustrate the invention.

Example 1. In the reaction flask are placed 0.5 g galactosidases of the polysaccharide carrageenan, add 50 g of water and the mixture was heated with constant stirring on a magnetic stirrer until the temperature� 40°C until complete dissolution of the polysaccharide, then the solution was cooled to room temperature and poured 5 ml of an aqueous solution containing 0.0037 g of selenium dioxide, or 0.0042 g se acid, or 0.0043 g of sodium Selenite. The mixture was stirred for 5 minutes, then add 0.0050 g of sodium borohydride. After 15 minutes, make the allocation Se0-containing nanocomposite deposition reaction mixture in a four-fold excess of acetone or ethanol, followed by filtration, washing on the filter with the same solvent, and then air drying. The obtained nanocomposite is a water-soluble powder orange-red color, with a mass content of selenium 0.5% and polysaccharide 99.5%. The output of the nanocomposite is 97% (in terms of selenium from its precursor).

The quantitative content of selenium in the resulting nanocomposite was determined by elemental analysis, as well as regino-energy-dispersive microanalysis. Received Se0-containing nanocomposites were analyzed by transmission electron microscopy and x-ray diffraction analysis. The presence of all the obtained nanocomposites nano-sized particles of elemental selenium, identified by the method of x-ray diffraction analysis (Bruker D8 ADVANCE Diffractometer), broadened by the reflexes in the region of 21.6°; 29.7°; and 42° 2θ, which correspond to (110), (101) and (200) plane�the spines of the crystal lattice of nanoscale elemental selenium. Spherical morphology of nanosized particles of selenium in the composition of nanocomposites on the basis of arabinogalactan, galactomannan and carrageenan established on the basis of transmission electron microscopy.

Example 2. Carried out analogously to example 1, but take increased 120 times the amount of selenium dioxide, or se acid, or sodium Selenite and sodium borohydride. The output of the nanocomposite is 99% (in terms of selenium from its precursor). The resulting materials are water-soluble powder orange-red color, with selenium 60% and polysaccharide 40%. The quantitative content of selenium in the resulting nanocomposite, the nature and morphology of the obtained nanocrystals selenium has been proven as in example 1.

Example 3. In the reaction flask are placed 0.5 g galactosidases of of polysaccharide galactomannan, add 50 g of water and the mixture was heated with constant stirring on a magnetic stirrer to a temperature of 40°C until complete dissolution of the polysaccharide, and then the solution was cooled to room temperature and add 5 ml of a solution containing 0.022 g of selenium dioxide, or 0.025 g se acid, or 0.026 g of sodium Selenite. The mixture was stirred for 5 minutes, then add 0.029 g of sodium borohydride. After 15 minutes, make the allocation Se0-containing nanocomposite (Se0 -GM) by precipitation of the reaction mixture in a four-fold excess of acetone or ethanol, followed by washing on the filter with the same solvent, filtering and drying in air. The output of the nanocomposite is 98% (in terms of selenium from its precursor). The resulting materials are water-soluble powder orange-red color, with selenium 3%, of polysaccharide 97%. The quantitative content of selenium in the resulting nanocomposite, the nature and morphology of the obtained nanocrystals selenium has been proven as in example 1.

Example 4. Carried out analogously to example 3, but take increased 20 times the amount of selenium dioxide, or se acid, or sodium Selenite and sodium borohydride. The output of the nanocomposite is 99% (in terms of selenium from its precursor). The resulting materials are water-soluble powder orange-red color, with selenium 60% and polysaccharide 40%. The quantitative content of selenium in the resulting nanocomposite, the nature and morphology of the obtained nanocrystals selenium has been proven as in example 1.

Example 5. In the reaction flask were placed 1.0 g of polysaccharide arabinogalactan and 0.08 g of organlzational or ammonium, or alkali or alkaline earth metal, then add 50 g of water and stirred on a magnetic stirrer until complete dissolution of the poly�of Farida. After which the solution was further incubated for 3 h at 35-40°C and add concentrated hydrogen peroxide (Perhydrol). The allocation of Se0-containing nanocomposite and cleaning nanocomposite formed from diphenylphosphinite is carried out by pouring the reaction mixture in a four-fold excess of acetone or ethanol, followed by washing on the filter with the same solvent, filtering the precipitate and drying in air. The output of the nanocomposite is 97% (in terms of selenium from its precursor). The resulting materials are water-soluble powder orange-red color, with selenium 2%, and the polysaccharide 98%. The quantitative content of selenium in the resulting nanocomposite, the nature and morphology of the obtained nanocrystals selenium has been proven as in example 1.

Example 6. In a three-neck reaction flask equipped with reflux condenser, thermometer and a hole for entry of the reactants, are placed 0.224 g of sodium hydroxide, add 0.5 g of hydrazine hydrate and the mixture was heated with constant stirring on a magnetic stirrer to a temperature of 70°C, then added 0.176 g or powdered, or granulopenia, or in the form of fragmented pieces of the elemental selenium. The mixture was stirred at the same temperature for 1.5 hours and cooled to room temperature, thus there is RA�creation of elemental selenium and education vysokoreaktsionnosposobnyh selenide anions.

Next, an aliquot of the resulting solution with a volume of 0.1 ml, bring to room temperature and vigorous stirring in a 1% aqueous solution galactosidases of the polysaccharide carrageenan, constantly barbotine air through the mixture for the selective oxidation of the selenide anions to atomic elemental selenium, followed by condensation to nanoparticles. Thus galactosidase polysaccharide carrageenan acts as a stabilizing ligand for forming nanoparticles Se0. After 15 minutes, make the allocation Se0-containing nanocomposite (Se0KG) deposition of the reaction mixture in a four-fold excess of acetone or ethanol, followed by washing on the filter with the same solvent, filtering and drying in air. The output of the nanocomposite is 98% (in terms of selenium from its precursor). The resulting materials are water-soluble powder orange-red color, with selenium 6%, of polysaccharide 94%. The quantitative content of selenium in the resulting nanocomposite, the nature and morphology of the obtained nanocrystals selenium has been proven as in example 1.

Example 7. Carried out analogously to example 6, but as a stabilizing ligand used galactosidase polysaccharide galactomannan. The output of the nanocomposite is 97% (in terms of selenium from its pre�of urara). The resulting materials are water-soluble powder orange-red color, with selenium 6%, of polysaccharide 94%. The quantitative content of selenium in the resulting nanocomposite, the nature and morphology of the obtained nanocrystals selenium has been proven as in example 1.

Example 8. Carried out analogously to example 6, but as a stabilizing ligand used galactosidase polysaccharide arabinogalactan. The output of the nanocomposite is 99% (in terms of selenium from its precursor). The resulting materials are water-soluble powder orange-red color, with selenium 6%, of polysaccharide 94%. The quantitative content of selenium in the resulting nanocomposite, the nature and morphology of the obtained nanocrystals selenium has been proven as in example 1.

Obtained Se0-containing nanocomposites prepared with antioxidant drugs for the treatment of redox-related pathologies. This is illustrated by the following examples.

Example 9.

Antioxidant with hepatoprotective effect on the basis of selenium nanoparticles and galactosidases of arabinogalactan polysaccharide or galactomannan or carrageenan and other polysaccharides prepared by dissolving the obtained selenium-containing nanocomposites in distilled water at room temperature. Tool performance�possessing a homogeneous clear solution light orange color, aggregately stable over a long period of time. The ratio of components in the received tool varies in the following ranges (wt.%).

selenium nanocomposite0.0001 to 1.0
distilled waterelse

In the preparation of the funds originally come from the selenium content in the dry nanocomposite, certain elemental analysis, x-ray energy-dispersive microanalysis, and other sensitive for the determination of selenium species analysis. Then calculate the ratio of the nanocomposite and water so that in the prepared antioxidant tool, the selenium concentration was close to the effective dose of selenium in sodium Selenite used for the prevention and treatment caused by selenium deficiency in the body white muscle disease (0.1-0.2 mg Na2SeO31 kg weight of the animal, or 0.0457-0.0913 mg of pure selenium per 1 kg of animal weight, or 3.0 mg once daily dose).

Example 10.

Determination of antioxidant activity in vitro.

For the assessment of antioxidant activity (AOA) of selenium nanocomposite with arabinogalactan used a spectrophotometric method in which� radical cation 2,2'-azinobis(3-eleventyseven-6-sulfonic acid) diammonium salt (ABTS) having a characteristic absorption in the region 600-850 nm, generated by potassium persulfate and measured the percentage of inhibition of the absorption of added antioxidants (Re R., Pellegrini N., Proteggente A., Pannala A., Yang M., Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation decolorization ASSAY // Free Radical Biology & Medicine, Vol.26, Nos. 9/10, pp.1231-1237, 1999)

The technique of definition. Antioxidant activity was determined by the method described in the work (Ilyasov R. I., Beloborodov V. L., Tyukavkina N. And., Verovskom E. V. Application of the radical cation ABTS·+in the evaluation of the antiradical activity of flavonoids. // Farmacia, 2008, No. 6, Pp. 15-18). To determine the AOA used a clean solution of arabinogalactan and its nanocomposites with selenium concentrations of 1.9% and 3% in distilled water. Solutions of antioxidants: ascorbic acid in water and quercetin in ethanol were prepared with concentrations of 0.002 mol/l and 0,00057 mol/l, respectively.

The percentage of inhibition was calculated by the formula:

% inhib. = 100·(1-A2/A1),

where A1- the optical density of a solution of ABTS·+at a wavelength of 730 nm without addition of the test sample; A2- the optical density of a solution of ABTS·+after 4 minutes after addition of the test sample.

On the basis of the results obtained were building plots of percentage inhibition against the concentration of antioxidant and determination�Yali IC50 - the concentration at which neutralized 50% of the radical-cations, also counted the most versatile relative characterization of ARA - teas - equivalent antioxidant activity in terms of trolox, whose activity is taken as 1. Data for ARA of the investigated compounds are presented in table 1.

Table 1.
Characterization of the antiradical activity of the test compounds to their molar concentrations
ConnectionInhibiting concentration IC50, µmol/lTeamIC50
ArabinogalactanHas noHas no
Nanocomposite of arabinogalactan and selenium18±2.90.91
s=2.5; n=6
The standard (ascorbic acid)16.1±1.10.98
s=0.96; n=5

The reliability of the obtained IC50 values were evaluated by the root mean square error (s) and confidence interval CP�Diego value (Δx), calculated for n measurements with 95% probability using equations (K. Doerffel, Statistics in Analytical Chemistry, Deut. Verlag Grundstoffind., Leipzig (1966).).

As the table shows, the values of the ARA standard antioxidant (ascorbic acid) coincide with the literature, therefore the methodology used to assess ARA can be considered adequate. The tabular data indicate that ARA obtained nanocomposite selenium with arabinogalactan is comparable with ascorbic acid. Since the experiments showed that the source arabinogalactan not able to inhibit ABTS·+at considerably higher concentrations than the nanocomposite based on it, you can believe that ARA is dened by the presence of Se.

The values of antioxidant activity of Se0-containing nanocomposites based on galactomannan and carrageenan in vitro, which was determined by us in a similar way, was the same as for Se0-containing nanocomposite on the basis of arabinogalactan.

Example 11.

Determination of antioxidant activity in vivo.

To determine the antioxidant activity of the preparation used healthy rats, weighing 180-220 g. the study was performed according to the "Manual on experimental (preclinical) study of new pharmacological substances" under the General editorship of corresponding member of RAMS, Professor R. W. Habre�VA (Publishing house "Medicine", 2005). Study drug was administered to animals orally by gavage at a dose of 2 µg/100 g animal weight (based on the information about the content of selenium and effective doses of sodium Selenite). The results of the study on the content of products of lipid peroxidation (LPO) and antioxidant activity (AOA) are shown in table 2.

Table 2.
The content of LPO and AOA
The timing of specimen collectionThe experimental group of animalsStudied parameters serum
Double bond (CONV. u)Diene conjugates (µm/l)Conjugated dienes Zobrazenie triene (us. units)Malonic dialdehyde (µm/l)Antioxidant activity (us. units)
7 daysNano-Se1.300±0.0921.950±0.1290.786±0.1612.290±0.287* 20.26±1.664
AG0.776±0.1452.300±0.2331.043±0.1362.960±0.141se♦13.50±1.726
CCl40.378±0.0813.424±0.6312.425±0.3503.575±0.5129.020±1.630
intact1.613±0.248Is 1.326±0.1550.770±0.1071.763±0.22919.90±1.784
14 daysNano-Se1.560±0.1441.753±0.2590.793±0.1592.426±0.33718.43±0.860•♦
AG0.586±0.201•♦2.616±0.2801.200±0.2213.156±0.26111.43±1.368
CCl40.374±0.0293.631±0.3852.370±0.2214.092±0.393 inch7706±1.746
intact1.430±0.0811.256±0.1480.466±0.1171.886±0.21719.96±3.442
21 daysNano-Se1.093±0.1231.257±0.062*•♦0.670±0.1471.938±0.24720.22±1.616
AG0.755±0.1292.110±0.304se1.340±0.2373.325±0.48112.18±2.242
CCl40.473±0.0782.993±0.3131.898±0.1893.204±0.4048.702±0.853
intact1.440±0.2061.460±0,2270.553±0.1822.100±0.25420.00±1.363
Note:+•- p<0.05 in comparison with intact animals; * - p<0.05 in comparison with the group of arabinogalactan; ♦ - p<0.05 in comparison with group CCl4.

DC is nontoxic and hormone-free�hydrated intermediate product FLOOR, and upon further oxidation DK form MDA, which has a negative impact on the body, known as redox-associated lesions. During all periods of the experiment, the content of intermediate products (DC) in the group of Nano-Se is lower than in the comparison groups, and getting close to the group of intact animals. Consequently, the formation of MDA from DC was significantly lower than in other groups. This figure as AOA - indicator protect the body from free radicals and reactive oxygen species - has always remained at the same level in the group of Nano-Se and even exceeded that of intact animals, while in the groups with hypertension and CCl4has been greatly reduced on all days of the experiment.

The activity of selenium-containing enzymes reduced (GSH) and oxidized (GSSG) glutathione, and superoxide dismutase (SOD) are shown in table 3.

Table 3.
The activity of selenium-containing enzymes reduced and oxidized glutathione and superoxide dismutase in serum
The timing of specimen collectionThe experimental group of animalsThe investigated parameter�
ODS (us.u)GSH (mm/l)GSSG (mm/l)
7 daysNanoSe1.620±0.084*1.826±0.069*•1.380±0.105*
AG1.343±0.040se♦1.144±0.177se♦1.380±0.032se♦
CCl41.078±0.1301.144±0.0861.233±0.099
intact1.637±0.0712.013±0.1721.436±0.059
14 daysNano-Se1.730±0.0971.940±1.1301.686±0.092
AG1.390±0.0831.276±0.153•♦1.473±0.056
CCl41.100±0.0911.093±0.0771.204±0.069
intact1.606±0.1182.026±0.062 1.660±0.093
21 daysNanoSe1.705±0.069*1.790±0.641*•♦1.548±0.158
AG1.362±0.166se♦1.235±0.086se1.290±0.106
CCl41.213±0.0671.160±0.0641.014±0.083
intact1.650±0.1022.030±0.0651.700±0.073
Note:- p<0.05 in comparison with intact animals; * - p<0.05 in comparison with the group of arabinogalactan; ♦ - p<0.05 in comparison with group CCl4

Superoxide dismutase (SOD) - an enzyme is a measure of protection of cell membranes from the damaging effects of free radicals generated during activation of the FLOOR is quite high in the group of Nano-Se. Also a slight increase in SOD activity is a compensatory mechanism for the protection of cells from oxidative stress (Gerasimenko M. N. Titova N. M. Zukov R. A. et al. lipid Peroxidation and antioxidant system in erythrocytes, pain�'s kidney cancer. / M. N. Gerasimenko, N. M. Titov, R. A. Zhukov, J. A. Diano, E. S. Overflow // Receipt. experiment, Biol. and honey. - 2012. - No. 3. - P. 693).

Selenium-containing enzyme glutathione reduced (GSH) is the major intracellular antioxidant (Pompella A., A. Visvikis, A. Paolicchi, De Tota V., Casini, A. F. 2003. The changing faces of glutathione, a cellular protagonist. Biochem. Phermacol. 66: 1499-1503.) and its activity depends on the presence in the body of the elemental selenium and vitamin E. In the same cells glutathione restores any disulfide bond. While GSH is converted to GSSG. The content of GSH in the group of Nano-Se is quite high and is approaching the performance of the group of intact animals, indicating a high level of antioxidant protection of the cell during all periods of the experiment.

The change in the concentration of fat-soluble vitamins, which are components of total antioxidant activity and may play a critical role in the homeostasis of viscous properties of the membranes are given in table 4.

α-tocopherol (µm/l)
Table 4.
The content of fat-soluble vitamins in serum
The timing of specimen collectionThe experimental group of animalsStudied parameters
retinol (µm/l)
7 daysNano-Se9.000±0.746*0.83±0.159
AG11.886±1.717se1.563±0.225
CCl45.208±1.0840.454±0.127
intact7.010±1.0200.776±0.085
14 daysNano-Se9.456±1.7370.930±0.152
AG14.403±1.2412.016±0.087
CCl45.554±1.1280.374±0.103
intact8.420±1.6981.040±0.220
21 daysNano-Se7.215±1.0821.185±0.163
AG14.440±1.916•♦1.362±0.148
CCl 46.569±0.8540.600±0.100
intact11.230±0.7911.140±0.355
Note:- p<0.05 in comparison with intact animals; * - p<0.05 in comparison with the group of arabinogalactan; ♦ - p<0.05 in comparison with the group of CL4

Thus, the study of a new nanocomposite preparation of selenium in vitro and in vivo showed his expressed antiradical properties and vitaminenergy effect, which is manifested in the hepatoprotective effect in toxic liver damage.

The present invention compared with other known technical solutions have the following advantages:

- directed mechanism of action;

- prevention of the onset of oxidative stress.

- the reduction of toxic action on the organism of selenium;

- high digestibility and availability in the body;

- easy and atraumatic insertion.

1. The selenium nanocomposites based on natural hepatotropic galactosaemia polysaccharide matrices representing a water-soluble powders orange-red color, containing nanoparticles of zero-valent selenium (Se0) with a particle size of 1-100 nm with the quantitative content of 0.5-60 wt., possess antioxidant activity, for the treatment and prevention of redox-related pathologies.

2. The selenium nanocomposites based on natural hepatotropic galactosaemia polysaccharide matrices representing a water-soluble powders according to claim 1, possessing antioxidant activity, for the treatment of toxic liver damage.

3. A method of producing a nanocomposite according to claim 1, characterized in that they are derived from selenium-containing raw materials directly in aqueous solution galactosaemia polysaccharides, which are both stabilizers formed of selenium nanoparticles and the allocation of nanocomposite powders is carried out by planting in water-miscible organic solvent followed by filtration and drying.

4. Method of producing nanocomposite according to claim 3, characterized in that it is produced by reduction of an aqueous solution of selenium dioxide, or se acid or its salts using sodium borohydride in aqueous solution galactosidase polysaccharide.

5. Method of producing nanocomposite according to claim 3, characterized in that the first aqueous solutions selenide - and policeline anions recovery of powder, granules or large chunks of elemental selenium hydrazine hydrate in aqueous-alkaline medium, and then the resulting selenide anions oxidize in aqueous solution �lactobacteria polysaccharides, blowing air at room temperature.

6. Method of producing nanocomposite according to claim 3, characterized in that it is obtained by oxidation of organiltsinkgalogenidov either ammonium or alkali metals or alkaline earth metals by hydrogen peroxide at room temperature or heated in aqueous solutions galactosaemia polysaccharides.

7. Antioxidant for the prevention and treatment of redox-dependent pathologies of humans and animals, characterized in that as an active pharmacologically active substances contains nanocomposite according to claim 1 and water, purified with the high ratio of these components to be prepared in the antioxidant agent concentration of selenium contained in an effective therapeutic dose 0.0457-0.0913 mg of pure selenium per 1 kg weight of the animal or 3.0 mg once daily dose of a person.



 

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2 cl, 1 tbl, 4 ex

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FIELD: chemistry.

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2 cl, 3 dwg, 2 tbl, 5 ex

FIELD: chemistry.

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7 cl, 3 dwg

FIELD: medicine.

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EFFECT: invention provides simplifying and accelerating the process of nanoencapsulation in xanthane gum and higher weight yield.

2 ex

FIELD: medicine.

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EFFECT: high effectiveness of the chelation process and low toxicity of Mexidol that makes it promising for biomedical application.

4 dwg, 3 tbl, 3 ex

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