RU2519760C1 - Antioxidant and method for preparing it - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns an antioxidant representing the amino acid glycine immobilised on the detonation-synthesised nanodiamond particles of 2-10 nm in size.

EFFECT: higher efficacy.

4 cl, 5 dwg, 7 tbl, 3 ex

Description

The invention relates to medicine, in particular to pharmacology, and relates to an antioxidant, which is glycine immobilized on particles of detonation nanodiamonds.

Currently, it is considered proven that the emergence and development of a wide range of pathological processes in the body is accompanied or caused by the activation of free radical lipid peroxidation (LPO) reactions, protein and nucleic acid denaturation. Free radicals formed in the cell can initiate secondary free radical reactions, interacting with various cellular components: proteins, nucleic acids and lipids [1].

The participation of free radicals in the pathogenesis of many diseases (shock of various origins, atherosclerosis, cerebral, coronary and peripheral circulation disorders; diabetes mellitus and diabetic angiopathy; rheumatoid, inflammatory and degenerative diseases of the musculoskeletal system; eye damage; pulmonary diseases; oncological pathology; thermal lesions; various intoxications; reperfusion lesions) and premature aging. In addition, drugs with prooxidant properties, a number of treatment procedures (oxygen therapy, hyperbaric oxygenation, ultraviolet irradiation, laser vision correction, radiation therapy), and various environmentally unfavorable environmental factors lead to increased formation of free radicals in the body [2]. Substances that inhibit free radical oxidation are called antioxidants. Their medicinal effect is realized either by direct binding of free radicals - this is how direct antioxidants act, or through activation of the body's antioxidant system (a group of indirect antioxidants). The action of antioxidants has a number of features: in therapeutic doses, they do not cause physiological or biochemical changes in a healthy body; their action is not specific, manifests itself in the most pathological processes. In the USSR, the first effective antioxidant Mexidol was created in the early 1980s and successfully introduced into medical practice. To date, Mexidol is one of the most used antioxidant drugs [3].

It is known that the replaceable amino acid glycine (NH ₂ CH ₂ COOH), being the central neurotransmitter of the inhibitory type of action, exhibits sedative activity and improves metabolic processes in brain tissues [1, p. 661]. In modern therapeutic practice, glycine is used as a drug that reduces the craving for alcohol, reduces withdrawal symptoms, depressive disorders, increased irritability, normalizes sleep, and also in the complex treatment of cerebrovascular disorders [1]. The pharmacological action of glycine is based on the effect of amplification of metabolic and neurotransmitter processes that occur due to an increase in its endogenous synthesis. It is possible to increase the intracellular synthesis of glycine only using signal transmission pathways due to interaction with receptor systems. Its interaction with glycine receptors leads to the opening of chlorine channels, hyperpolarization of the membrane and the spread of inhibition. Along with this, glycine is able to act as an allosteric coagonist of glutamate receptors. By binding at a specific site, it enhances the ability of glutamate and N-methyl-D-asparagus (NMDA) to open the cation channel [4, 5].

The pharmacopoeial glycine used is prescribed in the form of tablets (0.1 g each) under the tongue 3-4 times a day.

In [6], the effect of glycine on lipid peroxidation and the antioxidant system of the body was studied. It was shown that glycine reduces the quantitative content of lipid peroxidation products and increases the activity of antioxidant enzymes. But the research results were statistically unreliable. The antioxidant effect of glycine on lipid peroxidation was confirmed in [7]. The antioxidant properties of glycine are shown in models of ischemic stroke and alcohol intoxication [8-10].

Known glycine immobilized on particles of detonation nanodiamonds with a size of 2-10 nm, used as a binder component in polymer composites [11, 12]. The method of obtaining it is as follows [12]. A portion of the nanodiamond is placed in a constant helium current reactor and annealed at a temperature of 150-470 ° C for 3-4 hours. Then, fluorination of the nanodiamond samples is carried out at a temperature of 50-500 ° C for 1-24 hours by contacting with a mixture of gaseous fluorine and hydrogen . To obtain glycine immobilized on nanodiamond particles, fluorinated nanodiamonds are sonicated in o-dichlorobenzene for 20-30 minutes, glycine ethyl ester hydrochloride (NH ₂ CH ₂ COOCH ₂ CH ₃ · HCl) and a few drops of pyridine are added. The resulting mixture was stirred at a temperature of 130-140 ° C for 8-12 hours. The resulting product was filtered, washed with ethanol and dried under vacuum at 70 ° C.

An additional characteristic of this substance is the size of its particles in suspension, equal, according to dynamic light scattering (DLS), to 310 nm [12].

A feature of this substance is the presence on the surface of nanodiamond particles, in addition to glycine molecules, also fluorine atoms. Although the authors declare their number to be less than 1% at., In reality it has been experimentally established that the fluorine concentration on the surface of a nanodiamond can reach 14% at. and more. This is due to the fact that the CF bond (E _st = 115 kcal / g-atom) is strong and the fluorine derivatives of carbon are inert with respect to many substances. Therefore, during the chemical immobilization of glycine on the surface of a nanodiamond containing fluorine atoms, glycine molecules are replaced by fluorine atoms only partially. Moreover, it is known that the presence of fluorine and its derivatives in organic matter increases its toxicity and can change the indices of the microsomal xenobiotic biotransformation system in the liver [13]. Thus, the presence of fluorine atoms in the nearest nanostructured analog of nanodiamond — fullerene (C ₆₀ ) increases its general toxicity by 2.4–5 times [14]. In addition, fluorine and its compounds are able to accumulate in various environmental objects and be present in various amounts [15]. Therefore, glycine immobilized on nanodiamond particles that contain fluorine atoms is undesirable in medical practice as a medicine.

The use of glycine immobilized on detonation nanodiamond particles as an antioxidant is not described in the scientific and patent literature.

Therefore, obtaining an antioxidant, which is glycine, immobilized on particles of detonation nanodiamonds that do not contain fluorine atoms, with increased dispersion in a suspension used as a medicine, as well as reducing the level of environmental and endoecological danger, simplifying and cheapening the method of producing antioxidant, are relevant and practically significant task.

The purpose and objective of the invention is to expand the arsenal of drugs with antioxidant and antiradical properties without the manifestation of any side effects and toxic effects.

The problem is solved by using the antioxidant described in accordance with the invention, which is glycine immobilized on detonation nanodiamond particles 2-10 nm in size, not containing fluorine atoms and having a shell thickness of up to 1 nm, with glycine content of up to 21 ± 3% by weight. , and how to obtain it.

The described antioxidant in the form of glycine immobilized on particles of detonation nanodiamonds that do not contain fluorine atoms on its surface is an ultrafine powder (Figure 1) of dark gray or dark gray color with a greenish or dark blue shade with a particle size of 2 up to 10 nm, having a shell with a thickness of up to 1 nm (Figure 2), the size of the aggregates in an aqueous suspension of up to 100 nm (Figure 3) and a glycine content of up to 21 ± 3% by weight, which is part of the surface shell.

Figure 1 clearly shows the presence of the described antioxidant ultrafine structure of particles with a size less than the resolution of the used device (from 20 nm).

A micrograph of the particles of the described antioxidant was obtained on a Zeiss Ultra Plus field emission scanning electron microscope (Carl Zeiss, Germany).

Figure 2 shows that the particle size of the described antioxidant, coated with a thickness of up to 1 nm, is 2-10 nm.

A micrograph of the particles of the described antioxidant was obtained on a Jeol 1011 transmission electron microscope (JEOL, Japan).

Figure 3 shows the particle size distribution curve in the suspension of the described antioxidant, from which it follows that the particle sizes in the suspension do not exceed 100 nm.

The particle size distribution of the described antioxidant in suspension was measured by the DLS method on a ZetaSizer instrument (Malvern Instruments, USA). The abscissa represents the logarithmic scale of particle size in nm. The ordinate axis is the percentage of particles with specific sizes.

The elemental composition of the particle surface of the described antioxidant according to X-ray photoelectron spectroscopy (XPS) is given in Table 1.

Table 1 The elemental composition and binding energies of the surface atoms of the described antioxidant. Name of characteristic Chemical elements C O N Atomic% 77.5-94.5 4-14 1,5-8,5 Communication energies, eV 285.2 ± 0.5 530.7 ± 0.5 399.8 ± 0.5

The surface of the described antioxidant was studied on a LAS-3000 instrument (Riber, France) equipped with an OPX-150 hemispherical analyzer. To excite photoelectrons, nonmonochromatized x-ray radiation of an aluminum anode (AlK _α = 1486.6 eV) was used at a tube voltage of 12 kV and an emission current of 20 mA. The photoelectron peaks were calibrated using the C1s carbon line with a binding energy of 285 eV. The vacuum in the working chamber was 6.7 · 10 ^-8 Pa. To obtain a high vacuum, an ion pump was used.

The amount of glycine in the described antioxidant is determined as follows. Mixtures of nanodiamonds with different glycine contents are prepared. Take samples of each mixture of equal weight. Their IR spectra are recorded, the most intense characteristic signals are selected on them, which are correlated with the IR spectrum bands of the initial glycine. Then build calibration curves of the dependence of the signal intensity in the IR spectrum on the glycine content in the sample. Further, the quantitative content of glycine in it is determined from the intensity of the selected characteristic bands of the studied antioxidant from the calibration curves. According to the data obtained, the average value of the glycine content in the described antioxidant is determined.

The described method of producing an antioxidant is as follows. Detonation nanodiamonds are annealed in a stream of hydrogen gas at a temperature of 500-1200 ° C for 1-8 hours, then subjected to liquid-phase chlorination with molecular chlorine by photochemical treatment with visible light at a temperature of 50-70 ° C for 36-60 hours, followed by washing with carbon tetrachloride by centrifugation and drying under vacuum. Chlorine-modified nanodiamonds are dissolved in a polar solvent to form a suspension. Tertiary amine and glycine are added and the resulting mixture is sonicated for 5-60 minutes, followed by aging at 50-80 ° C for 12-48 hours, centrifugation, washing with solvent and drying. Ultrasound treatment is carried out for 5-60 minutes, triethylamine is used as a tertiary amine and pyridine, a lower aliphatic alcohol, a water-alcohol mixture or water are used as a polar solvent.

The described antioxidant does not contain fluorine atoms (Table 1), as well as atoms of other halogens in an amount exceeding the error of the device (0.1% at.), Since in the process of obtaining the antioxidant all chlorine atoms are replaced by glycine molecules and leave the surface of the nanodiamond in the form of HCl molecules.

A study of the antioxidant activity of the preparations was carried out in an in vitro system with the assessment of lipid peroxidation (lipid peroxidation) by the levels of diene conjugates and malondialdehyde and antiradical activity in the chemiluminescent system, consisting of unsubstituted 3-hydroxypyridine, horseradish root peroxidase and hydrogen peroxide.

For this, we used the method for preparing phospholipid liposomes [16–18], the method for determining the products of lipid peroxidation, the method for determining diene conjugates (DC), the method for determining malonic aldehyde (MDA), and the method of chemiluminescence [19].

The described antioxidant was used in various concentrations in the range from 10 μg / ml to 100 μg / ml. Pharmacopeia glycine and mexidol were used as reference substances.

LPO products were measured on a DU-50 spectrophotometer (Beckman, USA) in 10-mm semi-microcuvettes (Opton, Germany). The kinetics and intensity of chemiluminescence was determined on a luminometer with a digital display and a 500 μl initiator injection system (model 1250, LKB, Sweden) and a two-channel recorder (model BD-41, Kipp & Zonen, Germany).

It was found that the described antioxidant at a concentration of 10 μg / ml caused a significant decrease (2 times) in the level of MDA. At a concentration of 50 μg / ml, the described antioxidant statistically significantly reduced the level of DC by 2.4 times, and the level of MDA by 2 times (Table 3). At a concentration of 100 μg / ml, the effect of the described antioxidant was enhanced: under the influence of a substance, the DC level decreased by 5.8 times, and the MDA level - by 3.1 times (Table 3).

It was shown that glycine and mexidol dose-dependently inhibit lipid peroxidation, decreasing both the level of diene conjugates and malondialdehyde (Table 3), which corresponds to the data obtained previously [20].

Thus, the described antioxidant has a clear effect on the intensity of lipid peroxidation (LP) in the suspension of model liposome membranes, which is reflected in a statistically significant decrease in the levels of diene conjugates and malondialdehyde. The LPO-inhibiting effect of the described antioxidant is dose-dependent and increases with increasing concentration of the substance. According to the severity of LPO inhibition, the described antioxidant has similar activity with pharmacopoeial glycine. According to the severity of inhibition of lipid peroxidation in some respects, the antioxidant described is superior to the known LPO inhibitor - mexidol.

It was shown that the described antioxidant in the concentration range of 20-100 μg / ml has a distinct antiradical activity, significantly inhibiting chemiluminescence in the 3-hydroxypyridine-peroxidase-hydrogen peroxide system, and exceeds pharmacopoeial glycine and the drug mexidol in this indicator (Table 4). So, at a concentration of 100 μg / ml, the described antioxidant reduced quenching of chemiluminescence by 25.75 times.

The acute toxicity study of the described antioxidant was carried out according to the guidelines for the study of acute toxicity described in [21].

During the experiment, the following indicators were recorded: the nature of the coat, changes in the condition of the mucous membranes, ptosis of the upper eyelid, increased urination, increased bowel movements, increased salivation, piloeraction, vocalization, lateral position, rhythm and depth of respiratory movements, aggressiveness, timidity, tremor, convulsions, change in thresholds of pain reaction, change in posture, catalepsy, impaired coordination of movements in the test of a rotating rod, holding for 5 seconds on an inverted mesh platform, erelezanie with inverted mesh platform up, the presence of pineal, corneal reflex, sedation, stereotypes and grooming, the death of the animal.

Statistical processing of the results was carried out using statistical packages "BioStat" for Windows. Group averages and standard error indicators were calculated.

The obtained results convincingly show that the described antioxidant when administered intraperitoneally to mice at doses of 75, 150 and 225 mg / kg, as well as pharmacopeia glycine, does not cause signs of intoxication and death of animals during the 14 days of observation.

It is known that in stroke, as in other diseases accompanied by neurodegeneration, activation of free radical reactions (CPP) of lipid peroxidation (LPO) is observed [22, 23], which is a pathogenetic justification for using various natural or synthetic CPP inhibitors in the clinic as antioxidant therapy drugs (mexidol, α-tocopherol, carotenoids, flavonoids, probucol, etc.) [8, 9].

The data obtained indicate that the described antioxidant in the concentration range from 20 to 100 μg / ml has a clear inhibitory effect on the intensity of lipid peroxidation (LPO) in the suspension of liposome model membranes, which is expressed in a statistically significant decrease in the levels of diene conjugates and malondialdehyde, and has a distinct antiradical activity, inhibiting chemiluminescence in the system - 3-hydroxypyridine-peroxidase-hydrogen peroxide. The effect of the described antioxidant is dose-dependent in nature and increases with increasing concentration of the substance.

Based on the results obtained, it can be assumed that the antioxidant effect of the described antioxidant is based on its ability to inhibit the initiation of CPP, lipid peroxidation due to the formation of reactive oxygen species and the appearance of catalytically active iron ions, which suggests, in particular, the effectiveness of the described antioxidant as an inhibitor CPP in the acute period of development of neurodegenerative processes, including stroke, accompanied by an inflammatory reaction, when initiation stage efficiency is greatest.

A brief description of the graphic materials.

Figure 1 is an electron micrograph of the described antioxidant obtained on a scanning electron microscope.

Figure 2 is an electron micrograph of the described antioxidant obtained by a transmission electron microscope.

Figure 3 - particle size distribution of the described antioxidant in an aqueous suspension according to the method of DLS.

Figure 4 - IR spectra of mixtures of nanodiamonds with glycine used to build calibration curves. I, II, III - spectra of mixtures with a glycine content of 1: 1.75: 2.5, respectively. Within the framework, characteristic peaks are highlighted.

Figure 5 - calibration curves for each characteristic band of the IR spectrum of a mixture of nanodiamonds with glycine, a, b, c - calibration curves for the bands 1407, 1332 and 504 cm ^-1 , respectively.

The invention is illustrated by the following examples.

Example 1

300 mg of the initial detonation nanodiamond are annealed in a stream of hydrogen gas at a rate of 3.0 l / h at a temperature of 1000 ° C for 6 hours. Then, the annealed nanodiamond is subjected to liquid phase chlorination with molecular chlorine dissolved in 40 ml of CCl ₄ to 6 wt%. Cl ₂ . The chlorination reaction is carried out by photochemical exposure to visible light for 60 hours at a temperature of 60 ° C. Then, the sample was washed with CCl ₄ by centrifuging the suspension at 6000 rpm and dried under a pressure of 0.1 mm Hg. to constant weight. Then, a suspension is prepared from the chlorinated nanodiamond using 40 ml of a water-alcohol mixture (water: methanol = 1: 1), into which 300 mg of glycine as a free amino acid (NH ₂ CH ₂ COOH) is added with 1 ml of triethylamine. The resulting mixture was sonicated (50 W) for 60 minutes and kept under constant stirring at a temperature of 65 ° C for 30 hours. The resulting product was washed with a large amount of ethanol, centrifuged and dried under vacuum at 70 ° C overnight. The residual moisture content of the product is 2.2%. The yield of the target product is 279 mg (93%). The product is a dark gray with a bluish tint ultrafine powder (Figure 1) with primary particle sizes of 2-10 nm (Figure 2) having a shell of the surface layer up to 1 nm. In suspension, the particle size of the powder does not exceed 100 nm (Figure 3). The elemental composition of the particle surface of the obtained product is given in table.2.

table 2 XPS data of the obtained product. Name of characteristic Chemical elements C O N Atomic% 80.1 ± 0.1 11.5 ± 0.1 8.4 ± 0.1 Communication energies, eV 285.2 ± 0.5 530.7 ± 0.5 399.6 ± 0.5

To determine the mass fraction of glycine in the resulting product, 3 mixtures of nanodiamond with glycine are prepared, with a content of the latter 1: 1.75: 3.5, respectively. For each mixture, a weighed mass of 0.0035 g is taken and carefully ground in a mortar with 0.090 g of KBr. 0.070 g of the resulting mixture is pressed into a tablet and its PC spectrum is removed (Figure 4). Characteristic bands are selected at 1407, 1332 and 504 cm ^-1 , respectively, and calibration graphs are constructed for them (Figure 5). The intensity of the corresponding characteristic bands in the IR spectrum of the obtained sample with a mass of 0.0035 g was 0.23, 0.22, and 0.10 pu, respectively. From the calibration curves a, b, c in FIG. 5, the glycine content in the obtained sample is determined, which is 0.00057 ± 8 · 10 ^-5 g. Therefore, the mass fraction of glycine in the sample sample is 21 ± 3% of the mass.

Example 2

Study of the effect of the described antioxidant on the intensity of lipid peroxidation.

The following reagents were used in the work: 2-thiobarbituric acid (Fluka AG, Switzerland); Ionol (2,6-Di-tert-butyl-p-cresol, Fluka AG, Switzerland); 2-propanol (isopropyl alcohol, Himmed, Russia); Heptane normal reference (Himmed, Russia); Hexane H.H. (Himmed, Russia); Horseradish horse peroxidase (Boehringer Manheim GmbH, Germany); Hydrogen peroxide 3% solution (Samaramedprom OJSC, Russia); Ethanol H.H. (ethyl alcohol for chromatography, Himmed, Russia); Acetone H.H. (Himmed, Russia); Chromatography methanol (methyl alcohol, Chemapol, Poland); Chloroform H.H. (Himmed, Russia); Ferrous sulphate (FeSO ₄ · 7H ₂ O, Himmed, Russia); Salt Mora (Himmed, Russia); Acetic acid (Himmed, Russia).

The effect of the described antioxidant on the intensity of lipid peroxidation (LPO) was studied using estimates of the levels of diene conjugates (DCs) and malondialdehyde (MDA) in a suspension of model liposome membranes. Used the following methods:

1. The method of preparation of multilayer phospholipid liposomes. A heterogeneous system — a suspension of model liposome membranes [16] was prepared from the total fraction of egg yolk phospholipids. Phospholipids were extracted according to the method [17]. One volume of egg yolk was homogenized for 30 min in 20 volumes of chloroform-methanol mixture (2: 1, by volume), the homogenate was filtered through a defatted paper filter to separate the aggregated protein. The resulting lipid extract was washed by adding 1/3 of the total volume of a 0.74% aqueous KCl solution to it. After settling for 12 hours at 0-4 ° C, the upper water-methanol phase was carefully removed, the lower phase was transferred to a round bottom flask and evaporated using a rotary evaporator. The dry lipid film was washed with 3-4 ml of hexane and 20-fold amount of chilled acetone was added. In this case, phospholipids precipitated in the form of white flakes. The precipitation of phospholipids with acetone was carried out 5 times. The resulting phospholipids were dissolved in a chloroform-methanol mixture. The concentration of phospholipids was determined gravimetrically. The preparation of multilayer phospholipid liposomes was performed according to the method [18]. For this, the required amount of a chloroform-methanol solution of phospholipids was placed in a round bottom flask and the solvent was evaporated on a vacuum rotary evaporator. The dry lipid film was washed from the walls of the flask with a specific volume of 20 mM Tris-HCl buffer, pH 7.4. In order to form liposomes that are more uniform in size, the obtained liposome suspension was subjected to cryolytic treatment by three freezing-thawing. Before use, the liposome suspension was aged for at least 1 hour at 37 ° C.

2. Method for the determination of lipid peroxidation products. The initiation of lipid peroxidation was carried out by introducing a solution of FeSO ₄ to a final concentration of 0.1 mM for 30 minutes. The LPO reaction was stopped by the addition of ionol to a final concentration of 0.1 mmol / L. A solution of the described antioxidant at various concentrations in ethanol was incubated with a suspension of liposomes until initiation of LPO by ferrous sulfate for 30 minutes. Control samples contained an equivalent volume of ethanol instead of compounds. All measurements were carried out in 3 parallels.

3. Method for the determination of diene conjugates (DC). To 100 μl of the final reaction suspension (liposome-compound-ionol) was added 1000 μl of a mixture of 2-propanol-heptane (1: 1, by volume). Samples were vigorously shaken 2 times for 10 sec (Bio Vortex VI, BioSan, Latvia) and centrifuged at 3000 rpm for 10 min (J-6B, Beckman, USA). To 900 μl of the supernatant, 100 μl of distilled water was added to separate the phases and was vigorously shaken 2 times for 10 s and centrifuged at 3000 rpm for 10 min. 300 μl of the upper heptane phase were taken and 1200 μl of 95% ethanol was added. The optical density of the samples was determined on a DU-50 spectrophotometer (Beckman, USA) in a semi-microcuvette at 233 nm. The calculation of the number of DC was carried out on the basis of the molar extinction coefficient of 2.2 · 10 ⁵ M ^-1 cm ^-1 .

4. Method for the determination of malondialdehyde (MDA). To determine the MDA, 20 μl of 0.495 M Mora salt was added to 50 μl of the final reaction suspension (liposome-compound-ionol) and incubated at 37 ° C for 30 min. Then, 1030 μl of a 0.9% solution of 2-thiobarbituric acid in 50% acetic acid was added to the samples, shaken vigorously and incubated at 80 ° C for 60 min. After cooling, the optical density of the samples was measured on a DU-50 spectrophotometer (Beckman, USA) in a semi-microcuvette at 532 nm. The calculation of the amount of MDA was carried out on the basis of the molar extinction coefficient of 1.56 · 10 ⁵ M ^-1 cm ^-1 .

It was found that the described antioxidant at a concentration of 10 μg / ml caused an insignificant, statistically unreliable decrease in comparison with the control of DC levels, but a significant decrease (2 times) in the MDA level. At a concentration of 50 μg / ml, the described antioxidant statistically significantly reduced the level of DC by 2.4 times, and the level of MDA by 2 times. At a concentration of 100 μg / ml, the effect of the described antioxidant was enhanced: under the influence of a substance, the DC level decreased by 5.8 times, and the MDA level - by 3.1 times (Table 3).

Table 3
The effect of the described antioxidant, pharmacopoeial glycine and mexidol on the intensity of lipid peroxidation (LPO) by the level of diene conjugates and malondialdehyde in a suspension of model liposome membranes. The concentration of the substance, mcg / ml Diene conjugates, µmol / l Malondialdehyde, μmol / L The control - 81.248 ± 8.418 1.709 ± 0.306 Described Antioxidant 10 63.381 ± 5.912 0.954 ± 0.069 * fifty 20.143 ± 4.812 * 0.492 ± 0.073 * one hundred 8.174 ± 1.247 * 0.263 ± 0.041 * Pharmacopoeial Glycine 10 63,431 ± 6,469 0.794 ± 0.092 * fifty 35.896 ± 3.452 * 0.423 ± 0.098 * one hundred 11.779 ± 3.034 * 0.303 ± 0.029 * Mexidol 10 68.877 ± 8.543 68.877 ± 8.543 fifty 42.326 ± 5.714 * 42.326 ± 5.714 * one hundred 23.345 ± 3.017 * 23.345 ± 3.017 * * significance of differences at p <0.05 compared with the control.

Glycine in the substance at a concentration of 10 μg / ml, compared with the control, slightly reduces the levels of DC and MDA (Table 3). At a concentration of 50 μg / ml, Glycine in the substance statistically significantly reduced DC levels by 4 times, and MDA levels by 2.25 times. At a concentration of 100 μg / ml, Glycine in the substance reduced the level of DC by 6.86 times, and the MDA level by 5.67 times (Table 3).

Mexidol at a concentration of 10 μg / ml statistically significantly reduces compared with the control levels of diene conjugates (DC) and malondialdehyde (MD). At a concentration of 50 μg / ml, Mexidol statistically significantly reduced the level of DC by 1.86 times, and the level of MDA by 1.81 times (Table 3). At a concentration of 100 μg / ml, Mexidol reduced the level of DC by 3.37 times, and the level of MDA by 3.91 times (Table 3).

The study of the antiradical activity of the described antioxidant.

To determine the antiradical activity of the described antioxidant, the method of chemiluminescence was used (Zolotov N.N. Zalilov K.Yu., Mukhtarov B.E., Kuzmin V.I., Smirnov L.D., Dyumaev K.M. Chem. - pharmaceutical. ., 1989, No. 2. - S. 120-127). The method of chemiluminescence is based on the achievement of the effect of superweak glow accompanying free radical oxidation reactions. For this purpose, the oxidation reaction of unsubstituted 3-hydroxypyridine in the peroxidase – hydrogen peroxide system was used. The luminescence of the reaction mixture is associated with the formation of free radicals of 3-hydroxypyridine. When the studied compounds are added to this system, chemiluminescence quenching is observed due to their interaction with unsubstituted 3-hydroxypyridine radicals. The following reagents were used to carry out the CPO reaction: 0.1 M KH ₂ PO ₄ —KOH buffer with a pH of 7.4; a freshly prepared solution of horseradish root peroxidase (Boehringer Manheim GmbH, Germany) in phosphate buffer with a concentration of 2.5 mg / ml; 20 mM solution of unsubstituted 3-hydroxypyridine in ethanol; ethanol for monitoring or the test compound dissolved in ethanol of various concentrations from 10 μg / ml to 100 μg / ml (5 concentrations in total). Chemiluminescence was initiated by adding a freshly prepared aqueous solution of hydrogen peroxide at a concentration of 0.015%.

The antiradical effectiveness of the compounds, determined by the method of quenching chemiluminescence, is presented in table 4. It was found that the described antioxidant at a concentration of 20 μg / ml statistically significantly reduced quenching of chemiluminescence (in% of control) by 1.58 times (Table 4). With a further increase in the concentration of the described antioxidant, its effect intensified. At a concentration of 50 μg / ml, the described antioxidant reduced quenching of chemiluminescence by 3.32 times, at a concentration of 75 μg / ml - by 9.19 times, at a concentration of 100 μg / ml - by 25.75 times (Table 4). The data obtained indicate that the described antioxidant has a pronounced anti-radical activity, and its effect is dose-dependent in nature, increasing with increasing concentration of the compound. Pharmacopoeial glycine in the substance in the used chemiluminescent system did not show antiradical activity (Table 4).

Table 4 Quenching of chemiluminescence in a system of unsubstituted 3-hydroxypyridine, peroxidase and hydrogen peroxide under the action of the described antioxidant, pharmacopoeial glycine and mexidol. s, mcg / ml Quenching of chemiluminescence (% of control) / Substances Described Antioxidant Pharmacopoeial Glycine Mexidol 10 91.82 ± 9.14 95.89 ± 9.01 93.81 ± 5.34 twenty 65.82 ± 5.98 * 91.13 ± 8.18 78.46 ± 9.37 fifty 31.02 ± 2.96 * 89.15 ± 7.16 38.18 ± 5.29 * 75 11.72 ± 2.75 * 86.27 ± 6.89 17.62 ± 4.36 * one hundred 3.98 ± 0.78 * 83.85 ± 9.03 9.18 ± 0.75 * The control 103.04 ± 8.12

* significance of differences at p <0.05 compared with the control.

Thus, the obtained data showed that the described antioxidant in the concentration range of 20-100 μg / ml has a distinct antiradical activity, significantly inhibiting chemiluminescence in the 3-hydroxypyridine-peroxidase-hydrogen peroxide system, and exceeds pharmacopoeial glycine and the drug mexidol in this indicator (table .four). So, at a concentration of 100 μg / ml, the described antioxidant reduced quenching of chemiluminescence by 25.75 times.

Example 3

The study of the side effects and toxic effects of the described antioxidant.

The study was conducted on white mongrel sexually mature male mice weighing 20-24 g, 2-3 months old.

Animals were obtained from the Central laboratory animal nursery "Pillar", Moscow region. The keeping of animals corresponded to the rules of laboratory practice when conducting preclinical studies in the Russian Federation (GOST Z 51000.3-96 and 51000.4-96), the normative document "Sanitary rules for the design, equipment and maintenance of vivariums" approved by the Chief State Sanitary Doctor on 04/06/1973 No. 1045 -73, and Order of the Ministry of Health of the Russian Federation No. 267 of 06/19/2003 “On approval of laboratory practice rules” (GLP) in compliance with the International Recommendations of the European Convention for the Protection of Vertebrate Animals used in experimental studies (1997). The animals were kept in a vivarium at a temperature of 20-22 ° C, with a light cycle of 12 hours light and 12 hours dark periods, in T / 4A plastic cages measuring 580 × 375 × 200 mm with a stainless steel top lid and dust-free wooden bedding shavings. The animals were kept with constant access to feed and water using the full ration of extruded briquetted feed (GOST for feed P 50258-92) and drinking water. When conducting the experiments, the requirements of the Commission on the Ethics of Attitude to Animals of the Russian National Committee on Bioethics at the Russian Academy of Sciences and the ethical standards set forth in the "International Recommendations for Biomedical Research Using Animals" (1985) were taken into account. The experiments were carried out in the first half of the day. A total of 42 animals were used - 7 groups of 6 mice. Possible side effects, signs of intoxication, and death of animals were recorded 1 h to 14 days after the intraperitoneal administration of the described antioxidant in comparison with pharmacopoeial glycine in the same doses.

As a result of the study, it was found that the described antioxidant when administered intraperitoneally to mice at doses of 75, 150 and 225 mg / kg did not cause signs of intoxication and death of animals over 14 days (Tables 5-7). At the same time, the described antioxidant also did not cause changes in the coat of mice, the condition of the mucous membranes in mice. There was also no ptosis of the upper eyelid, increased urination, defecation, salivation, piloerection, vocalization, lateral position. Within the normal range, there was a rhythm and depth of respiratory movements, there was no aggressiveness, timidity, tremor, convulsions, catalepsy, stereotype and grooming. No change in posture was observed. The animals retained pineal, corneal and pain reflexes. During all 14 days of observation, the animals were kept on an inverted mesh platform for 5 s (Tables 5-7).

Table 5 The study of possible side effects and death of mice 1 hour after the administration of pharmacopoeial glycine and the described antioxidant (indicator of change in the group in%). Indicators Groups The control Antioxidant / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 Presence of piloerection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 33.3 * 0 0 33.3 * Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 66.7 * one hundred* one hundred* 0 one hundred* one hundred* Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 16.7 fifty* fifty* 33.3 * 33.3 * fifty* 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred 66.7 * 66.7 * fifty* 66.7 * 66.7 * 66.7 * The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 66.7 * one hundred* one hundred* 0 one hundred* one hundred* The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 6 Study of possible side effects and death of mice 24 hours after the administration of pharmacopoeial glycine and the described antioxidant (indicator of change in the group in%). Indicators Groups The control Antioxidant / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 Presence of piloerection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 0 0 0 0 Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 0 0 0 0 0 0 Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 0 0 0 33.3 * 16.7 33.3 * 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred 66.6 * The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 0 0 0 0 0 0 The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

Table 7 The study of possible side effects and death of mice 14 days after the administration of pharmacopoeial glycine and the antioxidant described (indicator of change in the group in%). Indicators Groups The control Antioxidant / dose, mg / kg Pharmacopoeial glycine / dose, mg / kg 75 150 225 75 150 225 Changing the nature of the coat 0 0 0 0 0 0 0 Change in the condition of the mucous membranes 0 0 0 0 0 0 0 Ptosis of the upper eyelid 0 0 0 0 0 0 0 Increased urination 0 0 0 0 0 0 0 Increased bowel movements 0 0 0 0 0 0 0 Increased salivation 0 0 0 0 0 0 0 Presence of piloerection 0 0 0 0 0 0 0 The presence of vocalization 0 0 0 0 0 0 0 Lateral position 0 0 0 0 0 0 0 Violation of the rhythm and depth of respiratory movements 0 0 0 0 0 0 0 Aggressiveness 0 0 0 0 0 0 0 Change in response to tapping a cell 0 0 0 0 0 0 0 Increased shyness 0 0 0 0 0 0 0 Tremor 0 0 0 0 0 0 0 Cramps 0 0 0 0 0 0 0 Change in pain response thresholds 0 0 0 0 0 0 0 Posture change, catalepsy 0 0 0 0 0 0 0 Lack of coordination in the test of a rotating rod 0 0 0 0 0 0 0 5 s holding on an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred one hundred Climbing from an inverted mesh platform one hundred one hundred one hundred one hundred one hundred one hundred 66.7 * The presence of a pineal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred The presence of a corneal reflex one hundred one hundred one hundred one hundred one hundred one hundred one hundred Sedation 0 0 0 0 0 0 0 The presence of stereotype 0 0 0 0 0 0 0 The presence of grooming 0 0 0 0 0 0 0 Animal death 0 0 0 0 0 0 0 * - P <0.05 - relative to the control by the criterion χ2

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Claims (4)

1. The antioxidant, which is glycine, immobilized on particles of detonation nanodiamonds with a size of 2-10 nm, having a shell thickness of up to 1 nm, with a glycine content of up to 21 ± 3% of the mass. 2. The method of producing the antioxidant according to claim 1, characterized in that the detonation nanodiamond is annealed in a stream of hydrogen gas at a temperature of 500-1200 ° C for 1-8 hours, subjected to liquid-phase chlorination with molecular chlorine by photochemical exposure to visible light at a temperature of 50-70 ° C for 36-60 hours, followed by washing with carbon tetrachloride, centrifugation and drying, the resulting chlorine-modified nanodiamond is dissolved in a polar solvent to form a suspension, tertiary amine and glycine are added, This mixture was sonicated, followed by aging at 50-80 ° C, centrifugation, washing with solvent and drying. 3. The method according to claim 2, where the ultrasound treatment is carried out for 5-60 minutes and keeping at 50-80 ° C is carried out for 12 to 48 hours 4. The method according to claim 2 or claim 3, wherein triethylamine is used as a tertiary amine and pyridine, a lower aliphatic alcohol, a water-alcohol mixture, or water are used as a polar solvent.

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