RU2183124C1 - Method to obtain radioprotector - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: aqueous solutions of oxycarboxylic acids, obtained out of natural raw material as humic substances, are treated with ammonium molybdate. Humic substances could be obtained by treating humified material with alkaline solutions under normal conditions. As humified material one should take composts, sapropels, peat from raised and transitional bogs, humic layers of different-type soils. Humic substances could be obtained out of oxidized woody lignin due to its treating with oxygen-containing gas in alkaline medium at 170 ± 20 C, 1.9-2.5 MPa pressure for 1-3 h and subsequent cooling the reaction mass, separation of a solid phase against a solution and acidification of the latter up to pH = 2-3. Treatment by ammonium molybdate is conducted at 40 ± 5 C. It is, also, possible to carry out ammonium molybdate treatment under ultrasound action at radiation power being 40 W/cm², 22 kHz frequency for 4-8 min. EFFECT: higher efficiency of substances based upon natural raw material with properties of radioprotectors. 8 cl, 4 tbl

Description

 The invention relates to medicine, in particular, relates to radioprotectors obtained on the basis of natural raw materials.

 Radioprotectors include substances that can, when used prophylactically, have a protective effect, which is manifested in preserving the life of the irradiated living organism or weakening the severity of radiation damage with prolonging the state of legal capacity and life expectancy (V.I. Legeza, V. G. Vladimirov. New classification of preventive radiation protection Radiation Biology, Radioecology, vol. 38, issue 3, p. 416, 1998).

 To reduce the effects of radiation, radioprotectors are used, which, as a rule, are introduced into the body before irradiation (Bak Z., Alexander P. Fundamentals of Radiology, M .: Publishing House of Foreign Literature, 1963, p. 427). The disadvantages of such substances include the need for their presence in the body before irradiation, a certain toxicity at a radioprotective dose, as well as a limited residence time (usually 2-5 hours) in the body.

 The results of the liquidation of the consequences of the Chernobyl accident and other radiation situations in different countries convincingly showed the currently limited base of biologically active substances with radio-modifying activity.

 Currently, an intensive search is under way for the correction of radiation injuries. It is proposed, for example, the use of a complex of vitamins and microelements (V. Sukolinsky et al. Radioprotective and radiotherapeutic agent "AK". Theses of the republican conference "Scientific and practical aspects of maintaining the health of people exposed to radiation in the area of the Chernobyl accident", 12- March 14, 1991, Minsk, p. 239). Preparations of this type are usually offered in the form of substances of plant origin and other biologically active compounds. Drugs based on them usually work directly through the body and help accelerate the processes of post-radiation restoration of the tissues of the irradiated body. However, the therapeutic effect of these drugs is poorly expressed.

 Also known radioprotective agent obtained from natural raw materials and representing zeolite-containing tuffs - shivyrturin and hongurin (RF Patent 2028800, CL A 61 K 31/00, publ. B.I. 5, 1995). Shivyrturin and hongurin are zeolite-bearing rocks of the Shivyrtuisky and Khongurin deposits (the type of mineral is clinoptilolite). They are allowed as feed additives in the diets of farm animals. To obtain minerals as food additives, they are ground. The finest grinding fineness of less than 1 mm is most effective. The addition of these minerals to food increases the survival of animals in conditions of radioactive damage and they are recommended for use in areas contaminated and contaminated with radionuclides.

 The objective of the invention is to develop a method that allows to obtain on the basis of natural raw materials substances with the properties of radioprotectors.

 The problem is solved in that a method for producing radioprotectors is proposed in which solutions of hydroxycarboxylic acids obtained from raw materials of plant origin in the form of humic substances are treated with ammonium molybdate.

 Humic substances can be obtained by treatment with alkaline materials under normal conditions of humified material, which can be used sapropels, humus horizons from various types of soils, compost, peat from upland and transitional bogs, etc.

Humic substances can also be obtained by oxidation of wood lignin, while wood lignin in the form of a water-alkaline suspension is oxidized with an oxygen-containing gas at a temperature of 170 ^° C ± 20 ^° C and a pressure of 1.9-2.5 MPa for 1-3 hours and after After cooling the reaction mass and separating the solid phase from the solution, humic acids are released from the acidified solution, which, after neutralization, is treated with ammonium molybdate.

Before oxidation of wood lignin, the reaction mass can be processed under the influence of ultrasound with a radiation power of 4-6 W / cm ² , with a frequency of 22 kHz for 6-10 minutes.

Treatment with ammonium molybdate is carried out at a temperature of 40 ^o C ± 5 ^o C.

 It is possible to treat ammonium molybdate under the action of ultrasound with a radiation power of 40.0 ± 0.5 W, a sound frequency of 22.0 ± 0.5 kHz for 20-25 minutes.

 Lignin, which is part of wood, is a valuable medicinal raw material, the reserves of which are practically unlimited. The medicinal properties of lignin are comprehensively considered in the monograph (Levanova VP, Medical lignin. - Ed. By N. A. Belyakov: SP (b) - 1992, p. 136). Although lignin and preparations based on it in medicine have so far been mainly considered as sorbents, many researchers in addition to sorption properties noted antiseptic, hypocholesterolemic and other properties.

 As wood lignin, a product of alkaline wood hydrolysis, lignin-based enterosorbents such as Polyphepan, Lignosorb, etc. can be used.

 Polyphepan, a non-specific enterosorbent (Peg. 80/1211/3), is a brown powder, odorless and tasteless, with a moisture content of 65%, consists mainly of lignin and contains no more than 20% residual polysaccharides (hydrocellulose).

 Lignosorb (polyphepan paste) VFS 42-2203-93.

 All substances used as feedstock are approved for oral administration.

 Special studies have shown that during the oxidation of lignin under the conditions of the proposed method, the formation of substances harmful to the body, such as polyaromatic hydrocarbons, nitrosoamines and polychlorinated dicyclo-p-dioxins, does not occur.

 The resulting products are concentrated dark brown liquids containing from 0.05 to 1.00% molybdenum with a specific taste and smell.

 It is well known that the process of humification (the process of formation of humic substances) is planetary and the resulting humic substances (otherwise, humic acids, which are a mixture of humic acids and fulvic acids) are characterized by similar spectra in the infrared region, regardless of how they were obtained.

 The authors used the obtained substances for the first time as radioprotectors. The radioprotective effect of substances of a similar nature is not described in the literature and was discovered by the authors for the first time. Currently, work is underway to clarify the structure of the proposed substances and their mechanism of action.

 The radioprotective activity of the obtained product was evaluated by its effect on outbred male mice weighing 18-20 g, since mice, being a standard object in biological studies, have a sensitivity to experimental radiation factors that are comparable with other types of laboratory animals and humans.

 The following examples explain in detail the method of producing radioprotectors and their effect.

EXAMPLE 1
The initial aqueous-alkaline suspension of lignin (Polyphepan brand, reg. 80/1211/3), a non-specific enteric sorbent, was oxidized with atmospheric oxygen.

Suspension composition
Polyphepan content - 1 kg
Alkali content (sodium hydroxide) - 100 g
The density of the suspension 1: 8
The oxidation was carried out in a reactor with mechanical stirring at a temperature of 160 ± 5 ^o C at a pressure of 2.5 MPa for 1 hour. Air consumption was 5 l / min.

The reaction mass was cooled to room temperature and the precipitate was separated from the solution by filtration. The filtrate was acidified with sulfuric acid to pH 2-3. The precipitate was separated by filtration, washed with distilled water, and then with a water-alcohol mixture until pH 6.0-6.5 was established and dried at 105 ^° C to constant weight. The obtained humic acids were neutralized with 5% aqueous ammonia at the rate of 1 g of humic acids per 80 ml of the indicated solution, thermostatted in a boiling water bath until excess ammonia was removed, filtered through a paper filter and 30 vol. % distilled water. 0.2 wt. % ammonium molybdate [(NH ₄ ) ₆ Mo ₇ O ₂₄ • 4H ₂ O] per 1 g of humic acids and created the process of developed acoustic cavitation under the action of ultrasound with a radiation power of 40 W / cm ² and a frequency of 22 kHz for 1 min. Next, the solution was adjusted with a label to 100 ml.

The elemental composition of the selected product in terms of organic matter, wt. %: C 54%, H 12%, N 2%, S + O 32% (by difference). The substance decomposes without melting starting at 168 ^o C.

The results of infrared Fourier spectra show that the product obtained contains bands of close intensity in intensity of 1700 cm ^-1 and 1600 cm ^-1 , characteristic of carboxyl groups, an intense band in the region of 1200 cm ^-1 , a broad band in structure with local maxima of 3400, 3200 , 3100 and 2600 cm ^-1 , as well as bands 2926, 2815, 1460 cm ^-1 and bands 2955 and 2870 cm ^-1 characteristic of methylene groups.

The resulting product has paramagnetism. Symmetrical EPR signals are observed in the product with a q factor of 2,000 ± 0.001 close to the value for a free electron (q = 2.00) and a width of H = 6.1 ± 0.1 Oersted. The concentration of PMC is at the level of (2.5 ± 0.4) • 10 ¹⁸ per gram.

 The organic portion of the product contains 2.9-3.4 mEq. COOH groups in 1 g of the product and 6.0-6.4 mEq. phenolic hydroxyls in 1 g of the product and in structure can be classified as polyoxycarboxylic acids.

EXAMPLE 2
Lake sapropel with an organic matter content of 32% per dry matter was subjected to alkaline treatment with a 0.1 M potassium hydroxide solution.

For this, 5.6 g of potassium hydroxide was added to 200 g of lake sapropel with a moisture content of 95% and the total volume of the mixture was brought to 1 L, after which the mixture was shaken for 5 minutes and left for 6 hours. After the indicated period, the precipitate was separated by filtration. The filtrate was acidified with phosphoric acid to pH 1-2. The precipitate was separated by filtration, washed with distilled water, and then with a water-alcohol mixture until a pH of 6.0-6.5 was established and dried at 105 ^° C to constant weight. The obtained humic acids were neutralized with 5% aqueous ammonia at the rate of 1 g of humic acids per 80 ml of the indicated solution, thermostatted in a boiling water bath until excess ammonia was removed, filtered through a paper filter and 30 vol. % distilled water. 0.2 wt. % ammonium molybdate per 1 g of humic acids and created the process of developed acoustic cavitation under the action of ultrasound with a radiation power of 40 W / cm ² and a frequency of 22 kHz for 1 minute. Next, the solution was adjusted with a label to 100 ml.

The elemental composition of the selected product in terms of organic matter, wt. %: C 60%, H 6%, N 2%, S + O 32% (by difference). The substance decomposes without melting starting at 168 ^o C.

The results of infrared Fourier spectra show that the product obtained contains bands of close intensity in intensity of 1700 cm ^-1 and 1600 cm ^-1 , characteristic of carboxyl groups, an intense band in the region of 1200 cm ^-1 , a broad band in structure with local maxima of 3400, 3200 , 3100 and 2600 cm ^-1 , as well as bands 2926, 2815, 1460 cm ^-1 and bands 2955 and 2870 cm ^-1 characteristic of methylene groups.

The resulting product has paramagnetism. Symmetrical EPR signals are observed in the product with a q factor of 2,000 ± 0.001 close to the value for a free electron (q = 2.00) and a width of H = 6.1 ± 0.1 Oersted. The concentration of PMC is at the level of (2.5 ± 0.4) • 10 ¹⁸ per gram.

 The organic portion of the product contains 3.0-3.6 mEq. COOH groups in 1 g of the obtained product and 6.0-6.5 mEq. phenol hydroxyls in 1 g of the obtained product and in structure can be classified as polyoxycarboxylic acids.

EXAMPLE 3
Carried out analogously to example 2, while vermicompost with an organic matter content of 23% per dry matter was subjected to alkaline treatment with a 0.1 M sodium hydroxide solution. 10 g of vermicompost (in terms of a completely dry sample) was poured into 1 liter of 0.1 M sodium hydroxide solution, the mixture was shaken for 1 hour, after which the precipitate was separated by filtration. The filtrate was acidified with sulfuric acid to pH 1-2 and humic acids were isolated.

0.17 wt.% Was added to the humic acid solution. % ammonium molybdate per 1 g of humic acids and heated to 40 ^o C.

 The elemental composition of the selected product in terms of organic matter, wt. %: C 57%, H 7%, N 2%, O + S 34% (by difference).

 The organic portion of the product contained 3.2-3.8 mEq. COOH groups in 1 g and 6.2-6.7 mEq. phenolic hydroxyls in 1 g.

 Studies of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that the obtained humic substances can be classified as polyoxycarboxylic acids in structure.

EXAMPLE 4
Carried out analogously to example 2, while peat from a high bog with an organic matter content of 73% dry matter was alkaline treated with a 0.2 M potassium hydroxide solution. 5 g of dry peat was poured with 0.2 M potassium hydroxide solution, and then left for 6 hours, after which the precipitate was separated by filtration. The filtrate was acidified to pH 2-3 with phosphoric acid. The treatment of the isolated humic acids with ammonium molybdate was carried out at 45 ^o C.

 The elemental composition of the selected product, calculated on the organic substance, wt. %: C 51%, H 12%, N 2%, O + S 35% (by difference).

 The organic portion of the product contains 3.4-4.0 mEq. COOH groups in 1 g and 6.3-6.9 mEq. phenolic hydroxyls in 1 g.

 Studies of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that the obtained humic substances can be classified as polyoxycarboxylic acids in structure.

EXAMPLE 5
Carried out analogously to example 2, while to 500 ml of lake sapropel with a moisture content of 90% and a solids content of 31% was added 500 ml of distilled water, 5.6 g of caustic potassium (0.1 M), 6 g of urea (0.1 M) and 37 g of Trilon B (0.1 M). After stirring for 1 hour under normal conditions, the precipitate was separated by filtration. The filtrate was acidified with sulfuric acid to pH 1-2.

0.17 wt.% Was added to the humic acid solution. % ammonium molybdate per 1 g of humic acids and heated to 40 ^o C.

 The elemental composition of the selected product in terms of organic matter, wt. %: C 52%, H 16%, N 2%, O + S 30% (by difference).

 The organic portion of the product contains 3.0-3.4 mEq. COOH groups in 1 g and 6.0-6.4 mEq. phenolic hydroxyls in 1 g.

 Studies of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that the obtained humic substances can be classified as polyoxycarboxylic acids in structure.

EXAMPLE 6
Carried out analogously to example 2, while to 100 g of the humus horizon of the soil (horizon A from ordinary black soil) with a total carbon content of 7% was added 500 ml of distilled water, and then 1.12 g of caustic potassium (0.02 M), 6 g of urea (0.1 M) and 7.4 g Trilon B (0.02 M). After stirring for 10 minutes, the mixture was kept under normal conditions for 6 hours and the precipitate was separated by filtration. The filtrate was acidified with sulfuric acid to pH 1-2.

0.2 wt.% Was added to the humic acid solution. % ammonium molybdate per 1 g of humic acids and heated to 40 ^o C.

 The elemental composition of the selected product in terms of organic matter, wt. %: C 56%, H 8%, N 3%, O + S 33% (by difference).

 The organic portion of the product contains 3.4-4.0 mEq. COOH groups in 1 g and 6.4-7.0 mEq. phenolic hydroxyls in 1 g.

 Studies of infrared spectra and determination of the content of COOH groups and phenolic hydroxyls showed that the obtained humic substances can be classified as polyoxycarboxylic acids in structure.

EXAMPLE 7
The radioprotective effectiveness of the substances obtained by the proposed method was tested on outbred male mice weighing 18-20 g.

 Irradiation was carried out on the RUM-17 installation under the following conditions: voltage of 180 kV. Current strength 15 mA, filter 0.5 mm Cu + 1.0 mm Al, focal length 70 cm, dose rate 0.355 Gy / min, irradiation direction: back-chest. Absorbed doses for animals of various groups were 2,4,6,8,10,15 and 20 Gy.

 To irradiate the mice, 10 animals were placed in plastic cases. False-irradiated animals were used to control the effectiveness of radiation exposure, which were placed in canisters under the RUM-17 apparatus with the anode tube turned off at the same time as the irradiated ones. The animals of the experimental and control groups were irradiated simultaneously, after which they were kept under the same conditions as the non-irradiated control.

 Dosimetric control was carried out using an individual dosimeter ID-11 with subsequent evaluation of the readings on the device GO-32.

 The substance obtained in example 1, in the form of a 1% solution in physiological saline in a volume of 0.2 ml was administered to experimental animals intramuscularly at a dose of 100 mg / kg (2 mg per individual). The drug was administered 1 time per day for 5, 4, 3 and 2 days before radiation exposure. The total dose of the drug for 4 days was 400 mg / kg (8 mg per individual).

 To identify the radio modifying efficacy of the studied drugs, we used groups of mice that were injected with physiological saline according to the same schemes as in the animals of the experimental groups, after which the mice were subjected to radiation exposure, as described above. Mice of another group were exposed to false radiation without the introduction of any drugs and were considered as biological controls.

 Observation of the animals of the experimental and control groups was carried out for a month before irradiation and 30 days after radiation exposure.

 The research results showed that the obtained substance has radioprotective efficacy in conditions of a general relatively uniform exposure of mice in the bone marrow and intestinal dose range.

 The results are presented in tables 1-4. Table 1 presents data on the dynamics of the death of mice after irradiation with different doses. Table 2 presents data on the effect of the drug on the dynamics of the death of mice after irradiation with different doses. Table 3 presents the indicators of death and survival of mice after irradiation with different doses. Table 4 shows the indicators of radioprotective efficacy of the drug when mice are irradiated with different doses.

 The following indicators were used as criteria for assessing the radiomodifying efficacy of drugs.

 The percentage of death of animals (% of death) was calculated by dividing the absolute number of dead animals by their total number in the study group, followed by multiplication by 100.

 Percentage of animal survival (% survival) is the remaining proportion of the percentage of death.

 The average life expectancy (LSS) of dead animals was calculated by adding the number of days lived by each dead animal after irradiation, and then dividing this indicator by the number of dead animals in the study group. The index of life expectancy was expressed in days.

 The percent protection of the drug (% protection) was calculated as the difference between the percent survival of animals of the experimental and control groups.

 The survival index (And survival) was calculated as the ratio of the percentage of survival of mice in the experimental group to the same indicator in the control group.

Фактор изменения дозы (ФИД) рассчитывался как отношение доз облучения, вызывающих одинаковый биологический эффект (в частности, СД_50/30 или СД_50/5) при применении предложенного средства и без него:

Из данных таблицы 2 видно, что гибель животных, не подвергшихся фармацевтической защите, определялась начиная с дозы 2 Гр, а при дозах 8 Гр выживших после облучения животных не было. При определении показателей смертельных доз выявлено, что СД_16/30 составила 3,50 Гр, СД_50/30 - 5,33 Гр, СД_84/30 - 7,15 Гр. Средняя продолжительность жизни погибших животных после облучения в костно-мозговом диапазоне доз находилась в пределах от 16 (4Гр) до 9 суток (8Гр). После облучения в кишечном диапазоне доз (10, 15, 20 Гр) наблюдалась 100% смертность, а животные жили в среднем 3,17 суток.The protection coefficient (K protection) was calculated as the ratio of the difference between the percentage of death of mice in the control (% death of control) and experimental (% death of experience) groups to the percentage of death in control (% death of control):

The dose change factor (PID) was calculated as the ratio of radiation doses causing the same biological effect (in particular, SD _50/30 or SD _50/5 ) with and without the use of the proposed agent:

From the data of table 2 it can be seen that the death of animals that were not subjected to pharmaceutical protection was determined starting with a dose of 2 Gy, and at doses of 8 Gy there were no survivors of the animals. When determining the indicators of lethal doses, it was revealed that SD _16/30 was 3.50 Gy, SD _50/30 - 5.33 Gy, SD _84/30 - 7.15 Gy. The average life expectancy of dead animals after irradiation in the bone marrow dose range ranged from 16 (4Gy) to 9 days (8Gy). After irradiation in the intestinal dose range (10, 15, 20 Gy), 100% mortality was observed, and the animals lived on average 3.17 days.

As the data in table 4, the prophylactic use of the proposed tool was accompanied by a decrease in animal death rates. With its prophylactic use, SD _16/30 amounted to 4.71 Gy, SD _50/30 - 6.56 Gy, SD _84/30 - 8.41 Gy. The PID of the proposed drug when irradiating mice in a minimally lethal dose was 1.28, with a semi-lethal dose of 1.26, with a minimum absolutely lethal dose of 1.25, i.e. upon irradiation of mice in the bone marrow dose range (from 2 to 8 Gy), the PID did not change with an increase in the absorbed dose.

 The prophylactic use of the resulting preparation increased the average life expectancy of a dead animal after irradiation at a dose of 10 Gy by 1.6 times and almost 1.5 times prolonged the life of animals after irradiation at a dose of 15 Gy. Tv

Claims (9)

 1. A method of producing a substance having the properties of a radioprotector from natural raw materials, characterized in that humic substances are obtained from natural raw materials and treated with ammonium molybdate. 2. The method according to p. 1, characterized in that the treatment with ammonium molybdate is carried out at a temperature of 40 ± 5 ^o C. 3. The method according to p. 1, characterized in that the treatment with ammonium molybdate is carried out under the influence of ultrasound with a radiation power of 40 W / cm ² , with a frequency of 22 kHz for 4-8 minutes  4. The method according to p. 1, characterized in that the humic substances are obtained by treating the humified material with alkaline solutions under normal conditions.  5. The method according to p. 4, characterized in that the humified material is treated with an alkaline solution in the presence of urea and complexon.  6. The method according to p. 4 or 5, characterized in that as a humified material using peat from bogs or transitional marshes, or sapropel, or composts, or humus horizons of different types of soils.  7. The method according to p. 1, characterized in that use humic substances obtained from oxidized wood lignin. 8. The method according to p. 7, characterized in that before the oxidation of wood lignin, the reaction mass is treated with ultrasound with a radiation power of 4-6 W / cm ² , with a frequency of 22 kHz for 6-10 minutes 9. The method according to p. 7 or 8, characterized in that the humic substances are obtained by oxidizing wood lignin in an alkaline environment with an oxygen-containing gas at a temperature of 170 ± 20 ^o C, a pressure of 1.9-2.5 MPa for 1-3 hours and subsequent cooling the reaction mass, separating the solid phase from the solution and acidifying the latter to a pH of 2-3.

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