Method of using superoxide dismutase 2 as an antioxidant in ultra-low doses

Abstract

The invention relates to the field of medicine, biotechnology, biology and agriculture and concerns a method of using superoxide dismutase 2 in ultra-low doses of from 10-12 to 10-200 as an activator of SOD2 gene expression, resulting in the systemic response of increased synthesis of native intracellular SOD and the effect of stimulating antioxidant defence, which manifests itself at a systemic level in terms of life expectancy, metabolic reactions and detoxification capacity.

Description

[0001] Method of using superoxide dismutase 2 as an antioxidant in ultra-low doses

[0002] Field of technology

[0003] The invention relates to the field of biotechnology, biology and agriculture and concerns the use of superoxide dismutase 2 (SOD2) as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of one's own intracellular SOD2 and the effects of stimulating antioxidant protection in ultra-low doses.

[0004] This drug can be effectively used to treat inflammatory, infectious, cardiovascular, oncological and neurodegenerative diseases.

[0005] In agriculture, the drug can be used in animal husbandry, increasing the immune resistance of animals. In plant growing, the drug can increase stress resistance to environmental factors, activate the yield of green mass and cause an increase in the yield of agricultural crops.

[0006] The drug can be used in the form of aqueous solutions, dry form for enteral use. In the form of sterile solutions for parenteral administration. In agriculture in the form of a dry substance for the preparation of aqueous solutions for irrigation and use in solutions for hydroponic cultivation of plants.

[0007] Superoxide dismutase (SOD, CCFF 1.15.1.1) belongs to the group of antioxidant enzymes. It protects the human body from constantly forming highly toxic oxygen radicals. Superoxide dismutase catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide. Consequently, the enzyme plays a major role in the antioxidant protection of many cells that are in contact with oxygen.

[0008] The enzyme is present in all aerobic organisms. In mammals, SOD2 is localized in the mitochondrial matrix. In humans, superoxide dismutase 2 is encoded by the SOD2 gene on chromosome 6.

[0009] State of the art

[0010] The use of superoxide dismutase as an antioxidant is widely described in the literature. However, nowhere in the literature is there a mention of the effect of ultra-low doses on living organisms.

[0011] The use of the enzyme superoxide dimutase in various industries is described.

[0012] Thus, the patent literature discloses the use of superoxide dismutase in medicine, cosmetology, agriculture, the food industry, etc.

[0013] Patent RU2600843, published on 27.10.2016, discloses a composition for the prevention or reduction of the risk of developing metabolic dysfunctions associated with obesity, containing Saccharomyces cerevisiae evarboulardii in an amount between 106 and 109 colony-forming units (CFU) per daily administration and the enzyme superoxide dismutase in an amount between 1 and 100 mg.

[0014] Patent RU2557894, published on 27.07.2015, discloses a cosmetic product for rapid skin restoration, containing superoxide dismutase, B vitamin, succinic acid, characterized in that it additionally contains a water-soluble analogue of vitamin E trolox and L-arginine, the activity of superoxide dismutase is 200,000-300,000 U / mg, the B vitamin is represented by dexpanthenol, the ratio of components, wt. %: superoxide dismutase 0.01-32, dexpanthenol 0.1-20, succinic acid 0.5 - up to pH 6.0, trolox 0.13-45, L-arginine 0.9-64, water for injection - the rest.

[0015] Patent RU2789458, published on 03.02.2023, describes a method for increasing the immune status of bee colonies infected with nosematosis, using a top dressing obtained by enriching sugar syrup 1:1 with a high-molecular antioxidant preparation SOD - superoxide dismutase + catalase + glutathione peroxidase, previously dissolved in water, intended for 3-fold top dressing with a frequency of 12 days with the preparation in dosages of 100 mg, 200 mg and 300 mg.

[0016] Patent GB2 183658, published June 10, 1987, describes a manganese-dependent superoxide dismutase, MnSOD (SOD2), which can be used to catalyze the reduction of superoxide radicals, reduce reperfusion injury, prolong the survival time of isolated organs, or treat inflammation.

[0017] Patent CN101420973, published on 29.04.2009, relates to compositions adapted for pharmaceutical administration, comprising at least one superoxide dismutase and at least one prolamin-based peptide fragment.

[0018] Disclosure of the essence of the invention.

[0019] Unexpectedly, the authors of the present invention have discovered the ability of the enzyme superoxide dismutase in ultra-low doses to be used as an activator of the expression of the SOD2 gene with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant protection, i.e. with the preservation of antioxidant properties even in a dilution of 10 -12 up to 10 -200. Thus, the present invention relates to a method for using superoxide dismutase 2 as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of one's own intracellular SOD and the effects of stimulating antioxidant protection, which consists in introducing superoxide dismutase 2 into a living organism in a dilution of 10 -12 up to 10 -200

[0020] The technical result of the present invention consists in the efficiency of using superoxide dismutase 2 as an activator of the expression of the SOD2 gene with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant protection, in ultra-low doses in a dilution of 10 -12 up to 10 -200 .

[0021] The mechanism of action of the drug in this dilution is based on the ability to restore and maintain the expression of the Superoxide dismutase 2 gene at an optimal level, thereby increasing the enzyme’s own intracellular synthesis, ensuring pronounced systemic biological reactions.

[0022] Recombinant human mitochondrial superoxide dismutase 2 (SOD2) protein (NM_00 1024465) MVPro, manufactured by OriGeneTechnologies Inc, USA, was used as superoxide dismutase 2. The dose used was 1.0 mg. The specified dose was dissolved in a 3 ml glass vial with a screw cap in 99 drops of ethyl alcohol. Primary dynamization of the matrix solution was performed by striking the vial clenched in a fist on a leather cushion 10 times. 1 ml of the matrix solution was collected and transferred to a 200 ml vial with a screw cap, dissolved in 99 ml of bidistilled water, mixed and shaken on the cushion 11 times. the process is repeated in new disposable vials until the entire spectrum of centesimal potencies from matrix to SP 200 (domestic potency C200) is obtained, each subsequent potency adds one hit to 40. Thus, the enzyme was obtained in the required dilution from 10 -12 up to 10 -200 .

[0023] Implementation of the invention

[0024] Experiments were conducted with dilution of superoxide dismutase 2, preparation B1 - dilution 10 -12 and the drug B2-10 -200 , test object of ceriodaphnia, species Ceriodaphnia dubia.

[0025] The object of the experiment is ceriodaphnia, species Ceriodaphnia dubia (Since 2016, this is the name given to a group of difficult to distinguish species of ceriodaphnia, which includes C. dubia, C. affinis and several other species. In all domestic methods, this species is called C. affinis, but according to WoRMS and the opinion of Doctor of Biological Sciences A.A. Kotov, it is correct to call it C. dubia.). They are smaller than daphnia, which complicates work with them, but their lifespan is shorter, only about 2 months, and therefore we were able to obtain results faster than when conducting experiments on daphnia, fish or mice.

[0026] Each line used 20 ceriodaphnia, individually placed in 10 ml penicillin vials.

[0027] The following lines participated in the experiment:

[0028] KO Clean control (with additions of clean water 2 times a week for uniformity of manipulations);

[0029] K Control with additions of saline solution 2 times a week for a month (for comparison with B 1, B2);

[0030] K+ Control with additions of saline solution 2 times a week throughout the experiment (for comparison with B1+, B2+); B1 Experimental replicates with additions of the B1 preparation 2 times a week for a month;

[0031] B2 Experimental repetitions with additions of the B2 preparation 2 times a week for a month;

[0032] B1+ Experimental repetitions with additions of the B1 preparation 2 times a week throughout the experiment (58 days);

[0033] B2+ Experimental repetitions with additions of the B2 preparation 2 times a week throughout the experiment (58 days).

[0034] Total: 7 lines of 20 penicillin vesicles = 140 penicillin vesicles = 140 ceriodaphnia.

[0035] The main indicators of changes in the organism's condition were the parameters of survival, fertility and lifespan compared to the control animals. These parameters were measured 2-3 times a week. Twice a week the medium in the experiment was replaced with a new one, and, accordingly, the preparation was added. The experiment continued until the death of the last ceriodaphnia in the observed samples. The total duration of the experiment was 58 days.

[0036] Results obtained during the experiment

[0037] Table 1. Changes in the number of Ceriodaphnia dubia (survival rate) in each of the studied lines.

[0038] Table 2. Change in the number of Ceriodaphnia dubia (in %) in each of the studied lines. During the first two weeks of the experiment, the best survival was observed in lines with B2 additives (95-100%) (Table 2). On the 31st day of the experiment, the number of crustaceans in all lines under study dropped to 50% and below (Table 2). At the same time, the survival in replicates with B1 and B2 additives was higher than with the addition of saline.

[0039] Table 3. Change in the number of Ceriodaphnia dubia (in %) in the studied lines up to 29 days of the experiment. Lines K and K+, B1 and B 1+ , B2 and B2+ are combined due to the unity of additives at this stage of the experiment.

[0040] On the 29th day of the experiment, the survival rate of C. Dubia with the addition of pure saline was higher than in the zero control without its addition (Table 3).

[0041] Table 4. Duration index of Ceriodaphnia dubia in the studied lines obtained during the experiment. Comparison with the zero control (K0) and individual controls (K and K+).

[0042] According to the average lifespan of Ceriodaphnia dubia in the studied lines, by the 30th day of the experiment we found a statistically significant stimulating effect of 13.7 and 12.4%, respectively, for preparations B 1 and B2 compared to the addition of pure saline solution.

[0043] The maximum lifespan of individuals throughout the experiment was 58 days for the B1+ preparation and 47 days for pure saline solution.

[0044] Thus, the effect of increasing the maximum duration of ceriodaphnia individuals in a 58-day experiment was discovered.

[0045] Table 5. Changes in the maximum lifespan of C. dubia in the studied lines.

[0046] Thus, we studied the preparations B1 and B2 using the ceriodaphnia dubiaB as a test object during 1- and 2-month experiments by adding them to the medium in the addition mode 2 times a week for 1 and 2 months.

[0047] According to the results of biotesting on the 29th day of the experiment, the effect on the survival of C. dubia crustaceans with the addition of the preparations B2 and B1 was expressed better than with the addition of pure saline solution.

[0048] In terms of the average lifespan of Ceriodaphnia dubia, a statistically significant stimulating effect of 13.7 and 12.4%, respectively, was found for preparations B1 and B2 in a 1-month experiment compared to the addition of pure saline.

[0049] It should be noted that for such an important general biological integral indicator as average life expectancy, the increase in the life expectancy of the crustaceans Ceriodaphnia dubia by 12-14% that we noted is very significant.

[0050] According to the indicator of the maximum lifespan of ceriodaphnia individuals in a 58-day experiment, the effect of increasing the maximum lifespan of individuals by 10 days was found for the B1+ preparation compared to pure saline solution.

[0051] The next experiment was carried out with microalgae.

[0052] B1 - superoxide dismutase 2 in 10 dilution -12

[0053] B2- superoxide dismutase 2 in 10 dilution -200

[0054] The test object of the study was an algologically pure culture of the green chlorococcal microalga Scenedesmusquadricauda (Tiger.)

[0055] Breb.(=Desmodesmuscommunis (E.Hegew.)E. Hegew.), widely distributed in fresh water bodies of the Southern and Northern Hemispheres and is an important link in their trophic chains.

[0056] S. quadricauda was obtained from the algae culture collection of the Department of Microbiology, Faculty of Biology, Lomonosov Moscow State University (DMMSU, strain S-3). This algae species belongs to coenobial organisms. 2- and 4-cell coenobia are most common, 8- and 16-cell coenobia are less common. During reproduction, autospores are formed in each cell, which are formed into a young colony inside the mother cell. This species is widely used in world practice in biotesting to assess the quality of the aquatic environment and the toxicity of various compounds and materials.

[0057] The culture was grown on Uspensky medium No. 1 (composition, g / l: 0.025 KNO3, 0.025 MgSO4, 0.1 KH2PO4, 0.025 Ca(NO3)2, 0.0345 K2CO3, 0.002 Fe2(SO4)3; pH 7.0-7.3) in a luminostat at an illumination of 3.5 klx with a day / night cycle (12:12 h), a temperature of 22±2°C and stirred 2 times a day to avoid cell sedimentation.

[0058] The test culture was pre-adapted to substances B 1 (the enzyme superoxide dismutase 22 in a dilution of 10-12 ) and B2 (the enzyme superoxide dismutase 2 in a dilution of 10 -200 . For this moment of sowing the NNAA culture, the nutrient medium with an initial quantity of 40 thousand cells / m l of the substance was added to the culture according to the following scheme:

[0059] B1 was added to flasks with the culture (50 ml volume) every working day (on days 0, 1, 2, 3, 4, 7, 8, 9, 10, 11, 14 of adaptation) at 0.1 ml.

[0060] B2 was added in the same volume, but twice a week, on Monday and Friday (0.4, 7, 11, 14 days of adaptation).

[0061] The data on the growth of the culture during the addition of substances B 1 and B 2 are presented in Table 6. Table 6. Change in the number of culture cells (in % of controls) upon the addition of substance B 1 and substance B2 during 14-day adaptation to substances B 1 and B2 (control - culture growth in a clean medium without the addition of substances)

[0062] As can be seen from Table 6, the growth of the culture during adaptation to substances B1 and B2 was practically no different from the control non-adapted culture during the entire 14-day adaptation period. At the same time, in all variants, including the control, the absolute number of cells in cells / ml increased approximately 60 times over 14 days of adaptation, from 40 thousand cells / ml (on the day of sowing) to 2.5 million cells / ml.

[0063] According to fluorescence microscopy data, the proportion of living cells in all variants and at all observation periods was at the control level and amounted to

[0064] 97-99%.

[0065] On the seventh day of adding the substances, stimulation of the efficiency of photosynthesis was recorded compared to the control by 10% for substance B2 and by 24% for substance B 1 (Table 7).

[0066] Table 7. Change in the photosynthesis index of culture cells (in % of controls) upon addition of substance B1 and substance B2 on the 7th day of culture growth (control - culture growth in a clean medium without the addition of substances)

[0067] Experiment with the reference toxicant potassium dichromate.

[0068] On the 14th day of adaptation, an inoculum was taken from the flasks with the pure control and the flasks with algae cells adapted to substances B1 and B2 to conduct the main part of the experiment with the toxicant. On the same day, an experiment was conducted to study the responses of the pure and B1 and B2-adapted Scenedesmus quadricaudan cultures. The standard toxicant was potassium dichromate. The toxicant was taken in concentrations of 0.1; 1 and 5 mg / l (low, medium and highly toxic concentrations) of potassium dichromate. The number of algae cells in each flask was 40 thousand cells / ml. The volume of the nutrient medium with the culture in each flask was 50 ml. For each studied concentration of the toxicant (0.1, 1 and 5 mg / l) and each type of culture (pure, adapted to B1, adapted to B2), three replicates were taken, a total of 27 flasks. Three flasks with pure culture without added toxicant were taken as a control. A total of 30 flasks.

[0069] Measurements were carried out on the 1st, 3rd, 7th, 10th and 14th days of the chronic experiment.

[0070] The studied parameters are the number of cells, the efficiency of photosynthesis and the proportion of living cells in the culture.

[0071] Table 8. Changes in the Qy photosynthesis index of a culture during growth on days 1 and 3 after the addition of the potassium dichromate toxicant in % of the control (control - photosynthesis of non-adapted cultures with the toxicant).

[0072] As can be seen from Table 8, significant stimulation of photosynthesis of the culture (by 15-76%) was noted for all cases of the experiment with different concentrations of the toxicant in the cultures adapted to substances B1 and B2 already on the first day of the experiment. At the same time, the stimulating effect was more pronounced in the cultures adapted to B2. Thus, the difference with the control in the cultures adapted to B2 with different concentrations of the toxicant was 41-76%. The controls in this case were photosynthesis in the presence of the corresponding concentrations of the toxicant in cultures not adapted to the substance. And the difference with the control in the cultures adapted to B 1 at different concentrations of the toxicant was 15-29%.

[0073] Thus, we have revealed the effect of decreasing toxicity at all studied concentrations of potassium dichromate in cultures adapted to substances B1 and B2 by the physiological indicator of the value of photosynthesis efficiency, which may indicate a rapid (already on the first day of the experiment) process of intensification of metabolic processes in cultures adapted to substances for detoxification of the heavy metal chromium (in the composition of potassium dichromate). In toxicology, this indicator allows us to identify an earlier response to toxic effects compared to the integral indicators of the number or survival of individuals that make up the population (in this case, the population of algae cells).

[0074] It is known that stimulation of the photosynthesis process can serve as an indicator of acceleration of the removal of excess toxic metal from the cell. This process is one of the important intracellular mechanisms of toxic metal detoxification.

[0075] Thus, preparations B1 and B2 have antioxidant properties, reducing the toxic effect of the heavy metal chromium and decreasing its toxicity for the studied test object at different levels of intoxication.

[0076] Experimentally, in a long-term experiment on a large number of generations of test object cells, a decrease in the toxic effect of potassium dichromate at different levels of its exposure under the influence of preparations B 1 and B2 was shown.

[0077] The following experiment was conducted on layers of the Lohmann White LSL cross aged 96-100 weeks. The experimental and control groups were formed according to the principle of pairs-analogues taking into account egg production and egg weight, the birds were divided into 2 groups of 41 heads each.

[0078] The chickens of the control and experimental groups received complete feed in accordance with the recommendations of the cross manufacturer. The birds of the experimental group received an experimental feed additive twice a week, which was the B2 preparation, i.e. superoxide dismutase 2 in a dilution of 10 -200 , applied to a grain of 38-41 pieces per 1 gram, and the birds of the control group received a placebo.

[0079] At the end of the experiment, 5 individuals from each group were euthanized. Liver and myocardial tissue samples were collected to assess superoxide dismutase gene expression.

[0080] Molecular genetic studies were performed.

[0081] Total RNA from the samples was isolated manually using the RNeasyMiniKit (QIAGEN, Germany) according to the protocol. Quantitative analysis of the isolated RNA was performed on a Qubit 3.0 fluorimeter (ThermoFisherScientific, USA) using the Qubit RNA HS (HighS ensitivity) AssayKit (ThermoFisherScientific, USA). cDNA synthesis from the RG matrix was performed using the iScriptcDNAsynthesiskit (Bio-Rad, USA) using a Gnome thermostat (DNA-Technology, Russia). Real-time polymerase chain reaction was performed on an iScriptcCycler 96 amplifier (Roche, Switzerland) using 96-well plates and using the PowerUp SYBR master mix.

[0082] GreenMasterMix (AppliedB iosystems, ThermoFisherScientific,

[0083] The temperature profile of the amplification reaction consisted of: 95°C for 10 minutes; 40 cycles at 95°C for 15 seconds, including annealing of primers at individual temperatures for 30 seconds and 72°C for 30 seconds. Each sample was tested in triplicate on a PCR plate. The amount of cDNA for each PCR reaction was 2 μl at a primer concentration of 0.32 μM. Calculation of SOD gene expression relative to the control gene (housekeeping gene) was performed manually using Microsoft Office Excel using the method. The obtained zootechnical and molecular genetic data were considered reliable at p<0.05.

[0084] Table 9. Expression of the superoxide dismutase 2 gene in the myocardium *p<0.05

[0085] From the data in Table 9, it can be concluded that the expression of the superoxide dismutase 2 gene in the myocardium in the experimental group is 4% higher than in the control group.

[0086] Table 10. Expression of the superoxide dismutase 2 gene in the liver From the data in Table 10, it can be concluded that the expression of the superoxide dismutase 2 gene in the liver parenchyma in the experimental group is 28% higher than in the control group.

[0087] Thus, as a result of the experiment, the following result was obtained: The expression of the superoxide dismutase 2 gene in the experimental group increased by 4% in myocardial tissue and by 28% in the liver parenchyma.

[0088] Thus, the use of superoxide dismutase 2 (SOD2) as an antioxidant in ultra-low doses, namely, in a dilution of 10 -12 up to 10-200 allows to restore and maintain at an optimal level the expression of the superoxide dismutase 2 gene, thereby increasing the enzyme’s own intracellular synthesis, ensuring pronounced systemic biological reactions.

[0089] Summarizing the above, the experiments conducted ALLOW us to state that superoxide dismutase 2 (SOD2) can be used as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant protection in any living organisms in ultra-low doses, namely, in a dilution of 10 -12 up to 10 -200

[0090] Consequently, the possibility of exerting external influence on the expression of superoxide dismutase genes in a relatively simple way, not associated with genetic engineering, has been proven, which opens up new prospects in biology, medicine, veterinary science, and plant growing.

Claims

Invention formula A method for using superoxide dismutase 2 (SOD2) as an activator of SOD2 gene expression with the development of subsequent systemic reactions of increasing the synthesis of its own intracellular SOD and the effects of stimulating antioxidant protection, which consists in introducing superoxide dismutase 2 into a living organism in a dilution of 10 -12 up to 10 -200 .

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