Pharmaceutical composition for treating African swine fever and its use

Abstract

The present invention provides a pharmaceutical composition for producing a safe amount of nitric oxide in the body, and uses thereof, which comprises an NO attenuator, an NO enhancer, and a nitric oxide synthase inducer. The pharmaceutical composition is highly versatile and highly effective in treating pathogenic microbial infections. The NO attenuator is selected from 5-methyltetrahydrofolic acid, NMN, and dehydroascorbic acid, the NO enhancer is selected from arginine, and the nitric oxide synthase inducer is selected from phytohemagglutinin. The composition provides a novel medicinal activity of 5-methyltetrahydrofolic acid, exerts various active effects on the immune system caused by pathogen infection, and can be used to treat or prevent viral infections and other diseases caused by pathogen infections. In particular, the composition can be used to prevent or treat African swine fever.

Description

[Technical field]

[0001] This application claims priority to a prior application bearing the patent number 201910719544.6 and entitled "Safe Nitric Oxide Composition and Use Thereof," filed with the State Intellectual Property Office of China on August 6, 2019, the entire contents of which are incorporated herein by reference. The present invention relates to the field of medicine, specifically to a pharmaceutical composition capable of producing nitric oxide in an animal body, which can provide a safe and sufficient amount of nitric oxide for the prevention and treatment of diseases. [Background technology]

[0002] African swine fever (ASF) is an acute, febrile, highly contagious, and fatal epizootic disease caused by the African swine fevervirus (ASFV). The main hosts of ASFV are various breeds of domestic pigs, African and Eurasian wild boars, and ticks, among which warthogs and wild boars are both infected but do not show clinical symptoms. African wild boars such as warthogs and ticks are hosts of ASFV. Depending on the virulence of ASFV, there are significant differences in the clinical symptoms and infection process of ASF, and the mortality rate of the most acute and acute infections is 100%.

[0003] There is no effective preventive vaccine for African swine fever, nor is there a specific treatment. Domestic pigs, wild boars and soft ticks at all stages are the natural hosts of African swine fever. It can be transmitted directly between domestic pigs and wild boars, or through tick bites, and can also be transmitted between countries and regions through food waste, feed and pork products such as dry-cured ham that are contaminated by the virus. When an outbreak is discovered, the pigs must be culled. It is one of the most serious infectious diseases that harm pigs in the world, and is the number one foreign animal disease that China focuses on in prevention.

[0004] In human history, new viruses have constantly emerged and known viruses have continued to mutate. Due to the lack of specific antibodies in the human body against new infectious viruses, large-scale infections have occurred quasi-periodically. Several influenza virus pandemics in humans have claimed many lives. In 2009, the H1N1 influenza virus was prevalent in the United States and Mexico, and in 2020, the COVID-19 virus has been prevalent worldwide. Depending on the individual, the same influenza virus can infect them, but the outcome is different, with some patients losing their lives while others have almost no symptoms. The virulence of the virus may differ, but the immune status of the host is also important.

[0005] When influenza virus infection needs to be prevented and treated, antibodies are the best tool, but influenza viruses evolve quickly, and the selective pressure of antibodies against seasonal influenza viruses can lead to escape mutants, which can cause epidemics in early strain immune communities, which is why seasonal influenza vaccines need to be constantly updated. Unfortunately, the specificity of antibody responses can also lead to influenza pandemics. In the last century, there have been multiple influenza virus or coronavirus epidemics, including A(H1N1), A(H2N2), A(H3N2), A(H1N1), SARS coronavirus, and COVID-19, in 1918, 1957, 1968, 2009, 2001, and 2020, respectively. Interestingly, during the above-mentioned influenza virus infection epidemics, the severity of the host varies greatly. According to one scholar, it has been proven that differences in the infection and activation of immune cells, especially T cells, result in differences in their ability to resist influenza viruses [Kelso,Anne.CD4+T cells limit the damage in influenza[J].Nature Medicine,2012,18(2):200-202.].

[0006] Although T cells can mediate cross-protective immunity and cannot prevent viral infection, there is evidence that they can sense infected cells by recognizing viral protein (epitope) fragments that are complexed with human leukocyte antigen (HLA) molecules on the surface of infected epithelial cells or antigen-presenting cells. Because T cells preferentially recognize epitopes derived from internal conserved proteins of the virus, cross-protective immunity is thought to be due to pre-existing cytotoxic CD8+ T cells, which kill virus-infected cells presenting these conserved epitopes, reducing the duration and severity of pandemic virus infection due to lack of antibody protection.

[0007] Due to the unique properties of African swine fever virus, it does not induce neutralizing antibodies in pigs recovering from infection to further resist the recurrence of the virus. The presence of non-neutralizing antibodies may cause an ADE (antibody-dependent enhancement) effect in African swine fever, i.e., the presence of antibodies promotes the pathological process of viral infection and associated diseases rather than preventing viral infection. At the same time, the presence of non-neutralizing antibodies has led to persistent antigen positivity in pigs immune-protected by attenuated vaccines. In the report [Dixon LK, Islam M, Nash R, et al. African swine fever virus evasion of host defences [J]. Virus research, 2019.], the authors described and analyzed the immune evasion strategy of African swine fever virus. SFV pA179 L Bcl-2 family protein binds to and inhibits several BH3-only domain proapoptotic proteins. pA224 L IAP family protein binds to and inhibits caspase 3, activates NF-kB signaling, and increases the expression of anti-apoptotic genes (including cFLIP, cIAP2, and c-rel). The existence of the above mechanisms allows African swine fever virus to inhibit apoptosis in infected cells and ensure continuous viral replication, while TNF-α induces apoptosis in uninfected lymphocytes. Acute ASFV disease is characterized mainly by massive apoptosis of B and T lymphocytes in lymphoid tissues and blood.

[0008] Due to the discovery of antibiotics, there are good clinical treatments for bacterial infections, but there are still no good treatments for viruses. Currently, the drugs for treating viruses are mainly divided into two categories: M2 ion channel blockers and neuraminidase inhibitors. M2 ion channel blockers have a general virus resistance effect and side effects on the nervous system, so their clinical application is not ideal. Although neuraminidase inhibitors can induce viruses, they have a weak effect. In recent years, a large number of viruses such as avian influenza virus, African swine fever virus, and SARS virus have become explosively prevalent, and the toxicological effects of these viruses are very serious, so that doctors cannot provide good treatments for patients or animals. Not only are there no good treatments for new viruses, but there are also no countermeasures for many viruses that have existed for a long time, including dengue virus and AIDS virus. The best way to treat viruses is prevention, that is, vaccines, which achieve the effect of preventing viruses through the human immune system. The above facts also demonstrate that the idea of ​​developing drugs that directly kill or inhibit pathogens, such as antibiotics, to treat viruses is actually twice the effort with half the results.

[0009] New ideas are needed for the development of antiviral drugs. The use of the human immune system to achieve the effect of treating viral infections is an important direction to realize versatile antiviral effects, especially for NO and immune-related drugs.

[0010] Nitric oxide gas is colorless and odorless, and soluble in water, alcohol, and fat. Before the 1980s of the last century, nitric oxide was just a normal and unnecessary chemical gas, and was only known to exist in automobile exhaust and gas pollutants from certain chemical processes. 27 years before 1980, a substance produced by endothelial cells (called "endothelium-derived relaxing factor") was discovered, and in 1986, the first experimental paper submitted by Ignarro claimed that endothelium-derived relaxing factor (EDRF) is nitric oxide. These results triggered a great deal of interest in and research into NO. NO can enter and leave cells quickly, transmit signals to regulate vasodilation, neurotransmission, brain development, and even learning and memory, strengthen the immune system, kill some foreign microorganisms, lower blood pressure, and prevent stroke, heart disease, tumors, and senile dementia.

[0011] NO is produced by the reduction of L-arginine with NADPH catalyzed by nitric oxide synthase (NOS), which is divided into endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS). They are involved in the regulation of the cardio-cerebrovascular system, immune regulation, and nervous system, respectively, in different human tissue cells.

[0012] NO, which is involved in immunity, is produced by various immune cells (dendritic cells, NK cells, macrophages, eosinophils and neutrophils), and when iNOS is expressed, a large amount of NO is generated, which becomes an active defense mechanism in humans. There is evidence that NO can suppress virus copying, and the related mechanisms include reducing palmitoylation of viral spike proteins, inhibiting viral proteases, and inhibiting the synthesis of viral proteins and nucleic acids.

[0013] Nitric oxide synthase is a dimer that uncouples under oxidative conditions, converting the reaction pathway that normally synthesizes NO to O 2 -, NO 3 - (PON) and other reactive oxygen radicals (ROS). NO itself can also react with reactive oxygen radicals (ROS) to generate reactive nitrogen species (RNS).

[0014] NO reacts rapidly with superoxide anion in the body to produce peroxynitrite. Under acidic conditions, it decomposes rapidly to produce hydroxyl radicals. Peroxynitrite is a highly oxidizing substance, which can lead to protein nitration and DNA strand breaks. Due to various causes, many oxidizing radicals containing both active oxygen and active nitrogen are produced in the body. The presence of these radicals has destroyed the previous balance to some extent. Of these radicals, the one with the greatest impact is the peroxynitrite anion (PON). Its production pathway is mainly obtained by the reaction between nitric oxide and superoxide anion.

[0015] PON has mostly negative effects in the human body, including but not limited to: 1. Oxidation: PON itself is a strong oxidant and is rapidly decomposed into nitrogen dioxide and hydroxyl radicals under acidic conditions. Hydroxyl radicals are even stronger oxidants and can oxidize and decompose almost all organic matter. In vivo, PON reacts with iron / sulfur centers, mercapto, lipids, etc. in many enzymes, proteins, and cytokines, causing oxidative damage, resulting in damage to cell function and apoptosis, and further reducing the radical scavenging mechanism by glutathione, resulting in a vicious cycle. The oxidative action of PON can cause various diseases, such as acute and chronic inflammation, sepsis, traumatic local ischemia, arteriosclerosis, and neuroregenerative dysfunction. 2. Nitration: PON reacts with tyrosine in proteins to generate nitrotyrosine, which can affect protein function and result in DNA breakage. 3. Effects on energy metabolism: The activity of thymoproteins is reduced by oxidation and nitration. For example, the activity of mitochondrial ATP synthase and aconitase is inhibited, resulting in a decrease in energy. PON is a strong activator of poly ADP-ribose synthase. When activated, this enzyme initiates an ineffective repair cycle, leading to rapid depletion of energy cells. Cell metabolism and membrane integrity are disrupted, causing cell death. 4. Interference with calcium transport: Na + / Ca2+ The sulfhydryl groups of exchange proteins are oxidized, causing dysfunction, leading to intracellular calcium overload and resulting in dysfunction. Of course, even tolerable doses of PON show positive effects, such as resisting harm to the human body caused by viruses, bacteria, pathogens, cancer cells, etc.

[0016] NO, the star molecule of 1992, is actually present in various parts of the body. NO is a messenger of the immune system, plays an important role in regulating blood flow, neurotransmission, and brain development, and can kill pathogens, viruses, pathogenic parasites, and cancer cells, making it a very important component of non-specific immunity. Foreign microorganisms or abnormal cells killed by NO self-decompose and then release a large amount of antigenic substances, initiating specific immunity. NO also causes the body to release many cytokines, such as interleukins, interferons, tumor necrosis factors (TNF), and colony-stimulating factors (CSF), to regulate immune responses.

[0017] NO reacts with radicals such as superoxide anion to produce peroxynitrite (PON), which has strong oxidizing properties and special nitration ability. When accumulated to a certain extent, it will cause inflammation and release cytokines that affect the pathological process. PON destroys protein function through protein nitration, breaks DNA, promotes viral mutations, destroys immune balance, activates proto-oncogenes, and induces cancer.

[0018] NO is involved in immune regulation. The acute inflammatory response is a complex and highly orchestrated sequence of events involving molecular, cellular and physiological changes, and when the host fails to respond to infection, further abnormal immune responses occur, resulting in a syndrome of organ dysfunction, i.e., sepsis. Research into the treatment of sepsis reflects progress in human understanding of pathophysiology and host-microbe interactions. With an early focus on microbes and their pathogenicity, and with the implementation of molecular cloning and sequencing of human inflammatory genes in the 1980s, research into sepsis has focused more on the host's response to invading pathogens.

[0019] According to the Third International Consensus Definitions for Sepsis in 2016, pyemia is defined as a life-threatening organ dysfunction due to a compromised host response to infection, with clinical symptoms of fever, tachypnea, altered level of consciousness, and hypotension, along with associated symptoms of the disease, such as pneumonia due to pulmonary infection, kidney infection, and urinary tract infection.

[0020] Although there is a much greater understanding of the origin and development of pyemia, the mortality rate from pyemia remains very high, and according to the article [Hotchkiss RS, Moldawer LL, Opal SM, et al. Sepsis and septic shock [J]. Nature reviews Disease primers, 2016, 2(1):1-21.], initial estimates based on data from high-income countries indicate that there may be 31.5 million cases of pyemia and 19.4 million cases of severe pyemia occurring annually worldwide, with 5.3 million deaths annually. In many cases, especially in patients with chronic diseases (e.g. cancer, congestive heart failure, and chronic obstructive pulmonary disease), official death records generally report the underlying disease rather than the immediate cause of death (pyemia), which may result in a significant underestimation of the mortality rate from pyemia. Furthermore, due to the lack of relevant incidence and mortality records from pyemia in low- and middle-income countries, these values ​​are only estimates.

[0021] Inflammation is a host defense response against invading pathogens, so using antibiotics or antiviral drugs to reduce the foreign stimulation of pathogen antigens is a treatment method that is clinically prioritized to eliminate pathogens. When viral infectious diseases progress to the stage of immune dysfunction, severe inflammation occurs. Some therapies that stop or resist inflammation reduce the number of macrophages in the inflamed area, so it is not always clear whether to improve or reduce the immune response. Common anti-inflammatory drugs include nonsteroidal anti-inflammatory drugs, glucocorticoids, etc. When severe inflammation occurs, glucocorticoids are generally used clinically, but the use of cortisol for pyemia has no substantial benefit. A randomized controlled trial [Annane D, Cariou A, Maxime V, et al. Corticosteroid treatment and intensive insulin therapy for septic shock in adults: a randomized controlled trial[J]. Jama, 2010, 303(4):341-348.] shows that fludrocortisone does not reduce mortality in patients with pyemia. A subsequent selective analysis [Wang C, Sun J, Zheng J, et al. Low-dose hydrocortisone therapy attenuates septic shock in adult patients but does not reduce 28-day mortality: a meta-analysis of randomized controlled trials[J]. Anesthesia & Analgesia, 2014, 118(2):346-357.] also shows that hydrocortisone does not reduce mortality in patients with severe infection or pyemia. Therefore, the use of steroids in patients with severe infection is currently controversial.

[0022] In the past 20 years, attempts have been made to clarify the relationship between vitamin C and pyemia. Patients with pyemia generally have very low serum vitamin C levels, and low vitamin C levels in critically ill patients are thought to be related to vascular hypertension, kidney damage, multiple organ dysfunction and increased mortality. Research into the mechanism of action of vitamin C has revealed a variety of mechanisms that may affect pyemia, including antioxidant, anti-inflammatory, microcirculatory, antithrombotic, increased adrenal sensitivity, and accelerated wound healing. However, unexpectedly, the clinical use of vitamin C has not shown any significant effect, and statistics from [Chang Xue● (● is a female radical with a ni), Li Min, Zhang Zhengxin et al. Meta-analysis of the effect of vitamin C in the treatment of patients with pyemia and pyogenic shock [J]. Chinese Journal of Critical Care Medicine (Electronic Edition), 2019, 012 (001): 37-41.] show that intravenous infusion of vitamin C cannot improve the mortality rate of patients with pyemia and pyogenic shock.

[0023] 5-Methyltetrahydrofolate is the active form of folic acid in the human body, and does not have a direct antiviral effect. At present, the direct relationship between folic acid and viruses is mainly through folate receptor alpha (FRalpha), which has been described as a factor mediating the entry of viruses, including Ebola, into cells. It is known that 5-methyltetrahydrofolate has a direct antioxidant effect and promotes the conversion of BH2 to BH4 through the action of dihydrofolate reductase, and BH4 is a necessary cofactor for eNOS. It has already been proven that 5-methyltetrahydrofolate is advantageous for the prevention and protection of cardiovascular diseases by promoting eNOS, but there have been few studies and reports on the effects of 5-methyltetrahydrofolate on iNOS and NO secretion by macrophages under conditions of innate immune activation.

[0024] L-arginine is a precursor for the endogenous synthesis of NO, which reacts with nitric oxide synthase to produce NO and L-citrulline; a small portion of L-arginine is metabolized in the body through this pathway, but in acute inflammation, the amount of NO produced by iNOS in macrophages far exceeds the normal amount in humans. L-arginine is a non-essential amino acid, and is endogenously synthesized in the metabolic pathway of proline, glutamine, or glutamic acid (through the whole-body proteolytic process); in the kidney, citrulline is converted to arginine by the action of arginine succinate synthase and arginine succinate degrading enzyme, but it is very important under different pathophysiological conditions when the endogenous synthesis of arginine cannot meet the metabolic needs of the body. Summary of the Invention

[0025] According to the present invention, it has been found that 5-methyltetrahydrofolic acid has different physiological activity at "pharmacological" concentrations than at "nutritional support" low concentrations, and compositions containing 5-methyltetrahydrofolic acid have the effect of treating viral infections, and have been further found to have therapeutic effects against any of a variety of different pathogens, including bacteria, fungi, etc. According to the present invention, it has been further found that the activity of dehydroascorbic acid and NMN is similar to that of 5-methyltetrahydrofolic acid.

[0026] Based on the above findings, the present invention provides the following technical solutions: A pharmaceutical composition capable of producing a safe amount of nitric oxide in an animal body, i.e., controlling or reducing the proportion of reactive nitrogen in the body, and capable of producing an amount of nitric oxide in the body necessary for the prevention and treatment of diseases.

[0027] The pharmaceutical composition of the present invention comprises an NO attenuator selected from antioxidants that scavenge peroxynitrite or its salts (PON) at a dosage, and an optional NO enhancer. Preferably, the attenuator does not suppress the expression of inducible nitric oxide synthase (iNOS) at a concentration of 10 μmol / L or more, for example, does not suppress the expression of iNOS in macrophages induced by LSP.

[0028] The NO attenuating agent of the present invention is selected from antioxidants that do not affect the activation of iNOS synthase and selectively quench peroxynitrite, such as one or more selected from 5-methyltetrahydrofolic acid or its salts, dehydroascorbic acid, and NMN.

[0029] The NO enhancer of the present invention is selected from an enzyme-derived NO substrate selected from L-arginine or a salt thereof, citrulline or a salt thereof, or an arginine-activating additive.

[0030] The pharmaceutical composition of the present invention comprises 5-methyltetrahydrofolic acid or a salt thereof and arginine or a salt thereof, and may further comprise phytohemagglutinin.

[0031] In the pharmaceutical composition of the present invention, the 5-methyltetrahydrofolic acid is present in a single dose of 15 mg or more, and the arginine is present in a single dose of 50 mg.

[0032] The present invention further provides the use of the above-mentioned pharmaceutical composition for preparing a medicament for preventing or treating a disease caused by a pathogenic microbial infection. Preferably, the pathogenic microbial infection is a viral infection.

[0033] The pharmaceutical composition of the present invention can be used to increase the levels of T cells, especially CD4 and CD8 T cells, and reduce the expression of inflammatory factors in virus-infected hosts, and can be used for anti-viral infection.

[0034] According to the use of the pharmaceutical composition of the present invention, the virus is influenza virus, herpes virus, African swine fever virus, coronavirus such as COVID-19.

[0035] In particular, the present invention provides a pharmaceutical composition for preventing and treating swine fever, comprising an NO attenuator and an NO enhancer, wherein the NO attenuator is one or more selected from 5-methyltetrahydrofolic acid or its salt, dehydroascorbic acid, and NMN, and the NO enhancer is one or more selected from arginine, citrulline, or arginine activating additive.

[0036] The composition for treating swine fever according to the present invention includes 5-methyltetrahydrofolic acid and arginine, and may further include phytohemagglutinin, the mass ratio of the three being 2:8:1.

[0037] According to the composition for treating swine fever of the present invention, the swine fever is African swine fever.

[0038] According to the composition for treating classical swine fever of the present invention, the composition can enhance the level of T cells, especially CD4 and CD8 T cells, and reduce the expression of inflammatory factors in the virus-infected host, and can be used for anti-viral infection.

[0039] According to the composition for treating swine fever of the present invention, the composition contains at least 30 mg / kilogram of active ingredient per dose, for example 50 mg / kilogram. According to the use of the pharmaceutical composition of the present invention, the composition is used to prepare a drug for preventing and treating pyemia and systemic inflammatory response syndrome caused by infection.

[0040] The pharmaceutical composition according to the present invention contains 5-methyltetrahydrofolic acid or a salt thereof and vitamin C. Preferably, the mass ratio of calcium 5-methyltetrahydrofolate to vitamin C is 2:1 to 5:1, for example, 3:1, 4:1.

[0041] The present invention further provides the use of the aforementioned pharmaceutical composition for preparing a medicament for treating systemic inflammatory response syndrome, pyemia caused by non-infectious causes.

[0042] According to the use of the pharmaceutical composition of the present invention, said pyemia is caused by Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, or influenza virus infection.

[0043] According to the pharmaceutical composition of the present invention, it may be prepared from an active ingredient and a pharma- ceutically acceptable auxiliary ingredient, for example, the pharmaceutical formulation may be selected from tablets, capsules, granules, injections, external patches or sprays.

[0044] The pharmaceutical composition according to the present invention is an immune adjuvant.

[0045] In the present invention, a safe amount of nitric oxide means that the ratio of nitric oxide converted into toxic radicals such as peroxynitrite and active nitrogen can be controlled so as to satisfy the need for safety in preventing and treating diseases using nitric oxide. These radicals greatly affect the metabolism of substances and energy in the body, affect the functions of cells and tissues, and even destroy the functions of cells and tissues, significantly increasing the probability of gene mutation, and causing the occurrence of many diseases.

[0046] By controlling toxic radicals, the composition of the present invention effectively increases the production of nitric oxide to meet the needs of disease prevention and treatment.

[0047] In the present invention, the pharmaceutical composition that produces a safe amount of nitric oxide has the prospect of being applied to the treatment of various diseases. The composition of the present invention can promote the proliferation and activation of T cells, increase the levels of CD4 and CD8 cells in the host during infection, block the apoptosis of CD4 and CD8 T cells, significantly improve the survival rate of the host, and improve the inflammatory response during infection.

[0048] In the present invention, a composition containing 5-methyltetrahydrofolic acid and arginine is administered to mice infected with influenza virus, resulting in a high cure rate and significantly shortening the course of the disease.

[0049] The main function of folic acid is carbon transfer, which is involved in DNA methylation, the synthesis of purines and thymine, and further synthesizes DNA and RNA. Viruses have a DNA or RNA structure, and it is thought that by replicating in large quantities in host cells and supplying sufficient folic acid, they should contribute to virus replication and transmission. However, unexpectedly, experimental results show that 5-methyltetrahydrofolic acid, in cooperation with nitric oxide increasers, conversely suppresses viruses. The present invention first proposes applying compositions such as 5-methyltetrahydrofolic acid and arginine to microbial infections, particularly virus infections.

[0050] iNOS is a key enzyme that generates NO in the immune system. It is known from prior art that oxidation of this enzyme causes dimer decoupling and converts the reaction pathway for generating NO into a reaction pathway for generating radicals and reactive nitrogen species. 5-methyltetrahydrofolic acid is an endogenous antioxidant that can activate NADPH, achieve a good antioxidant effect, and has a direct antioxidant action. According to the composition of the present invention, in a living body infected with a pathogen, it is possible to produce NO and not produce radicals that are harmful to the living body, including reactive oxygen species (ROS) and reactive nitrogen species (RNS). According to the present invention, antioxidants containing 5-methyltetrahydrofolic acid or its salts, dehydroascorbic acid, and NMN can remove peroxynitrite without affecting the function of iNOS expression.

[0051] The present invention provides a method for producing a sufficient amount of nitric oxide in the body. The composition of the present invention suppresses the generation of peroxynitrite, induces improvement without suppressing the activity of nitric oxide synthase, and further increases arginine, an enzyme-derived nitric oxide substrate, and its precursors, thereby producing a sufficient amount of nitric oxide in the body.

[0052] In the present invention, the concept of a sufficient amount means achieving or exceeding the minimum dosage of nitric oxide required for disease prevention and treatment.

[0053] The present invention provides a systematic method for providing sufficient amounts of nitric oxide, which can be selected and optimized as needed. In order to enhance the production of nitric oxide, the composition may contain a nitric oxide synthase inducer, such as phytohemagglutinin (PHA). Phytohemagglutinin (PHA) is a mitogen and an efficient and safe nitric oxide synthase inducer, which has already been produced on a large scale by extraction techniques from legumes. Another object of the present invention is to provide various uses of the above-mentioned safe nitric oxide composition.

[0054] The active ingredient in the composition of the present invention includes 5-methyltetrahydrofolic acid or a salt thereof, the salt being selected from, but not limited to, calcium salt, arginine salt, glucosamine salt, and sodium salt.

[0055] In one preferred embodiment, the amount of 5-methyltetrahydrofolic acid or a salt thereof in a single dose of the composition of the present invention is 15 mg or more, preferably 25 mg or more, more preferably 50 to 1000 mg.

[0056] In one embodiment, the composition contains 5-methyltetrahydrofolic acid or a salt thereof, or dehydroascorbic acid or NMN, and arginine. The amount of 5-methyltetrahydrofolic acid or a salt thereof contained in the composition per dose is 15 mg or more, preferably 25 mg or more, preferably 50 to 1000 mg, more preferably 50 to 500 mg. For example, the amount of arginine is 50 to 5000 mg, preferably 100 to 1000 mg.

[0057] In one embodiment, the composition includes 5-methyltetrahydrofolic acid or a salt thereof, arginine, and phytohemagglutinin PHA. The amount of 5-methyltetrahydrofolic acid or a salt thereof contained in the composition per unit dose is 15 mg or more (in terms of 5-methyltetrahydrofolic acid), preferably 25 mg or more, preferably 50 to 1000 milligrams, and more preferably 50 to 500 milligrams. The amount of arginine in the composition per unit dose is 50 to 5000 milligrams, preferably 100 to 1000 milligrams. The amount of phytohemagglutinin in the composition per unit dose is 10 to 500 milligrams, preferably 20 to 100 milligrams.

[0058] The pharmaceutical formulation may be selected from tablets, capsules, granules, injections, external poultices or gas formulations. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0059] NO-induced stabilization of HIF-1α and phosphorylated p53 levels are reduced by reactive oxygen species [Thomas DD, Ridnour LA, Espey MG, et al. Superoxide fluxes limit nitric oxide-induced signaling. J Biol Chem. 2006;281(36):25984-25993.] Indeed, the addition of antioxidants has a protective effect against nitrosative signaling [Edirisinghe I, Arunachalam G, Wong C, et al.Cigarette-smoke-induced oxidative / nitrosative stress impairs VEGF- and fluid-shear-stress-mediated signaling in endothelial cells [retracted in:Rahman I.Antioxid Redox Signal.2013 Apr 2018(12):1535].Antioxid Redox Signal.2010;12(12):1355-1369.]. Therefore, NO levels and subsequent downstream signaling are regulated by ROS, which is also a factor in regulating redox signaling.

[0060] The expression of iNOS requires simultaneous activation of STAT and NF-κB, and NF-κB acts as a master switch for inflammation and is involved in H 2 O 2 The production of iNOS is regulated by NF-κB through redox regulation. Most reducing agents or antioxidants have anti-inflammatory effects to some extent, and can inhibit the expression of iNOS by inhibiting the NF-κB pathway. In one embodiment, the effects of different antioxidants on the expression of iNOS in macrophages induced by LPS were compared, and 5-methyltetrahydrofolate, dehydroascorbic acid, BH 4It is shown that , glutathione, and nicotinamide mononucleotide have little effect on the expression of iNOS at a concentration of 10 μmol / L. Considering the reactivity of the above-mentioned antioxidants to peroxynitrite, it is shown that 5-methyltetrahydrofolic acid, dehydroascorbic acid, and NMN all have high peroxynitrite removal ability. It has already been proven that under hypoxic conditions, the immune function of lymphocytes is suppressed and the apoptosis rate increases. The lack of active oxygen inhibits the synthesis of iNOS, destroys the bond between iNOS and α-actinin4, and prevents iNOS from attaching to the actin cytoskeleton. Therefore, the presence of antioxidants may down-regulate iNOS. However, the present invention has found that the antioxidants 5-methyltetrahydrofolic acid, dehydroascorbic acid, and NMN have unique properties and have good peroxynitrite removal ability without reducing the expression of iNOS at a certain concentration. All the above antioxidants do not reduce the immune response ability after immune activation by antigens, especially do not adversely affect the expression of iNOS during the infection process, and reduce the production of peroxynitrite. NO has the effect of inhibiting cell apoptosis, inhibiting caspases-8, caspases-9 or caspases-3 through S-nitrosation, while peroxynitrite promotes cell apoptosis through DNA damage and upregulation of p53.

[0061] NO has direct and indirect effects on infectious microorganisms, NO can directly destroy the enzyme structure of pathogenic microorganisms, especially the [Fe-S] cluster, and in viral infection, the expression of NO can inhibit the enzyme activity of the virus and inhibit the virus copy. The direct toxicity of NO, especially the extracellular antiviral activity, has been well proven, but the indirect regulatory effect of NO on immune function is quite complicated. Research has demonstrated that iNOS-deficient mice infected with influenza virus have no histopathological evidence of pneumonia, so the scholar believes that host iNOS contributes more to pneumonia than viral copy [Karupiah G, Chen JH, Mahalingam S, Nathan CF, MacMicking JD.Rapid interferon gamma-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice.J Exp Med.1998;188(8):1541-1546.]. In endotoxemia, early results of treatment with iNOS inhibitors in preclinical models were disappointing [Hauser B, Bracht H, Matejovic M, et al.Nitric oxide synthase inhibition in sepsis? Lessons learned from large-animal studies[J].Anesthesia & Analgesia, 2005, 101(2):488-498.]. Beneficial and detrimental effects have been described, and it is unclear whether NO is positive or negative for infection.

[0062] It was found that exogenous NO inhibits the proliferation of T lymphocytes, and exogenous NO (i.e., not NO itself produced by T cells) inhibits proliferation and even causes T cell death [Bogdan C. Regulation of lymphocytes by nitric oxide. [J]. Methods Mol Biol, 2011, 677: 375-393.]. Mice lacking key antioxidant mechanisms (i.e., GSNOR) show a marked deficiency of T and B cells in the periphery due to excessive S-nitrosation and lymphocyte apoptosis. Meanwhile, a small amount of NO-branched T cell subsets, especially Th1 cells and FoxP3 negative regulatory T cell population, can effectively suppress the differentiation of Th17 cells. Recent studies have also shown that exogenous NO regulates Th9 and Th17 cells.

[0063] In one embodiment of the present invention, we found that 5-methyltetrahydrofolic acid at a concentration of 15.625 μm in cell culture medium had little effect on NO secretion by macrophages, but more interestingly, in the absence of LPS stimulation, 5-methyltetrahydrofolic acid was found to be able to promote NO secretion at low concentrations.

[0064] It is shown that the combination of the NO attenuator and NO enhancer selected in the present invention can significantly improve the proliferation activity of antigen-stimulated CD4+ T cells. Previous studies have shown that while virus clearance is mediated by antigen-specific CD8+ effector T cells, memory CD4+ T cells play an important role in maintaining the memory response of CD8+ T and B cells [Stambas J, Guillonneau C, Kedzierska K, et al. Killer T cells in influenza [J]. Pharmacology & therapeutics, 2008, 120(2):186-196.]. Recent studies have also shown that both CD4+ and CD8+ T cells are involved in the control of pneumonia and limit excessive tissue damage by producing interleukin-10. Therefore, the pharmaceutical composition containing the above-mentioned NO attenuator and NO enhancer in the present invention can be used for virus clearance and anti-inflammatory treatment.

[0065] In the present invention, arginine, as an NO enhancer, has shown unexpected antiviral and pyometabolic disease treatment effects when used together with 5-methyltetrahydrofolic acid. In one embodiment, the composition of the present invention can significantly stimulate the proliferation of T cells in the thymus and spleen of mice, and compared with the addition of arginine alone, the combination of arginine and 5-methyltetrahydrofolic acid significantly enhances the proliferation of CD4+ T cells, demonstrating that the composition can improve the proliferation ability of effector CD4+ T cells. As described in the Background Art, virus-specific memory CD4 + The number of T cells can predict the severity of human infection by influenza virus, and the number of virus-specific T cells is inversely proportional to the severity of the disease. Therefore, the composition of the present invention has the prospect of treating influenza virus infection and can reduce the severity of the pathology. It is known that peroxynitrite affects the immune response of cells, and research supports that peroxynitrite inhibits the feedback ability to suppress inflammatory response and repair, and is prone to cause immune dysfunction of the host during infection. The composition used can not only improve the immune capacity of the host, but also maintain the negative feedback mechanism of inflammation and protect the host from infection, especially viral infection.

[0066] Most of the traditional viral cold medicines are used to relieve symptoms and reduce the pain of cold, but they cannot reliably and significantly shorten the course of the disease. The above-mentioned pharmaceutical composition of the present invention has a revolutionary effect on the treatment of cold. It has a fast effect, and after re-examination of more than 40 subjects, the cold symptoms generally disappeared within 48 hours after the composition was administered. Although no double-blind controlled clinical trial has been conducted, the feedback results of the relevant trial composition are also unexpected.

[0067] Furthermore, the present invention has verified the anti-viral infection effect of the composition in an animal model, which shows that the composition can protect the immune function of mice, alleviate the pathological state of pulmonary infection by influenza virus, and reduce lung tissue damage. The composition of 5-methyltetrahydrofolic acid and arginine can significantly reduce the level of inflammatory factors caused by infection and significantly reduce the viral titer in the lungs 5 ​​days after infection, suggesting that the composition has a certain anti-viral effect. In addition, the use of the composition can significantly increase the CD4 count in the spleen and thymus of infected mice. + and CD8 + The level of T cells can be significantly improved, suggesting that the composition reduces inflammatory factors but does not reduce the host's immunity. The results of lung tissue sections show that the composition can reduce lung tissue damage and inflammatory conditions, and shows very good therapeutic effect in the host model against cold virus.

[0068] Recent studies have shown that NO can promote immune synapse (IS) signals mediated by T cell receptor (TCR) [Garcia-Ortiz A, Martin-Cofreces NB, Ibiza S, et al. eNOS S-nitrosylates β-actin on Cys374 and regulates PKC-θ at the immune synapse by impairing actin binding to profilin-1[J]. PLoS biology, 2017, 15(4):e2000653.]. IS is very important in regulating T cell activation, secretion and immune signal communication between cells, which may also be the reason why the composition can significantly improve T cell numbers.

[0069] In one embodiment of the present invention, the composition was applied to the treatment of pigs infected with African swine fever virus, and a very good effect was obtained, significantly improving the survival rate of pigs infected with African swine fever virus, further proving the anti-viral prospects of the composition.

[0070] In addition, the present invention has found that the above-mentioned composition of the present invention can significantly protect the survival of the host in a high-dose virus challenge experiment, and shows promise for the treatment of certain types of pyemia.

[0071] In the past three decades, more than 100 Phase II and III clinical trials have been conducted to test various new drugs and therapeutic interventions, hoping to improve the prognosis of patients with severe pyemia and septic shock. However, all these efforts have failed to produce new drugs that can reduce organ failure and improve the survival rate of patients with pyemia [Artenstein AW, Higgins TL, Opal SM. Sepsis and scientific revolutions. Crit Care Med. 2013; 41 (12): 2770-2772.]. All these studies have used single drugs of a specific molecule or pathway, and it is not easy to select drugs with such a concept, because it involves a very complex immunometabolic pathway and more than a thousand possible targets.

[0072] Supplementation of exogenous arginine is controversial in the treatment of pyemia. NO-mediated peroxidation is important in the pathogenesis of pyemia. It is assumed that a pharmacological blockade of the NO production process can treat pyemia, so NOS synthase inhibitors have been developed, but clinical results show that NOS-suppressing therapy is generally not beneficial. On the other hand, arginine levels in patients with pyemia are reduced, but this may have a negative effect of increasing oxidative stress due to an increase in endogenous donors of NO. The combination of 5-methyltetrahydrofolic acid and arginine in the composition of the present invention unexpectedly provides very good therapeutic effects in preclinical animal models.

[0073] In the present invention, we found that 5-methyltetrahydrofolic acid can significantly reduce the mortality rate of LPS-induced pyomechanical mice, suggesting that 5-methyltetrahydrofolic acid may be beneficial in the treatment of severe allergic pyomechanism.

[0074] In the present invention, it was found that a composition of 5-methyltetrahydrofolic acid and arginine significantly reduced the mortality rate of mice with pyemia caused by infection with microorganisms (e.g., Staphylococcus aureus). Pyemia is a highly lethal disease characterized by extensive cell apoptosis induced immune cell depletion and subsequent immunosuppression. In the present invention, the combination of 5-methyltetrahydrofolic acid and arginine significantly improved the survival rate of the host and blocked the apoptosis of CD4 and CD8 T cells, and the therapeutic effect of the composition was demonstrated in a variety of bacterial and viral pyemia models.

[0075] It should be understood that in the human body, certain oxidative signals are beneficial to infectious diseases, and a delicate balance exists between protective oxidative signals and the harmful effects of reactive oxygen species. The antioxidants used in the compositions of the present invention have unique properties, and can alleviate the symptoms of pathogenic microbial infection while increasing the level of NO, so that the pathogen killing effect mediated by NO can overcome the oxidative stress mechanism induced by NO.

[0076] Indeed, T cells play an important role in cross-protection, and in the field of vaccines, inactivated virus vaccines provide protection by inducing specific antibodies against a certain virus, and have little effect on enhancing the cross-protective T cell response. The composition of the present invention has the effect of enhancing the immune response, and in one embodiment, use of the composition significantly increases the antibody levels in immunized animals following rabies vaccination.

[0077] The present invention utilizes the immune system of the human and animal body, which is a natural and powerful antiviral tool. African swine fever is a severe infectious disease with a mortality rate of 95% to 100%, and nothing can be done with conventional techniques, but by using the present invention, a clear therapeutic effect is shown, indicating that the composition has an unexpected antiviral infection ability. [Oura, CA L. In vivo depletion of CD8+T lymphocytes abrogates protective immunity to African swine fever virus [J]. Journal of General Virology, 2005, 86 (9): 2445-2450.] reported an attenuated virus isolate OUR / T88 / 3, and when this virus is used as a vaccine, infection with the avirulent ASFV isolate OUR / T88 / 3 can protect distantly related pigs from attack by the highly virulent Portuguese ASFV isolate OUR / T88 / 1. However, pigs exposed to OUR / T88 / 3 and depleted of CD8(+) lymphocytes were not fully protected from challenge with OUR / T88 / 1, indicating that CD8+ lymphocytes play an important role in the protective immune response to ASFV infection.

[0078] In recent years, the understanding of the effect of NO on immunity has progressed greatly, and in addition to direct inhibition of pathogenic microorganisms, it also widely regulates immune function. In addition to iNOS, eNOS is also required as a source of NO in immune conditions. Therefore, it is recognized that the inhibition or activation of NOS subclasses is difficult to apply to clinical practice due to the supplementary understanding of the various functions and targets of NO. However, the results of the composition of the present invention in preclinical animal models are encouraging, and NO shows promise in antiviral and pyometra treatment.

[0079] The present invention is the first to propose the concept of applying safe and sufficient amount of nitric oxide to anti-viral infectious diseases, and the composition used shows synergistic effect and good effect. There are complex regulatory mechanisms in the immune system, and many signal pathways or target targets have beneficial or harmful effects. There are tens of thousands of research papers on nitric oxide, but the research conclusions are not consistent, contradictory, and difficult to clarify. Some researchers even introduced the concept of "yin and yang balance" in Eastern civilization to explain the double-edged sword-like action of various target mechanisms [Burke AJ, Sullivan FJ, Giles FJ, et al. The yin and yang of nitric oxide in cancer progression [J]. Carcinogenesis, 2013, 34(3):503-512.]. The present invention provides a composition invented from the whole immune system that provides a fully remarkable technical effect. That is, 1. It can significantly improve the host's immune cell level or prevent the apoptosis of immune cells. 2. It can effectively reduce inflammatory factors and reduce inflammatory damage. 3. It can resist viruses by regulating immune function, and the host can better resist secondary infections. 4. The safety of each component in the composition is good.

[0080] Noun interpretation: NO, nitric oxide. NOS, nitric oxide synthase. SNO, safe nitric oxide, refers to nitric oxide with a controllable content of toxic radicals such as peroxynitrite, which satisfies the need for safety when using nitric oxide to treat and prevent diseases. PON: Peroxynitrite and its salts. NO attenuator: A reducing agent used to reduce the production of toxic nitrogen-containing radicals such as peroxynitrite. NO enhancers: precursor substances that produce NO. They are divided into chemical substances that can release NO in the body and substances such as arginine and arginine activator additives that produce enzyme-derived NO. NO synthase inducer: A substance that induces the production of NO synthase. Folic acid: Calcium 6S-5-methyltetrahydrofolate. The salts mentioned in this invention refer to pharma- ceutically acceptable salts. [Brief description of the drawings]

[0081] [Figure 1] 1 shows the weight change curves for each group in Example 7. [Diagram 2] 1 shows the body temperature change curves for each group in Example 7. [Diagram 3] 1 shows a curve of change in food intake for each group in Example 7. [Figure 4] 1 shows a curve of change in water intake for each group in Example 7. [Diagram 5] 1 shows the survival curves of each group in Example 7. [Figure 6] 1 shows the effect of various antioxidants in Example 8 on the expression of iNOS in macrophages induced by LPS. [Figure 7] 1 shows the removal effect of various antioxidants on peroxynitrite in Example 9. [Figure 8] The effect of the composition in Example 10 on the proliferation of CD4 T cells stimulated for three days. [Figure 9] 1 shows the results of the quantitative composition in Example 10 on the proliferation of CD4 T cells stimulated for three days. [Figure 10] 1 shows the detection results of inflammatory factors in Example 12. [Figure 11] 1 shows the detection results of inflammatory factors in Example 12. [Figure 12] 1 shows the detection results of inflammatory factors in Example 12. [Figure 13] 13 shows the change in spleen index and the total number of each immune cell in the spleen in Example 13. [Figure 14] 13 shows the change in spleen index and the total number of each immune cell in the spleen in Example 13. [Figure 15] 13 shows the change in spleen index and the total number of each immune cell in the spleen in Example 13. [Figure 16] 13 shows changes in thymic index and the total number of each immune cell in the thymus in Example 13. [Figure 17] FIG. 13 is a tissue section of the lung in Example 13. [Figure 18] 13 shows changes in the secretion of peripheral blood inflammatory factors in Example 13. [Figure 19] Virus titers in mice of each group in Example 13. [Figure 20] FIG. 13 is a graph showing splenic lymphocyte counts in a pyometra model mouse in Example 17. [Figure 21] This is a graph showing changes in LYMP / NEUP in routine ear vein blood tests of farm pigs due to the composition in Example 24. EXAMPLES

[0082] The above and other characteristics and advantages of the present invention will be further explained in detail by the following examples of the present invention. It should be understood that the following examples are merely illustrative of the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention, which is limited to the claims and equivalent solutions. Unless otherwise specified, all materials and reagents herein are commercially available or may be prepared by those skilled in the art using conventional techniques.

[0083] Example 1 Capsules Calcium 6s-methyltetrahydrofolate 200mg Vitamin C 200mg Filler (appropriate amount) Adhesive (appropriate amount) Disintegrant Appropriate amount

[0084] Example 2 Capsules Calcium 6s-methyltetrahydrofolate 100mg Arginine 400mg Filler (appropriate amount) Adhesive (appropriate amount) Disintegrant Appropriate amount

[0085] Example 3 Tablets Calcium 6S-5-methyltetrahydrofolate 100 milligrams 50 milligrams of phytohemagglutinin Arginine 400 mg Filler (appropriate amount) Adhesive (appropriate amount) Disintegrant Appropriate amount

[0086] Example 4 Tablets Calcium 6S-5-methyltetrahydrofolate 200 milligrams Vitamin C 600 milligrams Filler (appropriate amount) Adhesive (appropriate amount) Disintegrant Appropriate amount

[0087] Example 5 Tablets Calcium 6S-5-methyltetrahydrofolate 400 milligrams Sodium Vitamin C 100 mg Fructose-1,6-bisphosphate 2 milligrams Filler (appropriate amount) Adhesive (appropriate amount) Disintegrant Appropriate amount

[0088] Example 6 Lyophilized powder for injection Calcium 6S-5-methyltetrahydrofolate 800 milligrams Arginine 3 grams This can be accomplished by dissolving, filtering, and lyophilizing.

[0089] Example 7 Anti-cold test in mice Mice: 25 Balb / c mice, female, 6 weeks old, 15-17g. A, Drug-treated group. 10 mice were infected and given the drug. B, model group, infected but without drug, 10 mice. C, normal group, neither infected nor treated, 5 mice.

[0090] Infection method: Mice were anesthetized by intraperitoneal injection of 150 μl of 5% chloral hydrate, and the PR8 influenza virus (1 × 10 6 pfu / mouse) via nasal drop. Administration method: 5-methyltetrahydrofolate calcium was mixed with distilled water to a concentration of 6 mg / ml, and 200 μl (1.2 mg / mouse) was administered intragastrically to each mouse 32 hours after infection in this experiment.

[0091] From the day of infection, body weight, body temperature, water intake, and food intake were measured. Body weight, food intake, and water intake of the mice were measured once a day at a fixed time. Body temperature was measured twice a day at 12-hour intervals for three days after infection, and once a day at a fixed time from the fourth day after infection. In the experiment, the body weight of the mice was basically restored by the 15th day after infection.

[0092] Results: The weight change curves for each group are shown in Figure 1, the body temperature change curves in Figure 2, the food intake change curves in Figure 3, the water intake change curves in Figure 4, and the survival curves in Figure 5. Each figure shows that the condition of the drug-administered group was significantly improved. The ordinates in Figures 1 to 4 are relative values, with the value on the first day being 1.

[0093] Example 8 Screening of attenuating agents in compositions 1. Experimental materials 1. Cell line: macrophage RAW264.7. 2. Reagents: LPS (Sigma); iNOS detection kit (Stressgen); MTT (Biotopped).

[0094] II. Experimental Design 1, Cell culture: Mouse macrophages RAW264.7 were incubated at 37°C, 5% CO 2 The cells were cultured in DMEM high glucose medium containing 10% FBS in an incubator.

[0095] 2. Drug addition processing: Cell density was 5 x 10 4 Adjust the cell suspension to 100 cells / mL, add 100 μL / well of a 96-well plate, and incubate with CO. 2 The cells were cultured in an incubator for 24 hours. LPS-induced inflammation model: LPS induction: 40 μL of LPS was added to all wells (to a final concentration of 0.1 μg / mL). Vitamin C group: Vitamin C and LPS were added to each well (final concentrations were 10 μmol / L for vitamin C and 0.1 μg / mL for LPS). Vitamin E group: Vitamin E and LPS were added to each well (final concentrations were 10 μmol / L for vitamin E and 0.1 μg / mL for LPS). Glutathione group: glutathione and LPS were added to each well (final concentrations were 10 μmol / L for glutathione and 0.1 μg / mL for LPS). 5-Methyltetrahydrofolate group: Calcium 5-methyltetrahydrofolate and LPS were added to each well (final concentrations were 10 μmol / L for calcium 5-methyltetrahydrofolate and 0.1 μg / mL for LPS). Dehydroascorbic acid group: dehydroascorbic acid and LPS were added to each well (final concentrations were 10 μmol / L for dehydroascorbic acid and 0.1 μg / mL for LPS). Anthocyanidin group: anthocyanidin and LPS were added to each well (final concentrations were 10 μmol / L for anthocyanidin and 0.1 μg / mL for LPS). Curcumin group: curcumin and LPS were added to each well (final concentrations were 10 μmol / L for curcumin and 0.1 μg / mL for LPS). Resveratrol group: resveratrol and LPS were added to each well (final concentrations were 10 μmol / L for resveratrol and 0.1 μg / mL for LPS). Andrographolide group: andrographolide and LPS were added to each well (final concentrations were 10 μmol / L for andrographolide and 0.1 μg / mL for LPS). Baicalin group: baicalin and LPS were added to each well (final concentrations were 10 μmol / L for baicalin and 0.1 μg / mL for LPS). NMN group: NMN and LPS were added to each well (final concentrations were 10 μmol / L for NMN and 0.1 μg / mL for LPS). Tetrahydrobiopterin group: tetrahydrobiopterin and LPS were added to each well (final concentrations were 10 μmol / L for tetrahydrobiopterin and 0.1 μg / mL for LPS). Normal group: 50 μL of complete medium was added to each well. Mix uniformly and add CO 2 The culture was continued for 24 h in an incubator.

[0096] 3. Detection of iNOS iNOS protein levels in macrophages were measured by ELISA using polyclonal antibody anti-human iNOS (Stressgen).Macrophage numbers were determined using an automated flow cytometer.

[0097] 4, results The results are shown in Figure 6. The results showed that 5-methyltetrahydrofolic acid, glutathione, NMN, and tetrahydrobiopterin did not affect the expression of iNOS at a concentration of 10 μmol / L.

[0098] Example 9 Comparison of peroxynitrite removal by different antioxidants The peroxynitrite donor 3-morpholino-sydnonimine (SIN-1) was added to 15 test tubes at a concentration of 1 μmol / L. To each tube containing SIN-1 solution, calcium 5-methyltetrahydrofolate was added to final concentrations of 1 μmol / L, 10 μmol / L, and 100 μmol / L, dehydroascorbic acid to final concentrations of 1 μmol / L, 10 μmol / L, and 100 μmol / L, glutathione to final concentrations of 1 μmol / L, 10 μmol / L, and 100 μmol / L, NMN to final concentrations of 1 μmol / L, 10 μmol / L, and tetrahydrobiopterin to final concentrations of 1 μmol / L, 10 μmol / L, and 100 μmol / L. The concentration of peroxynitrite was measured spectrophotometrically, with a detection wavelength of 302 nm. The results are shown in Figure 7. The results show that dehydroascorbic acid, 5-methyltetrahydrofolic acid, and NMN all have an extremely good effect on removing peroxynitrite.

[0099] Example 10: T cell proliferation and differentiation experiment using the composition Mice were killed by cervical dislocation, and the spleens and lymph nodes of the mice were aseptically isolated and placed in Hank's solution, and CD4 T cells were purified from the spleens and lymph nodes of the mice using immunomicrospheres (CD4+ cell extraction kit; Miltenyi Biotec, USA). Mouse CD3 monoclonal antibody 4μg / mL and anti-CD28 (Biolegend) 2μg / mL were added to a 96-well plate, respectively, and Dulbecco's modified Eagle's medium (without L-arginine), trace penicillin, glycine, and 10% fetal bovine serum were added.

[0100] Composition group A: 5-methyltetrahydrofolate calcium was added to the cell culture medium to a final concentration of 10 μmol / L, and arginine was added to a final concentration of 40 μmol / L. Composition group B: Dehydroascorbic acid was added to the cell culture medium to a final concentration of 10 μmol / L, and arginine was added to a final concentration of 40 μmol / L. Composition group C: NMN was added to the cell culture medium to a final concentration of 10 μmol / L, and arginine was added to a final concentration of 40 μmol / L. Arginine group: Arginine was added to the cell culture medium to a final concentration of 40 μmol / L. Blank: Initial cell culture medium (not containing L-arginine) was used.

[0101] The purified T cells were stained with a Cell Violet Trace Proliferation kit (Invitrogen) and cultured for three days, and proliferation was analyzed and measured using a flow cytometer. The results are shown in Figures 8 and 9. The results indicate that all of the selected compositions can enhance the proliferation of CD4 cells after stimulation to a certain extent, and can improve the cellular immune competence of the infected host.

[0102] Example 11 Preclinical study of the composition on cold patients Folic acid (calcium 6S-5-methyltetrahydrofolate) capsules containing 400 mg each were prepared and administered to patients with a cold, and the time (hours) until each symptom disappeared after administration is shown in Table 1. The disappearance of all symptoms except for tonsillitis and sore throat was defined as basic recovery, and the disappearance of all symptoms, including tonsillitis and sore throat, was defined as complete recovery.

[0103] Table 1 Clinical statistics of cold patients (time to disappearance of symptoms, hours) [Table 1]

[0104] The cold patients were given a trial of prescription GK301 (300 mg folic acid, 100 mg L-arginine) capsules, and the results are summarized in Table 2.

[0105] Table 2. Clinical analysis of cold patients (time to symptom resolution) [Table 2]

[0106] Comparing with Table 1, it can be seen that the addition of arginine shortened the course of the disease. The anti-cold effect of the composition was superior to that of 5-methyltetrahydrofolic acid alone. However, the data in Tables 1 and 2 both showed that after administration of 5-methyltetrahydrofolic acid, most of the patients' colds were cured within 2 days.

[0107] Example 12 Effects of 5-methyltetrahydrofolic acid on some inflammatory factors and on NO secretion 1. Experimental materials 1. Cell line: macrophage RAW264.7. 2. Drugs: LPS (Sigma); MTT (Biotopped); folic acid is calcium 5-methyltetrahydrofolate (Lianyungang Jinkang Hexin Pharmaceutical Co., Ltd.); NO detection kit (Beyotime)

[0108] II. Experimental Design 1. Cell culture: Mouse macrophage RAW264.7 cells were cultured at 37°C and 5% CO 2 The cells were cultured in DMEM high glucose medium containing 10% FBS in an incubator. The inflammatory factors (TNF-α, IL-1α, IL-6) in the supernatant were detected by ELISA. 2. Drug addition treatment: 1) Cell density is increased to 2 x 10 5 Adjust the cell suspension to 100 cells / mL, add 100 μL / well of a 96-well plate, and incubate with CO. 2 The cells were cultured in an incubator for 24 hours. 2) In the folic acid group, 50 μL of folic acid was added to each well (to final concentrations of 15.625, 62.5, and 250 μmol / L). In the LPS + folic acid group, 50 μL of LPS was added to each well (to a final concentration of 0.1 μg / mL), and after incubation in an incubator for 6 h, 10 μL of folic acid was added to each well (to final concentrations of 15.625, 62.5, and 250 μmol / L). In the LPS group, 50 μL of LPS was added (to a final concentration of 0.1 μg / mL). In the normal group, 50 μL of complete medium was added to each well. 3) Mix uniformly and add CO 2 The cells were cultured in an incubator for 24 hours. The absorbance value at 520 nm was expressed as the average number ± standard difference. NO secretion rate = (OD sample well - OD blank well) / (OD normal well - OD blank well) × 100% was calculated, and the amount of NO secretion was calculated from the standard curve.

[0109] 3. Experimental results Table 3. Measurement of OD value at 520 nm using NO detection kit. Shown as average value (n=6). [Table 3]

[0110] From the results, it can be seen that 5-methyltetrahydrofolic acid did not suppress the expression of NO in macrophages induced by LPS at a concentration of 15.625 μmol / L. The results of the inflammatory factors are shown in Figure 10, Figure 11, and Figure 12 of the specification. From the above results, it can be seen that calcium 5-methyltetrahydrofolate did not significantly affect the expression of inflammatory factors induced by macrophages and LPS.

[0111] Example 13 Consideration of the early protective effect on mice infected with influenza virus by a single administration of compositions with different dosages 1.1 Materials and methods 1.1.1 Mice Twenty Balb / c mice (5 mice per group), female, 6 weeks old, 15 - 17 g, purchased from Vital River Laboratory Animal Technology Co., Ltd. 1.1.2 Drug preparation The drug was dissolved using deionized water and used within 30 minutes after preparation.

[0112] 1.1.3 Administration method Group G1: Blank control group Group G2: Model group Group G3: Low-dose administration group (calcium 5-methyltetrahydrofolate: arginine = 1:4, 0.173 g / kg) Group G4: High-dose administration group (calcium 5-methyltetrahydrofolate: arginine = 1:4, 0.346 g / kg) In the low-dose group and high-dose group, intragastric administration was performed. In the model group, only model construction was carried out without drug administration, and an equal volume of deionized water was administered. In the blank control group, an equal volume of deionized water was administered. It was administered once. 1.1.4 Infection method Mice were anesthetized by intraperitoneal injection with 150 μl of 5% chloral hydrate and infected by intranasal instillation with PR8 influenza virus (1×10 6 pfu / mouse).

[0113] 1.1.5 Mouse treatment method Body weights were measured from the day of infection, and the body weights of the mice were measured once a day at a fixed time. On the third day after infection, 100 μl of venous blood was collected from the orbit, and serum was prepared and frozen. On the fifth day after infection, the mice were killed, and the lungs, thymus, spleen, and peripheral blood were removed. Blood: Serum was prepared, a portion was used for cytokine detection (external detection), and the remainder was frozen and stored. Lung: The lung tissue was divided into two, one of which (right lobe) was used to measure the virus titer, and the other (upper tip of the left lobe) was fixed, embedded in paraffin, sectioned, and stained with HE. Spleen and thymus: were weighed, photographed, cells were counted and stained for immune cells.

[0114] 1.1.6 Indicator observation Body weight was changed and measured daily. Sample storage: On the 5th day, serum samples were used for detection of inflammatory factors (detection using Biolegend's LEGENDplex Mouse Inflammation Panel, external detection). Lungs: Virus titers were measured, pathological sections of lung tissue were made, and changes in inflammatory factors in lung tissue were detected. Thymus: Weighed, photographed, thymus total cells were counted, and lymphocytes were stained (CD4+, CD8+ T cells). Spleen: Weighed, photographed, spleen total cells were counted, and splenic lymphocyte staining analysis was performed (surface staining, B cells, CD4+ T cells, CD8+ T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, neutrophils).

[0115] 1.2 Experimental results 1.2.1 Spleen index and changes in the total number of each immune cell in the spleen This is shown in Figures 13, 14, and 15 of the specification. 1.2.3 Changes in thymic index and total number of each immune cell This is shown in Figure 16 of the specification. 1.2.2 Pulmonary pathological changes This is shown in FIG. 17 of the specification. 1.2.6 Changes in peripheral blood inflammatory factor secretion This is shown in Figure 18 of the specification. 1.2.7 Changes in pulmonary virus titers (5 dpi) This is shown in FIG. 19 of the specification.

[0116] From these results, after infection with influenza virus, all mice in each group except the normal control group had a decrease in splenic lymphocytes and a decrease in the size of the thymus, and the high-dose group showed a smaller change in the thymus. Both the spleen and the thymus are immune organs and are related to the immune function of mice, and a decrease in the size of the thymus is one of the causes of a decrease in immune function, suggesting that the composition contributes to the protection of immune organs and may have a certain protective effect against immune depression caused by resistance to viral infection. After HE staining of lung pathological sections, the model group and the treatment group both showed similar degrees of lymphocyte infiltration and changes in lung tissue structure, and the high-dose treatment group had a slightly less severe degree of lung tissue damage than the model group. This suggests that the composition contributed to the alleviation of the early lung pathological state infected with influenza virus and reduced lung tissue damage.

[0117] After infection with influenza virus, the model group showed a significant increase in various cytokines in peripheral blood 5 days after infection, and the drug-administered group was able to significantly reduce the secretion of inflammatory factors caused by infection. It was proven that the drug can effectively reduce the level of inflammatory factors, which can contribute to attenuating the inflammatory factor storm, and can prevent and treat lung damage caused by the inflammatory factor storm. Regarding the pulmonary virus titer 5 days after infection, the treatment groups all showed a tendency to decrease to different degrees compared to the model group. In particular, the decrease in virus titer in the high-dose treatment group approached the threshold of statistical significance compared to the model group. It was suggested that the composition can reduce the virus titer, suppressing the copying of the virus in the body, and has a certain antiviral effect.

[0118] Example 14 Treatment of mice with herpesvirus type I encephalitis using the composition Sixty male Kunming mice weighing 14-18g were used, and HeLa cells were challenged with HSV-1. HSV-1 was cultured in the HeLa cells for 48 hours, and the virus was harvested and the viral titer was measured. The mass fraction was 100TCID 50 10 -5The virus was inoculated into mice. The mice were divided into a control group, a model group, a saline treatment group, an acyclovir treatment group (10 mg / kg), and a composition group (5-methyltetrahydrofolic acid 14 mg / kg, arginine 50 mg / kg, and phytohemagglutinin 7 mg / kg). The control group was injected with 0.03 ml of sterile saline, and the model group and each treatment group were injected with 0.03 ml of HSV-1 virus solution, which was then administered intragastrically for 4 consecutive days. The death status and other changes in each group were observed. After 7 days, 0.5 ml of blood was collected from the eyeball, stored in a 35°C incubator for 2 hours, and centrifuged at 1000 r / min for 5 minutes. The results of detecting NO and 1L-1β are shown below.

[0119] Table 4. Number of deaths and mortality rate in each group [Table 4]

[0120] [Table 5] This experiment shows that the composition can significantly reduce the mortality rate of mice infected with herpes virus, and can enhance the release of NO and reduce the levels of inflammatory factors in mice during the infection process.

[0121] Example 15 Screening of 5-methyltetrahydrofolic acid compositions for antipyretic formulations Male C57 mice aged 6-8 weeks and weighing 18-22g were used to observe the general physiological indices, body weight, and feeding status of the animals. They were allowed to acclimate for one week. They were fed standard pelleted food and allowed free access to water. The mice were illuminated with natural day and night light, with room temperature at 18-26°C and relative humidity at 40%-70%. LPS was purchased from Sigma, product number: L2880. Forty-nine C57 male mice were divided into seven groups, seven mice per group, six drug-treated groups and one model group, and administered LPS at 13 mg / kg by intraperitoneal injection (a dose determined in a preliminary experiment; because an LPS dose of 20 mg could not be observed for 120 hours, a dose of 13 mg / kg was confirmed in a preliminary experiment to extend the survival time of the model group).

[0122] Group A: 5-methyltetrahydrofolate calcium:vitamin C = 3:1 (human dose: 300 mg 5-methyltetrahydrofolate calcium, 100 mg vitamin C); Group B: calcium 5-methyltetrahydrofolate:vitamin C = 1:3 (human dose: calcium 5-methyltetrahydrofolate 100 mg, vitamin C 300 mg); Group C: calcium 5-methyltetrahydrofolate:vitamin C = 1:12 (human dose: calcium 5-methyltetrahydrofolate 50 mg, vitamin C 600 mg); Group D: calcium 5-methyltetrahydrofolate:vitamin C = 12:1 (human dose: calcium 5-methyltetrahydrofolate 600 mg, vitamin C 50 mg); Group E: calcium 5-methyltetrahydrofolate:vitamin C = 4:1 (human dose: calcium 5-methyltetrahydrofolate 1200 mg, vitamin C 300 mg); Group F: 5-methyltetrahydrofolate calcium:vitamin C = 3:1 (human dose: 1200 mg 5-methyltetrahydrofolate calcium, 400 mg vitamin C; Group H: model group. In all drug groups, the drug was administered once 9 hours after model construction (9 p.m. on the first day), once the next morning, and once the third morning, for a total of three times, and the administration volumes were consistent. The results are shown below.

[0123] Table 6. Animal phenotype and mortality at each time point after intraperitoneal injection of LPS [Table 6] In groups C and D, some mice showed shivering and lethargy during the administration process. Groups E and F showed the best condition, followed by groups A and B. No animals died in any group after 144 hours.

[0124] The results showed that the compositions of 5-methyltetrahydrofolic acid and vitamin C with different doses and ratios could suppress the mortality rate of mice induced by LPS to different degrees and improve the survival rate of mice, and the optimal ratio was 3:1. With increasing doses, the effect improved significantly, and the human dose of 1200mg of 5-methyltetrahydrofolic acid could interact with 400mg of vitamin C to make the survival rate of LPS-induced pyemia model mice 100%, which is of great clinical value.

[0125] Example 16 Attempt to protect mice against Staphylococcus aureus pyemia model using 5-methyltetrahydrofolic acid composition Using SPF grade Kunming mice weighing approximately 20 grams, a single colony of Staphylococcus aureus was inoculated into the culture medium and cultured overnight at 37°C with shaking. The bacterial liquid was collected and centrifuged at 4000 rpm for 3 minutes, and the precipitate was collected and washed twice with sterile saline. The bacterial liquid was approximately 5 × 10 9 Preliminary experiments showed that intraperitoneal injection of 2 ml of the bacterial solution resulted in a 7-day mortality rate of over 90%.

[0126] Mice were randomly divided into male and female halves as follows: Group A: high dose group, calcium 5-methyltetrahydrofolate:vitamin C=3:1 (human dose 1200mg / day, i.e. 192mg / kg / day); Group B: medium dose group, calcium 5-methyltetrahydrofolate:vitamin C=3:1 (human dose 600mg / day, i.e. 96mg / kg / day); Group C: low dose group, calcium 5-methyltetrahydrofolate:vitamin C=3:1 (human dose 300mg / day, i.e. 48mg / kg / day); Group D: combination treatment group, calcium 5-methyltetrahydrofolate:vitamin C=3:1 (human dose 600mg / day, i.e. 96mg / kg / day) + oxacillin 30mg / kg / d; Model group. Starting 4 h after intraperitoneal injection of the bacterial solution, the drug was administered in three doses at alternate days (days 0, 2, and 4) at the doses mentioned above.

[0127] Table 7. Treatment of Staphylococcus aureus pyemia model [Table 7] Surprisingly, the experimental results showed that the composition that was definitely protective in the LPS model group of mice was unable to alleviate the disease in the Staphylococcus aureus model mice, with no significant difference between the two.

[0128] Example 17 Treatment of pyemia and antibacterial test by formulation of Composition C Calcium 5-methyltetrahydrofolate, arginine and phytohemagglutinin were mixed in a ratio of 2:8:1 to obtain the composition of Formula C. 120 healthy ICR mice, half male and half female, weighing 18-24 g, were used, and Staphylococcus aureus and Streptococcus pneumoniae were used as test bacterial species. They were randomly divided into a normal group, a model group, a low-dose composition group (40 mg / kg), a medium-dose composition group (80 mg / kg), a high-dose composition group (160 mg / kg), and an amoxicillin group (120 mg / kg) based on body weight. Each of the above groups was intraperitoneally administered once daily at 20 ml / kg, and Staphylococcus aureus liquid (5 x 10 9 CFU / ml) was intraperitoneally injected. (The infection method and grouping of Streptococcus pneumoniae were the same as those for Staphylococcus aureus.) The mortality rate of mice in each group was observed within 4 days after the injection of the bacterial liquid, and the differences between the groups were compared to calculate the survival rate. The experimental results are shown below.

[0129] [Table 8] Note: Compared to the model control group, * p<0.05. Compared with the model control group, ** p<0.01.

[0130] The results show that in a clearly infected animal model, it is necessary to add L-arginine to the composition.

[0131] The composition was able to significantly improve the survival rate of the host. To further verify the effect of the composition on lymphocytes, an independent experiment was conducted. That is, ICR mice were inoculated with Staphylococcus aureus liquid (5×10 9 0.5ml of 5-methyltetrahydrofolic acid / arginine solution was injected intraperitoneally into 10 model mice at the same time, and 24 hours after constructing the model, the mice were killed and the spleens were collected. Total splenic cells were counted, and staining analysis of splenic lymphocytes (surface staining, B cells, CD4+ T cells, CD8+ T cells, NK cells) was performed.

[0132] The results are shown in Figure 20. The results show that the composition can prevent apoptosis of CD4 and CD8 T cells in sepsis, and 24 hours after the model was established, sepsis mice induced apoptosis of all types of immune effector cells in sepsis, and the composition prevented apoptosis of CD4 T, CD8 T cells and B cells, but did not prevent the decrease of NK cells (n=11). There have been no reports on the action of such a composition, and the therapeutic prospects of the composition for pyemia should be thoroughly considered.

[0133] Example 18 Inhibitory effect of the composition on gram-negative bacteria in the body Calcium 5-methyltetrahydrofolate, arginine and phytohemagglutinin were mixed in a ratio of 2:8:1, and after thorough mixing, the composition of formula C was obtained. Fifty male BAL B / C mice weighing 18-22g were divided into six groups, eight mice per group, two of which were experimental groups, and the remaining groups were various control groups, namely, a low-dose composition group (40mg / kg), a high-dose composition group (80mg / kg), a normal group, a model group, a penicillin group (450mg / kg), and a meropenem group (75mg / kg). After streaking the Pseudomonas aeruginosa solution on LB solid medium, a typical colony was selected and inoculated into a normal LB liquid medium, and after shaking culture at 37°C overnight for about 12 hours, the medium was centrifuged at 4000r / min for 3 minutes, the supernatant was discarded, and the bacteria were resuspended in physiological saline until use. A lethal dose of Pseudomonas aeruginosa solution was intraperitoneally injected into BAL B / C male mice in each experimental group and three control groups at 500μL / mouse. 30 min after infecting the BAL B / C male mice in the experimental group with the bacteria, the medium dose composition group and the high dose composition group were intragastrically administered with the bacteria, the penicillin group and the meropenem group were intragastrically administered with the bacteria, and purified water was intragastrically administered to the normal group. 24 h after administration, the BAL B / C male mice in each experimental group and the control group were administered with the bacteria again, and the administration type and dose were the same as the first administration. After administration to the BAL B / C male mice, they were observed every 24 h, the survival status was recorded, and all the animals were killed on the 15th day.

[0134] The results show that the composition of the present invention, formulation C, has an inhibitory effect on Gram-negative bacteria in animals and has low toxicity. The composition of the present invention can maintain the survival rate of mice infected with a lethal dose of Pseudomonas aeruginosa at 100% for 14 days. In the meropenem group, the survival rate of mice after 14 days was 100%, while all mice in the penicillin group and all mice in the model group died.

[0135] Example 19: Protective effect of therapeutic and prophylactic administration of the composition on the mortality of mice infected with H1N1 (FM1) influenza virus 1.1 Test Samples Composition granules (5-methyltetrahydrofolic acid calcium:arginine = 1:4), Lianyungang Jin Kang Hexin Pharmaceutical Co., Ltd. 1.2 Test animals ICR mice, SPF grade, body weight 13 - 15 g, half male and half female. Provided by Beijing Vital River Laboratory Animal Technology Co., Ltd., license number: SCXK(Beijing)2016 - 0006, animal certificate number: 1100111911082385. 1.3 Inoculated virulent strain FM1 virulent strain (concentration 100.TICD 50 ) was purchased. Subcultured in this laboratory and stored in a refrigerator at -80°C.

[0136] 2 Test methods and results 2.1 Dosage design In the test, for the test articles, mice were inoculated intranasally with a 1000-fold dilution of the FM1 virulent strain, and the compositions were divided into three dosage groups: high, medium, and low. In addition, a prophylactic administration group was set up, administered once at a low dose, and a vitamin group and a medium-dose group were set up for comparison. 2.2 Preparation of bacterial solution After thawing 0.2 ml of the FM1 virulent strain by freezing and thawing, it was serially diluted with physiological saline to obtain the concentration required for the test (1000-fold volume).

[0137] 3.3 Determination of animal infection dose Mice were inoculated intranasally with different concentrations of FM1 virus solution, 10 mice in each concentration group at 45 μl / mouse, and the death status of the animals within 12 days after infection was observed. The virus solution concentration that caused 80 ± 5% death of the mice was taken as the infection concentration for this test. The results are shown in Table 9.

[0138] Table 9 Determination of the lethal infection virus solution concentration of influenza virus FM1 in mice

Table 9

[0139] 4.1 Animal infection and grouping 130 ICR mice were used, and the animals were randomly divided into 7 groups according to body weight: normal control group, model control group, high, medium and low dose composition groups, composition prevention group, and composition post-treatment group. 10 mice were used as normal control group, and the remaining 20 animals in each group were infected with H1N1 influenza virus by nasal drop at 45μl / animal. After infection, each drug administration group was intragastrically administered at 0.1ml / 10g.

[0140] 4.2 Dose planning for therapeutic and prophylactic administration Normal control group: an equal volume of saline was administered. Model control group: an equal volume of saline was administered. High-dose group: The composition (calcium 5-methyltetrahydrofolate:arginine = 1:4) was administered at 0.346 g / kg body weight, once at 12 hours and once at 24 hours after infection, with the second dose being half the amount of the first dose. Medium dose group: The composition (calcium 5-methyltetrahydrofolate:arginine = 1:4) was administered at 0.173 g / kg body weight, once at 12 hours and once at 24 hours after infection, with the second dose being half the amount of the first dose. Low-dose group: The composition (calcium 5-methyltetrahydrofolate:arginine = 1:4) was administered at 0.087 g / kg body weight, once at 12 hours and once at 24 hours after infection, with the second dose being half the amount of the first dose. Prevention group: prevention effect. That is, 12 hours before model construction, the low dose group, that is, the composition (calcium 5-methyltetrahydrofolate: arginine = 1:4) was administered once at 0.087 g / kg body weight. Post-treatment group: 0.173 g / kg body weight of the composition (5-methyltetrahydrofolic acid:arginine=1:4) was administered once 12 hours and 24 hours after infection, with the second dose being half the amount of the first dose. 0.173 g / kg body weight of the composition (5-methyltetrahydrofolic acid:arginine:vitamin C=3:12:1) was administered once 3 days and 6 days after infection.

[0141] The mortality of the animals was observed for 14 days after infection, and the mortality rate and mortality protection rate (control group mortality rate - experimental group mortality rate) / control group mortality rate were calculated. Lung index = wet lung weight (g) / body weight (g). The results were statistically processed by X2 test and t test for intergroup comparison. The results are shown in the table below.

[0142] Table 10. Protective effect against lethality in mice following primary FM1 influenza virus infection [Table 10]

[0143] Table 11. Effect of FM1 influenza virus on lung inflammation in mice [Table 11]

[0144] 5. Mortality protection against superinfection in surviving mice after treatment with the composition In the above experiment, on the 15th day after administration, the mice that survived the above influenza virus infection or drug intervention were subjected to a superinfection experiment, and the surviving animals were reinfected with the same influenza virus, and the mortality rate within 7 days after reinfection was observed, and the effects of different treatment groups on the mortality rate and mortality protection rate of mice reinfected with influenza virus were compared. No drug intervention was performed on each group in the superinfection experiment.

[0145] Table 12. Protective effect of the composition against death in mice superinfected with FM1 influenza virus [Table 12] The results showed that the high dose group of the composition had a protective effect against animal death after primary infection and superinfection. The prophylactic administration group also showed a certain protective effect. It was suggested that the composition not only reduced the mortality rate of mice caused by influenza virus and had good therapeutic and death protective effects on animals, but also showed a certain protective effect through its prophylactic administration, and could extend the survival time of mice.

[0146] Example 20: Inhibitory effect of the composition of formulation A on endotoxin-induced fever Preparation of the composition of formula A: 5-methyltetrahydrofolate calcium and vitamin C were mixed in a mass ratio of 1:1, and then thoroughly mixed by three-dimensional mixing to prepare the composition of formula A. Preparation of endotoxin: According to previous literature reports, in this experiment, the endotoxin calorific value was adjusted to 250 ng / ml / kg after a preliminary experiment, and was prepared with physiological saline before the experiment. Selection of rabbits: Thirty-five New Zealand rabbits weighing 2.0-3.0 kg were used, and their rectal temperatures were measured once a day for two consecutive days to allow the rabbits to become accustomed to this temperature measurement procedure. Those with body temperatures ranging from 37.5 to 38.5°C and with body temperature fluctuations within 0.5°C were selected for use in the experiment.

[0147] Endotoxin was injected into each rabbit through the ear vein, and rectal temperature was measured 1 hour after injection. According to the change in body temperature, the rabbits were divided into a model group, a positive drug group, and a high (40 mg / kg), medium (20 mg / kg), and low (10 mg / kg) dose group of the present invention's drug composition A formulation. Each drug administration group was intragastricly administered once at 2 ml / kg, and the model group was administered distilled water under the same conditions. Rectal temperature was measured 0.5 h, 1 h, 1.5 h, and 2 h after administration. The experimental results are shown in Table 13.

[0148] Table 13. Effect of the composition of formulation A of the present invention on the change in body temperature of endotoxin-induced fever rabbits (n=6) [Table 13] The results showed that after endotoxin injection, the body temperature of all the rabbits in each group increased significantly, and after the drug was administered, the body temperature of all the rabbits in each group decreased, indicating that the composition has a certain antipyretic effect.

[0149] Example 21 In vivo anti-Mycoplasma pneumoniae test using formulation C Mycoplasma pneumoniae international reference strain (ATCC FH15531) was purchased from the American Type Culture Collection. Composition of formula C: It was self-prepared in the laboratory, and calcium 5-methyltetrahydrofolate, arginine, and phytohemagglutinin were mixed in the ratio of 2:8:1, and after total mixing, the composition of formula C was obtained. Fifty BALB / C mice, half male and half female, weighing 16–20 g, were purchased from the Guangdong Provincial Medical Laboratory Animal Center. Positive drug group: azithromycin dispersible tablets, Ha Yao Group Pharmaceutical Factory No. 6, batch number 160303, specification 0.25g / tablet.

[0150] After acclimatizing and raising BALB / C mice for one week, half of the mice were randomly divided into five groups: a normal control group, a model control group, a positive drug control group (40 mg / kg), a high dose group of C composition (80 mg / kg), and a low dose group of C composition (40 mg / kg). Mice in each group except the normal control group were anesthetized with ethyl ether, and then immersed in ethyl ether at a concentration of 10 6 Mice were infected with 50μL of Mycoplasma pneumoniae (MP) solution (CCU / ml) by nasal drop for three consecutive days. Then, intragastric administration was performed once a day for 10 consecutive days. Four hours after the last administration, the mice were killed by blood sampling from the eyeball, and the lungs, spleen, and thymus were removed and weighed, and then pathological observations were performed. Separately, a small piece of lung tissue was taken, polished, and the MP content was quantitatively detected by PCR. The results are shown below.

[0151] Table 14. Effects on mouse spleen and thymus indices [Table 14] Note: Compared with the model control group, * p<0.05.

[0152] Compared with the model group, the spleen index of the mice in the blank control group was significantly different, and the spleen index of the mice in each drug administration group was significantly different, suggesting that after administration, MPs in the body were killed, the stimulation of immune organs was reduced, and the spleen index was reduced. Pathological examination of mouse lung tissue showed that the lesions of the lung tissue of the model group were more severe than those of the normal group at the time of dissection, the appearance of the lungs showed hyperemia and edema, and the lung lobes were unevenly scattered with necrotic foci. Pathological examination showed that the lesions were mainly in the lungs, mainly interstitial pneumonia and bronchiolar pneumonia, and significant lymphocytic infiltration was observed in the bronchi, while the lung tissue of the blank was basically normal. Mild interstitial pneumonia was observed in the azithromycin control group, while inflammation was significantly reduced in the C composition group, with slight inflammatory cell infiltration around the bronchiolar, and the degree of interstitial pneumonia was gradually reduced with increasing dose. The results show that C composition has the effect of controlling the infection of mice with Mycoplasma pneumoniae, and the degree of lesions in the lung tissue was reduced.

[0153] Example 22: Effect of nitric oxide composition as an immune adjuvant on rabies virus vaccine efficacy Thirty adult Kunming mice, weighing 20–28 g and of half male and half female, were purchased from the Laboratory Animal Center of Xinjiang Medical University and were infected with rabies rSRV. 9Attenuated oral freeze-dried live vaccine was purchased from Beijing Zhongliankang Biotechnology Co., Ltd. For the preparation of formulation B of the nitric oxide composition, calcium 5-methyltetrahydrofolate and arginine were mixed in a mass ratio of 1:4 to prepare the composition of formulation B. Thirty mice were divided into three groups, half male and half female, with 10 mice per group: a blank control group, a virus oral immunization group, and a formulation B + virus oral immunization group. The vaccines were orally administered on the 1st, 7th, and 14th days of the test, and 300μL / mouse blood was collected from the orbit on the 0th, 14th, 21st, 35th, 42nd, and 70th days after immunization. After leaving the blood for 1 hour, it was centrifuged at 5000r / min for 5 minutes to absorb the serum. Approximately 0.05g of mouse feces was collected synchronously, placed in 500μL of PBS (pH about 7.4), crushed to form a turbid liquid, the supernatant was centrifuged and absorbed, and stored in a refrigerator at -20℃. Serum IgG antibodies were detected by an ELISA detection kit, and fecal IgA antibodies were detected by an ELISA detection kit using mouse serum rabies-specific IgA antibodies. The results are shown in the table below.

[0154] Table 15. Detection of serum anti-rabies specific IgG levels (U / ml) at different times after primary immunization of mice in each group. [Table 15] The results show that Composition B increased serum IgG antibody levels, and the combination of oral vaccine with Composition B resulted in sufficient antibody levels on the 14th day, with significant differences in antibody levels between different groups 21 days after immunization.

[0155] Table 16. Detection of fecal SIgA levels (U / ml) at different times after primary immunization of mice in each group. [Table 16] From the above results, it was found that the composition of Formula B improved the immune activity of the vaccine, and the composition of Formula B showed a synergistic effect as an immune adjuvant, and rSRV 9It has been found that the oral attenuated virus vaccine can significantly improve antibody expression in mice, reduce the number of immunizations, and improve the immune effect.

[0156] Example 23 Application of the composition of Formula C in the treatment of African swine fever A case of African swine fever was confirmed by the China Animal Health and Epidemiology Research Center from a sample sent by the Jiangsu Provincial Animal Disease Prevention Center. The positive sample came from a farm in Ganyu District, Lianyungang, Jiangsu Province, where 300 live pigs were kept, of which 130 became ill and more than 120 died. Pathological examinations of the dead infected pigs revealed symptoms such as pulmonary hemorrhage and interstitial pneumonia. Spleen dissection revealed severe splenomegaly, with some splenomegaly reaching seven times its size. Stomach dissection revealed diffuse hemorrhage on the surface of the gastric serosal membrane. Renal enlargement was also prominent. These symptoms matched those of African swine fever.

[0157] Blood was taken from three infected pigs and ten healthy pigs from the farm, centrifuged at 3000r / min, the serum was added to a Roche containing ceramic beads, PBS buffer was added, and DNA was extracted and detected using a virus DNA kit, which confirmed that it was ASFV African swine fever genotype II, a genus of virus that was prevalent in Far East Russia and Eastern Europe in 2017. The composition of prescription C was used to treat African swine fever.

[0158] Preparation of the composition injection of formula C: 5-methyltetrahydrofolate calcium, L-arginine and phytohemagglutinin were mixed in a ratio of 2:8:1, and after thorough mixing, the composition of formula C was obtained. The composition of formula C was sterilized, dissolved in saline, filtered through a microfiltration membrane, and the heat source was adsorbed with activated carbon, and then prepared into an injection.

[0159] 18 early infected pigs were isolated, and the associated feed, wastewater, and feces were detoxified. The average body temperature of the infected pigs was 40°C, some of the infected pigs showed hyperemia and cyanosis in the skin, and multiple bleeding or red spots appeared on the ears and under the abdomen. All of the infected pigs had abnormal feeding habits and lost their appetite. Blood samples from the infected pigs showed that the white blood cell levels were lower than those of normal pigs.

[0160] Based on the above, intervention treatment with the formulation C composition was carried out on 18 infected pigs, with each pig receiving an injection containing formulation C at a dose of 50 mg / kilogram every day for two consecutive days, during which the body temperature of each pig was monitored. The results are shown below.

[0161] Table 17. Summary of 7-day survival rates of 18 infected pigs [Table 17] The results showed an unexpected effect on pigs infected with African swine fever, suggesting that the composition of Formula C has a very good antiviral effect. The results show that only one pig died in 7 days. Then, to meet the policy requirements, all the infected pigs were killed, and the remaining infected pigs on the farm all died within 3 to 4 days after onset of symptoms.

[0162] Autopsy detection of African swine fever fatalities When the pigs died of the disease, their spleens were severely enlarged and congested, becoming brittle and easily crumbling, and there was extensive bleeding in the lungs, which was defined as interstitial pneumonia based on the observation of the lung tissue. The stomach also had similar bleeding, with diffuse bleeding on the surface of the gastric serosal membrane and significant kidney enlargement.

[0163] After the blood was left to stand for 1 h, it was centrifuged at 5000 r / min for 5 min and the serum was absorbed. The IgG antibody level was detected. The blood was added with Krebs-HEPES buffer and kept at 37°C for 30 min, and L-NAME (100 μM) was added. The contents of superoxide, nitrite and NO were detected by electrochemical methods.

[0164] The cured pigs were killed and dissected for pathological observation. In addition to a slight increase in the spleen, localized hemorrhage was observed in the lungs. The blood was left to stand for 1 h, then centrifuged at 5000 r / min for 5 min, and the serum was absorbed. The IgG antibody level was detected. The blood was added with Krebs-HEPES buffer and left to stand for 30 min at 37°C, and L-NAME (100 μM) was added, and the nitrite and NO contents were detected by photochemical method. The results are shown below.

[0165] Table 18. Biochemical indicators of dead pigs and recovered pigs [Table 18] The results show that there were a large amount of antibodies in the bodies of the cured pigs, and the improvement of Composition C can improve the pigs' immune system level and play an antiviral role.

[0166] And while the NO levels of the diseased pigs were not low, the reactive nitrogen levels were significantly increased, suggesting that acute symptoms and death from the virus are related to the reactive nitrogen levels. A strong immune system can actually promote the death of an individual, as is often seen in the treatment of viruses. In contrast, the survival time of immune gene knockout mice against a certain virus was far longer than that of normal mice. Therefore, it is speculated that death from African swine fever is related to an overreaction of the immune system. In comparison, it was found that the blood RNS / NO ratio of diseased pigs was more than three times that of cured pigs.

[0167] It is suggested that the composition of the present invention can reduce the death caused by malignant viruses through immune overexpression by reducing RNS, and can achieve the purpose of maintaining the normal operation of the immune system and eliminating the virus. The practical effect of the composition of the present invention has far exceeded expectations.

[0168] Example 24: Effect of the composition on immune cells in domestic pigs Three normal pigs, about 3 months old and weighing about 25 kg, were administered a composition containing 5-methyltetrahydrofolic acid. The composition was prepared by mixing 5-methyltetrahydrofolic acid calcium, L-arginine and phytohemagglutinin in a ratio of 1:4:0.1 to obtain a drug composition. Before administration of the composition, blood was taken from the ear and a blood routine test was performed. Then, the composition was orally administered at 30 mg / kilogram according to the weight of the pig, and blood was taken from the ear in the first and second weeks and a blood routine test was performed. The main indicators of the blood routine test were LYMP (lymphocytes) and NEUP (neutrophils).

[0169] Indicators for routine blood tests TIFF0007681000000019.tif34155 Patients with high immune index LYMP / NEUP had relatively mild symptoms caused by viral infection. The article [Zhang B, Zhou X, Qiu Y, et al. Clinical characteristics of 82 death cases with COVID-19 [J]. MedRxiv, 2020.] states that clinical analysis of patients with COVID-19 showed that the patients who died basically had a low lymphocyte / neutrophil ratio. Figure 21 shows that the composition can increase the LYMP / NEUP ratio and limit the severity of symptoms caused by viral infection. It has been explained to some extent that the composition has a preventive effect against viruses.

[0170] Although the embodiments of the present invention have been described above, the present invention is not limited to the above-mentioned embodiments. Any amendments, equivalent replacements, improvements, etc. within the spirit and principle of the present invention are included in the protection scope of the present invention. Preferred aspects of the present invention are as follows. [1] A pharmaceutical composition for preventing and treating swine fever, comprising an NO attenuator and an NO enhancer, wherein the NO attenuator is selected from one or more of 5-methyltetrahydrofolic acid and its salts, dehydroascorbic acid, and NMN, and the NO enhancer is selected from one or more of arginine, citrulline, or arginine activating additives. [2] The composition described in [1] above, comprising 5-methyltetrahydrofolic acid and arginine. [3] The composition described in [2] above, further comprising a phytohemagglutinin. [4] The composition described in [3] above, wherein the mass ratio of 5-methyltetrahydrofolate:arginine:phytohemagglutinin is 2:8:1. [5] The composition described in [1], wherein the swine fever is African swine fever. [6] The composition described in [1], wherein the composition can increase the levels of T cells, particularly CD4 and CD8 T cells, in a host infected with a virus, reduce the expression of inflammatory factors, and resist viral infection. [7] The composition according to [1] or [4], wherein the single dose of the active ingredient of the composition is at least 30 mg / kilogram, for example 50 mg / kilogram. [8] Use of the composition according to any one of [1] to [7] above in the preparation of a pharmaceutical composition for the prevention or treatment of classical swine fever.

Claims

1. A pharmaceutical composition for preventing or treating African swine fever, comprising 5-methyltetrahydrofolic acid or a salt thereof, arginine or a salt thereof, and phytohemagglutinin.

2. 2. The pharmaceutical composition of claim 1, wherein the mass ratio of calcium 5-methyltetrahydrofolate:arginine:phytohemagglutinin is 2:8:

1.

3. The pharmaceutical composition of claim 1, which can enhance the level of T cells in a virus-infected host, reduce the expression of inflammatory factors, and resist viral infection.

4. The composition of claim 3 , wherein the T cells are CD4 and CD8 T cells.

Images