Drug composition for producing a safe amount of nitric oxide in vivo and its use

Abstract

The present invention provides a pharmaceutical composition for producing a safe amount of nitric oxide in the body, comprising an NO attenuator, an optional NO enhancer, and a nitric oxide synthase inducer, and uses thereof. The present invention provides a pharmaceutical composition that is highly versatile and highly effective in treating pathogenic microbial infections. New medicinal activities have been discovered for 5-methyltetrahydrofolic acid, NMN, and dehydroascorbic acid, which have a variety of active effects on the immune system caused by pathogen infections, and can be used to treat or prevent diseases caused by viral infections and other pathogen infections.

Description

Technical Field

[0001] This application claims the priority of a prior application filed with the China National Intellectual Property Administration on August 6, 2019, with the patent number 201910719544.6 and the invention title "Safe Nitric Oxide Composition and Its Use", and all the contents of the above-mentioned prior application are incorporated herein by reference. The present invention belongs to the field of medicine, and specifically relates to a drug composition capable of producing nitric oxide in a living body, which can provide a safe and sufficient amount of nitric oxide for use in the prevention and treatment of diseases.

Background Art

[0002] Throughout human history, new viruses have constantly emerged and known viruses have continued to mutate. For new infectious viruses, there are no corresponding specific antibodies in the human body, so large-scale infections occur quasi-periodically. In some human influenza virus pandemics, many human lives have been taken. In 2009, the H1N1 influenza virus prevailed in the United States and Mexico, and in 2020, the COVID-19 virus has been prevalent throughout the world. For individuals, although they are infected with the same influenza virus, the results are different. While some patients lose their lives, some patients have almost no symptoms. The virulence of the virus may vary, but the immune status of the host is also important.

[0003] When it is necessary to prevent and treat influenza virus infection, antibodies are the optimal means. However, the influenza virus evolves rapidly, and escape mutants emerge due to the selective pressure of antibodies against seasonal influenza viruses, causing epidemics in the early strain immune community, which is why seasonal influenza vaccines need to be constantly updated. Unfortunately, the specificity of the antibody response also underlies influenza pandemics. In the previous century, influenza viruses or coronaviruses, including A(H1N1) in 1918, A(H2N2) in 1957, A(H3N2) in 1968, A(H1N1) in 2009, SARS coronavirus in 2001, and COVID-19 in 2020, pandemic occurred multiple times. Interestingly, during the epidemics of the above-mentioned influenza virus infections, there are huge differences in the severity of the host. According to a certain scholar, among immune cells, it has been proven that there are differences in the ability to resist influenza virus due to the differences in the infection and activation of T cells in particular [Kelso, Anne. CD4+T cells limit the damage in influenza[J]. Nature Medicine, 2012, 18(2): 200-202.].

[0004] T cells can mediate cross-protective immunity. Although they cannot prevent virus infection, there is evidence that they can sense infected cells by recognizing viral protein (epitope) fragments that complex with human leukocyte antigen (HLA) molecules on the surface of infected epithelial cells or antigen-presenting cells. Since T cells preferentially recognize epitopes derived from the 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 and reduce the time and severity of pandemic virus infection due to lack of antibody protection.

[0005] For the discovery of antibiotics, there are good clinical treatment means for bacterial infections, but there are still no good treatment means for viruses. Currently, antiviral drugs are mainly classified into two categories: M2 ion channel blockers and neuraminidase inhibitors. M2 ion channel blockers have overall antiviral resistance effects and side effects on the nervous system, so their clinical application is not ideal. Neuraminidase inhibitors can induce viruses, but their effects are weak. In recent years, a large number of viruses such as avian influenza virus, African swine fever virus, and SARS virus have exploded in popularity, and the toxicological effects of these viruses are very serious, and doctors cannot provide good treatment means for patients or diseased animals. There are not only no good treatment means for new viruses, but also no countermeasures for many long-existing viruses including dengue virus and AIDS virus. The optimal solution for treating viruses is prevention, that is, vaccines, which achieve the effect of preventing viruses by the human immune system. The above facts also prove that for the treatment of viruses, the idea of developing drugs that directly kill or suppress pathogens such as antibiotics is actually twice as laborious but only half as effective.

[0006] New concepts 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 for realizing a highly versatile antiviral effect, especially for drugs related to NO and immunity.

[0007] Nitric oxide gas is colorless and odorless and soluble in water, alcohols, and fats. Before the 1980s of the previous century, nitric oxide was usually just an unwanted chemical gas and was only known to exist in automobile exhaust and gas pollutants in certain chemical processes. 27 years before 1980, a substance produced by endothelial cells (referred to as "endothelium-derived relaxing factor") was discovered. In 1986, from the first experimental paper submitted by Ignarro, it was claimed that the endothelium-derived relaxing factor (EDRF) is nitric oxide. These results have induced great interest in the attention and study of NO. NO can quickly enter and exit cells, transmit signals, regulate vasodilation, neurotransmission, brain development, and thus learning and memory, enhance immunity, kill some foreign microorganisms, and prevent blood pressure drops, strokes, heart diseases, tumors, and senile dementia.

[0008] NO is reduced from L-arginine by the catalysis of nitric oxide synthase (NOS) with NADPH. Nitric oxide synthase is divided into endothelial nitric oxide synthase (eNOS), inducible nitric oxide synthase (iNOS), and neuronal nitric oxide synthase (nNOS). They are respectively involved in the regulation of the cardiovascular system, immune regulation, and nervous system regulation in different human tissue cells.

[0009] NO involved in immunity is produced by various immune cells (dendritic cells, NK cells, macrophages, eosinophils, and neutrophils). When iNOS is expressed, a large amount of NO is generated, which becomes the active defense mechanism of humans. There is evidence that NO can suppress the replication of viruses, and the related mechanisms include the reduction of palmitoylation of viral spike proteins, the inhibition of viral proteases, and the inhibition of the synthesis of viral proteins and nucleic acids.

[0010] Nitric oxide synthase is a dimer, which becomes uncoupled under oxidative conditions, converting the original reaction pathway for synthesizing NO to generate reactive oxygen species (ROS) such as O2−, NO3− (PON). Also, NO itself can react with reactive oxygen species (ROS) to generate reactive nitrogen species (RNS).

[0011] NO reacts rapidly with superoxide anions in the body to produce peroxynitrous acid. Under acidic conditions, it decomposes rapidly to produce hydroxyl radicals. Peroxynitrous acid is a highly oxidizing substance that leads to protein nitration and DNA strand breakage. Due to various reasons, many oxidizing radicals containing both reactive oxygen and reactive nitrogen are produced in vivo. The presence of these radicals has disrupted the previous balance to some extent. Among these radicals, the most influential one is peroxynitrite anion (PON). Its production pathway is mainly obtained by the reaction of nitric oxide and superoxide anion.

[0012] PON has mostly negative effects in the human body, including but not limited to the following. 1. Oxidizing effect: PON itself is a strong oxidant and decomposes rapidly into nitrogen dioxide and hydroxyl radicals under acidic conditions. Hydroxyl radicals are stronger oxidants and can oxidize and decompose almost all organic substances. In vivo, PON reacts with many enzymes, proteins, iron / sulfur centers of cytokines, mercaptans, lipids, etc. to cause oxidative damage, resulting in cell function damage and apoptosis. Furthermore, it reduces the radical removal mechanism by glutathione, leading to a vicious cycle. Due to the oxidizing effect of PON, it may cause various diseases such as acute and chronic inflammation, sepsis, traumatic local ischemia, arteriosclerosis, and nerve regeneration disorders. 2. Nitrating effect: PON can react with tyrosine in proteins to produce nitrotyrosine, which can affect the function of proteins and result in DNA breakage and other consequences. 3. Effect on energy metabolism: Thymoprotein has its activity reduced by the oxidizing and nitrating effects. For example, the activities of mitochondrial ATP synthase and aconitase are inhibited, resulting in a decrease in energy. PON is a strong activator of poly ADP-ribose synthase. When this enzyme is activated, it initiates an ineffective repair cycle, and the energy cells are rapidly depleted. Cell metabolism and membrane integrity are disrupted, leading to cell death. 4. Interference with calcium transport: Na + / Ca 2+The mercapto group of the exchange protein is oxidized, resulting in dysfunction, leading to intracellular calcium overload and causing dysfunction. Of course, the tolerance amount of PON also shows positive effects such as resisting the harm to the human body caused by viruses, pathogenic bacteria, protozoa, cancer cells, etc.

[0013] NO, the star molecule in 1992, actually exists everywhere in the living body. NO is a messenger of the immune system, plays important roles in blood flow regulation, neurotransmission, brain development, etc., can kill pathogenic bacteria, viruses, protozoa, cancer cells, and is a very important component of non-specific immunity. The foreign microorganisms or abnormal cells killed by NO release a large amount of antigenic substances after autolysis, initiating specific immunity. NO also causes the living body to release many cytokines such as interleukin, interferon, tumor necrosis factor TNF, colony-stimulating factor CSF, etc., and regulates the immune response.

[0014] NO reacts with radicals such as superoxide anion to generate peroxynitrite (PON). PON has extremely strong oxidizing properties, has a special nitrating ability, and when it accumulates to a certain extent, it causes inflammation and releases cytokines that affect the pathological process. PON destroys protein functions through protein nitration, breaks DNA, promotes virus mutation, destroys immune balance, activates proto-oncogenes, and induces cancer.

[0015] NO is involved in immune regulation. The acute inflammatory response is a complex and highly coordinated sequence of events. Regarding molecular, cellular, and physiological changes, when the host's response to infection is dysregulated, an abnormal immune response further occurs, resulting in a syndrome of organ dysfunction, that is, sepsis. The research on the treatment of sepsis reflects the progress of human understanding of pathophysiology and host-microorganism interactions. Initially, microorganisms and their pathogenicity were the focus. With the implementation of molecular cloning and the sequencing of human inflammatory genes in the 1980s, the research on sepsis has focused on the host's response to invading pathogens.

[0016] According to the Third International Consensus Definitions for Sepsis in 2016, sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Its clinical symptoms include fever, tachypnea, changes in the level of consciousness, and hypotension, and are accompanied by related symptoms of the disease, such as pneumonia caused by lung infection, kidney infection, urinary tract infection, etc.

[0017] Although the understanding of the origin and development of sepsis has been significantly enhanced compared to the past, the mortality rate of sepsis remains very high. According to the article [Hotchkiss R S, Moldawer L L, Opal S M, et al. Sepsis and septic shock [J]. Nature reviews Disease primers, 2016, 2(1): 1-21.], initial estimates of data from high-income countries indicate that globally, there are 31.5 million cases of sepsis and 19.4 million cases of severe sepsis each year, and 5.3 million people may die each year. In many cases, especially in patients with chronic diseases (such as cancer, congestive heart failure, and chronic obstructive pulmonary disease), official death records generally report the underlying disease rather than the direct cause of death (sepsis), so the mortality rate of sepsis may be significantly underestimated. In addition, due to the lack of relevant morbidity rates and mortality records of sepsis in low-income and middle-income countries, these values are only estimates.

[0018] Since inflammation is a host defense response against invading pathogens, reducing the foreign stimulus of pathogen antigens using antibiotic drugs or antiviral drugs is a clinically preferred treatment option for eliminating pathogens. When viral infectious diseases progress to the stage of immune dysregulation, severe inflammation occurs. With some therapies that stop or resist inflammation, the number of macrophages in the inflamed area decreases, making it always difficult to decide whether to enhance or reduce the immune response. Commonly seen anti-inflammatory drugs include non-steroidal anti-inflammatory drugs and glucocorticoids. When severe inflammation occurs, glucocorticoids are generally used clinically, but for sepsis, the use of cortisol 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 the mortality of septic patients. A subsequent extraction 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 cannot reduce the mortality of severely infected patients or sepsis. Therefore, the current clinical use of steroids in severely infected patients is controversial.

[0019] In the past 20 years, attempts have been made to clarify the relationship between vitamin C and sepsis. Sepsis patients generally have very low serum vitamin C levels, and the low vitamin C levels in critically ill patients are thought to be related to increased vasopression, kidney injury, multiple organ dysfunction, and mortality. Studies on the mechanism of action of vitamin C have discovered various mechanisms that may act on sepsis, including antioxidant, anti-inflammatory, microcirculation, antithrombotic, increased adrenal sensitivity, and promotion of wound healing. However, unexpectedly, no significant effect has been seen in the clinical use of vitamin C. According to the statistics by [Chang Xue ● (● is the female variant of Ni), Li Min, Zhang Zhengxin et al. Meta-analysis of the effect of vitamin C in the treatment of sepsis and septic shock patients [J]. Chinese Journal of Critical Care Medicine (Electronic Edition), 2019, 012(001): 37-41.], intravenous injection of vitamin C cannot improve the mortality rate of sepsis and septic shock patients.

[0020] 5-Methyltetrahydrofolate is the active form of folic acid in the human body and does not show a direct antiviral effect. Currently, the direct relationship between folic acid and viruses mainly lies in folate receptor α (FRalpha), which has been described as a factor mediating the entry of viruses such as Ebola into cells. 5-Methyltetrahydrofolate has a direct antioxidant effect and promotes the conversion from BH2 to BH4 through the action of dihydrofolate reductase. It is known that BH4 is a cofactor required for eNOS. Although it has been proven that 5-Methyltetrahydrofolate is beneficial for the prevention and protection of cardiovascular diseases by promoting eNOS, there are few studies and reports on the effect of 5-Methyltetrahydrofolate on iNOS and NO secretion by macrophages under conditions of innate immune activation.

[0021] L-arginine is a precursor of the endogenous synthesis of NO. It reacts under the action of nitric oxide synthase to produce NO and L-citrulline. Only a small part of L-arginine is metabolized in the body through such a pathway. However, in the case of acute inflammation, the NO produced by iNOS of macrophages far exceeds the normal amount in humans. L-arginine is a non-essential amino acid and is endogenously synthesized in the metabolic pathways of proline, glutamine or glutamic acid (by the whole-body proteolysis process). In the kidney, citrulline is converted to arginine under the action of argininosuccinate synthetase and argininosuccinate lyase. However, if the endogenous synthesis of arginine cannot meet the metabolic needs of the living body, it becomes very important under different pathophysiological conditions.

Summary of the Invention

[0022] According to the present invention, 5-methyltetrahydrofolic acid has a physiological activity different from the low concentration of "nutritional support" under "pharmacological" concentrations, and it has been found that a composition containing 5-methyltetrahydrofolic acid has an effect of treating viral infections, and it has further been found that it has a therapeutic effect on any of different pathogens including bacteria, fungi, etc. According to the present invention, it has further been found that the activities of dehydroascorbic acid and NMN are similar to those of 5-methyltetrahydrofolic acid.

[0023] Based on the above findings, the present invention provides the following technical solutions. A drug composition that produces a safe amount of nitric oxide in an animal body, that is, can control or reduce the proportion of reactive nitrogen in the body, and can make the nitric oxide produced in the body reach the amount required for the prevention and treatment of diseases.

[0024] The drug composition according to the present invention includes a NO attenuator selected from antioxidants that remove peroxynitrous acid or its salt (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 higher, for example, does not suppress the expression of iNOS in macrophages induced by LSP.

[0025] The NO attenuating agent according to the present invention is selected from antioxidant substances that do not affect the activation of iNOS synthase and selectively quench peroxynitrite. Examples include one or more selected from 5-methyltetrahydrofolic acid or its salts, dehydroascorbic acid, and NMN.

[0026] The NO increasing agent according to the present invention is selected from enzyme-derived NO substrates selected from L-arginine or its salts, citrulline or its salts, or arginine activation additives.

[0027] The drug composition according to the present invention contains 5-methyltetrahydrofolic acid or its salts and arginine or its salts. Furthermore, it may contain phytohemagglutinin.

[0028] In the drug composition according to the present invention, the 5-methyltetrahydrofolic acid is 15 mg or more in a single dose, and the arginine is 50 mg in a single dose.

[0029] The present invention further provides the use of the above-described drug composition for preparing a drug for preventing or treating a disease caused by pathogenic microorganism infection. Preferably, the pathogenic microorganism infection is a viral infection.

[0030] According to the use of the drug composition according to the present invention, it 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 be used for antiviral infection.

[0031] According to the use of the drug composition according to the present invention, the virus is an influenza virus, a herpes virus, an African swine fever virus, or a coronavirus such as COVID-19.

[0032] According to the use of the drug composition according to the present invention, the composition is used for preparing a drug for preventing and treating sepsis and systemic inflammatory response syndrome caused by infection.

[0033] According to the pharmaceutical composition of the present invention, it contains 5-methyltetrahydrofolic acid or its salt and vitamin C. Preferably, the mass ratio of the above-mentioned calcium 5-methyltetrahydrofolate to vitamin C is 2:1 to 5:1, for example, 3:1, 4:1.

[0034] The present invention further provides the use of the above-mentioned pharmaceutical composition for preparing a drug for treating systemic inflammatory response syndrome and sepsis caused by non-infectious causes.

[0035] According to the use of the pharmaceutical composition of the present invention, the sepsis is caused by Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, or influenza virus infection.

[0036] According to the pharmaceutical composition of the present invention, it may be prepared from an active ingredient and a pharmaceutically acceptable auxiliary material. For example, the pharmaceutical preparation is selected from tablets, capsules, granules, injections, topical patches (topical ointments), or sprays.

[0037] According to the pharmaceutical composition of the present invention, it is an immunoadjuvant.

[0038] In the present invention, a safe amount of nitric oxide means that the ratio of nitric oxide converted into toxic radicals such as peroxynitrous acid and reactive nitrogen species can be controlled so as to meet the safe requirements for 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, further destroy the functions of cells and tissues, significantly increase the probability of gene mutations, and cause the occurrence of many diseases.

[0039] The composition of the present invention effectively improves the production amount of nitric oxide until the needs for preventing and treating diseases are met by controlling toxic radicals.

[0040] In the present invention, a drug 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 host CD4 and CD8 cells during the infection process, block the apoptosis of CD4 and CD8 T cells, significantly improve the survival rate of the host, and improve the inflammatory response during the infection process.

[0041] In the present invention, by administering a composition containing 5-methyltetrahydrofolic acid and arginine to mice infected with influenza virus, a high cure rate is obtained, and the course of the disease is significantly shortened.

[0042] 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, are copied in large quantities in host cells, and should contribute to virus copying and transmission by supplying sufficient folic acid. However, from the experimental results, unexpectedly, 5-methyltetrahydrofolic acid cooperates with a nitric oxide increaser and conversely suppresses the virus. The present invention first proposes to apply compositions such as 5-methyltetrahydrofolic acid and arginine to microbial infections, particularly virus infections.

[0043] iNOS is a key enzyme that generates NO in the immune system. From the prior art, it is known that the oxidation of this enzyme causes dimer decoupling and converts the reaction pathway that generates NO into a reaction pathway that generates 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 effect. 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 disadvantageous 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 salt, dehydroascorbic acid, and NMN can remove peroxynitrite without affecting the function of iNOS expression.

[0044] 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 precursor, thereby producing a sufficient amount of nitric oxide in the body.

[0045] In the present invention, the concept of a sufficient amount means achieving or exceeding the minimum dose of nitric oxide required for the prevention and treatment of diseases.

[0046] The present invention provides a systematic plan for providing a sufficient amount of nitric oxide, which can be selected and optimized as needed. To increase the production amount of nitric oxide, a nitric oxide synthase inducer such as phytohemagglutinin may be used in the composition. Phytohemagglutinin PHA is a mitogenic factor and an efficient and safe nitric oxide synthase inducer, and has already been mass-produced by the technology of extraction from leguminous plants. Another object of the present invention is to provide various uses of the above-mentioned safe nitric oxide composition.

[0047] The active ingredient in the composition of the present invention includes 5-methyltetrahydrofolic acid or a salt thereof. The salt is selected from, but not limited to, calcium salt, arginine salt, glucosamine salt, and sodium salt.

[0048] In one preferred embodiment, the dose of 5-methyltetrahydrofolic acid or a salt thereof in the composition per single dose in the present invention is 15 mg or more, preferably 25 mg or more, more preferably 50 to 1000 milligrams.

[0049] In certain embodiments, 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 single dose is 15 mg or more (in terms of 5-methyltetrahydrofolic acid), preferably 25 mg or more, preferably 50 to 1000 milligrams, more preferably 50 to 500 milligrams. By way of example, the amount of arginine is 50 to 5000 milligrams, preferably 100 to 1000 milligrams.

[0050] In certain embodiments, the composition contains 5-methyltetrahydrofolic acid or a salt thereof, arginine, and phytohemagglutinin PHA. The dosage of 5-methyltetrahydrofolic acid or a salt thereof contained in the composition per unit dose is 15 mg or more, preferably 25 mg or more, preferably 50 to 1000 milligrams, 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.

[0051] The pharmaceutical preparation may be selected from tablets, capsules, granules, injections, topical patches, or gas preparations.

Mode for Carrying Out the Invention

[0052] The p53 levels of NO-induced HIF-1α stabilization and phosphorylation are decreased 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 protects against nitrosative signals [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 signal transduction are regulated by reactive oxygen species, which are also elements that regulate redox signaling.

[0053] The expression of iNOS requires the simultaneous activation of STAT and NF-κB. NF-κB serves as the main switch for inflammation, and regarding the production of H2O2, NF-κB is regulated by redox. Most reducing agents or antioxidants have an anti-inflammatory effect to some extent and can suppress the expression of iNOS by inhibiting the NF-κB pathway. In one example, when comparing the effects of different antioxidants on the expression of iNOS in macrophages induced by LPS, it is shown that 5-methyltetrahydrofolate, dehydroascorbic acid, BH4, 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 towards peroxynitrite, it is shown that 5-methyltetrahydrofolate, dehydroascorbic acid, and NMN all have a high ability to remove peroxynitrite. Under hypoxic conditions, it has already been proven that the immune function of lymphocytes is suppressed and the apoptosis rate increases. Due to the lack of reactive oxygen species, the synthesis of iNOS is inhibited, the binding between iNOS and α-actinin4 is disrupted, and the attachment of iNOS to the actin cytoskeleton is prevented. Therefore, the presence of antioxidants may downregulate iNOS. However, the present invention has found that the antioxidants 5-methyltetrahydrofolate, dehydroascorbic acid, and NMN have unique properties and have a good ability to remove peroxynitrite without reducing the expression of iNOS at a certain concentration. All of the above-mentioned antioxidants do not reduce the immune response ability after activating the immunity with an antigen, do not particularly affect the expression of iNOS during the infection process, and reduce the production of peroxynitrite. NO has the effect of suppressing cell apoptosis and inhibits caspases-8, caspases-9, or caspases-3 by S-nitrosylation, while peroxynitrite promotes cell apoptosis through DNA damage and upregulation of p53.

[0054] NO has direct and indirect effects on infectious microorganisms. NO can directly destroy the enzyme structure of pathogenic microorganisms, especially the [Fe-S] cluster. In viral infections, the expression of NO can suppress the enzyme activity of the virus and inhibit virus replication. Although the direct toxicity of NO, especially its antiviral activity outside the cell, has been well demonstrated, the indirect regulatory effect of NO on immune function is quite complex. According to research, it has been proved that iNOS-deficient mice infected with influenza virus have no histopathological evidence of pneumonia. Therefore, the scholar believes that the host iNOS contributes more to pneumonia than virus replication [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, the results of preclinical models of treatment with iNOS inhibitors used early 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.]. So far, both beneficial and harmful effects have been described, and it is not known whether NO is positive or negative for infection.

[0055] Exogenous NO suppresses the proliferation of T lymphocytes. It has been found that exogenous NO (i.e., not the NO produced by T cells themselves) suppresses proliferation and even causes the death of T cells [Bogdan C. Regulation of lymphocytes by nitric oxide. [J]. Methods Mol Biol, 2011, 677: 375 - 393.]. Mice lacking an important antioxidant mechanism (i.e., GSNOR) show significant deficiencies in peripheral T and B cells due to excessive S-nitrosylation and lymphocyte apoptosis. On the other hand, a small subset of NO-producing T cells, particularly Th1 cells and the negative regulatory T cell population of FoxP3, can effectively suppress the differentiation of Th17 cells. Additionally, recent research indicates that exogenous NO also regulates Th9 and Th17 cells.

[0056] In one embodiment of the present invention, in cell culture medium, 5-methyltetrahydrofolic acid at a concentration of 15.625 μm has little effect on NO secretion by macrophages. More interestingly, it has been found that in the absence of LPS stimulation, 5-methyltetrahydrofolic acid can promote NO secretion at low concentrations.

[0057] It is shown that the combined use of the NO attenuator and NO enhancer selected in the present invention can significantly improve the proliferative activity of antigen-stimulated CD4+ T cells. According to previous research, virus elimination is mediated by antigen-specific CD8+ effector T cells, but memory CD4+ T cells have been shown to play an important role in maintaining the memory responses 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.]. Also, recent research indicates 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 is used for virus elimination and anti-inflammatory treatment.

[0058] In the present invention, when arginine is used together with 5-methyltetrahydrofolic acid as an NO incrementer, it showed an unexpected antiviral and sepsis treatment effect. In one example, the composition of the present invention can significantly stimulate the T cell proliferation in the thymus and spleen of mice. When administering a combination of arginine and 5-methyltetrahydrofolic acid as compared with the case of adding only arginine, the proliferation of CD4 cells is significantly improved, which explains that the composition can improve the proliferation ability of effector CD4+ T cells. As described in the background art, the severity of human infection by influenza virus can be predicted from the number of virus-specific memory CD4 + T cells, 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 disease state. 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 likely to cause immune dysregulation in the host during infection. The composition used can not only improve the immune ability of the host, but also maintain the negative feedback mechanism of inflammation and protect the host from infection, especially virus infection.

[0059] Most of the conventional antiviral cold medicines are used to relieve symptoms and reduce the pain of colds, but they cannot surely and significantly shorten the course of the disease. The above-mentioned drug composition of the present invention brings a revolutionary effect to the treatment of colds. It has a quick effect. When more than 40 subjects were reexamined, the cold symptoms generally disappeared within 48 hours after the composition was administered. Although a double-blind controlled clinical experiment has not been conducted, the feedback results of the related trial compositions are also unexpected.

[0060] Furthermore, when the anti-viral infection effect of the composition was verified in an animal model, it was shown that the composition could protect the immune function of mice, reduce the pathological state of lung infection caused by influenza virus, and reduce lung tissue damage. The composition of 5-methyltetrahydrofolic acid and arginine can significantly reduce the level of inflammatory factors due to 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, by using the composition, the levels of CD4 + and CD8 + T cells in the spleen and thymus of infected mice can be significantly improved, suggesting that the composition reduces inflammatory factors but does not reduce the host's immunity. From the results of lung tissue sections, the composition can reduce lung tissue damage and inflammatory conditions, showing a very good therapeutic effect in the host model against the cold virus.

[0061] According to recent research, NO can promote the immune synapse (IS) signal mediated by the T cell receptor (TCR) [Garcia-Ortiz A, Martin-Cofreces N B, 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 for the activation, secretion of T cells and the regulation of intercellular immune signal communication, which is also a possible reason why the composition can significantly improve the number of T cells.

[0062] In one embodiment of the present invention, when the composition was applied to the treatment of guinea pigs infected with African swine fever virus, very good effects were obtained, significantly improving the survival rate of guinea pigs infected with African swine fever virus and further proving the anti-viral prospect of the composition.

[0063] In addition, in the present invention, it has been found that the above-described composition according to the present invention can significantly protect the survival of the host in a high-dose virus challenge experiment and shows a certain prognosis for the treatment of sepsis.

[0064] 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, with the expectation of improving the prognosis of patients with severe sepsis and septic shock. However, despite all these efforts, no new drugs have been produced that can reduce organ failure and improve the survival rate of sepsis patients [Artenstein AW, Higgins TL, Opal SM. Sepsis and scientific revolutions. Crit Care Med. 2013;41(12):2770-2772.]. Since all these studies use single drugs for specific molecules or pathways and relate to very complex immunometabolic pathways and more than a thousand possible targets, it is not easy to select drugs based on such concepts.

[0065] The supplementation of exogenous arginine is controversial in the treatment of sepsis. Peroxidation mediated by NO is important for the progression of sepsis. Since it is assumed that a pharmaceutical blocker in the process of NO production can treat sepsis, NOS synthase inhibitors have been developed. However, looking back at the clinical results, the therapy of suppressing NOS is not beneficial as a whole. On the other hand, although the arginine level in sepsis patients decreases, it may have an adverse effect of increasing oxidative stress due to an increase in the endogenous donor of NO. The combined use of 5-methyltetrahydrofolic acid and arginine in the composition of the present invention unexpectedly obtains a very good therapeutic effect in a preclinical animal model.

[0066] In the present invention, it has been found that 5-methyltetrahydrofolic acid can significantly reduce the mortality of sepsis mice induced by LPS, suggesting that 5-methyltetrahydrofolic acid may be beneficial for the treatment of sepsis caused by severe allergy.

[0067] In the present invention, it has been found that a composition of 5-methyltetrahydrofolic acid and arginine can significantly reduce the mortality rate of septic mice caused by microbial (e.g., Staphylococcus aureus) infection. Sepsis is a highly lethal disease, characterized by depletion of immune cells induced by extensive cell apoptosis and subsequent immunosuppression. In the present invention, a combination of 5-methyltetrahydrofolic acid and arginine can significantly improve the survival rate of the host, block apoptosis of CD4 and CD8 T cells, and the therapeutic effect of the composition has been proven in sepsis models caused by various bacteria and viruses.

[0068] It should be understood that in the human body, a certain oxidative signal is beneficial for infectious diseases, and there is a delicate balance between protective oxidative signals and the harmful effects of reactive oxygen species. The antioxidants used in the composition of the present invention have unique properties, can relieve symptoms caused by pathogenic microorganism infection, improve the level of NO, and the pathogen-killing effect mediated by NO can exceed the oxidative stress mechanism induced by NO.

[0069] Certainly, T cells play an important role in cross-protection. 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 response of cross-protective T cells. The composition of the present invention has the effect of enhancing the immune response. In one example, the use of the composition can significantly improve the antibody level of immunized animals after rabies vaccination.

[0070] The present invention utilizes the immune systems of humans and animals, which are natural and powerful anti-viral tools. African swine fever is a severe infectious disease with a lethality rate of 95% - 100%, and nothing could be done with the prior art. However, by using the present invention, a clear therapeutic effect is shown, indicating that the anti-viral infection ability of the composition is unexpectedly high. [Oura, C.A 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. When using this virus as a vaccine, infection with the non-toxic ASFV isolate OUR / T88 / 3 can protect distantly related pigs from the attack of the Portuguese ASFV virulent isolate OUR / T88 / 1. However, pigs exposed to OUR / T88 / 3 and depleted of CD8(+) lymphocytes cannot be completely protected from the attack of OUR / T88 / 1, indicating that CD8+ lymphocytes play an important role in the protective immune response to ASFV infection.

[0071] In recent years, the understanding of the role of NO in immunity has advanced significantly. In addition to directly suppressing pathogenic microorganisms, it widely regulates immune functions. Besides iNOS, eNOS needs to be considered as a source of NO under immune conditions. Therefore, it is recognized that it is difficult to apply the suppression or activation of NOS subclasses to clinical practice due to the complementary understanding of the various functions of NO and its target targets. However, the results in the preclinical animal model of the composition of the present invention are encouraging, and NO shows promise in anti-viral and sepsis treatment.

[0072] The present invention first proposes the concept of applying a safe and sufficient amount of nitric oxide to anti-viral infectious diseases. The composition used shows a synergistic effect and exhibits good effects. In the immune system, there are complex regulatory mechanisms, and many signal pathways or target targets have beneficial or harmful effects. There are also tens of thousands of research papers on nitric oxide, but the research conclusions do not agree, contradict each other, and are difficult to clarify. Some researchers have even introduced the concept of "yin-yang balance" in Oriental civilization to explain the double-edged sword-like effects of various target mechanisms [Burke A J, Sullivan F J, Giles F J, et al. The yin and yang of nitric oxide in cancer progression [J]. Carcinogenesis, 2013, 34(3): 503-512.]. The composition invented from the overall immune aspect of the present invention brings about a sufficiently remarkable technical effect. That is, 1. It can significantly improve the level of host immune cells or prevent apoptosis of immune cells. 2. It can effectively reduce inflammatory factors and reduce inflammatory damage. 3. By regulating immune function, it can resist viruses and enable the host to resist secondary infections well. 4. The safety of each component in the composition is good.

[0073] Noun interpretation: NO, nitric oxide. NOS, nitric oxide synthase. SNO, safe nitric oxide. It refers to nitric oxide with a controllable content of toxic radicals such as peroxynitrous acid, and can meet the safety requirements when using nitric oxide to treat and prevent diseases. PON: peroxynitrous acid and its salts. NO attenuator: a reducing substance for reducing the generation of toxic nitrogen-containing radicals such as peroxynitrous acid. NO enhancer: a precursor substance that produces NO. It can be divided into substances such as chemical substances that can release NO in the body and arginine, an arginine activation additive that produces enzyme-derived NO. NO synthase inducer: a substance that induces the production of NO synthase Folic acid: 6S-5-methyltetrahydrofolic acid calcium. The salts described in the present invention refer to pharmaceutically acceptable salts.

Brief Description of the Drawings

[0074]

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Example

[0075] Hereinafter, the above and other characteristics and advantages of the present invention will be further explained and described in detail with reference to the examples of the present invention. It should be understood that the following examples are illustrative of the technical solutions of the present invention and are not intended to limit the protection scope of the present invention defined by the claims and equivalent solutions. Unless otherwise stated, the materials and reagents in the text are all commercially available products or can be prepared by those skilled in the art according to the prior art.

[0076] Example 1 Capsule Calcium 6s-methyltetrahydrofolate 200 mg Vitamin c 200 mg Filler Appropriate amount Adhesive Appropriate amount Disintegrant Appropriate amount

[0077] Example 2 Capsule Calcium 6s-methyltetrahydrofolate 100 mg Arginine 400 mg Filler Appropriate amount Adhesive Appropriate amount Disintegrant Appropriate amount

[0078] Example 3 Tablet Calcium 6S-5-methyltetrahydrofolate 100 milligrams 50 milligrams of phytohemagglutinin 400 milligrams of arginine Appropriate amount of filler Appropriate amount of adhesive Appropriate amount of disintegrant

[0079] Example 4 Tablet 200 milligrams of 6S-5-methyltetrahydrofolic acid calcium 600 milligrams of vitamin C Appropriate amount of filler Appropriate amount of adhesive Appropriate amount of disintegrant

[0080] Example 5 Tablet 400 milligrams of 6S-5-methyltetrahydrofolic acid calcium 100 milligrams of sodium ascorbate 2 milligrams of fructose-1,6-bisphosphate Appropriate amount of filler Appropriate amount of adhesive Appropriate amount of disintegrant

[0081] Example 6 Lyophilized powder for injection 800 milligrams of 6S-5-methyltetrahydrofolic acid calcium 3 grams of arginine It can be dissolved, filtered, and lyophilized.

[0082] Example 7 Anti-influenza test in mice Mice: Balb / c mice, female, 6 weeks old, 15 - 17 g, 25 mice. A, Drug administration group. Infected and administered drugs. 10 mice. B, Model group. Infected but not administered drugs. 10 mice. C, Normal group. Neither infected nor administered drugs. 5 mice.

[0083] 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). Administration method: Calcium 5-methyltetrahydrofolate and distilled water were formulated 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.

[0084] From the day of infection, body weight, body temperature, water intake, and food intake were measured. The 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 every 12 hours within 3 days after infection, and once a day at a fixed time from the 4th day after infection. In the experiment, the body weight of the mice basically returned to normal by the 15th day after infection.

[0085] Results: The body weight change curves of each group are shown in Figure 1, the body temperature change curves are shown in Figure 2, the food intake change curves are shown in Figure 3, the water intake change curves are shown in Figure 4, and the survival curves are shown in Figure 5, respectively. It was found from each figure that the situation of the drug administration group was significantly improved. Among them, the vertical coordinates of Figures 1 to 4 are relative values with the value on the first day being 1.

[0086] Example 8 Selection of Attenuating Agents in Compositions I. Experimental Materials 1. Cell line: Macrophage RAW264.7. 2. Reagents: LPS (Sigma); iNOS detection kit (Stressgen); MTT (Biotopped).

[0087] II. Experimental Protocol 1. Cell culture: Mouse macrophage RAW264.7 was cultured in DMEM high-glucose medium containing 10% FBS in a 37°C, 5% CO2 incubator.

[0088] 2. Drug addition treatment: The cell density was adjusted to 5×10 4 cells / mL, and 100 μL / well of the cell suspension was added to a 96-well plate and cultured in a CO2 incubator for 24 h. Inflammatory model induced by LPS: LPS induction: 40 μL of LPS (up to a final concentration of 0.1 μg / mL) was added to all wells. Group of vitamin C: Vitamin C and LPS were added to each well (with the final concentrations of vitamin C being 10 μmol / L and LPS being 0.1 μg / mL). Group of vitamin E: Vitamin E and LPS were added to each well (with the final concentrations of vitamin E being 10 μmol / L and LPS being 0.1 μg / mL). Group of glutathione: Glutathione and LPS were added to each well (with the final concentrations of glutathione being 10 μmol / L and LPS being 0.1 μg / mL). Group of 5-methyltetrahydrofolic acid: Calcium 5-methyltetrahydrofolate and LPS were added to each well (with the final concentrations of calcium 5-methyltetrahydrofolate being 10 μmol / L and LPS being 0.1 μg / mL). Group of dehydroascorbic acid: Dehydroascorbic acid and LPS were added to each well (with the final concentrations of dehydroascorbic acid being 10 μmol / L and LPS being 0.1 μg / mL). Group of anthocyanidin: Anthocyanidin and LPS were added to each well (with the final concentrations of anthocyanidin being 10 μmol / L and LPS being 0.1 μg / mL). Group of curcumin: Curcumin and LPS were added to each well (with the final concentrations of curcumin being 10 μmol / L and LPS being 0.1 μg / mL). Group of resveratrol: Resveratrol and LPS were added to each well (with the final concentrations of resveratrol being 10 μmol / L and LPS being 0.1 μg / mL). Group of andrographolide: Andrographolide and LPS were added to each well (with the final concentrations of andrographolide being 10 μmol / L and LPS being 0.1 μg / mL). Group of baicalin: Baicalin and LPS were added to each well (with the final concentrations of baicalin being 10 μmol / L and LPS being 0.1 μg / mL). Group of NMN: NMN and LPS were added to each well (with the final concentrations of NMN being 10 μmol / L and LPS being 0.1 μg / mL). Group of tetrahydrobiopterin: Tetrahydrobiopterin and LPS were added to each well (with the final concentrations of tetrahydrobiopterin being 10 μmol / L and LPS being 0.1 μg / mL). Normal group: 50 μL of complete medium was added to each well. It was uniformly mixed and continuously cultured in a CO2 incubator for 24 h.

[0089] 3. Detection of iNOS Using the polyclonal antibody anti-human iNOS (Stressgen), the iNOS protein level in macrophages was measured by ELISA. The number of macrophages was determined using an automatic flow cytometer.

[0090] 4. Results The results are shown in Figure 6. From the results, it was shown that 5-methyltetrahydrofolic acid, glutathione, NMN, and tetrahydrobiopterin did not affect the expression of iNOS at a concentration of 10 μmol / L.

[0091] Example 9 Comparison of peroxynitrite removal by different antioxidants 3-Morpholino-sydnonimine (SIN-1), a peroxynitrite donor, was placed in 15 test tubes at a concentration of 1 μmol / L each. To the test tubes containing the SIN-1 solution, calcium 5-methyltetrahydrofolate was added to a final concentration of 1 μmol / L, 10 μmol / L, 100 μmol / L, dehydroascorbic acid was added to a final concentration of 1 μmol / L, 10 μmol / L, 100 μmol / L, glutathione was added to a final concentration of 1 μmol / L, 10 μmol / L, 100 μmol / L, NMN was added to a final concentration of 1 μmol / L, 10 μmol / L, 100 μmol / L, and tetrahydrobiopterin was added to a final concentration of 1 μmol / L, 10 μmol / L, 100 μmol / L. The concentration of peroxynitrite was measured by spectrophotometry, and the detection wavelength was 302 nm. The results are shown in Figure 7. From the results, it was shown that dehydroascorbic acid, 5-methyltetrahydrofolic acid, and NMN all had a very good peroxynitrite removal effect.

[0092] Example 10 Proliferation and differentiation experiment of T cells by the composition Mice were sacrificed by cervical dislocation, and the spleens and lymph nodes of the mice were aseptically isolated and placed in Hank's solution. CD4 T cells were purified from the spleens and lymph nodes of the mice using an immunomicrosphere (CD4+ cell extraction kit; Miltenyi Biotec, USA). Mouse CD3 monoclonal antibody at 4 μg / mL and anti-CD28 (Biolegend) at 2 μg / mL were added to a 96-well plate, and Dulbecco's modified Eagle medium (without L-arginine), trace penicillin, glycine, and 10% fetal bovine serum were added.

[0093] Composition A group: Calcium 5-methyltetrahydrofolate and L-arginine were added to the cell culture medium at final concentrations of 10 μmol / L and 40 μmol / L, respectively. Composition B group: Dehydroascorbic acid and L-arginine were added to the cell culture medium at final concentrations of 10 μmol / L and 40 μmol / L, respectively. Composition C group: NMN and L-arginine were added to the cell culture medium at final concentrations of 10 μmol / L and 40 μmol / L, respectively. Arginine group: L-arginine was added to the cell culture medium at a final concentration of 40 μmol / L. Blank: The initial cell culture medium (without L-arginine) was used.

[0094] The purified T cells were stained with the Cell Violet Trace Proliferation kit (Invitrogen) and cultured for three days, and the proliferation was analyzed and measured using a flow cytometer. The results are shown in Figures 8 and 9. From the results, it was shown that all the selected compositions could improve the proliferation of CD4 cells after stimulation to a certain extent, indicating that they could improve the cellular immune ability of the infected host.

[0095] Example 11 Preclinical experiment of applying the composition to patients with colds 400 milligrams per capsule of folic acid (6S-5-methyltetrahydrofolic acid calcium) were prepared and tested on cold patients, and the disappearance time (in hours) of each symptom after administration is shown in Table 1. When all symptoms other than tonsillitis and sore throat disappeared, it was defined as basic recovery, and when all including tonsillitis and sore throat disappeared, it was defined as complete recovery.

[0096] Table 1 Clinical statistics of cold patients (symptom disappearance time, in hours)

Table 1

[0097] Cold patients were given prescription GK301 (300 milligrams of folic acid, 100 milligrams of L-arginine) capsules for trial, and the results were summarized in Table 2.

[0098] Table 2 Clinical analysis of cold patients (symptom disappearance time)

Table 2

[0099] Comparing with Table 1, it can be seen that adding arginine shortened the course of the disease. The anti-cold effect by administering the composition was superior to that of 5-methyltetrahydrofolic acid alone. However, from the data in Table 1 and Table 2, it was shown that after administering 5-methyltetrahydrofolic acid, the colds of the patients were mostly cured within 2 days.

[0100] Example 12 Effects of 5-methyltetrahydrofolic acid on some inflammatory factors and on NO secretion I. Experimental materials 1. Cell line: macrophage RAW264.7. 2. Drugs: LPS (Sigma); MTT (Biotopped); folic acid is 5-methyltetrahydrofolic acid calcium (Lianyungang Jin Kang Hexin Pharmaceutical Co., Ltd.); NO detection kit (Beyotime)

[0101] II. Experimental protocol 1. Cell culture: Mouse macrophage RAW264.7 was cultured in DMEM high-glucose medium containing 10% FBS in a 37°C, 5% CO2 incubator. Supernatant inflammatory factors (TNF-α, IL-1α, IL-6) were detected by ELISA. 2. Drug addition treatment: 1) The cell density was adjusted to 2×10 5 cells / mL, and 100 μL / well of the cell suspension was added to a 96-well plate and cultured in a CO2 incubator for 24 h. 2) In the folic acid group, 50 μL of folic acid was added to each well (up to final concentrations of 15.625, 62.5, 250 μmol / L). In the LPS + folic acid group, 50 μL of LPS was added to each well (up to a final concentration of 0.1 μg / mL), and after culturing in an incubator for 6 h, 10 μL of folic acid was added to each well (up to final concentrations of 15.625, 62.5, 250 μmol / L). In the LPS group, 50 μL of LPS was added (up to a final concentration of 0.1 μg / mL). In the normal group, 50 μL of complete medium was added to each well. 3) They were mixed uniformly and cultured in a CO2 incubator for 24 h. The absorbance value at 520 nm was expressed as the mean ± standard deviation. The NO secretion rate = (OD sample well - OD blank well) / (OD normal well - OD blank well) × 100% was calculated, and the NO secretion amount was calculated from the standard curve.

[0102] III. Experimental results Table 3 Measurement of OD values at 520 nm by the NO detection kit. Shown as the mean value (n = 6)

Table 3

[0103] From the results, it can be seen that 5-methyltetrahydrofolic acid did not inhibit 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.

[0104] 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.

[0105] 1.1.3 Administration method Group G1: Blank control group Group G2: Model group Group G3: Low-dose administration group (5-methyltetrahydrofolate: arginine = 1:4, 0.173 g / kg) Group G4: High-dose administration group (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. A single administration was performed. 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).

[0106] 1.1.5 Mouse treatment method The body weight was measured from the day of infection, and the body weight of the mice was measured once a day at a fixed time. On the 3rd day after infection, 100 μl of venous blood was collected from the orbital sinus, serum was prepared, and stored frozen. On the 5th day after infection, the mice were sacrificed, and the lungs, thymus, spleen, and peripheral blood were excised. Blood: Serum was prepared, a part of which was used for cytokine detection (external detection), and the rest was stored frozen. Lung: The lung tissue was divided into two parts. The viral titer was measured for one part (right lung lobe), and the other part (upper tip of the left lung lobe) was fixed, paraffin-embedded, sectioned, and stained with HE. Spleen and thymus: Weighed, photographed, and the cells were counted and immunocytes were stained.

[0107] 1.1.6 Index observation Body weight changed, and body weight was measured daily. Sample storage: Serum samples were used for the detection of inflammatory factors on the 5th day (detection using Biolegend's LEGENDplex Mouse Inflammation Panel, external detection). Lung: The viral titer was measured, pathological sections of lung tissue were made, and changes in inflammatory factors in lung tissue were detected. Thymus: Weighed, photographed, total thymus cells were counted, and lymphocytes were stained (CD4+ and CD8+ T cells). Spleen: Weighed, photographed, total spleen cells were counted, and staining analysis of spleen lymphocytes was performed (surface staining, B cells, CD4+ T cells, CD8+ T cells, NK cells, NKT cells, monocytes, macrophages, dendritic cells, neutrophils).

[0108] 1.2 Experimental results 1.2.1 Changes in spleen index and total number of each immune cell in the spleen Shown in Figures 13, 14, and 15 of the specification. 1.2.3 Changes in thymus index and total number of each immune cell Shown in Figure 16 of the specification. 1.2.2 Pulmonary pathological changes Shown in Figure 17 of the specification. 1.2.6 Changes in secretion of peripheral blood inflammatory factors Shown in Figure 18 of the specification. 1.2.7 Changes in pulmonary viral titer (5dpi) Shown in Figure 19 of the specification.

[0109] From these results, after infection with influenza virus, in each group other than the normal control group, the spleen lymphocytes of all mice decreased, the thymus became smaller, and the changes in the thymus were less in the high-dose group. Both the spleen and thymus are immune organs, related to the immune function of mice. The shrinkage of the thymus is one of the causes of immune function decline, suggesting that the composition may contribute to the protection of immune organs and may have a certain protective effect against immune deficiency caused by viral infection resistance. After HE staining of lung pathological sections, both the model group and the treatment group showed similar degrees of lymphocyte infiltration and changes in lung tissue structure. The degree of lung tissue damage in the high-dose treatment group was slightly less than that in the model group. This suggests that the composition contributed to the alleviation of the early pathological state of the lungs infected with influenza virus and reduced lung tissue damage.

[0110] After infection with influenza virus, in the model group, various cytokines in peripheral blood were significantly improved 5 days after infection, and in the drug administration group, the secretion of inflammatory factors due to infection could be significantly reduced. It was proved that the drug could effectively reduce the level of inflammatory factors, contribute to attenuating the inflammatory factor storm, and prevent and treat lung damage caused by the inflammatory factor storm. Regarding the lung virus titer 5 days after infection, the treatment groups all showed a downward trend to varying degrees compared with the model group. Especially in the high-dose treatment group, the decrease in virus titer was close to the threshold of statistical significance compared with the model group. This suggests that by reducing the virus titer with the composition, the replication of the virus in the body can be suppressed, indicating that it has a certain antiviral effect.

[0111] Example 14 Treatment of Herpes Simplex Virus Type I Encephalitis Mice with the Composition Sixty Kunming male mice weighing 14 - 18 g were used. Hela cells were attacked with HSV-1, and HSV-1 was cultured in Hela cells for 48 hours. The virus was recovered and the virus titer was measured. The mass fraction was 100 TCID 50 10 -5The virus was inoculated into mice. The mice were divided into a control group, a model group, a physiological saline treatment group, an acyclovir treatment group (10 mg / kg), and a composition group (14 mg / kg of 5-methyltetrahydrofolic acid, 50 mg / kg of arginine, 7 mg / kg of phytohemagglutinin). The control group was injected with 0.03 ml of sterile physiological saline, and after the model group and each treatment group were injected with 0.03 ml of HSV-1 virus solution, they were administered intragastrically for 4 consecutive days, and the death status and other changes of each group were observed. After 7 days, 0.5 ml of blood was collected from the eyeballs, stored in a 35 °C incubator for 2 h, centrifuged at 1000 r / min for 5 min, and the results of detecting NO and IL-1β are shown below.

[0112] Table 4 Number of deaths and mortality rates in each group

Table 4

[0113]

Table 5

[0114] Example 15 Selection of anti-sepsis prescriptions for 5-methyltetrahydrofolic acid compositions C57 male mice aged 6-8 weeks and weighing 18-22 g were used to observe the general physiological indicators, body weight, and feeding status of the animals. They were bred for one week to get used to the environment. They were fed with standard pellet feed and allowed to drink water freely. They were illuminated with natural day and night light, the room temperature was set at 18-26 °C, and the relative humidity was set at 40%-70%. LPS was purchased from Sigma, and the product number was: L2880. Forty-nine C57 male mice were divided into seven groups of seven mice each: six drug administration groups and one model group. LPS was administered by intraperitoneal injection at a dose of 13 mg / kg (the dose determined in the preliminary experiment. At a LPS dose of 20 mg, the condition could not be observed for 120 hours, so the dose was confirmed to be 13 mg / kg by the preliminary experiment in order to extend the survival time of the model group).

[0115] Group A: Calcium 5-methyltetrahydrofolate: Vitamin C = 3:1 (300 mg of calcium 5-methyltetrahydrofolate and 100 mg of vitamin C at human dose); Group B: Calcium 5-methyltetrahydrofolate: Vitamin C = 1:3 (100 mg of calcium 5-methyltetrahydrofolate and 300 mg of vitamin C at human dose); Group C: Calcium 5-methyltetrahydrofolate: Vitamin C = 1:12 (50 mg of calcium 5-methyltetrahydrofolate and 600 mg of vitamin C at human dose); Group D: Calcium 5-methyltetrahydrofolate: Vitamin C = 12:1 (600 mg of calcium 5-methyltetrahydrofolate and 50 mg of vitamin C at human dose); Group E: Calcium 5-methyltetrahydrofolate: Vitamin C = 4:1 (1200 mg of calcium 5-methyltetrahydrofolate and 300 mg of vitamin C at human dose) Group F: Calcium 5-methyltetrahydrofolate: Vitamin C = 3:1 (1200 mg of calcium 5-methyltetrahydrofolate and 400 mg of vitamin C at human dose Group H: Model group. In all drug administration groups, the drugs were administered three times at 9 hours after model establishment (9 pm on the first day), once in the morning of the next day, and once in the morning of the third day, with the same administration volume each time. The results are shown below.

[0116] Table 6 Animal manifestations and death status at each time point after intraperitoneal injection of LPS

Table 6

[0117] From the results, compositions of 5-methyltetrahydrofolic acid and vitamin C with different dosages and mixing ratios can suppress the mortality rate of mice induced by LPS to different degrees, improve the survival rate of mice, and can achieve the effect of reducing the mortality rate of septic model mice only by oral gavage, and their optimal mixing ratio was 3:1. With the increase of dosage, the effect was significantly improved. At the dosage of 1200 mg of 5-methyltetrahydrofolic acid at the human dosage, it interacted with 400 mg of vitamin C to make the survival rate of septic model mice induced by LPS reach 100%, which has extremely great clinical value.

[0118] Example 16 Attempts to protect Staphylococcus aureus septic model mice with a 5-methyltetrahydrofolic acid composition SPF-grade Kunming mice weighing about 20 grams were used. Staphylococcus aureus was inoculated into the culture medium with a single colony and cultured overnight with shaking at 37°C. The bacterial solution was collected and centrifuged at 4000 rpm for 3 min, the precipitate was collected and washed twice with sterile physiological saline. The bacterial solution was about 5×10 9 CFU / ml. From the preliminary experiment, it was found that when 2 ml of the bacterial solution was intraperitoneally injected, the mortality rate within 7 days was over 90%.

[0119] The mice were randomly divided into the following groups with equal numbers of males and females. Group A: high-dose group, calcium 5-methyltetrahydrofolate: vitamin C = 3:1 (1200 mg / day at the human dosage, that is, 192 mg / kg / day); Group B: medium-dose group, calcium 5-methyltetrahydrofolate: vitamin C = 3:1 (600 mg / day at the human dosage, that is, 96 mg / kg / day); Group C: low-dose group, calcium 5-methyltetrahydrofolate: vitamin C = 3:1 (300 mg / day at the human dosage, that is, 48 mg / kg / day); Group D: combined treatment group, calcium 5-methyltetrahydrofolate: vitamin C = 3:1 (600 mg / day in human dosage, i.e., 96 mg / kg / day) + oxacillin 30 mg / kg / d; Model group. From 4 h after intraperitoneal injection of the bacterial solution, the drugs were administered every other day in three divided doses at the above-mentioned dosages (days 0, 2, and 4).

[0120] Table 7 Treatment of Staphylococcus aureus sepsis model [Table 7] Surprisingly, from the experimental results, the composition that surely has protective effects on the mice in the LPS model group could not relieve the disease in the staphylococcus model mice, and there was no significant difference between the two groups.

[0121] Example 17 Treatment of sepsis and antibacterial test with the prescription of composition C Calcium 5-methyltetrahydrofolate, arginine, and phytohemagglutinin were mixed at a ratio of 2:8:1 to obtain the composition of prescription C. Using 120 healthy ICR mice weighing 18 - 24 g, half male and half female, with Staphylococcus aureus and Streptococcus pneumoniae as the 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) according to body weight. For each of the above groups, they were intraperitoneally administered once a day at 20 ml / kg. For each mouse in each group other than the normal group, a Staphylococcus aureus solution (5×10 9 CFU / ml) was intraperitoneally injected at 0.5 ml / mouse. (The infection method and grouping situation of Streptococcus pneumoniae were the same as those of Staphylococcus aureus) The death situation of each group of mice within 4 days after injection of the bacterial solution was observed, the differences between groups were compared, and the survival rate was calculated. The experimental results are shown below.

[0122] [Table 8] Note: Compared with the model control group, *p < 0.05. Compared with the model control group, ** p < 0.01.

[0123] From the results, it can be seen that for the clearly infected model animals, it is necessary to add L-arginine to the composition.

[0124] The composition was able to significantly improve the survival rate of the host. To further verify the effect of the composition on lymphocytes, independent experiments were conducted. That is, ICR mice were injected with 0.5 ml of Staphylococcus aureus bacterial solution (5×10 9 CFU / ml) each, and simultaneously, 20 mg / kg of the composition solution of 5-methyltetrahydrofolic acid and arginine was intraperitoneally injected into 10 model mice. 24 hours after model establishment, the mice were sacrificed and the spleens were collected. Total cell count of the spleen and staining analysis of splenic lymphocytes (surface staining, B cells, CD4+ T cells, CD8+ T cells, NK cells) were performed.

[0125] The results are shown in Figure 20. From the results, it is shown that the composition can prevent apoptosis of CD4 and CD8 T cells in sepsis, and septic mice induce apoptosis of all types of immune effector cells in sepsis 24 hours after model establishment. 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 has been no report on such an effect of the composition so far, and the treatment perspective of the composition for sepsis should be fully considered.

[0126] Example 18 Inhibitory effect of the composition on Gram-negative bacteria in vivo Calcium 5-methyltetrahydrofolate, arginine, and phytohemagglutinin were mixed at a ratio of 2:8:1. After complete mixing, the composition of Prescription C was obtained. Fifty BALB / C male mice weighing 18 - 22 g were divided into six groups of 8 mice each. Two groups were experimental groups, and the rest were various control groups, namely the low-dose composition group (40 mg / kg), the high-dose composition group (80 mg / kg), the normal group, the model group, the penicillin group (450 mg / kg), and the meropenem group (75 mg / kg). After culturing the Pseudomonas aeruginosa bacterial solution by streaking on an LB solid medium, typical colonies were selected and inoculated into a normal LB liquid medium. After culturing with shaking overnight at 37°C, about 12 h later, centrifugation was performed at 4000 r / min for 3 min. The supernatant was discarded, and the bacterial cells were resuspended in physiological saline until use. A lethal dose of the Pseudomonas aeruginosa bacterial solution was intraperitoneally injected into BALB / C male mice in each experimental group and three control groups at 500 μL / mouse. Thirty minutes after infecting the BALB / C male mice in the experimental groups with the bacterial cells, intragastric administration was performed on the medium-dose composition group and the high-dose composition group of the composition, and intragastric administration was similarly performed on the penicillin group and the meropenem group, while purified water was intragastrically administered to the normal group. Twenty-four hours after administration, re-administration was performed on the BALB / C male mice in each experimental group and control group, and the administration type and dose were the same as the first administration. After administering to the BALB / C male mice, observation was performed every 24 h, the survival status was recorded, and all animals were sacrificed on the 15th day.

[0127] From the results, it can be seen that the composition of Prescription C of the present invention has an inhibitory effect on Gram-negative bacteria in the animal body and has low toxicity. The said composition of the present invention could 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 was 100% after 14 days, but in the penicillin group, all died, and all also died in the model group.

[0128] Example 19 Death protection effect on mice infected with H1N1 (FM1) influenza virus by therapeutic and prophylactic administration of the composition 1.1 Test samples Composition granules (calcium 5-methyltetrahydrofolate:arginine = 1:4), Lianyungang Jin Kanghexin 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.

[0129] 2 Test methods and results 2.1 Dosage design In the test, for the test articles, all were used to infect mice by nasal instillation 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 required concentration (1000 - fold volume) for the test.

[0130] 3.3 Determination of animal infection dose Mice were infected by nasal instillation with 45 μl / mouse of FM1 virus solutions with different concentrations, 10 mice in each concentration group. The death status of the animals within 12 days after infection was observed, and 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.

[0131] Table 9 Determination of the lethal infection virus solution concentration of influenza virus FM1 in mice

Table 9

[0132] 4.1 Animal infection and grouping Using 130 ICR mice, the animals were randomly divided into 7 groups according to body weight levels: a normal control group, a model control group, high, medium, and low dose groups of the composition, a composition prevention group, and a composition post-treatment group. Ten mice were in the normal control group, and 20 mice in each of the remaining groups were intranasally infected with H1N1 influenza virus at 45 μl / mouse. After infection, each drug administration group was given intragastric administration at 0.1 ml / 10 g.

[0133] 4.2 Dosage design for therapeutic administration and prophylactic administration Normal control group: Administered an equal volume of physiological saline. Model control group: Administered an equal volume of physiological saline. High dose group: The composition (5-methyltetrahydrofolic acid calcium: arginine = 1:4) was administered at 0.346 g / kg body weight, once each at 12 h and 24 h after infection, and the second dose was half of the first dose. Medium dose group: The composition (5-methyltetrahydrofolic acid calcium: arginine = 1:4) was administered at 0.173 g / kg body weight, once each at 12 h and 24 h after infection, and the second dose was half of the first dose. Low dose group: The composition (5-methyltetrahydrofolic acid calcium: arginine = 1:4) was administered at 0.087 g / kg body weight, once each at 12 h and 24 h after infection, and the second dose was half of the first dose. Prevention group: Prophylactic effect. That is, the dose of the low dose group was administered once at 0.087 g / kg body weight 12 h before model construction, namely the composition (5-methyltetrahydrofolic acid calcium: arginine = 1:4). Post-treatment group: The composition (5-methyltetrahydrofolic acid: arginine = 1:4) was administered at 0.173 g / kg body weight, once each at 12 h and 24 h after infection, and the second dose was half of the first dose. The composition (5-methyltetrahydrofolic acid: arginine: vitamin C = 3:12:1) was administered at 0.173 g / kg body weight, once each on the 3rd and 6th days after infection.

[0134] The death situation of animals within 14 days after infection was observed, and the mortality rate and the death protection rate, (control group mortality rate - experimental group mortality rate) / control group mortality rate, were calculated. The lung index was equal to wet lung weight (g) / body weight (g). For the results, statistical analysis was performed by X2 test and t test for comparison between groups. The results are shown in the following table.

[0135] Table 10 Protective effect against mouse lethality caused by the first FM1 influenza virus infection

Table 10

[0136] Table 11 Influence of FM1 influenza virus on mouse lung inflammation

Table 11

[0137] 5. Death protection effect of the composition against superinfection in surviving mice after treatment In the above experiment, on the 15th day after administration, for the mice that survived after being infected with the above-mentioned influenza virus or receiving drug intervention, a superinfection experiment was conducted. The surviving animals were reinfected with the same influenza virus, and the death situation within 7 days after reinfection was observed. The effects of different treatment groups on the mortality rate and death protection rate of mice reinfected with the influenza virus were compared. No drug intervention was performed for each group in the superinfection experiment.

[0138] Table 12 Death protection effect of the composition against mice superinfected with FM1 influenza virus

Table 12

[0139] Example 20 Inhibitory effect of the composition of Prescription A on fever caused by endotoxin Preparation of the composition of Prescription A: Calcium 5-methyltetrahydrofolate and vitamin C were mixed at a mass ratio of 1:1, and after being completely mixed by three-dimensional mixing, the composition of Prescription A was prepared. Preparation of endotoxin: According to the reports in the literature, in this experiment, after preliminary experiments, the endotoxin fever amount was set to 250 ng / ml / kg and it was prepared with physiological saline before the experiment. Selection of rabbits: 35 New Zealand rabbits with a body weight of 2.0 - 3.0 Kg were used. The rectal temperature was measured once a day for 2 consecutive days to acclimatize the rabbits to this temperature measurement operation. Those with a body temperature range of 37.5 - 38.5 °C and a body temperature fluctuation range within 0.5 °C were selected for the experiment.

[0140] Endotoxin was injected into each rabbit via the auricular vein. One hour after the injection, the rectal temperature was measured. According to the body temperature changes, the rabbits were evenly divided into a model group, a positive drug group, and high (40 mg / kg), medium (20 mg / kg), and low (10 mg / kg) dose groups of the drug composition A prescription of the present invention. In each drug administration group, intragastric administration was performed once at 2 ml / kg, and in the model group, distilled water was administered under the same conditions. The rectal temperature was measured at 0.5 h, 1 h, 1.5 h, and 2 h after administration. The experimental results are shown in Table 13.

[0141] Table 13 Influence of the composition of Prescription A of the present invention on the body temperature changes of endotoxin-induced fever rabbits (n = 6)

Table 13

[0142] Example 21 In vivo anti - Mycoplasma pneumoniae test with Prescription C The international standard strain of Mycoplasma pneumoniae (ATCCFH15531) was purchased from the American Type Culture Collection. Composition of Prescription C: Prepared in the laboratory by mixing calcium 5 - methyltetrahydrofolate, arginine, and phytohemagglutinin in a ratio of 2:8:1. After complete mixing, the composition of Prescription C was obtained. Fifty BALB / C mice, half male and half female, weighing 16 - 20 g, were purchased from the Guangdong Provincial Medical Experimental Animal Center. Positive drug group: Azithromycin dispersible tablets, manufactured by Harbin Pharmaceutical Group Pharmaceutical Six Factory, batch number 160303, specification 0.25 g / tablet.

[0143] After acclimating the BALB / C mice for one week, they 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 Composition C (80 mg / kg), and a low - dose group of Composition C (40 mg / kg), with half male and half female. After anesthetizing the mice in each group except the normal control group with ethyl ether, they were nasally infected with 50 μL of Mycoplasma pneumoniae (MP) bacterial solution at a concentration of 10 6 CCU / ml for 3 consecutive days. Then, intragastric administration was carried out once a day for 10 consecutive days. Four hours after the last administration, the mice were bled from the eyeballs and sacrificed, and the lungs, spleens, and thymuses were excised, weighed, and then pathological observations were made. Separately, small pieces of lung tissue were taken, ground, and the MP content was quantitatively detected by PCR. The results are shown below.

[0144] Table 14 Effects on the spleen index and thymus index of mice

Table 14

[0145] Compared with the model group, there were significant differences in the spleen indices of the mice in the blank control group, and there were significant differences in the spleen indices of the mice in each drug administration group, suggesting that MP in vivo was killed after administration. The stimulation to the immune organs was reduced, and the spleen index decreased. In the pathological examination of mouse lung tissue, at the time of dissection, compared with the normal group, the lesions in the lung tissue of the model group were remarkable. Congestion and edema were observed in the appearance of the lungs, and uneven necrotic foci were scattered in the lung lobes. According to pathological examination, the lesions were mainly in the lungs, mainly interstitial pneumonia and bronchiolitis, and significant lymphocyte infiltration was observed in the bronchi. The lung tissue was basically normal in the blank group. Mild interstitial pneumonia was observed in the azithromycin control group, and the inflammation was significantly reduced in the C composition group. Only slight inflammatory cell infiltration was observed around the bronchioles, and the degree of interstitial pneumonia gradually decreased with the increase in dosage. From the results, it can be seen that the C composition has the effect of controlling the infection of mouse pneumonia mycoplasma, and the degree of lung tissue lesions was reduced.

[0146] Example 22 Influence of Nitric Oxide Composition on the Effect of Rabies Virus Vaccine as an Immunoadjuvant Thirty mature Kunming mice weighing 20 - 28 g and divided equally between males and females were purchased from the Experimental Animal Center of Xinjiang Medical University, and the rabies rSRV9 attenuated oral freeze-dried live vaccine was purchased from Beijing Zhonglian Kang Biotechnology Co., Ltd. Regarding the preparation of Formulation B of the nitric oxide composition, calcium 5-methyltetrahydrofolate and arginine were mixed at a mass ratio of 1:4 to prepare the composition of Formulation B. The 30 mice were divided equally between males and females, with 10 mice / group, into three groups: a blank control group, a virus oral immunization group, and a Formulation B + virus oral immunization group. The corresponding vaccines were orally administered on the 1st, 7th, and 14th days of the test respectively. Orbital blood sampling was performed at 300 μL / mouse on days 0, 14, 21, 35, 42, and 70 after immunization. After standing for 1 h, centrifugation was carried out at 5000 r / min for 5 min, and the serum was collected. About 0.05 g of the mice's feces was collected synchronously, placed in 500 μL of PBS (pH about 7.4), ground to form a turbid solution, the supernatant was centrifuged and collected, and stored in a refrigerator at -20 °C. Serum IgG antibodies were detected using an ELISA detection kit, and fecal IgA antibodies were detected using an ELISA detection kit with mouse serum rabies-specific IgA antibodies. The results are shown in the following table.

[0147] Table 15 Detection of serum anti-rabies specific IgG levels (U / ml) in mice of each group at different times after the first immunization

Table 15

[0148] Table 16 Detection of fecal SIgA levels (U / ml) in mice of each group at different times after the first immunization

Table 16

[0149] Example 23 Application of the Composition of Formulation C in the Treatment of African Swine Fever From the samples sent by the Jiangsu Provincial Animal Disease Prevention Center, the China Animal Health and Epidemiology Center confirmed one case of African swine fever. The positive sample was from a farm in Ganyu District, Lianyungang City, Jiangsu Province. The farm raised 300 live pigs, 130 of which became ill and more than 120 died. When the dead infected pigs were dissected for pathological examination, symptoms such as pulmonary hemorrhage and interstitial pneumonia were found. When the spleen was dissected, severe splenomegaly was found, and some were enlarged seven times. When the stomach was dissected, diffuse hemorrhage was found on the surface of the gastric serosa. Also, significant kidney enlargement was observed. These were consistent with the symptoms of African swine fever.

[0150] Blood was taken from 3 infected pigs and 10 healthy pigs from the farm, centrifuged at 3000 r / min, and the serum was added to Roche containing ceramic beads, PBS buffer was added, and DNA was extracted and detected using a viral DNA kit. It was confirmed that it was the African swine fever virus genotype II, belonging to the virus genus that prevailed in the Russian Far East and Eastern Europe in 2017. For the treatment of African swine fever, the composition of Formulation C was intervened.

[0151] Preparation of the composition injection of Formulation C: Calcium 5-methyltetrahydrofolate, L-arginine, and phytohemagglutinin were mixed at a ratio of 2:8:1. After complete mixing, the composition of Formulation C was obtained. After sterilizing the composition of Formulation C, it was dissolved in physiological saline, filtered through a precision filter membrane, adsorbed the heat source with activated carbon, and then prepared into an injection.

[0152] Eighteen pigs in the early stage of infection were isolated, and the related feed, drainage, and feces were harmlessly treated. The average body temperature of the infected pigs was 40°C. Some infected pigs showed skin congestion and cyanosis, and multiple bleeding spots or erythema appeared on the ears and underbelly. All infected pigs had abnormal feeding and loss of appetite. When the infected pigs were blood-tested, it was found that the white blood cell level was lower than that of normal pigs.

[0153] Based on the above, 18 infected pigs were treated with the composition of Prescription C by intervention. An injection containing Prescription C was continuously injected into each pig at a dose of 50 mg / kg per day for 2 days, and the body temperature of each pig was detected during that period. The results are shown below.

[0154] Table 17 Summary of the 7-day survival rate of 18 infected pigs

Table 17

[0155] Autopsy detection of death cases caused by African swine fever When the dead pigs were autopsied, the spleens of the dead pigs were severely swollen, congested, brittle and prone to collapse. Large-scale bleeding appeared in the lungs, and it was defined as interstitial pneumonia by observing the lung tissue. Also, the stomach was similarly bleeding, diffuse bleeding appeared on the surface of the gastric serosa, and kidney swelling was prominent.

[0156] 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. Krebs-HEPES buffer was added to the blood, maintained at 37°C and left to stand for 30 min, L-NAME (100 μM) was added, and the contents of superoxide, nitrite and NO were detected by an electrochemical method.

[0157] The cured pigs were sacrificed and dissected for pathological observation. It was found that in the cured pigs, except that the spleen was slightly enlarged, local bleeding appeared in the lungs. After the blood was allowed 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. Krebs-HEPES buffer was added to the blood, maintained at 37 °C and allowed to stand for 30 min, L-NAME (100 μM) was added, and the contents of nitrite and NO were detected by a photochemical method. The results are shown below.

[0158] Table 18 Biochemical indexes of the dead pigs and cured pigs

Table 18

[0159] And although the NO level of the dead pigs was not low, the level of active nitrogen among them was significantly improved, suggesting that the acute symptoms and death caused by the virus were related to the level of active nitrogen. A high-intensity immune system instead promoted the death of the individual, which was often seen in the virus treatment process. For a certain virus, the survival time of immune gene knockout mice far exceeded that of normal mice. Therefore, it is speculated that the death caused by African swine fever is related to the overreaction of the immune system. By comparison, it was found that the ratio of RNS / NO in the blood of the dead pigs was more than three times that of the cured pigs.

[0160] It is suggested that the composition of the present invention was able to achieve the purpose of reducing the death caused by malignant viruses due to overexpression of immunity by reducing RNS, maintaining the normal operation of the immune system, and removing the virus. The implementation effect of the composition of the present invention far exceeded expectations.

[0161] Example 24 Influence of the composition on the immune cells of breeding pigs Three normal breeding pigs, about 3 months old and weighing about 25 kg, were used, and each was administered a composition containing 5-methyltetrahydrofolic acid. For the preparation of the composition, calcium 5-methyltetrahydrofolate, L-arginine, and phytohemagglutinin were mixed at a ratio of 1:4:0.1 to obtain a drug composition. Before administration of the composition, blood was collected from the ear and routine blood tests were performed. Subsequently, the composition was orally administered at 30 mg / kg based on the weight of the pigs, and blood was collected from the ear at the first and second weeks, respectively, and routine blood tests were performed. The main indicators of the routine blood tests were LYMP (lymphocytes) and NEUP (neutrophils).

[0162] Indicators of routine blood tests TIFF0007709427000019.tif34155 Patients with a high immune index LYMP / NEUP had relatively mild symptoms due to viral infection. In the article [Zhang B, Zhou X, Qiu Y, et al. Clinical characteristics of 82 death cases with COVID-19[J]. MedRxiv, 2020.], a clinical analysis was performed on patients with novel coronavirus infection, and it was stated that the patients who died basically had a low ratio of lymphocytes / neutrophils. In Figure 21, it is shown that the composition was able to increase the LYMP / NEUP ratio and limit the severity of symptoms due to viral infection. This somewhat explains that the composition has a preventive effect against viruses.

[0163] As described above, the embodiments of the present invention have been described, but the present invention is not limited to the above-described embodiments. Any corrections, equivalent substitutions, improvements, etc. within the spirit and scope of the present invention are all included in the protection scope of the present invention. Preferred embodiments of the present invention are as follows. 〔1〕A drug composition for producing a safe amount of nitric oxide in an animal body, comprising a NO attenuating agent and an optional NO increasing agent, wherein the NO attenuating agent is selected from antioxidants that remove peroxynitrous acid or its salt (PON) at a dosage, and preferably, the attenuating agent 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, a drug composition. 〔2〕The drug composition according to 〔1〕 above, wherein the NO attenuating agent is selected from one or more of the following substances: 5-methyltetrahydrofolic acid or its salt, dehydroascorbic acid, and NMN. 〔3〕The drug composition according to 〔1〕 above, wherein the NO increasing agent is selected from enzyme-derived NO substrates, for example, the enzyme-derived NO substrate is selected from L-arginine or its salt, citrulline or its salt, or an arginine activation additive. 〔4〕The drug composition according to 〔1〕 above, comprising 5-methyltetrahydrofolic acid or its salt, and arginine or its salt, and may further comprise phytohemagglutinin. 〔5〕The drug composition according to 〔1〕 above, wherein the single dose of 5-methyltetrahydrofolic acid is 15 mg or more, and the single dose of arginine is 50 mg or more. 〔6〕The drug composition according to 〔1〕 above, wherein the composition comprises 5-methyltetrahydrofolic acid or its salt and vitamin C, and preferably, the mass ratio of 5-methyltetrahydrofolic acid to vitamin C is 2:1 to 5:1. 〔7〕The drug composition according to 〔1〕 above, comprising an active ingredient and a pharmaceutically acceptable adjuvant, for example, the pharmaceutical preparation is selected from tablets, capsules, granules, injections, topical ointments or sprays. 〔8〕An immunoadjuvant comprising the composition according to any one of 〔1〕 to 〔7〕 above. 〔9〕Use for the preparation of a drug for the prevention or treatment of a disease caused by pathogenic microorganism infection of the drug composition according to any one of 〔1〕 to 〔8〕 above, and preferably, the pathogenic microorganism infection is a viral infection. 〔10〕Use of the pharmaceutical composition according to 〔9〕, wherein the pharmaceutical composition can be used for antiviral infection by increasing the levels of T cells, particularly CD4 and CD8 T cells, in a host infected with a virus and reducing the expression of inflammatory factors. 〔11〕Use of the pharmaceutical composition according to 〔9〕, wherein the virus is an influenza virus, a herpes virus, and a coronavirus, such as COVID-19. 〔12〕Use of the pharmaceutical composition according to 〔9〕, wherein the composition is used to prepare a drug for preventing and treating sepsis and systemic inflammatory response syndrome caused by infection. 〔13〕Use of the pharmaceutical composition according to 〔12〕, wherein the sepsis is caused by Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and influenza virus infection. 〔14〕Use of the pharmaceutical composition according to 〔9〕, wherein the composition is used to prepare a drug for treating systemic inflammatory response syndrome and sepsis caused by non-infectious causes. 〔15〕Use of the pharmaceutical composition according to 〔14〕, wherein the composition is the one according to 〔6〕.

Claims

1. A pharmaceutical composition for preventing or treating a disease caused by a pathogenic microorganism, the composition comprising 5-methyltetrahydrofolic acid or a salt thereof, wherein the pathogenic microorganism is an influenza virus.

2. A pharmaceutical composition for preventing or treating a disease caused by a pathogenic microorganism, the composition comprising 5-methyltetrahydrofolic acid or a salt thereof, and further comprising arginine or a salt thereof.

3. The pharmaceutical composition according to claim 2, wherein the single dose of the 5-methyltetrahydrofolic acid is 15 mg or more, and the single dose of the arginine is 50 mg or more.

4. The pharmaceutical composition according to claim 2 or 3, further comprising phytohemagglutinin.

5. The pharmaceutical composition according to any one of claims 2 to 4, wherein the pathogenic microorganism is selected from the group consisting of an influenza virus, a herpes virus, Staphylococcus aureus, Streptococcus pneumoniae, Pseudomonas aeruginosa, and Mycoplasma pneumoniae.

6. A pharmaceutical composition for preventing or treating lipopolysaccharide-induced sepsis or endotoxin-induced fever, the composition comprising 5-methyltetrahydrofolic acid or a salt thereof and vitamin C.

7. The pharmaceutical composition according to claim 6, wherein the mass ratio of 5-methyltetrahydrofolic acid to vitamin C is 2:1 to 5:

1.

8. The pharmaceutical composition according to any one of claims 1 to 7, further comprising a pharmaceutically acceptable adjuvant, and being selected from tablets, capsules, granules, injections, topical ointments, or sprays.

9. An immunoadjuvant comprising 5-methyltetrahydrofolic acid or a salt thereof and arginine or a salt thereof.

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