A composition for enhancing immunity and resistance to pathogen infection and its use therein.

Abstract

This invention belongs to the pharmaceutical field, specifically relating to a composition for enhancing innate immunity and exerting anti-infective effects, and its uses. Specifically, this invention selects ALOX15 enzyme activity inhibitors, including baicalin; compounds that promote ALOX15 transcriptional activity, including aconitine; and compounds that inhibit ALOX15 protein degradation, including forsythoside A / E / I, and combines them in a multi-strategy scientific formulation. Experimental studies have shown that the composition containing ALOX15 enzyme activity inhibitors, ALOX15 transcriptional activity promoters, and ALOX15 protein degradation inhibitors can effectively enhance the body's innate immunity, increase the synthesis and secretion of IFN-β, and exert anti-pathogen infection effects. The composition of this invention can also inhibit the production of pathogen-induced inflammatory factors and has a therapeutic effect on pathogen-induced secondary inflammatory symptoms, showing great promise for clinical application.

Description

Technical Field

[0001] This invention belongs to the field of medicine, specifically relating to a composition for enhancing immunity and resisting pathogen infection, and its uses. Background Technology

[0002] Infectious diseases are caused by numerous pathogens such as bacteria, fungi, rickettsiae, viruses, and parasites, triggering inflammatory storms that lead to tracheitis, bronchitis, pneumonia, arthritis, otitis media, and even other gastrointestinal diseases and neurological symptoms. The innate immune system, as the body's first line of defense against external pathogens, is activated upon infection, subsequently recognizing and eliminating the corresponding pathogens. During the activation of the innate immune system, type I interferon (IFN), especially IFN-β signaling, responds to pathogens in a crucial anti-infection mechanism. Therefore, increasing the body's IFN levels and enhancing immunity is an important strategy for combating pathogenic infections.

[0003] The inventors discovered that 15-lipoxygenase (ALOX15) possesses strong anti-infective activity, and that ALOX15's anti-infective effect in the body is independent of its enzyme activity. Furthermore, the inventors found a positive correlation between ALOX15 levels and the level of the body's innate anti-infective factor IFN-β; that is, increasing ALOX15 expression promotes IFN-β synthesis and secretion. Therefore, ALOX15 can serve as a novel anti-infective target for developing new drugs against pathogenic infections.

[0004] Currently, there are numerous traditional Chinese medicine and Western medicine drugs and methods for treating pathogens, including various viruses, bacteria, fungi, and parasites. However, whether targeting novel viruses, influenza viruses, or viruses, bacteria, fungi, and parasites that have long coexisted with the host, existing drugs suffer from drawbacks such as low efficacy, significant adverse reactions, and even a lack of effective drugs. Therefore, the development of new treatment methods and drugs is urgently needed. Most of the traditional Chinese medicine compositions or natural product compositions reported in currently published patents and related literature suffer from drawbacks such as unclear therapeutic targets, complex compositions, complex sources, and high costs, which greatly limit their clinical translation.

[0005] Therefore, based on the inventors' previous research findings, this application uses ALOX15 as a drug development target, selecting compounds that promote ALOX15 transcriptional activity, inhibit ALOX15 protein degradation, and inhibit ALOX15 enzyme activity, and scientifically combining them to increase the protein level of ALOX15 in the body or lesion site and inhibit lipid peroxidation in the body or lesion area. This results in a composition with a clear target, good efficacy, few side effects, and low cost. This composition has effects such as enhancing immunity, resisting pathogen infection, and inhibiting inflammation. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems, the purpose of this invention is to provide an active ingredient composition that increases the content of ALOX15 protein but inhibits its enzyme activity and its new use in enhancing the body's immunity and resisting pathogen infection.

[0007] Specifically, the present invention is achieved through the following technical solutions:

[0008] The present invention provides the use of a composition in the preparation of a medicament for enhancing immunity and resisting pathogen infection, the composition comprising an ALOX15 enzyme activity inhibitor, an ALOX15 transcription activity promoter and / or an ALOX15 protein degradation inhibitor, and / or other protein degradation inhibitors.

[0009] Alternatively, in the above-described uses, the ALOX15 protein degradation inhibitor is selected from ALOX15 protein ubiquitination degradation inhibitors, and / or ALOX15 protein autophagy degradation inhibitors, and / or other protein degradation inhibitors.

[0010] Alternatively, in the above-described uses, the composition may further comprise one or more other active ingredients selected from: 5-O-caffeoylquinic acid, secoxyloganin, forsythoside, caffeic acid, chlorogenic acid, isochlorogenic acid, caffeine, ursodeoxycholic acid, baicalin, quercetin, rosmarinic acid, or derivatives of the above compounds, or traditional Chinese medicinal materials and / or extracts containing the above compounds, or any combination thereof.

[0011] Alternatively, in the above-described uses, the composition may further comprise one or more anti-inflammatory active ingredients selected from the following: ursodeoxycholic acid, atractylone, paeonol, perillaldehyde, or derivatives of the above compounds, traditional Chinese medicinal materials and / or extracts containing the above compounds, or any combination thereof.

[0012] Preferably, as an optional method, in the above-mentioned uses, the ALOX15 enzyme activity inhibitor is selected from one or more of the following: baicalein, rosmarinic acid, quercetin, chlorogenic acid, isochlorogenic acid, caffeic acid, caffeine, PD146176, daidzein, berberine, ferulic acid, hesperidin, quercetin, hesperidin, ML351, or derivatives of the above compounds, or traditional Chinese medicinal materials and / or extracts containing the above compounds, or any combination thereof.

[0013] Preferably, as an optional approach, in the above-described uses, the ALOX15 transcription activity promoter is selected from one or more of the following: songorine, neochlorogenic acid, strychnos nux-vomica glycoside, or derivatives of the above compounds, traditional Chinese medicinal materials and / or extracts containing the above compounds, or any combination thereof.

[0014] Preferably, as an optional method, in the above-described uses, the ALOX15 protein degradation inhibitor is selected from one or more of the following: forsythoside A, forsythoside E, forsythoside I, or derivatives of the above compounds, Chinese medicinal materials and / or extracts containing the above compounds, or any combination thereof.

[0015] Preferably, as an optional method, in the above-described uses, the composition mainly comprises aconitine, baicalin, and forsythoside E.

[0016] Preferably, as an optional method, in the above-described uses, the composition consists of aconitine, baicalin, and forsythoside E.

[0017] Alternatively, in the above uses, the active ingredient is artificially synthesized and / or extracted from plants, and / or a natural product modified product.

[0018] Alternatively, in the above-mentioned uses, the pathogen is selected from one or more of the following pathogenic microorganisms: influenza virus, common coronavirus, novel coronavirus (COVID-19), SARS virus, MERS virus, hepatitis C virus, Japanese encephalitis virus, rhinovirus, poliovirus, Coxsackie virus, dengue virus, rotavirus, Ebola virus, HIV, Marburg virus, Mycobacterium tuberculosis, Legionella pneumophila, pathogenic Escherichia coli, Salmonella typhimurium, Streptococcus flexneri, Group B streptococci, Staphylococcus aureus, Streptococcus pneumoniae, Candida albicans, Aspergillus fumigatus, Chlamydia trachomatis, Chlamydia pneumoniae, or any combination thereof.

[0019] Preferably, as an optional method, in the above-described uses, the pathogen is an influenza virus, a common coronavirus, a novel coronavirus, Mycobacterium tuberculosis, Chlamydia trachomatis, or any combination thereof.

[0020] Alternatively, in the above-described uses, the composition can also prevent or treat diseases associated with the pathogen infection, which are inflammatory diseases caused by pathogen infection, selected from one or more of the following: pneumonia and its complications, tracheitis, bronchitis, laryngitis, encephalitis, pharyngitis, nephritis, hepatitis, enteritis, gastritis, arthritis, conjunctivitis, otitis media, or any combination thereof.

[0021] Alternatively, in the above-described uses, the composition maintains the protein level of the anti-pathogen infection protein ALOX15 and inhibits its enzymatic activity, promotes the synthesis and secretion of the innate immune factor IFN-β, enhances innate immunity, inhibits pathogen invasion of the host, and suppresses host inflammation.

[0022] Alternatively, in the above-described uses, the composition inhibits the expression / secretion of at least one of the inflammatory factors TNF-α, IL-6, IL-1β, and IFN-γ, and / or reduces the number of inflammatory infiltrating cells.

[0023] Alternatively, in the above-described uses, the composition is prepared by adding conventional excipients and following conventional processes to produce a clinically acceptable drug. The dosage form of the drug is selected from liquid, solid, or semi-solid dosage forms.

[0024] Preferably, the dosage form of the drug is a gel, cream, tablet, capsule, powder, mixture, pill, granule, oral liquid, syrup, decoction, suppository, aerosol, plaster, ointment, injection, spray, liniment, tincture, wet compress, paste, or lotion.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] (1) This invention uses ALOX15 as a drug development target. For the first time, it selects compounds that promote ALOX15 transcriptional activity, inhibit ALOX15 protein degradation and inhibit ALOX15 enzyme activity, and scientifically combines them to increase the protein level of ALOX15 in the body or lesion site and inhibit the lipid peroxidation level in the body or lesion area. It has successfully developed a drug composition with a clear target, good efficacy, few toxic side effects, low cost and the effects of enhancing immunity, resisting pathogen infection and inhibiting inflammation.

[0027] (2) Pharmacological experiments have shown that the various active ingredients in the drug composition of the present invention have significant synergistic effects in enhancing immunity, resisting pathogen infection and inhibiting inflammation, and have no toxic side effects, and have good clinical application prospects.

[0028] (3) The various natural product monomers used in the pharmaceutical compositions of the present invention are widely available and can all be produced by mature methods, making the pharmaceutical compositions of the present invention easy to produce on a large industrial scale. In addition, by adding a pharmaceutically acceptable carrier, the pharmaceutical compositions of the present invention can be formulated into conventional preparations, which makes the efficacy of the drugs stable and convenient to use. Attached Figure Description

[0029] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0030] Figure 1 The composition of this invention increases the survival rate of mice infected with influenza virus.

[0031] Figure 2The composition of this invention reduces the lung index in mice infected with influenza virus.

[0032] Figure 3 The composition of this invention reverses lung damage caused by influenza virus infection.

[0033] Figure 4 The composition of this invention inhibits the secretion of inflammatory factors caused by influenza virus.

[0034] Figure 5 The composition of this invention promotes IFN-β secretion in mice.

[0035] Figure 6 The composition of this invention promotes the secretion of IFN-β by cells infected with Mycobacterium tuberculosis and Chlamydia trachomatis.

[0036] Figure 7 The composition of this invention increases ALOX15 protein levels. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the invention.

[0038] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0039] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available products.

[0040] Example 1: In vitro anti-influenza virus activity of the composition of the present invention

[0041] Human alveolar basal epithelial cells (A549) for lung cancer were treated with the following drugs for 12 hours: aconitine (10 μg / mL), forsythoside E (5 μg / mL), baicalein (5 μg / mL), or combination A (aconitine + forsythoside E, weight ratio 2:1), or combination B (aconitine + forsythoside E + baicalein, weight ratio 2:1:1), or combination C (ursodeoxycholic acid + isochlorogenic acid, weight ratio 1:1), or combination D (aconitine + forsythoside E + baicalein + ursodeoxycholic acid + isochlorogenic acid, weight ratio 2:1:1:0.5:0.5). The normal control group received an equal volume of DMSO. After 12 hours, the cells were adsorbed with 10 times the TCID50 of influenza virus H1N1 for 2 hours. After washing away the unadsorbed virus, the cells were continuously cultured for 24 hours. Twenty-four hours after viral infection, genes from each group were collected for RT-qPCR detection of NP levels. The synergy index was determined using the Jin Zhengjun q-value method, with the q-value calculated using the formula: q = E. A+B / (E) A +E B -E A ×E B In the formula, E A E B These represent the inhibition rates of the aconitine group and the forsythoside E group, or the inhibition rates of the combination group A and the baicalin group, or the inhibition rates of the combination group B and the combination group C. q<1 indicates an antagonistic effect when the two drugs are used together; q>1 indicates a synergistic effect when the two drugs are used together; and q=1 indicates an additive effect when the two drugs are used together.

[0042] The experimental results are shown in Table 1. Composition A significantly inhibited influenza virus replication in vitro, while the inhibitory effects of aconitine and forsythoside E at the same dosage were lower than those of composition A. Furthermore, composition B showed better antiviral replication inhibition than composition A and baicalin. Composition D was the most effective, with an inhibition rate as high as 95.51%. These results demonstrate that the antiviral effect of the compositions of this invention is the result of the synergistic and combined action of the components.

[0043] Table 1: In vitro anti-influenza virus activity of the compositions of the present invention

[0044]

[0045] Note: Data are expressed as mean ± SD. Statistical analysis was performed using one-way ANOVA in SPSS 25.0. Compared with the normal control group, .

[0046] Example 2: Protective effect of the composition of the present invention against influenza virus infection in mice.

[0047] 1. The composition of the present invention increases the survival rate of mice infected with influenza virus.

[0048] Male BALB / c mice aged 6-8 weeks were selected and divided into four groups: a normal control group, a model control group, and two other groups: a group consisting of aconitine + forsythoside E + baicalein (weight ratio 2:1:1, group A) and a group consisting of aconitine + forsythoside E + baicalein + ursodeoxycholic acid + isochlorogenic acid (weight ratio 2:1:1:0.5:0.5, group B), with 10 mice in each group. Mice in groups A (composition A) and B (composition B) were administered the corresponding composition solutions (10 mg / kg) by gavage, while the other groups were administered an equal volume of distilled water by gavage. This treatment was continued for 10 days. After 10 days, except for the normal control group, all other groups were infected with influenza virus strain A / FM / 1 / 47 (H1N1) at 2 times the LD50 viral load via nasal drop. Mice were observed for 21 consecutive days, and the survival status of each group was recorded.

[0049] Experimental results are as follows Figure 1 As shown, compared with the virus-infected group alone, the compositions of both groups A and B significantly increased the survival rate of virus-infected mice, with the composition of group B being more effective.

[0050] 2. The composition of the present invention reduces the lung index in mice infected with influenza virus.

[0051] Male BALB / c mice aged 6-8 weeks were selected and divided into four groups: a normal control group, a model control group, and two other groups: a group receiving aconitine + forsythoside E + baicalein (weight ratio 2:1:1, group A), and a group receiving aconitine + forsythoside E + baicalein + ursodeoxycholic acid + isochlorogenic acid (weight ratio 2:1:1:0.5:0.5, group B), with 10 mice in each group. Mice in groups A and B were administered the corresponding combination solutions (10 mg / kg) by gavage, while the other groups were administered an equal volume of distilled water by gavage. This treatment was continued for 10 days. After 10 days, except for the normal control group, all other groups were infected with influenza virus strain A / FM / 1 / 47 (H1N1) at 2 times the LD50 viral load via nasal drop. On day 5 of H1N1 infection via nasal drop, the body weight of the mice was measured and recorded. Immediately afterwards, the mice in each group were anesthetized with isoflurane, and lung tissue was harvested, weighed, and the lung index was calculated. The formula for calculating the lung index is: Lung Index = Lung Wet Weight (g) / Body Weight (g) × 100%.

[0052] Data were plotted using Graphpad Prism software (version 8) and expressed as mean ± SD. Statistical analysis was performed using SPSS software (version 25) through one-way ANOVA, and the statistical data conformed to the standard of a normal distribution with similar variances.

[0053] Experimental results are as follows Figure 2As shown, the lung index of the model control group was significantly increased compared with the normal control group. After treatment with the composition, the lung index of mice was significantly reduced.

[0054] 3. The composition of the present invention reverses lung damage caused by influenza virus infection.

[0055] Male BALB / c mice aged 6-8 weeks were selected and divided into four groups: a normal control group, a model control group, and two other groups: a group consisting of aconitine + forsythoside E + baicalein (weight ratio 2:1:1, group A) and a group consisting of aconitine + forsythoside E + baicalein + ursodeoxycholic acid + isochlorogenic acid (weight ratio 2:1:1:0.5:0.5, group B), with 10 mice in each group. Mice in groups A and B were administered the corresponding combination solutions (10 mg / kg) by gavage, while the other groups were administered an equal volume of distilled water by gavage. This treatment was continued for 10 days. After 10 days, except for the normal control group, all other groups were infected with influenza virus strain A / FM / 1 / 47 (H1N1) at 2 times the LD50 viral load via nasal drop. On day 5 of H1N1 infection via nasal drop, mice in each group were anesthetized with isoflurane, and lung tissue was perfused and fixed in paraformaldehyde. Subsequently, the tissue was embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to observe pathological changes in the lung tissue.

[0056] Experimental results are as follows Figure 3 As shown, compared with the normal control group, the model control group mice showed lung damage, including tissue necrosis and extensive inflammatory cell infiltration, while the lung tissue damage was significantly improved after the composition was administered for protection.

[0057] 4. The composition of the present invention inhibits the secretion of inflammatory factors induced by influenza virus.

[0058] Male BALB / c mice aged 6-8 weeks were selected and divided into four groups: a normal control group, a model control group, and two other groups: a group consisting of aconitine + forsythoside E + baicalein (weight ratio 2:1:1, group A) and a group consisting of aconitine + forsythoside E + baicalein + ursodeoxycholic acid + isochlorogenic acid (weight ratio 2:1:1:0.5:0.5, group B), with 10 mice in each group. Mice in groups A and B were administered the corresponding combination solutions (10 mg / kg) by gavage, while the other groups were administered an equal volume of distilled water by gavage. This treatment was continued for 10 days. After 10 days, except for the normal control group, all other groups were infected with influenza virus strain A / FM / 1 / 47 (H1N1) at 2 times the LD50 viral load via nasal drop. On day 5 of H1N1 infection via nasal drop, mice in each group were anesthetized with isoflurane, and serum was collected for ELISA detection of IL-1β levels.

[0059] Data were plotted using Graphpad Prism software (version 8) and expressed as mean ± SD. Statistical analysis was performed using SPSS software (version 25) through one-way ANOVA, and the statistical data conformed to the standard of a normal distribution with similar variances.

[0060] Experimental results are as follows Figure 4 As shown, the composition of the present invention can significantly inhibit the level of IL-β.

[0061] Example 3: In vitro antiviral activity of the composition of the present invention against coronaviruses

[0062] A mixture of aconitine, forsythoside E, baicalin, ursodeoxycholic acid, and isochlorogenic acid was prepared at a weight ratio of 2:1:1:0.5:0.5, weighed, and dissolved in DMSO for in vitro anticoronavirus efficacy testing. Vero cells (African green monkey kidney cell line) were evenly seeded into 96-well plates. After 12 hours, 100 μL of a 2-fold serially diluted composition was added to each well. An equal volume of culture medium was added to the blank control group and normal cell group, and the cells were incubated for 48 hours. Then, 20 μL of 5 mg / mL MTT solution was added to each well, and incubation continued for 4 hours. The supernatant was discarded, and 100 μL of DMSO was added to each well, with low-speed shaking for 5 minutes to fully dissolve the crystals. The absorbance was measured at 570 nm using a UV spectrophotometer, and the half-maximal toxicity concentration (TC) was calculated. 50 ).

[0063] Vero cells (African green monkey kidney cell line) were evenly seeded into 96-well plates and incubated at 100-fold TCID after 12 hours. 50 Human coronavirus hCoV-229E viral load was used to infect cells and adsorb the virus for 2 hours. After 2 hours, unadsorbed virus was washed away. Then, a 2-fold serial dilution of the extract was added, with four replicates per concentration, using the maximum non-toxic concentration as the starting concentration. Cells were continuously incubated for 48 hours. Cytopathic effect (CPE) was recorded daily. MTT staining was used to determine OD values ​​and calculate the effective concentration (IC50) of the drug. 50 ), calculate the selection index SI, the formula for SI is: SI = TC 50 / IC 50 [Refer to "Experimental Methods in Pharmacology", edited by Xu Shuyun], a therapeutic index (SI) > 1 indicates effectiveness.

[0064] The experimental results are shown in Table 2. The composition of the present invention has a significant inhibitory effect on human coronavirus hCoV-229E.

[0065] Table 2: Inhibitory effect of the compositions of the present invention on human coronavirus hCoV-229E

[0066]

[0067] Example 4: The composition of the present invention enhances the immunity of virus-infected mice.

[0068] Male BALB / c mice aged 6-8 weeks were selected and divided into four groups: a normal control group, a model control group, and two other groups: a group consisting of aconitine + forsythoside E + baicalein (weight ratio 2:1:1, group A) and a group consisting of aconitine + forsythoside E + baicalein + ursodeoxycholic acid + isochlorogenic acid (weight ratio 2:1:1:0.5:0.5, group B), with 10 mice in each group. Mice in groups A and B were administered the corresponding combination solutions (10 mg / kg) by gavage, while the other groups were administered an equal volume of distilled water by gavage. This treatment was continued for 10 days. After 10 days, except for the normal control group, all other groups were infected with influenza virus strain A / FM / 1 / 47 (H1N1) at 2 times the LD50 viral load via nasal drop. On day 5 of H1N1 infection via nasal drop, mice in each group were anesthetized with isoflurane, and serum was collected for ELISA detection of IFN-β levels.

[0069] Data were plotted using Graphpad Prism software (version 8) and expressed as mean ± SD. Statistical analysis was performed using SPSS software (version 25) through one-way ANOVA, and the statistical data conformed to the standard of a normal distribution with similar variances.

[0070] Experimental results are as follows Figure 5 As shown, the composition of the present invention promotes IFN-β secretion.

[0071] Example 5: The composition of the present invention promotes the secretion of IFN-β in cells infected with Mycobacterium tuberculosis and Chlamydia trachomatis.

[0072] (1) Extraction of mouse bone marrow mononuclear cell-derived macrophages (BMDM): Eight-week-old C57BL / 6 mice were collected, euthanized by cervical dislocation, and placed in a tray containing 75% ethanol. After soaking for 5 minutes, the mice were transferred to a laminar flow hood. Under aseptic conditions, the skin was cut open, and the femur and tibia of the hind legs were removed, and the muscle tissue was completely removed. The femur and tibia were placed in a culture dish containing sterile PBS. The ends of the femur and tibia were cut off. An appropriate amount of PBS was drawn up with a 5 mL syringe, and a 1 mL fine needle was used. The bone was held with forceps in the left hand, and the needle was inserted into the medullary cavity of the femur and tibia with the right hand for rinsing. The PBS containing cells was then filtered through a 50 mL centrifuge tube with a cell filter and rinsed until the bone cavity was colorless. The filtrate was aliquoted into 15 mL centrifuge tubes and centrifuged at 1200 rpm for 10 minutes. The supernatant was discarded, and the cells were resuspended in 1 mL of culture medium and combined into one tube. 10 μL of the cell suspension was placed in a cell counting chamber for counting. Calculate the number of cells, in 10 5The cells were plated at a density of 1 / mL and cultured in DMEM complete medium containing 50 ng / mL M-CSF.

[0073] (2) BMDM cell infection with Mycobacterium tuberculosis: Mycobacterium tuberculosis H37Rv was infected with the obtained BMDM cells at MOI 3 (multiplicity of infection) 3 for 4 hours. Simultaneously, composition A (20 μg / mL) and composition B (20 μg / mL) were added to the cell culture medium, respectively. Compositions A and B remained in the culture medium until IFN-β levels were detected. The culture medium was then discarded, and the extracellular bacteria were washed away with PBS. The medium containing the cells was replaced with a medium containing 100 μg / mL gentamicin. Finally, 20 hours post-infection (20 hpi), the supernatant was collected, and the IFN-β level in the supernatant was detected using an ELISA kit.

[0074] (3) HeLa cell infection with Chlamydia trachomatis (Ct): HeLa cells were seeded into 10 cm culture dishes and cultured overnight in a cell culture incubator in DMEM medium containing 10% fetal bovine serum. The next day, when the cell density was about 80%, the medium was discarded, and Ct suspension was added for infection for 24 hours. At the same time, composition A (20 μg / mL) and composition B (20 μg / mL) were added to the cell culture medium, and composition A and composition B were kept in the culture medium until the IFN-β content was detected. After 24 hours, the medium was discarded, and growth medium (containing 10% fetal bovine serum and 2 mg / L actinomycin) was added for further culture for 24 hours. Finally, the cell supernatant was collected, and the IFN-β level in the supernatant was detected using an ELISA kit.

[0075] Data were plotted using Graphpad Prism software (version 8) and expressed as mean ± SD. Statistical analysis was performed using SPSS software (version 25) through one-way ANOVA, and the statistical data conformed to the standard of a normal distribution with similar variances.

[0076] Experimental results are as follows Figure 6 As shown, the composition of the present invention promotes the secretion of IFN-β in cells infected with Mycobacterium tuberculosis and Chlamydia trachomatis.

[0077] Example 6: The composition of the present invention increases ALOX15 protein levels.

[0078] The inventors previously discovered that ALOX15 can stabilize the mitochondrial antiviral protein MAVS, inhibit its ubiquitination and degradation, and promote the generation of the antiviral molecule IFN-β. In this invention, it was found that the composition of this invention (aconitum carmichaelii + forsythoside E + baicalin, weight ratio 2:1:1) increases the level of ALOX15 protein.

[0079] Human alveolar basal epithelial cells (A549) for lung cancer were treated with the following drugs: a combination of aconitine, forsythoside E, and baicalin (weight ratio 2:1:1) (40 μg / mL), and the protein synthesis inhibitor actinomycin (CHX, 100 μM), with CHX + the combination for 12 hours. A normal control group was also included, treated with an equal volume of DMSO for the same duration. After 12 hours, cells were collected, proteins were extracted, and ALOX15 protein levels were detected by Western blot.

[0080] Experimental results are as follows Figure 7 As shown, the composition of the present invention can significantly increase the level of ALOX15 protein, indicating that the composition of the present invention may exert its immune-enhancing and antiviral infection-fighting effects through ALOX15.

[0081] In summary, the composition of the present invention containing ALOX15 enzyme activity inhibitor, ALOX15 transcription activity promoter, and ALOX15 protein degradation inhibitor can effectively enhance antiviral immunity, inhibit viral replication, increase the synthesis and secretion of IFN-β, and exert antiviral effects.

[0082] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Use of a composition in the manufacture of a medicament for improving immunity and resistance to pathogenic infection, characterized in that, The active ingredients of the composition are as follows: 2:1 of sengernine and forsythoside E by weight, 2:1:1 of sengernine, forsythoside E and baicalein by weight, and the pathogenic infection is influenza virus H1N1 infection.

2. Use of a composition in the manufacture of a medicament for improving immunity and resistance to pathogenic infection, characterized in that, The active ingredients of the composition are as follows: 2:1:1:0.5:0.5 of sengernine, forsythoside E, baicalein, ursodeoxycholic acid and isochlorogenic acid by weight, and the pathogenic infection is influenza virus H1N1 infection or human coronavirus hCoV-229E infection.

3. Use according to claim 1 or claim 2, characterized in that, The active ingredients are artificially synthesized and / or extracted from plants.

4. Use according to claim 1 or claim 2, characterized in that, The composition is added with conventional adjuvants, and prepared into a clinically acceptable pharmaceutical dosage form according to a conventional process. The active ingredients are artificially synthesized and / or extracted from plants. The composition is added with conventional adjuvants, and prepared into a clinically acceptable pharmaceutical dosage form according to a conventional process.

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