Composition for treating influenza virus pneumonia as well as preparation method and application thereof

Abstract

The invention discloses a composition for treating influenza virus pneumonia as well as a preparation method and application thereof. Belongs to the technical field of traditional Chinese medicine (veterinary medicine), the caffeic acid-iron nanoparticles are composed of caffeic acid and ferric chloride hexahydrate, and the caffeic acid-iron nanoparticles are prepared through the steps that raw materials to be used are placed in a beaker according to the proportion of 1: 1 to be mixed; after mixing, putting the mixture into a shaking table at 37 DEG C to react for 1 hour, and performing ultrasonic treatment after the reaction is completed; and centrifuging the solution subjected to ultrasonic treatment for 20 minutes at the speed of 10000 r / min, and centrifuging for three times to obtain the final caffeic acid-iron nanoparticles. The preparation disclosed by the invention has antibacterial, antiviral, antioxidant and anti-inflammatory effects, has a good treatment effect on mouse pneumonia induced by influenza A virus, can enhance the immunity of mice and improve the antiviral ability of organisms, and has no toxic or side effect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of traditional Chinese medicine (veterinary medicine), and relates to a composition for treating influenza virus pneumonia, a preparation method thereof and application. BACKGROUND

[0002] Influenza virus infection has become a major public health problem threatening public health. New influenza strains without pre-immunity in the population can cause local or wide-spread prevalence, and the mortality rate of patients with severe infection is still high. In addition, avian influenza has also occurred in humans, so the prevention and treatment of influenza viruses has attracted much attention.

[0003] Nowadays, medical treatment of influenza pneumonia mainly adopts the way of injection or aerosol inhalation of target antiviral drugs and antibiotics, but the price is high, the toxicity is large, and drug resistance is easy to occur. The commonly used antiviral drugs all have different degrees of adverse reactions, among which oseltamivir can cause gastrointestinal bleeding; the main toxic reactions of amantadine are headache, delirium, ataxia, and vomiting; the side effects of ribavirin are anemia, loss of appetite, and fatigue. In addition, due to the rapid virus variation, vaccines are difficult to provide real-time protection. The natural medicine is convenient and safe, has the advantages of multi-target, multi-effect, and multi-component, and has small toxicity and is not easy to produce drug resistance, which is a better choice than vaccines and chemical drugs for the prevention and treatment of influenza. SUMMARY

[0004] The present application aims to provide a composition for treating influenza virus pneumonia with the advantages of multi-target, multi-effect, and multi-component, and another object of the present application is to provide a preparation method of the composition for treating influenza virus pneumonia with small toxicity and not easy to produce drug resistance, and the present application also provides the application of the composition in treating influenza virus pneumonia.

[0005] The technical solution of the present application is: the composition for treating influenza virus pneumonia, the mass components of the composition are as follows: 3-7 mg / mL of caffeic acid and 10-20 mg / mL of ferric chloride hexahydrate.

[0006] Further, the mass components of the composition are as follows: 5 mg / mL of caffeic acid and 15 mg / mL of ferric chloride hexahydrate.

[0007] Further, the present application also provides a preparation method of the composition for treating influenza virus pneumonia, and the preparation steps are as follows:

[0008] (1) The raw materials (caffeic acid and ferric chloride hexahydrate) to be used are mixed in a beaker at a ratio of 1:1;

[0009] (2) After mixing, it is placed in a 37℃ shaking bed for 1h of reaction, and then ultrasonic is performed after the reaction is completed.

[0010] (3) After ultrasonic treatment, the solution is centrifuged at 10,000 r / min for 20 min, and the final coffee acid-iron nanoparticles are obtained after centrifugation for three times.

[0011] Further, the preparation method of the application can prepare a composition for treating influenza virus pneumonia in terms of appearance, particle size and Zeta potential.

[0012] Further, the preparation method of the application can prepare a composition for treating influenza virus pneumonia in terms of appearance, particle size and Zeta potential.

[0013] Further, the application also provides the use of the composition for treating influenza virus pneumonia in reducing the mortality of a H1N1 (PR8)-induced mouse pneumonia model.

[0014] Further, the application also provides the use of the composition for treating influenza virus pneumonia in reducing the lung pathological score of a H1N1 (PR8)-induced mouse.

[0015] Further, the application also provides the use of the composition for treating influenza virus pneumonia in preparing a drug for reducing the lung index of a pneumonia-infected mouse.

[0016] Further, the lung index refers to the collection of lung samples of the mouse on the last day and scoring; wherein, no obvious damage is recorded as 0 points; inflammatory cell infiltration around the bronchiole and lung tissue, interstitial capillary dilation and congestion, a small amount of exudation in part of the cells is recorded as 1 point; a large number of inflammatory cells in the bronchiole and mild squamous epithelial hyperplasia, increased alveolar exudate, part of which has been consolidated, and obvious interstitial fibrous tissue hyperplasia is recorded as 2 points; most or all of the lung tissue is consolidated, the bronchiole is obviously squamous epithelial, and the bronchiole is blocked by inflammatory exudation is recorded as 3 points.

[0017] Further, the application also provides the use of the composition for treating influenza virus pneumonia in preparing a drug for reducing the lung inflammatory factor of a pneumonia-infected mouse; wherein, the inflammatory factor includes TNF-α, IL-1β and IL-10.

[0018] Further, the application also provides the use of the composition for treating influenza virus pneumonia in preparing a drug for reducing the lung oxidative stress level of a pneumonia-infected mouse; wherein, the oxidative stress level includes SOD, GSH-Px and MDA.

[0019] Further, the application also provides the use of the composition for treating influenza virus pneumonia in preparing a drug for reducing the virus load in mouse alveolar epithelial cells (MLE-12).

[0020] Further, the application also provides application of the composition for treating influenza virus pneumonia in preparation of drugs for reducing oxidative stress level in mouse alveolar epithelial cells (MLE-12); wherein the oxidative stress level comprises SOD, GSH-Px and MDA.

[0021] Further, the application also provides application of the composition for treating influenza virus pneumonia in preparation of drugs for reducing inflammatory factor level in mouse alveolar epithelial cells (MLE-12); wherein the inflammatory factor comprises TNF-α, IL-1β and IL-10.

[0022] The iron ion has the effect of enhancing the immune function of the body.

[0023] The caffeic acid has the effects of anti-inflammation, anti-oxidation and promoting cell growth, has good effects of antibiosis, anti-inflammation and anti-oxidation, can affect inflammatory pathways such as NF-κB and Nfr2, has a good prevention and treatment effect on inflammatory damage of mammary epithelial cells induced by LPS, can significantly inhibit the apoptosis and pyroptosis of macrophages, and improve the survival rate of mice with LPS-induced sepsis, the caffeic acid has the effect of inhibiting the replication of influenza viruses, can reduce the expression of inflammatory factors and the oxidative stress level in infected mice, the iron ion has the effects of enhancing immunity and improving metabolism of the body, and the preparation can inhibit the replication of viruses or enhance the immunity of the body while playing the effect of preventing and treating diseases (under the correct guidance of veterinarians), and has the advantages of small toxic and side effects.

[0024] Beneficial effects: compared with the prior art, the composition has the following significant features: 1. the composition can effectively treat mouse pneumonia; the composition has the effects of clearing heat and resolving toxins, cooling blood and stopping diarrhea, promoting qi and removing stagnation, and drying dampness and invigorating the spleen, can act on drug-resistant virus strains, can significantly inhibit the reproduction of viruses in the body, can reduce the lung damage of infected mice, can improve the survival rate of infected mice and effectively restore the growth performance of the mice, and the treatment effect is better than the treatment level of the commonly used anti-viral drugs; 2. the composition provides a new choice for preventing and treating influenza pneumonia in the process of livestock and poultry breeding, and is expected to reduce or replace the addition of anti-viral drugs in livestock and poultry breeding, alleviate the generation of drug resistance of livestock and poultry, and reduce food safety problems such as drug residues. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is a particle size result schematic diagram of the caffeic acid-iron nanoparticles in the embodiment of the application;

[0026] Figure 2 is a Zeta potential result schematic diagram of the caffeic acid-iron nanoparticles in the embodiment of the application;

[0027] Figure 3is a transmission potential result comparison chart of caffeic acid-iron nanoparticles in the embodiment of the present application;

[0028] Figure 4 is a Fourier infrared spectrum analysis comparison chart of caffeic acid-iron nanoparticles in the embodiment of the present application;

[0029] Figure 5 is a survival rate and body weight change comparison chart of each test group in the embodiment of the present application; wherein, Figure A is a body weight change comparison chart of each test group within 7 days; Figure B is a survival rate comparison chart of each test group;

[0030] Figure 6 is a lung lesion score comparison chart of each test group in the embodiment of the present application;

[0031] Figure 7 is a lung tissue lesion observation comparison chart of each group of mice in the embodiment of the present application;

[0032] Figure 8 is a lung tissue pathological section result comparison chart of each group of mice in the embodiment of the present application;

[0033] Figure 9 is a lung index comparison chart of each test group of mice in the embodiment of the present application;

[0034] Figure 10 is a lung virus load result comparison chart of each group of mice in the embodiment of the present application;

[0035] Figure 11 is a peripheral blood inflammatory factor content level comparison chart of each group of mice in the embodiment of the present application; wherein, Figure A is an IL-1β content comparison chart in the serum of each test group; Figure B is a TNF-α content comparison chart in the serum of each test group; Figure C is an IL-10 content comparison chart in the serum of each test group;

[0036] Figure 12 is an oxidative stress index result comparison chart of each group of mice in the embodiment of the present application; wherein, Figure A is an MDA content comparison chart in the lung tissue of each test group; Figure B is an SOD content comparison chart in the lung tissue of each test group; Figure C is a GSH-Px content comparison chart in the lung tissue of each test group;

[0037] Figure 13 is a content chart of caffeic acid and caffeic acid-iron nanoparticles in the plasma of mice in the embodiment of the present application;

[0038] Figure 14 is a virus load result chart of mouse alveolar epithelial cells (MLE-12) in the embodiment of the present application;

[0039] Figure 15 is an inflammatory factor index result chart of mouse alveolar epithelial cells (MLE-12) in the embodiment of the present application;

[0040] Figure 16 Figure is the result chart of the mouse alveolar epithelial cell (MLE-12) oxidative stress index in the embodiment of the application. DETAILED DESCRIPTION

[0041] The specific technical solutions of the application will be further described in detail below in combination with specific examples.

[0042] As shown in the figure, the composition for treating influenza pneumonia in large-scale breeding according to the application is taken by proportion: one pair of coffee acid 5 mg / mL and ferric chloride hexahydrate 15 mg / mL, which are respectively dissolved and mixed in a ratio of 1:1; after mixing, it is put into a 37℃ shaking bed for reaction for 1h, and then ultrasonic treatment is performed; the solution after ultrasonic treatment is centrifuged at 10000 r / min for 20 min, and the final coffee acid-iron nanoparticles can be obtained after centrifugation for three times; each diseased animal is administered with a dose of 50 mg / kg per day, and a gavage tool is used for administration.

[0043] The preparation has the effects of clearing heat and drying dampness, cooling blood and stopping diarrhea, and promoting qi and removing stagnation, has a good therapeutic effect on H1N1 PR8 infection, and has small toxic and side effects.

[0044] Experimental results

[0045] 1. Method

[0046] 1.1. Experimental animals and materials

[0047] Experimental animals: ICR mice, male, 5 weeks old, weight 21±1g, purchased from the experimental animal center of a certain university (production license: SCXK(Su)2017-0007; use license: SCXK(Su)2017-0044); feeding conditions: room temperature 20±1℃, humidity 30%~40%, free feeding and drinking water, adaptive feeding for 1d before experiment.

[0048] Experimental materials: paraformaldehyde (Shanghai Biotechnology Co., Ltd.); glutaraldehyde, section paraffin (69019361) purchased from a group chemical reagent Co., Ltd.; section machine (MR2016), biological microscope; su-tai injection, physiological saline solution (both purchased from the animal hospital of a certain university), H1N1 (PR8) strain MLD 50 is 10 3.12 and 10 2.42 TCID 50 / 50 μL; Superoxide dismutase (SOD) kit, glutathione peroxidase (GSH-Px) kit, malondialdehyde (MDA) kit, all purchased from a company in Nanjing, the above indexes were detected by chemiluminescence method and according to the kit instructions; cytokine indexes (IL-1β, TNF-α, IL-10) were detected by ELISA kit (Shanghai Biotechnology Co., Ltd.); microplate reader (Epoch) was purchased from a company in the United States.

[0049] 1.2, preparation of water decoction

[0050] Coffee acid-iron nanoparticles were obtained by mixing coffee acid solution and FeCl3 solution in sterile water; 5 mg / mL coffee acid solution and 15 mg / mL FeCl3 solution were added to sterile water in turn, and each addition required shaking and mixing; then it was placed in a 37°C constant temperature water bath oven and stirred in the dark for 1 h, and the color of the reaction solution gradually changed to dark green; after the reaction was completed, centrifugation was performed at 10000 rpm / min for 20 min, the supernatant was removed, and the precipitate was collected; the precipitate was washed with sterile water and ultrasonically mixed, the supernatant was removed by centrifugation twice, and the precipitate was resuspended with sterile water, shaken and ultrasonically dispersed, and finally pure coffee acid-iron nanoparticles were obtained, which were stored at 4°C in the dark.

[0051] 1.3, experimental method

[0052] Experimental grouping: blank group (Control), model group (Model), coffee acid-iron nanoparticle low-dose group (Low), coffee acid-iron nanoparticle high-dose group (High), ribavirin group (RBV), 15 mice in each group.

[0053] Treatment method: except for the blank group, the rest of the mice were anesthetized by intraperitoneal injection of Turbuhaler, and 50 μL MLD 50 of H1N1 (PR8) strain was used for nasal drop modeling, and the blank group was anesthetized and dropped with 50 μL of normal saline; after modeling, the model group of mice was given intragastric injection of normal saline every day; the low and high dose groups of coffee acid-iron nanoparticles were given coffee acid-iron nanoparticle solution at doses of 25 and 50 g / kg, respectively; the ribavirin group was given intraperitoneal injection of ribavirin suspension at a dose of 18 mg / kg; all experimental groups of mice were treated once a day, and the dose was 0.2 mL / d, for 7 consecutive days; the mice were free to drink water and eat during the whole test period; the body weight and death of the mice in each group were recorded every day, and the samples were collected on the 8th day of the experiment; the peripheral blood and lung tissue of the mice were collected and frozen in a-80°C refrigerator, and the lung tissue was fixed in 4% paraformaldehyde solution without collecting the bronchoalveolar lavage fluid.

[0054] 1.4、Particle size and Zeta potential determination of caffeic acid-iron nanoparticles

[0055] Dilute the appropriate amount of caffeic acid-iron nanoparticles by 20 times, and then determine the particle size and Zeta potential by Malvern particle analyzer.

[0056] 1.5、Transmission electron microscope observation of caffeic acid-iron nanoparticles

[0057] After the prepared caffeic acid-iron nanoparticles are appropriately diluted with PBS, they are placed on a copper mesh, dried, and then 1% tungsten phosphate is added dropwise for negative staining for 5 minutes. After air-drying at room temperature, they are observed and photographed by transmission electron microscope (TEM).

[0058] 1.6、Fourier infrared spectrum analysis of caffeic acid-iron nanoparticles

[0059] Potassium bromide flakes are used as a blank background. An appropriate amount of sample is added to the potassium bromide powder, mixed and ground together, and then dried to form flakes. A Fourier infrared spectrometer is used to scan in the range of 500-4000 cm -1 to obtain the infrared spectrum of each sample.

[0060] 1.7、Observation of clinical symptoms of mice

[0061] The clinical symptoms of mice in each test group are closely observed at the beginning of the test, including mental state, water drinking status, and food intake status, etc. The respiratory status of mice is observed starting from the 3rd day after virus inoculation.

[0062] 1.8、Mortality rate

[0063] The mortality of mice in each group is counted on the 8th day after infection. The mortality rate (%) of each group = the number of dead mice in the group / the total number of test mice in the group x 100%.

[0064] 1.9、Lung lesion score and lung index determination of mice

[0065] On the 8th day after infection, the mice in each test group are sacrificed and their lungs are collected. The appearance of the lesions is observed and the lung pathological score is determined according to Table 1.

[0066] Determination of lung index and judgment of lung lesion degree: lung index = mouse lung weight (g) / mouse body weight (g) x 100%

[0067] Table 1: Lung pathological score determination criteria

[0068]

[0069] 1.10、Histological observation of lung tissue of mice

[0070] The mice in each test group were sacrificed 8 days after H1N1 PR8 infection, and the lungs were collected and scored for pathological changes. One side of the lung tissue was placed in 4% neutral formaldehyde for re-fixing to prepare conventional HE-stained sections. The other side of the lung tissue was stored in a -80°C refrigerator.

[0071] 1.11. Determination of inflammatory factors in lung tissue of mice

[0072] TNF-α, IL-1β, and IL-10 content detection: according to the kit instructions; after washing the microplate 3 times, centrifuge the tissue homogenate at 1000 r / min for 10 min, take 100 μL of supernatant and add it to the microplate, incubate at 37°C for 2 h, then wash the plate 3 times; add 100 μL of biotinylated antibody working solution, incubate at 37°C for 1 h, then wash the plate 3 times; add 100 μL of streptavidin-HRP working solution and incubate at 37°C for 0.5 h, then wash 3 times; add 100 μL of substrate solution and incubate at 37°C for 20 min in the dark; immediately after adding 50 μL of stop solution, use an enzyme-labeled instrument to detect the reading at 450 nm, with a correction wavelength of 630 nm; use the standard curve to make corresponding calculations and statistics.

[0073] 1.12. Determination of redox indicators in lung tissue of mice

[0074] After the end of the experiment, 5 mice were randomly selected from each group, sacrificed, and lung tissue was collected, quickly frozen in liquid nitrogen, and stored at -80°C.

[0075] Preparation of lung tissue homogenate: take 70 mg of lung tissue, rinse with pre-cooled normal saline, and wipe dry with filter paper, weigh, and add RIPA lysis buffer to prepare a 10% tissue homogenate.

[0076] SOD activity detection: centrifuge the 10% tissue homogenate at 3000 r / min for 10 min; take 20 μL of supernatant and add it to the microplate, according to the kit requirements, add the corresponding reaction solution prepared to the microplate, incubate at 37°C for 20 min, zero at 450 nm with distilled water, and then use an enzyme-labeled instrument to read the OD values of each tube, and use the recommended method in the instructions to make corresponding calculations and statistics.

[0077] GSH-Px activity detection: centrifuge the 10% tissue homogenate at 3000 r / min for 10 min; take 100 μL of supernatant and add it to a 4 mL centrifuge tube with the corresponding reaction solution prepared according to the kit requirements, stand at room temperature for 15 min, zero at 412 nm with distilled water, and then use an enzyme-labeled instrument to read the OD values of each tube, and use the recommended method in the instructions to make corresponding calculations and statistics.

[0078] MDA content detection: take 10% tissue homogenate 100 μL and the prepared reaction solution into 4 mL centrifuge tube, vortex mix, 95°C water bath for 40 min, cool with running water, 4000 r / min centrifuge for 10 min, take supernatant, adjust zero at 532 nm, read OD value of each tube by enzyme label instrument, use the recommended method to calculate and count.

[0079] 1.13, Drug content determination in mouse plasma

[0080] Two groups of mice were respectively given 5 mg / mL coffee acid and coffee acid-iron nanoparticles by gavage, and anticoagulated blood was collected at 5 min, 10 min, 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h and 24 h, and centrifuged at 3000 r / min for 10 min, and the supernatant was taken; 50 μL of plasma was added with 200 μL of methanol to precipitate protein, and centrifuged at 12000 r / min for 20 min, and the supernatant was taken; the drug content in plasma was detected.

[0081] 1.14, Determination of viral load in mouse alveolar epithelial cells (MLE-12)

[0082] After 72 h of virus infection, 1 mL of Trizol was added to each well of the 6-well plate, and after 10 min of action, 400 μL of DDW was added to each well, the cells were blown down and collected in a 1.5 mL centrifuge tube, centrifuged at 12000 r / min for 10 min at 4°C, the supernatant was taken, and the extraction was repeated twice, 600 μL of isopropanol was added to each tube, and it was mixed gently up and down; stand for 15 min; 4°C, 12000 r / min centrifugation for 10 min, discard the supernatant, add 500 μL of 75% ethanol to each tube, gently invert, reselect the precipitate, 4°C, 7500 r / min centrifugation for 10 min, wash twice, finally aspirate the alcohol, dry at room temperature for 10 min; add 20 μL of DDW to each tube, mix gently, detect the RNA concentration, use DDW to balance the RNA content in each tube; add 3 μL of 5X gDNA digester Mix to each tube, 42°C for 2 min, then add 5 μL of 4X Hifair Ⅲ Super Mix plus, and reverse transcribe to cDNA on the machine; add primers, SYPR and cDNA to form a suitable system, and detect the expression amount of H1N1 M1 mRNA on the machine.

[0083] 1.15, Inflammatory factor determination of mouse alveolar epithelial cells (MLE-12)

[0084] After 72 h of H1N1 PR8 infection of mouse alveolar epithelial cells (MLE-12), the cells were collected.

[0085] TNF-a, IL-1 b, IL-10 content detection: according to the kit instructions; after washing the microplate for 3 times, centrifuge the cell suspension at 1000 r / min for 10 min, take 100 mI_ supernatant and add it into the microplate, incubate at 37°C for 2 h, then wash the plate for 3 times; add 100 mI_ biotinylated antibody working solution, incubate at 37°C for 1 h, then wash the plate for 3 times; add 100 mI_ streptavidin-HRP working solution, incubate at 37°C for 0.5 h, then wash for 3 times; add 100 mI_ substrate solution and incubate at 37°C for 20 min in the dark; add 50 mI_ stop solution, then immediately detect by microplate reader at 450 nm, and the correction wavelength is 630 nm; use the standard curve for corresponding calculation and statistics.

[0086] 1.16, Redox index determination of mouse alveolar epithelial cells (MLE-12)

[0087] Collect the cells after H1N1 PR8 infection of mouse alveolar epithelial cells (MLE-12) for 72 h.

[0088] SOD activity detection: centrifuge the cell suspension at 3000 r / min for 10 min; take 20 mI_ supernatant and add it into the microplate, add the corresponding reaction solution prepared according to the kit requirements into the microplate, incubate at 37°C for 20 min, then read the OD values of each tube at 450 nm after zero adjustment with distilled water, and use the recommended method in the instruction for corresponding calculation and statistics.

[0089] GSH-Px activity detection: centrifuge the cell suspension at 3000 r / min for 10 min; take 100 mI_ supernatant and add it into 4 mL centrifuge tube with the corresponding reaction solution prepared according to the kit requirements, stand at room temperature for 15 min, then read the OD values of each tube at 412 nm after zero adjustment with distilled water, and use the recommended method in the instruction for corresponding calculation and statistics.

[0090] MDA content detection: take 100 mI_ cell suspension and add it into 4 mL centrifuge tube with the corresponding reaction solution prepared, vortex to mix, then incubate at 95°C water bath for 40 min, cool with running water, then centrifuge at 4000 r / min for 10 min, take the supernatant, read the OD values of each tube at 532 nm after zero adjustment with distilled water, and use the recommended method in the instruction for corresponding calculation and statistics.

[0091] 1.17, Data analysis

[0092] Use IBM SPSS Statistics 17.0 software for statistical analysis, use Graphpad Prism 9.5.1 to make charts for test data; the data are represented as mean ± standard deviation (x ± s), and one-way ANOVA is used for comparison between groups; the significant difference standard is P<0.05.

[0093] 2. Results

[0094] 2.1 Appearance, particle size and zeta potential of caffeic acid-iron nanoparticles

[0095] like Figure 1 and Figure 2 The caffeic acid-iron nanoparticles, as determined by a Malvern particle analyzer, had a particle size of 173.2 ± 8.34 nm, a polydispersity index (PDI) of 0.28 ± 0.03, and a zeta potential of 24.1 ± 2.14 mV. Figure 3 As shown, caffeic acid-iron nanoparticles appear as uniform gray particles under visual observation, and as uniform spherical particles under TEM.

[0096] 2.2 Infrared Spectroscopy Analysis

[0097] The infrared spectra of caffeic acid-iron nanoparticles, CA, and FeCl3 were measured respectively as follows: Figure 4 As shown; in the CA spectrum, the stretching vibrations of the two phenolic hydroxyl groups (OH) are located at 3418 cm⁻¹. -1 and 3231cm -1 The C=O stretching vibration of the carboxyl carbonyl group is at 1648 cm⁻¹. -1 In the caffeic acid-iron spectrum, after caffeic acid chelates with iron ions, only 3445 cm⁻¹ remains. -1 The absorption peak of the OH stretching vibration of the phenolic hydroxyl group is at 3418 cm⁻¹. -1 The disappearance of the OH signal peak at the phenolic hydroxyl group indicates that the hydroxyl group in the caffeic acid molecule has transformed from a free state to an associated state under the action of iron ion complexation, forming ionic bonds and hydrogen bonds, etc.; 1647cm -1 The presence of the C=O stretching vibration absorption peak at the carboxyl carbonyl group indicates that caffeic acid-iron nanoparticles have been successfully prepared.

[0098] 2.3. Caffeic acid-iron nanoparticles improve the survival rate of mice infected with H1N1 PR8.

[0099] Figure 5 As shown in Table 1, mice died in all experimental groups except the Control group. The survival rate was 60% in the Model group, 66.7% in the low-dose caffeic acid-iron nanoparticle group, and 86.7% in both the high-dose caffeic acid-iron nanoparticle group and RBV group. This indicates that there was no significant difference in the efficacy of 50 mg / kg caffeic acid-iron nanoparticles and 18 mg / kg ribavirin in treating H1N1PR8-infected mice. The changes in survival rate and relative weight gain showed that 50 mg / kg caffeic acid-iron nanoparticles had a better effect on treating pneumonia in H1N1PR8-infected mice.

[0100] 2.4, Lung tissue pathological score results of each group of mice

[0101] As shown in the table, the lesion score of the coffee acid-iron nanoparticle treatment group was reduced, and the treatment effect of the high-dose coffee acid-iron nanoparticle group was not significantly different from that of the RBV treatment group. Figure 6

[0102] 2.5, Lung tissue pictures of each group of mice

[0103] As shown in the table, the lung tissue of the model group of mice showed edema, hemorrhage and necrosis; the coffee acid-iron nanoparticle group showed varying degrees of edema and hemorrhage, and the high-dose group had the lightest lung lesions, which was not significantly different from the lung tissue of the RBV group. Figure 7

[0104] 2.6, Lung tissue pathological section results of each group of mice

[0105] As shown in the table, the lung tissue of the model group of mice lost the complete alveolar structure, the alveolar septum thickened, and the inflammatory cells and red blood cells in the tissue infiltrated seriously; compared with the model group, the lung tissue of the low and high dose coffee acid-iron nanoparticle groups of mice still had complete alveolar structure, the alveolar septum thickening was reduced, and the red blood cell infiltration in the tissue was significantly reduced; the lung tissue of the RBV group of mice had obvious red blood cell infiltration and alveolar septum thickening. Figure 8

[0106] 2.7, Body weight and lung index results of each group of mice

[0107] As shown in the table, compared with the control group, the lung index of the model group of mice was extremely significantly increased (P<0.001); compared with the model group, the lung index of the low-dose coffee acid-iron nanoparticle group was reduced (P<0.05), and the lung index of the high-dose coffee acid-iron nanoparticle group was extremely significantly reduced (P<0.001); compared with the model group, the lung index of the RBV treatment group was reduced (P<0.05); the results showed that coffee acid-iron nanoparticles can effectively reduce the lung index of viral pneumonia mice, and the treatment effect is not significantly different from that of RBV. Figure 9 2.8, Lung viral load results of each group of mice

[0108] As shown in the table, the lung viral load of the model group of mice was extremely significantly increased (P<0.001); compared with the model group, the lung viral load of the low-dose coffee acid-iron nanoparticle group was reduced (P<0.05), and the lung viral load of the high-dose coffee acid-iron nanoparticle group was extremely significantly reduced (P<0.001); compared with the model group, the lung viral load of the RBV treatment group was reduced (P<0.05); the results showed that coffee acid-iron nanoparticles can effectively reduce the lung viral load of viral pneumonia mice, and the treatment effect is not significantly different from that of RBV.

[0109] Figure 10 ​​​​As shown, compared with the Control group, the relative expression level of H1N1 in the Model group was significantly increased (P < 0.001); compared with the Model group, the relative expression level of H1N1 in the lung tissue of mice treated with caffeic acid-iron nanoparticles was significantly decreased (P < 0.001), and the relative expression level of H1N1 in mice treated with RBV was significantly decreased (P < 0.05); PCR results showed that both caffeic acid-iron nanoparticles and RBV could effectively reduce the viral content in the lung tissue of mice with viral pneumonia, and the difference between the two was not significant.

[0110] 2.9 Results of inflammatory factor indices in each group of mice

[0111] like Figure 11 As shown, caffeic acid-iron nanoparticles can effectively reduce the content of inflammatory factors in mouse serum, and there is no significant difference in lung tissue between the RBV group and the RBV group.

[0112] 2.10 Results of oxidative stress indices in each group of mice

[0113] like Figure 12 The results showed that, compared with the Control group, the levels of GSH-Px and SOD in the lung tissue of mice were significantly increased (P < 0.01), and the level of MDA was significantly decreased (P < 0.001). Compared with the Model group, the low-dose caffeic acid-iron nanoparticle group showed significantly increased GSH-Px and SOD levels (P < 0.01), and significantly decreased MDA levels (P < 0.01). The high-dose caffeic acid-iron nanoparticle group showed significantly increased GSH-Px levels (P < 0.01), increased SOD levels (P < 0.05), and significantly decreased MDA levels (P < 0.001). Compared with the Model group, the RBV group showed significantly increased GSH-Px levels and significantly decreased MDA levels (P < 0.01). The results indicated that caffeic acid-iron nanoparticles can effectively improve the level of oxidative stress in mice, and there was no significant difference in lung tissue between the RBV group and the Model group.

[0114] 2.11. Content of caffeic acid and caffeic acid-iron nanoparticles in mouse plasma

[0115] like Figure 13 The results showed that within 2 hours after gavage, the content of caffeic acid-iron nanoparticles in mouse plasma was higher than that of caffeic acid; after 2 hours, the content of each substance decreased significantly.

[0116] 2.12 Results of viral load in mouse alveolar epithelial cells (MLE-12)

[0117] like Figure 14The results showed that, compared with the control group, the relative expression level of H1N1 in the model group was significantly increased (P < 0.001); compared with the model group, the relative expression level of MLE-12 H1N1 in the caffeic acid-iron nanoparticle treatment group was significantly decreased (P < 0.001), and the relative expression level of MLE-12 H1N1 in the RBV treatment group was significantly decreased (P < 0.05); PCR results showed that both caffeic acid-iron nanoparticles and RBV could effectively reduce the viral load in MLE-12, and the difference between the two was not significant.

[0118] 2.13 Results of inflammatory factor markers in mouse alveolar epithelial cells (MLE-12)

[0119] like Figure 15 As shown, caffeic acid-iron nanoparticles can effectively reduce the content of inflammatory factors in MLE-12, and there is no significant difference in lung tissue between the RBV group and the RBV group.

[0120] 2.14 Results of Oxidative Stress Indicators in Mouse Alveolar Epithelial Cells (MLE-12)

[0121] like Figure 16 The results showed that, compared with the control group, the contents of GSH-Px and SOD in MLE-12 were significantly increased (P<0.01), and the contents of MDA were significantly decreased (P<0.001); compared with the model group, the contents of GSH-Px and SOD in the 100 μg / mL caffeic acid group were significantly increased (P<0.01), and the contents of MDA were significantly decreased (P<0.01), while the contents of GSH-Px in the 100 μg / mL caffeic acid-iron nanoparticle group were significantly increased (P<0.01); compared with the model group, the contents of GSH-Px in the RBV group were significantly increased, and the contents of MDA were significantly decreased (P<0.01). The results indicate that caffeic acid-iron nanoparticles can effectively improve the oxidative stress level in MLE-12.

[0122] 3. Summary

[0123] Caffeic acid-iron nanoparticles can significantly reduce the viral load in the lungs of influenza virus pneumonia, reduce pathological damage to the lung tissue of mice caused by H1N1PR8 infection, improve the lung index, increase the survival rate of infected chickens, reduce the content of MDA in lung tissue, increase the content of SOD and GSH, alleviate the inflammatory response in the lungs, reduce the viral load in alveolar epithelial cells of mice after infection, reduce the content of MDA in alveolar epithelial cells of mice, increase the content of SOD and GSH, and alleviate the inflammatory response of alveolar epithelial cells of mice, thereby effectively reducing the mortality rate of severe pneumonia induced by H1N1 in mice.

[0124] Table 2 is a comparison table of the survival rates of various experimental groups in the embodiments of the present invention:

[0125] Table 2

[0126]

Claims

1. A composition for treating influenza virus pneumonia, characterized in that, The composition by mass is as follows: caffeic acid 3-7 mg / mL, ferric chloride hexahydrate 10-20 mg / mL.

2. The method for preparing the composition for treating influenza virus pneumonia as described in claim 1, characterized in that, The preparation steps are as follows: (1) Mix the raw materials to be used in a beaker according to the proportion; (2) After mixing, place the mixture in a shaker at 37°C to react, and then sonicate after the reaction is complete. (3) The ultrasonic solution was centrifuged three times to obtain caffeic acid-iron nanoparticles.

3. The method for preparing the composition for treating influenza virus pneumonia according to claim 2, characterized in that, The mass ratio of the raw materials to be used in step (1) is 1:1; The reaction time in step (2) is 1 hour; The centrifugation conditions in step (3) are: 10000 r / min, 20 min.

4. The application of the composition for treating influenza virus pneumonia prepared according to the method described in claim 2 in reducing the mortality rate of an H1N1 (PR8)-induced mouse pneumonia model.

5. The application of the composition for treating influenza virus pneumonia prepared according to the method described in claim 2 in reducing the pathological score of lungs in mice induced by H1N1 (PR8).

6. The use of the composition for treating influenza virus pneumonia prepared according to the method described in claim 2 in the preparation of a drug for reducing the lung index of mice with pneumonia infection.

7. The application according to claim 6, characterized in that, The lung index refers to the lung samples collected from mice on the last day and scored accordingly.

8. The use of the composition for treating influenza virus pneumonia prepared according to the method described in claim 2 in the preparation of a drug that reduces the levels of inflammatory factors and oxidative stress in the lungs of pneumonia-infected mice.

9. The use of the composition for treating influenza virus pneumonia prepared according to the method described in claim 2 in the preparation of a drug for reducing viral load in mouse alveolar epithelial cells.

10. The use of the composition for treating influenza virus pneumonia prepared according to the method described in claim 2 in the preparation of drugs that reduce the level of oxidative stress and inflammatory factors in mouse alveolar epithelial cells.

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