Preparation method and application of Arbidol and chitosan-glycyrrhizic acid gel
By preparing Abidol@chitosan-glycyrrhizic acid gel and using chitosan and glycyrrhizic acid to cross-link Abidol, the problems of drug resistance and gastric juice release of chemical synthetic drugs were solved, high safety and multi-target antiviral effects were achieved, and lung viral load and adverse reactions were reduced.
Patent Information
- Application Number
- CN202510936074.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-08
- Publication Date
- 2025-10-17
AI Technical Summary
Existing chemically synthesized drugs may develop antiviral resistance during or after the treatment of influenza A, and the large-scale release of Abidol in gastric juice may cause adverse reactions. There is an urgent need to develop a highly safe, multi-target alternative or auxiliary drug.
Arbidol@chitosan-glycyrrhizic acid gel was prepared by cross-linking the natural polysaccharide chitosan with glycyrrhizic acid. The release of Arbidol in gastric juice was reduced through gel sustained release, and combined with the multi-target antiviral effect of glycyrrhizic acid, it inhibited phosphorylated Akt and phosphorylated NF-κB to weaken the cytokine storm.
The drug's anti-influenza A virus efficacy is improved, the lung viral load is reduced, adverse reactions are minimized, biosafety is enhanced, and large-scale production is facilitated.
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Figure CN120789081A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biopharmaceuticals, more particularly, it relates to a preparation method and application of arbidol@chitosan-licorice acid gel. BACKGROUND
[0002] At present, the treatment methods for influenza A virus include vaccine prevention, direct virus killing treatment, inhibition of viral gene replication and protein activity drugs, reduction of lung injury and "cytokine storm" caused by viral infection, and treatment of influenza virus drugs including small molecule compounds, macromolecular compounds, monoclonal antibody therapy, attenuated vaccine, inactivated vaccine and subunit vaccine. Small molecule drugs can be divided into chemical synthetic drugs and natural small molecule monomer compounds. At present, the FDA-approved small molecule chemical synthetic drugs include NA inhibitors oseltamivir, zanamivir and peramivir, which are administered orally, inhaled and intravenously, respectively, and act on NA to reduce the release of progeny viruses. M2 ion channel inhibitors amantadine and rimantadine inhibit ion channel proteins to inhibit viral acidification. In addition, the PA inhibitor baloxavir, which targets vRNP, has been marketed in the United States and Japan, and the candidate drug favipiravir has entered clinical trials. In addition, the FDA-approved drug for the treatment of postpartum depression, tetrahydrogestrinone, has the effect of targeting NP to play an anti-influenza virus role, protecting the NES3 region of NP, preventing NP from being exported, and causing NP to abnormally accumulate in the nucleus. Sialic acid glycan analogues and arbidol can target HA to play an anti-influenza virus role.
[0003] Due to the continuous evolution of influenza A virus, chemical synthetic drugs may develop antiviral drug resistance during or after treatment, and there is an urgent need to develop a drug that can replace or assist small molecule chemical synthetic drugs in antiviral action. Traditional Chinese medicine small molecule monomer compounds have attracted more and more attention in the field of antiviral drugs due to their good safety and multi-target effects. It has been reported that natural small molecule drugs with antiviral and anti-inflammatory effects include flavonoids such as apigenin, emodin, quercetin and hesperidin, phenolic acids such as rosmarinic acid, alkaloids such as berberine, and terpenoids such as oleanolic acid and glycyrrhizic acid. Natural high molecular materials have been gradually recognized due to their good biocompatibility and certain antiviral effects. Studies have shown that marine polysaccharides and oligosaccharides such as chitosan, carrageenan, alginate and fucoidan have antiviral activity.
[0004] Therefore, a natural polysaccharide chitosan is developed as a material, glycyrrhizic acid is used as a chitosan crosslinking agent and an anti-inflammatory and antiviral drug component, arbidol is loaded, and an arbidol@chitosan-licorice acid (Ab@CS-GL) gel is constructed to play a slow-release role, reduce the gastrointestinal irritation of arbidol, improve the safety of drug administration, and combine with the multi-target effect of glycyrrhizic acid to play an anti-influenza virus role. SUMMARY
[0005] The application aims to provide a preparation method and application of arbidol@ chitosan-glycyrrhizic acid gel, which realizes combination of drugs, improves the anti-influenza A virus efficacy of the drugs, and overcomes the adverse reactions caused by massive release of arbidol in gastric juice through gel sustained release.
[0006] The above technical objective of the application is achieved by the following technical scheme: a preparation method of arbidol@ chitosan-glycyrrhizic acid gel, comprising the following steps:
[0007] S1: (1) 500.0 mg of chitosan is weighed in a reaction bottle, dissolved by adding appropriate amount of 1% glacial acetic acid, and stirred at 150 rpm on a magnetic stirrer for 4 h until completely dissolved;
[0008] (2) 1.0 g of glycyrrhizic acid is weighed in another reaction bottle, dissolved by adding DMSO, 465.8 mg of EDC and 279.7 mg of NHS are added to the reaction bottle at 0℃, and stirred under argon protection for 0.5 h;
[0009] (3) After the reaction is completed, the glycyrrhizic acid after activation of the carboxyl group is slowly added to the chitosan reaction bottle, and stirred while adding, and after the addition is completed, the reaction is carried out overnight;
[0010] (4) The chitosan glycyrrhizic acid after the reaction is completed is added to a dialysis bag with a molecular weight of 8000 Da, and after dialysis for 12 h, the chitosan-glycyrrhizic acid gel, i.e., CS-GL gel, is obtained by freeze-drying;
[0011] S2: 500 mg of the freeze-dried CS-GL is weighed in a reaction bottle, and after stirring to a viscous gel state, arbidol hydrochloride hydrate is added, and after stirring for 12 h, the Ab@CS-GL drug-loaded gel is obtained by freeze-drying.
[0012] The application further provides application of arbidol@ chitosan-glycyrrhizic acid gel in preparation of a drug for treating H1N1 and H3N2 influenza virus strain infection.
[0013] The application is further provided as follows: the drug reduces cytokine storm, reduces lung viral load, and improves lung lesions by inhibiting phosphorylated Akt and phosphorylated NF-κB.
[0014] In summary, the application has the following beneficial effects:
[0015] The natural product glycyrrhizic acid is used to crosslink chitosan, the toxic side effects of other chemical crosslinking agents are avoided, arbidol is physically encapsulated in the gel network to reduce the adverse reactions caused by massive release of arbidol in the stomach, glycyrrhizic acid and arbidol are combined to exert anti-virus effect through multiple targets, and the efficacy is increased and the toxicity is reduced.
[0016] The glycol of arbidol@ chitosan-licorice acid provided by the application has good biological safety by adopting the mode of combined medication, can weaken cytokine storm by inhibiting phosphorylation Akt and phosphorylation NF-κB, reduce lung virus load, improve lung lesions, can reduce the infection rate of MDCK cells infected by influenza A virus and the degree of cytopathic effect, reduce the level of intracellular virus nucleoprotein and inhibit the replication of influenza A virus. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 Ab CS-GL and CS-GL synthesis route and structural change diagram.
[0018] Figure 2 A GL (500Hz, d6-DMSO), CS (500Hz, D2O), CS-GL (500Hz, D2O) nuclear magnetic hydrogen spectrum.
[0019] Figure 3 CS-GL, CS, GL infrared spectrum.
[0020] Figure 4 Ab CS-GL, CS-GL, GL, Ab ultraviolet spectrum.
[0021] Figure 5 Release of Ab CS-GL under simulated physiological conditions (SGF: artificial gastric juice; SIF: artificial small intestinal juice).
[0022] Figure 6 CS-GL and Ab CS-GL different magnification field emission scanning electron microscope images
[0023] Figure 7 Cytotoxicity of Ab CS-GL incubated MDCK cells for 48h and inhibition rate of influenza virus H1N1 infection.
[0024] Figure 8 Indirect immunofluorescence detection of Ab CS-GL each group inhibits H1N1 infection level.
[0025] Figure 9 Indirect immunofluorescence detection of Ab CS-GL each group inhibits H3N2 infection level.
[0026] Figure 10 Changes of rat weight and survival rate of H1N1 infected mice treated with Ab CS-GL each group drug for 14 days.
[0027] Figure 11 Changes of rat weight and survival rate of H3N2 infected mice treated with Ab CS-GL each group drug for 14 days.
[0028] Figure 12Virus load of lung tissue and nasal turbinate of H1N1 challenged mice treated with Ab@CS-GL for 4, 6 days.
[0029] Figure 13 Lung tissue lesion of H1N1 challenged mice treated with Ab@CS-GL for 4, 6 days.
[0030] Figure 14 Phosphorylated Akt and phosphorylated NF-κB protein levels of lung tissue of H1N1 challenged mice.
[0031] Figure 15 Related inflammatory factor levels of lung tissue of H1N1 challenged mice. DETAILED DESCRIPTION
[0032] The following description will be made in conjunction with the accompanying drawings as follows: Figures 1-15 The present application is further described in detail.
[0033] The main experimental reagents and instrument equipment used in the present application are briefly introduced as follows: chitosan (CS, viscosity), glycyrrhizic acid (GL), arbidol hydrochloride (Ab), 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI·HCl), N-hydroxysuccinimide (NHS), dialysis bag (MW 8000), fully digital superconducting nuclear magnetic resonance hydrogen spectrometer, freeze dryer, etc.
[0034] Example 1
[0035] The preparation method of arbidol@chitosan-glycyrrhizic acid (Ab@CS-GL) synthesis in the present embodiment is shown in the synthesis route of CS-GL and Ab@CS-GL as follows: Figure 1
[0036] The specific steps are as follows:
[0037] 1.1 Synthesis of CS-GL
[0038] (1) Take chitosan 500.0 mg in a reaction bottle, add appropriate amount of 1% glacial acetic acid to dissolve, and stir at 150 rpm on a magnetic stirrer for 4 h until completely dissolved.
[0039] (2) Take 1.0 g of glycyrrhizic acid and put it into another reaction bottle, dissolve in DMSO, and add 465.8 mg of EDC and 279.7 mg of NHS to the reaction bottle at 0°C, and stir for 0.5 h under argon protection.
[0040] (3) After the reaction is completed, slowly drop the glycyrrhizic acid after activation of the carboxyl group into the chitosan reaction bottle, and stir while dropping. After adding, the reaction is protected by argon overnight.
[0041] (4) The reaction completed chitosan glycyrrhizin acid was added into 8000 Da dialysis bag, and after dialysis for 12 h, the chitosan-glycyrrhizin acid gel, i.e. CS-GL gel, was obtained by lyophilization.
[0042] 1.2 Ab@CS-GL synthesis
[0043] 500.0 mg of lyophilized CS-GL was weighed in a reaction bottle, and after stirring to a viscous gel state, 196.0 mg of arbidol hydrochloride hydrate dissolved in purified water (CS-GL: Ab = 10.3) was added. After stirring for 12 h, Ab@CS-GL drug-loaded gel was obtained by lyophilization.
[0044] 1.3 Characterization method and result analysis
[0045] (1) The success of CS and GL connection was verified using a nuclear magnetic resonance instrument;
[0046] 5-10 mg of GL was dissolved in d6-DMSO, and CS and CS-GL were dissolved in D2O. The success of CS-GL connection was verified using a nuclear magnetic resonance instrument, as shown in Figure 2 .
[0047] (2) The structure of CS and GL was verified using a Fourier infrared spectrometer;
[0048] An appropriate amount of the solid sample to be tested GL, CS, CS-GL and dry potassium bromide were mixed according to a ratio of 1:100, ground and pressed into a tablet. The infrared spectrum was measured using a Fourier infrared spectrometer (FT-IR), as shown in Figure 3 , which proved that glycyrrhizin acid was crosslinked with chitosan by amide bond;
[0049] (3) The drug loading of Ab and GL was calculated using a UV-visible spectrometer;
[0050] A certain amount of Ab@CS-GL and CS-GL was dissolved in a cuvette, and the OD value was measured at 314 nm and 249 nm using a UV-visible spectrophotometer. The OD value was brought into the calibration curve, and the drug loading was calculated, as shown in Figure 4 , which proved that CS-GL successfully loaded Ab, and the glycyrrhizin acid drug loading was 33%, and the arbidol drug loading was 30%;
[0051] (4) In vitro release device was used to simulate in vitro release
[0052] The dialysis method was used to detect the release of Ab@CS-GL gel in different pH media, and the ultraviolet absorbance method was used to calculate the drug release rate. 2mL of uniformly dispersed Ab@CS-GL and CS-GL were respectively placed in dialysis bags with a retention capacity of 3600Da, and the dialysis bags were placed in 15mL of release medium (artificial gastric juice and artificial intestinal juice), and incubated in a 37°C constant temperature shaking box for 72h with a rotation speed of 150rpm. Samples were taken at certain time points, and 2mL of release medium was then aspirated and replenished in equal amounts. The release amounts of GL and Ab in the Ab@CS-GL and CS-GL in the samples were measured using a UV-visible spectrophotometer at 249nm and 314nm, respectively, and the cumulative release amount was calculated; the results are shown in FIG. Figure 5 As shown, the gel reduces the large-scale release of Ab in the stomach and reduces gastric irritation.
[0053] (5) Observe the surface structure of Ab@CS-GL and CS-GL using scanning electron microscopy;
[0054] After the hydrogel was freeze-dried, a gold film was sprayed on its surface, and the surface morphology and structure of the sample were observed using SEM (sirion 200). Figure 6 As shown, it shows that the synthesized CS-GL gel has a loose porous structure and still has a loose porous structure after encapsulating Ab;
[0055] Example 2
[0056] 1 Experimental Materials
[0057] The following materials were selected to study the level of inhibition of influenza A virus infection by the present invention: the H1N1 influenza virus mouse-adapted strain (A / Changchun / 01 / 2009) and the H3N2 influenza virus mouse-adapted strain (A / Baikalteal / Shanghai / SH-89 / 2013) were from the Virology Laboratory of the Military Veterinary Research Institute, Military Medical Research Institute, Academy of Military Sciences, CCK-8 was purchased from MCE, the viral nucleoprotein antibody (Ab128193) and related signaling pathway protein antibodies were purchased from Abcam and CST, the fluorescent secondary antibody was purchased from Beyotime, the primers were synthesized by Changchun Kumeihe Biotechnology Co., Ltd., the RNA extraction kit was produced by Magen, the RT-qPCR kit was TaKaRa (RR047A), the fluorescent dye was RR082A, and the 6- to 8-week-old female BALB / c mice used were purchased from Jintai Medi.
[0058] 2 Experimental methods
[0059] 2.1 CCK-8 assay for cytotoxicity of Ab@VS-GL to MDCK cells
[0060] MDCK cells in culture flask were washed with PBS, then trypsinized, resuspended, and added with DMEM complete medium to obtain cell suspension, which was blown and counted uniformly. 7000 cells per well were added to 96-well cell culture plates, and incubated in an incubator for 12 h. A series of different concentrations of drugs were prepared with the base medium. When the cells grew to 60-70% of the bottom area, 100 μL of each concentration of drugs in each group was added to each well, 100 μL of base medium was added to the blank group, and it was placed in a 37°C, 5% CO2 cell incubator for incubation for 24 h, 48 h, and three repeated wells were set for each concentration. CCK-8 kit detection.
[0061] Cell activity (%) = (OD value of cells in the drug administration group - OD value of cells in the blank group) / (OD value of cells in the control group - OD value of cells in the blank group) x 100%.
[0062] 2.2 CCK-8 measurement of Ab@CS-GL inhibition rate on influenza virus H1N1 infected MDCK cells
[0063] MDCK cells in culture flask were washed with PBS, then trypsinized, resuspended, and added with DMEM complete medium to obtain cell suspension, which was blown and counted uniformly. 7000 cells per well were added to 96-well cell culture plates, and incubated in an incubator for 10 h. H1N1, H3N2 were diluted to 100 TCID 50 The medium in the 96-well plate was discarded, the first column was added with base medium as a blank control group, and the remaining 100 μL of 100 TCID50 virus diluent was added to each well, and it was placed in a virus incubator for incubation for 2 h, and it was shaken once every certain time in a secondary biological safety cabinet. Ab@CS-GL was added to the base medium to prepare a series of different drug concentrations. This operation set up blank group (Control group), negative control group (Mock group), and different concentration drug administration groups. Three repeated wells were set for each concentration. It was placed in a virus incubator for incubation for 48 h. CCK-8 virus inhibition rate determination.
[0064] Virus inhibition rate = (OD value of drug experimental group - average OD value of virus control group) / (average OD value of cell control group - average OD value of virus control group)
[0065] Graphpad 9.0 software was used to draw the cytotoxicity of different concentrations of drugs on MDCK cells and the inhibition rate curve of virus infected MDCK cells, and to calculate the half cytotoxicity concentration (CC50), half inhibition concentration (IC50), and selection index (SI).
[0066] 2.3 Indirect immunofluorescence detection of H1N1, H3N2 virus nucleoprotein level after Ab@CS-GL treatment
[0067] Plating, challenge, dosing: MDCK cells were resuspended in complete medium and plated at a density of 20% per well in 24-well cell culture plates. When they reached 50-60% confluence, they were challenged. H1N1 and H3N2 virus was diluted logarithmically, and 100 TCID 50 Challenge 2h, change medium, give equivalent concentration of drug, incubate at 37°C, 5% CO2, for 24h. Fix, permeabilize, block, incubate antibody, stain nuclei, photograph, and analyze fluorescence intensity using Image J.
[0068] 2.4 Mouse challenge protection
[0069] Mice were fed for 3 days before challenge, and were marked with ear tags, randomly divided into groups, and the gel groups were divided into 8 groups, namely, blank control group, negative control group, positive control group, Ab@CS-GL, CS-GL, GL, Ab, and CS group, with 6 mice in each group. The initial body weight of the mice was weighed and recorded. 3 MLD 50 Nasal challenge of mice; the mice were anesthetized in a biological safety cabinet, and 50 μL of virus was challenged through a single nostril. The drug in the gel group was prepared at a concentration of Ab@CS-GL 40 mg / kg, and the drugs in the other groups were prepared according to the drug loading amount. Intragastric administration was performed 12h after challenge, twice a day, for 5 consecutive days. The state of the mice was observed, the body weight of the mice was recorded 14 days after administration, and the survival rate was calculated.
[0070] 2.5 Determination of virus load in lung tissue and nasal bone of mice by chicken embryo method
[0071] Grinding sample: lung tissue in a 2 mL grinding tube containing steel balls was added to 1 mL of opti-MEM, and nasal bone was added to 500 μL of opti-MEM. The sample was ground in a grinder with a program of 4 cycles at 4°C, 50 Hz, and 180 min, and centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was taken to a new centrifuge tube, and the sample was diluted: lung tissue grinding liquid and nasal bone grinding liquid were diluted by logarithmic dilution method. The lung tissue grinding liquid and nasal bone grinding liquid of the infected mice were taken with a syringe, 100 μL of virus diluent was added to each chicken embryo. After the virus was inoculated, the opening was sealed with glue. It was placed in a 37°C incubator for 48h. The air chamber of the chicken embryo was opened, and 50 μL of allantoic fluid of the chicken embryo was harvested with a pipette gun. Hemagglutination (HA) assay was performed, and the median infectious dose (EID 50 ) of each mouse lung tissue and nasal bone was analyzed to determine the virus titer of each group.
[0072] 2.6 Lung pathological changes
[0073] The mice were dissected 4 and 6 days after challenge in the Ab@CS-GL group, and the lung tissue of the mice was taken for photography.
[0074] 2.7WB analysis of the levels of inhibition of inflammation-related signaling pathways by various components of Ab@CS-GL
[0075] After 4 and 6 days of treatment, 1 mL of RIPA lysis buffer containing cocktail and phosphatase inhibitors was added to the grinding tube containing the steel beads and gel from each group of lung tissue. The tissue was ground at 4°C, 50 Hz, and 180 s for four cycles. The sample was then centrifuged at 4°C, 12,000 rpm, for 10 minutes. The supernatant was aliquoted into a fresh tube to obtain a protein sample. 80 μL of this protein sample was transferred to a 1.5 mL centrifuge tube, and 20 μL of 5× SDS was added. The protein was denatured in a metal bath at 100°C for 10 minutes. 2 μL of the protein sample was removed and diluted to 20 μL in PBS. The cooked protein was then subjected to gel electrophoresis, membrane transfer, blocking, antibody incubation, and development according to the protein quantification results.
[0076] 2.8 RT-qPCR analysis of Ab@CS-GL inhibition of inflammatory factors
[0077] RNA extraction and lung tissue RNA quantification were performed according to the Magen kit instructions. 1 μL of protein eluate was taken to calibrate the instrument, and ssRNA in the tissue was detected after loading. The system was prepared according to the RT-qPCR kit instructions, and the program was set on the Real Time PCR amplification instrument. After the reaction, a melting curve was performed to verify the specificity of the qPCR amplification product. The obtained Cq value was substituted into 2 -ΔΔct calculate.
[0078] All animal experimental conditions and procedures adhered to the ethical guidelines of the International Society for the Study of Pain and were approved by the Animal Care and Use Committee of the People's Liberation Army. All experiments were conducted in a biosafety level 2 laboratory, using approved viral strains and autoclaved medical instruments that came into contact with viruses.
[0079] 3 Results Analysis
[0080] 3.1 CCK-8 assay for Ab@CS-GL cytotoxicity and influenza virus H1N1 infection inhibition
[0081] The final Ab@CS-GL drug was diluted to 2 by half dilution method. 0 , 2 1 , 2 2 ,……,2 8 μg / mL, and cells were incubated for 48 h. Figure 7 As shown in the figure, the calculated CC50 value of Ab@CS-GL for MDCK cells was 4305 μg / mL. 6No obvious cytotoxicity in the range; the inhibition rate of Ab@CS-GL at 64 μg / mL was 63.27%, IC50=833.4 μg / mL, and the selectivity index (SI) was 5.16, indicating that Ab@CS-GL had certain antiviral activity in vitro.
[0082] 3.2 Ab@CS-GL treatment of infected cells reduces H1N1, H3N2 viral nucleoprotein levels
[0083] The level of viral nucleoprotein in cells infected with influenza virus was determined by indirect immunofluorescence, and the results are shown in Figure 8 , Figure 9 The results of infection with two different virus strains were consistent with the rule that the H1N1 influenza virus strain had low infection rate and high mortality, and the H3N2 influenza virus strain had high infection rate and low mortality. Compared with the Mock group without drug treatment, the drug treatment groups had certain inhibitory effect on viral infection in vitro; compared between the drug groups, the effect of the Ab@CS-GL treatment group was comparable to that of the positive drug and superior to that of the single drug group. This indicates that the gel can enable the two drugs to synergistically exert an anti-influenza virus effect.
[0084] 3.3 Ab@CS-GL challenge protection study
[0085] The results are shown in Figure 10 , Figure 11 Compared with the negative control group, the gel drug groups could improve the survival rate of infected mice and to some extent reduce the decrease in mouse weight. The survival rates of the Ab@CS-GL group and the CS-GL group in treating H1N1 infected mice were 66.7% and 50%, respectively, and the protection rate of the Ab@CS-GL group in treating H3N2 infected mice was 80%, which was superior to that of each single drug group. This hydrogel has a high protective effect in vivo.
[0086] 3.4 Ab@CS-GL reduces H1N1 infected mouse lung tissue and turbinate viral titers
[0087] The viral load in the lung tissue of the virus infected mice was higher than that in the turbinate. The lung tissue viral titers of the gel drug groups after challenge and treatment for 4 and 6 days are shown in Figure 12As shown. In the early stage of infection in mice, the hydrogel groups were orally administered with drugs. Compared with the Mock group, except for the CS group, which was ineffective, the other drug-treated groups could reduce the lung viral load, and the combined drug Ab@CS-GL group was better than the single drug groups in reducing the level of lung tissue viral load. Compared with the Mock group, the combined drug treatment can reduce the viral titer of the nasal concha in the early stage of infection, while there was no significant difference in the viral load of the nasal concha in the single drug groups compared with the Mock group. On the 6th day after treatment, the hydrogel groups reduced the viral titer of the nasal concha in infected mice. Among them, the GL and Ab groups reduced the viral titer of the nasal concha in one group of mice to a level close to that of the Control group, but due to the large difference in the titer of the nasal concha compared with the other two groups of mice, it shows that the effect of reducing the titer of the nasal concha was unstable, resulting in no significant difference compared with the Mock group. After the combined drug treatment, the difference between the three mice was reduced, and the drug efficacy was relatively stable.
[0088] 3.5Ab@CS-GL treatment of H1N1-infected mice significantly improved lung lesions
[0089] In the gel group, mice were dissected 4 and 6 days after the challenge treatment to observe the lung lesions. Figure 13 As shown in the figure. Four days after challenge treatment, the mock group showed a small amount of blood spots in the lungs compared to the control group. Six days after challenge treatment, the mock group showed significant lung lesions, with significant congestion and swelling in the lung tissue. Compared to the mock group, the gel-treated groups reduced congestion and swelling in the mice's lungs, and the combined treatment was more effective than either drug alone.
[0090] 3.6Ab@CS-GL inhibits phosphorylation of NF-κB and phosphorylated Akt proteins, thereby reducing the levels of pro-inflammatory factors
[0091] The effects of each group of drugs in the hydrogel on NF-κB protein and Akt protein are shown in the following table. Figure 14 As shown in the results, GL has an inhibitory effect on the phosphorylated NF-κB and phosphorylated Akt signaling pathways, and Ab has an inhibitory effect on NF-κB. After combined administration, Ab@CS-GL has an inhibitory effect on the phosphorylated NF-κB and phosphorylated Akt signaling pathways, and almost completely inhibits the phosphorylated NF-κB protein, thereby significantly reducing the level of pro-inflammatory factor (TNF-α) protein.
[0092] 3.7Ab@CS-GL significantly inhibited the levels of inflammatory factors in the lungs of mice and weakened the cytokine storm
[0093] The results are as follows Figure 15As shown in the gel, the main drug for anti-inflammatory effect in each group of drugs is GL, and the anti-inflammatory effect of Ab group may be caused by the self-inhibition of viral replication, and the anti-inflammatory effect of CS group is unstable, but it inhibits the replication of viral M gene and viral NP at the late stage of infection, so it is speculated that its anti-inflammatory effect is also caused by the reduction of inflammatory factor level caused by the inhibition of viral load. Ab@CS-GL group reduces the cytokine storm caused by influenza A virus infection by GL anti-virus and anti-inflammatory, and Ab reduces the secretion of pro-inflammatory factors by anti-virus.
[0094] The specific embodiments are only an explanation of the present application, which is not a limitation of the present application, and those skilled in the art can make modifications to the embodiments without creative contribution according to the needs after reading the specification, but as long as it is within the scope of the claims of the present application, it is protected by the patent law.
Claims
1. The preparation method of Abidol@chitosan-glycyrrhizic acid gel is characterized by: The following steps are involved: S1: (1) Weigh 500.0 mg of chitosan into a reaction flask, add appropriate amount of 1% glacial acetic acid to dissolve it, and stir on a magnetic stirrer at 150 rpm for 4 h until it is completely dissolved; (2) Weigh 1.0 g of glycyrrhizic acid into another reaction bottle, add DMSO to dissolve, add 465.8 mg of EDC and 279.7 mg of NHS to the reaction bottle at 0°C, and stir under argon for 0.5 h; (3) After the reaction is completed, slowly add the glycyrrhizic acid after the carboxyl group activation to the chitosan reaction bottle while stirring. After the addition is completed, the reaction is allowed to proceed overnight. (4) The chitosan-glycyrrhizic acid after the reaction was completed was added to an 8000Da dialysis bag, dialyzed for 12 hours, and then freeze-dried to obtain chitosan-glycyrrhizic acid gel, i.e., CS-GL gel; S2: Weigh 500 mg of freeze-dried CS-GL into a reaction bottle, stir until it becomes a viscous gel, add arbidol hydrochloride hydrate, stir for 12 hours, and then freeze-dry to obtain Ab@CS-GL drug-loaded gel.
2. Use of the Abidol@chitosan-glycyrrhizic acid gel according to claim 1 in the preparation of a medicament for treating H1N1 and H3N2 influenza virus infections.
3. The use of the Abidol@chitosan-glycyrrhizic acid gel according to claim 2 in the preparation of a drug for treating H1N1 and H3N2 influenza virus infections, characterized in that: The drug reduces cytokine storm by inhibiting phosphorylated Akt and phosphorylated NF-κB, thereby reducing lung viral load and improving lung lesions.