Application of penthorum chinense pursh in preparation of medicine for preventing and / or treating influenza virus
By using pharmaceutical preparations prepared from Huanghuangcao or its extract, the problem that existing drugs are difficult to effectively prevent and treat influenza viruses is solved, and the effects of significantly inhibiting virus replication and alleviating symptoms are achieved.
Patent Information
- Application Number
- CN202411706136.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-26
- Publication Date
- 2025-01-21
AI Technical Summary
There are fewer drugs available for preventing and treating influenza viruses and have drug resistance problems, making it difficult to effectively control the risk of an influenza pandemic.
The use of yellow grass or its extracts as active ingredients, combined with the pharmaceutical preparations prepared by pharmaceuticals to suppress the replication of the influenza virus and alleviate the symptoms caused by it.
Huangcao extract can significantly inhibit the replication of influenza viruses, alleviate weight loss and lung damage, prolong survival time, and effectively treat lung diseases caused by influenza viruses.
Smart Images
Figure CN119326807A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of new uses of traditional Chinese medicines, and particularly relates to an application of Herba Lycopodii in preparing a medicine for preventing and / or treating influenza virus. Background Art
[0002] Influenza (flu) caused by influenza virus infection is the first infectious disease to be monitored globally, and it is also a respiratory viral infectious disease that has not been completely controlled so far. According to statistics from the World Health Organization, there are 600 million to 1.2 billion cases of influenza worldwide each year, and 500,000 to 1 million deaths, of which 3 million to 5 million cases of severe influenza, and the mortality rate of severe influenza is 8% to 10%. Influenza is caused by influenza virus (IV) infection, and the main symptoms are high fever, headache, muscle aches, cough, fatigue, etc. According to the differences in nucleoprotein and matrix protein, influenza virus can be divided into three subtypes: A, B, and C. At present, there are few drugs related to the treatment of influenza, and drug resistance mutations are beginning to appear. The risk of influenza pandemics is getting higher and higher. Humans should consider preparing for future influenza pandemics, including early warning, medical systems, vaccines and drugs, among which vaccines and antiviral drugs are the main means.
[0003] At present, the chemical drugs that are currently on the market and in the research stage for the prevention and treatment of influenza in clinical practice mainly include M2 ion channel inhibitors, neuraminidase inhibitors, influenza virus polymerase inhibitors, nucleoprotein inhibitors, and envelope glycoprotein hemagglutinin inhibitors. However, influenza viruses have the characteristics of high mutation frequency, wide transmission range, high pathogenicity, and difficulty in prevention and control, which seriously threaten the quality of human life and life safety. Although my country has preliminary means of fighting influenza such as influenza vaccines and drugs, there are still problems with adverse reactions, solubility and bioavailability. On the other hand, viral mutations and viral resistance caused by drug abuse are becoming more and more serious, which limits clinical treatment methods.
[0004] Therefore, there is an urgent need to find a new drug that can be used to prevent and treat influenza, providing a more effective treatment for influenza virus. Summary of the invention
[0005] In view of the deficiencies in the prior art, the present invention provides an application of Herba Lycopodii in preparing a drug for preventing and / or treating influenza virus.
[0006] A use of ophiopogon japonicus or an extract thereof in preparing a medicine for preventing and / or treating influenza virus.
[0007] Preferably, the yellowing grass is the whole herb of PenthorumchinensePursh.
[0008] Preferably, the influenza virus is selected from H1N1 subtype influenza virus.
[0009] Preferably, the drug is capable of inhibiting the replication of influenza virus.
[0010] Preferably, the drug can alleviate weight loss caused by influenza virus infection.
[0011] Preferably, the drug can alleviate the damage caused by influenza virus to the lungs.
[0012] Preferably, the drug can prevent and / or treat lung diseases caused by influenza virus.
[0013] The present invention also provides a pharmaceutical composition for preventing and / or treating influenza virus, which is a preparation prepared by taking Herba Lycopodii or its extract as active ingredient and adding pharmaceutically acceptable auxiliary materials.
[0014] Preferably, the pharmaceutically acceptable excipient is selected from at least one of a diluent, an absorbent, a wetting agent, an excipient, a filler, a disintegrant, a surfactant, a colorant, a glidant, a lubricant, a binder, a stabilizer, a suspending agent, and a buffer.
[0015] Preferably, the preparation is at least one of an injection, a spray, an aerosol, and an irrigation agent.
[0016] The whole herb of the plant PenthorumchinensePursh belongs to the Saxifragaceae Juss., PenthorumGronov.ex L. It is now clinically used to treat hepatitis, edema, traumatic injury, swelling and pain, etc. The present invention firstly finds that the PenthorumchinensePursh has the effect of resisting influenza virus, and proves through in vitro and in vivo experiments that the PenthorumchinensePursh can significantly inhibit the replication of influenza virus, alleviate the weight loss caused by influenza virus infection, prolong the survival time, has a significant protective effect on lung tissue, and can treat lung diseases caused by influenza virus, and has broad application prospects.
[0017] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.
[0018] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 This is a diagram for the extraction, purification and identification of Herba Chrysanthemi Indici.
[0020] Figure 2 This is a diagram showing the effect of yellow grass on the safety of cell growth.
[0021] Figure 3 A diagram showing how the herb inhibits the replication of influenza virus H1N1 in cells.
[0022] Figure 4 This is a graph showing the effect of yellow grass on the body weight of influenza virus H1N1 model mice.
[0023] Figure 5 This is a graph showing the effect of yellow grass on the survival rate of influenza virus H1N1 model mice.
[0024] Figure 6 This is a diagram showing the effect of Herba Chrysanthemi Indici on the protection of lung tissue in mice infected with influenza virus H1N1 model.
[0025] Figure 7 This is a diagram showing the pathological changes in lung tissue of mice infected with influenza virus H1N1 by Gan Huang Cao.
[0026] Figure 8 This is a diagram of the effect of Huangcao on the expression of viral nucleoprotein in the lung tissue of mice infected with the influenza virus H1N1 model. DETAILED DESCRIPTION
[0027] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.
[0028] Example 1 Extraction, purification and identification of Herba Lycopodii
[0029] 1. Experimental Methods
[0030] The herb of Ganhuangcao from Gulin, Luzhou, Sichuan (the herb of Ganhuangcao was provided by the Medical Basic Research Center of Southwest Medical University) was dried, crushed and sieved, 5g of herb powder was taken, 100mL of water or 75% ethanol solution was added respectively, sealed with plastic wrap, shaken, left to stand for 0.5h, extracted with an ultrasonic extractor at 120W 35℃ for 30min, extracted twice in total, cooled to room temperature and filtered twice under reduced pressure, the filtrate was combined, concentrated and transferred to a volumetric flask and diluted to 50mL to obtain a water extract or alcohol extract of the herb solution. The herb solution water extract was obtained by vacuum freeze drying to obtain a dry powder sample, and 1g / mL sterile water was added to dissolve it; the herb solution alcohol extract was obtained by suspension evaporation to obtain a dry powder, 1g / mL sterile water was added to dissolve it, and analyzed by liquid chromatography HPLC spectroscopy.
[0031] 2. Experimental Results
[0032] The results are as follows Figure 1As shown: We conducted HPLC analysis on the extract of the Herba Glehniae and confirmed that the Herba Glehniae extract mainly contained flavonoids. By comparing the retention time with that of the standard, the five peaks corresponding to the Herba Glehniae ethanol extract or its water extract were identified as gallic acid, isoquercetin, quercetin, quercetin, and kaempferol.
[0033] Example 2 In vitro, the therapeutic effect of Herba Chrysanthemi on influenza virus was investigated
[0034] 1. Experimental Methods
[0035] 1. Cell culture: Canine kidney cells MDCK and human lung cancer cells A549 were obtained from the Basic Medical Research Center of Southwest Medical University. MDCK cells and A549 cells stored in liquid nitrogen were taken out, revived, and continuously propagated for three generations. The experiment was conducted after the cells grew well. Among them, canine kidney cells MDCK were cultured in Dulbecco's modified Eagle medium (DMEM, C11995500BT, Sigma-Invitrogen) supplemented with 5% fetal bovine serum (FBS, 10099141C, Gibco), 100U / mL penicillin and 100mg / mL streptomycin; human lung cancer cells A549 were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS, 10099141C, Gibco), 100U / mL penicillin and 100mg / mL streptomycin.
[0036] 2. Treatment method 1: MDCK cells and A549 cells were inoculated into 96-well plates and the cells were counted. The number of cells in each well was 1×10 5 , cultured overnight in a 37°C, 5% CO2 constant temperature cell incubator. When the cell density was 70% to 80%, different concentrations (5μg / mL, 10μg / mL, 20μg / mL, 40μg / mL, 80μg / mL, 100μg / mL, 6 concentrations in total) of water extract of the herb were added, and 6 replicates were measured for each concentration. A blank cell control and a PBS control group were also set up, with 6 replicates, and the culture was continued. The cell state was observed under a microscope every day. After about 36 hours of drug addition, the cell activity was detected by MTT method.
[0037] 3. Treatment method 2: First, place the H1N1-PR8 strain (from Beijing Sino Biological Technology Co., Ltd.) stored at -80℃ on ice to slowly thaw, and use the virus dilution with a content of 100TCID50 for subsequent inoculation experiments. MDCK cells and A549 cells were inoculated into 96-well plates respectively, and the cell count was performed. The number of cells in each well was 1×10 5, cultured in a 37°C, 5% CO2 constant temperature cell incubator for 12 hours. When the cells are in a monolayer state, add different concentrations of the yellow grass solution (5μg / mL, 10μg / mL, 20μg / mL, 40μg / mL, 80μg / mL, a total of 5 concentrations) to treat the cells in a 96-well plate for 6 hours, and then inoculate the cells with a virus dilution of 100TCID50. Set up blank cell control wells and virus control wells, with 3 replicates in each group. Place the inoculated cell plate in a 37°C, 5% CO2 incubator and continue to culture for 36 hours, and then use the MTT method to detect cell activity.
[0038] 4. MTT method: After the cells were exposed to the above treatment conditions and cultured for 36 hours, 10 μL of MTT solution (5 mg / mL) was added to each well and cultured in an incubator at 37°C and 5% CO2 for 4 hours. Then, 100 μL of dimethyl sulfoxide (DMSO) solution was added, mixed gently, and incubated at 37°C and 5% CO2 for 15 minutes before measuring OD. 570 Determination of value.
[0039] 5. IC50 value: Calculate the sensitivity of cells to drugs (IC50 value) based on the OD value. The calculation formula is: cell sensitivity = (drug control group OD / cell control group OD) × 100%.
[0040] 6. EC50 value: The half maximal effective concentration (EC50) of the drug against the influenza virus was calculated based on the OD value. The calculation formula is: infection rate = (OD of drug treatment group - OD of virus control group) ÷ (OD of cell control group - OD of virus control group)) × 100%.
[0041] 2. Experimental Results
[0042] 1. The effect of radix scutellariae on the safety of cell growth
[0043] The cells were treated according to treatment method 1 in the experimental method. The results are as follows Figure 2 As shown, the half inhibitory concentration of GHC on the growth of MDCK cells was 76.83 μg / mL, and the half inhibitory concentration on the growth of A549 cells was 83.2 μg / mL.
[0044] 2. Ganhuangcao inhibits the replication of influenza virus H1N1 in cells
[0045] The cells were treated according to treatment method 2 in the experimental method. The results are as follows Figure 3 As shown, the EC50 value of Herba Piperidis in MDCK cells was 25.26 μg / mL, while the EC50 value in A549 cells was 27.17 μg / mL, indicating that Herba Piperidis has a significant inhibitory effect on H1N1-PR8 strain at the cellular level.
[0046] Example 3 In vivo investigation of the therapeutic effect of Herba Chrysanthemi on influenza virus
[0047] 1. Experimental Methods
[0048] 1. Experimental group 1: BALB / c mice were purchased from Weitonglihua Experimental Animal Technology Co., Ltd.; influenza virus H1N1-PR8 strain was derived from the Medical Basic Research Center of Southwest Medical University; the mice were divided into blank control group (Control group), virus control group (Virus group), GHC (Stomach) gavage group (Virus + GHC (Abdomen) group) and Oseltamivir intraperitoneal injection group (positive control) (Virus + Oseltamivir group), with 6 mice in each group. The blank control group was treated with normal saline; the virus control group was treated with 10MLD50 / 50μL influenza virus dilution; the preventive treatment mode was adopted in the oral administration group of the herb: the mice were treated with herb (50μg / mL) in advance (oral administration, twice a day, 100μL each time), and 50μL of 10MLD50 influenza virus dilution was added 2 days later; the therapeutic treatment mode was adopted in the intraperitoneal injection group of the herb: 50μL of 10MLD50 influenza virus dilution was added first, and then the herb (50μg / mL) was injected intraperitoneally 12 hours later, and the intraperitoneal injection was once a day, 100μL each time; the oseltamivir intraperitoneal injection group was treated with 50μL of 10MLD50 influenza virus dilution first, and then oseltamivir (10μg / mL) was injected 12 hours later, once a day, 100μL each time. The weight of mice in each group was monitored every day.
[0049] 2. Experimental Group 2: BALB / c mice were purchased from Weitong Lihua Experimental Animal Technology Co., Ltd.; influenza virus H1N1-PR8 strain was derived from the Medical Basic Research Center of Southwest Medical University; the mice were divided into a blank control group (Control group), a virus control group (Virus group), a group given oral administration of GHC (Virus+GHC (Stomach) group), and a group given intraperitoneal injection of GHC (Virus+GHC (Abdomen) group). The H1N1-PR8 strain was placed on ice to slowly melt, and 50 μL of 10 MLD50 influenza virus dilution was added to the nose and inoculated into mice in groups other than the blank control group. The rest of the administration method was the same as in Experimental Group 2. The weight of mice in each group was monitored and the survival status was observed every day, and the lung tissues of mice were taken on the third and fifth days after virus inoculation.
[0050] 3. Lung index: The calculation formula is: Lung index = (weight of mouse lung tissue ÷ mouse body weight) × 100%.
[0051] 4. HE staining
[0052] 5. Immunohistochemical staining: The lung tissue of mice was fixed with formalin at 25°C for 72h, and the fixed mouse lung tissue was embedded in paraffin to prepare ultrathin sections. Subsequently, antigen retrieval, blocking of endogenous peroxidase and serum blocking were performed, and primary antibody was added for incubation (4°C overnight); then, secondary antibody was added for incubation (room temperature for 2h) before DAB color development and counterstaining of cell nuclei; finally, electron microscope (Nikon) was used for microscopic examination and image acquisition and analysis. Hematoxylin stained cell nuclei in blue, and DAB showed positive expression in brown-yellow. The calculation formula of the positive rate is as follows: Positive rate = (number of positive cells ÷ total number of cells) × 100%.
[0053] 2. Experimental Results
[0054] 1. Effect of Ganhuangcao on the body weight of influenza virus H1N1 model mice
[0055] The experiment was conducted using experimental group 1, and the results are as follows Figure 4 As shown, the mice in the blank control group were in good spirits, responsive, and had a small change in weight; the mice in the virus control group lost weight significantly and showed symptoms such as mental depression, decreased appetite, and shortness of breath. Treatment with the drug Ganhuangcao can alleviate the weight loss of mice caused by virus infection, and the therapeutic effect is not significantly different from that of oseltamivir.
[0056] 2. Effect of Herba Chrysanthemi on the Survival Rate of Influenza Virus H1N1 Model Mice
[0057] The experiment was conducted using experimental group 1, and the results are as follows Figure 5 As shown, the mice in the blank control group were in good spirits and responsive; the mice in the virus control group began to die 3 days after infection, with a mortality rate of 100%; the treatment of the group treated with Ganhuangcao gavage can prolong the survival time of mice and reduce the mortality rate of infected mice. The test results show that Ganhuangcao can prolong the survival time of influenza virus model mice and can be used for the prevention and treatment of influenza virus.
[0058] 3. Effect of Herba Chrysanthemi on lung tissue protection in mice infected with influenza virus H1N1
[0059] The experiment was conducted using experimental group 2, and the results are as follows Figure 6 As shown, influenza virus infection in mice causes pneumonia, swelling and bleeding in the lungs ( Figure 6 A), lung weight increases, lung index increases accordingly ( Figure 6 B), virus titer increased ( Figure 6 C); Adding ganhuangcao can alleviate the pathological damage of infected mice, and the therapeutic effect is better in the ganhuangcao gavage group than in the ganhuangcao intraperitoneal injection group. The results show that ganhuangcao can alleviate the damage caused by influenza virus to the host lungs.
[0060] 4. Effect of Ganhuangcao on pathological changes of lung tissue in mice with influenza virus H1N1 model
[0061] The experiment was conducted using experimental group 2. The results of lung tissue HE staining are shown in Figure 7 As shown in the figure, in the virus control group, there were scattered lymphocytes and neutrophils infiltrating on the alveolar wall, local alveolar wall thickening, multiple hemorrhages, and high pathological scores. Compared with the virus control group, after treatment with Gan Huang Cao, a small amount of lymphocyte infiltration was still visible, but the alveolar cavity was relatively intact, without a large amount of cell fragments, and the pathological score was reduced. The effect of the Gan Huang Cao gavage group was better than that of the Gan Huang Cao intraperitoneal injection group. This result shows that Gan Huang Cao can alleviate the pathological damage caused by influenza virus to the host's lungs.
[0062] 5. Effect of Herba Campestris on the Expression of Viral Nucleoprotein in Lung Tissue of Mice Infected with H1N1 Influenza Virus
[0063] The experiment was conducted using experimental group 2, and the results are as follows Figure 8 As shown in the figure, the expression of vNP protein in mice in the virus control group increased, indicating that the virus replicated in vivo. However, the expression of vNP protein in mice treated with Huangcao decreased ( Figure 8 A), the number of positive cells decreased to a certain extent after treatment with Gan Huang Cao, and the effect of Gan Huang Cao oral administration group was better than that of Gan Huang Cao intraperitoneal injection group ( Figure 8 B), indicating that Huangcao can inhibit the replication of influenza virus H1N1 in the lung tissue of model mice, and prevent and control influenza virus through this pathway.
[0064] Based on the above results, the experimental results show that Herba Lycopodii can inhibit the replication of influenza virus, alleviate the weight loss of mice caused by influenza virus infection, prolong survival time, and also has a protective effect on lung diseases caused by influenza virus. Therefore, Herba Lycopodii can be used for the prevention of influenza virus and the research and development of related therapeutic drugs, and has broad application prospects.
Claims
1. Use of Herba Lycopodii or its extract in the preparation of a medicament for preventing and / or treating influenza virus.
2. The use according to claim 1, characterized in that The yellowing grass is the whole herb of PenthorumchinensePursh.
3. The use according to claim 1, characterized in that The influenza virus is selected from H1N1 subtype influenza virus.
4. The use according to claim 1, characterized in that The drug can inhibit the replication of influenza virus.
5. The use according to claim 1, characterized in that: The medicine can alleviate weight loss caused by influenza virus infection.
6. The use according to claim 1, characterized in that The drug can alleviate the damage caused to the lungs by the influenza virus.
7. The use according to claim 1, characterized in that The medicine can prevent and / or treat lung diseases caused by influenza virus.
8. A pharmaceutical composition for preventing and / or treating influenza virus, characterized in that: The invention is a preparation prepared by taking the herb or its extract as active ingredients and adding pharmaceutically acceptable auxiliary materials.
9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutically acceptable excipient is selected from at least one of a diluent, an absorbent, a wetting agent, an excipient, a filler, a disintegrant, a surfactant, a colorant, a glidant, a lubricant, a binder, a stabilizer, a suspending agent, and a buffer.
10. The pharmaceutical composition according to claim 8, characterized in that The preparation is at least one of an injection, a spray, an aerosol, and an irrigation agent.