An anti-influenza virus drug combination, preparation and use thereof

Abstract

The present invention provides an anti-influenza virus drug combination, preparation and use thereof, belonging to the field of biomedicine technology. The drug combination includes andrographolide and tetrandrine, which has dual efficacy, can not only inhibit the replication of influenza virus in vivo and in vitro, but also can relieve inflammation caused by influenza virus, and has a synergistic effect. Moreover, the andrographolide and tetrandrine in the drug combination of the present invention are highly safe, have abundant raw materials, are easy to obtain and prepare, and are suitable for industrial production. The present invention provides a choice for clinical anti-influenza virus drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an anti-influenza virus drug combination, a preparation and use thereof. Background Art

[0002] Tetrandrin is a dibenzylisoquinoline alkaloid extracted from the tuberous roots of the Menispermaceae plant. It is one of the main active ingredients of Tetrandrin and has immunosuppressive, antipyretic, analgesic, diuretic and anti-inflammatory effects.

[0003] Andrographolide is extracted from Andrographis paniculata, an annual herb. Its secondary metabolite, andrographolide, is a diterpene lactone compound. Andrographolide has shown antibacterial, antiviral, anti-inflammatory, immunomodulatory and anti-cancer biological activities in in vivo and in vitro studies.

[0004] Influenza A virus (IAV) is a virus that can cause global pandemics and continues to pose a threat to human health. Influenza outbreaks are closely linked to season and geographic environment. Most influenza viruses continuously evolve through antigenic drift and antigenic shift, affecting people of all ages. Clinical manifestations include varying degrees of systemic and respiratory symptoms, such as high fever, headache, cough, and limb aches. Severe cases can cause complications and even death. The genetic instability of influenza viruses and their ability to adapt to their environment allow them to evolve rapidly. Despite the availability of vaccines targeting seasonal influenza viruses, this rapid evolution can lead to resistance to antiviral drugs, complicating prevention efforts. As influenza viruses evolve, it is difficult to guarantee that more drug-resistant strains will not emerge in the future. Furthermore, the complications caused by influenza virus-induced cytokine storms need to be mitigated. Therefore, an effective anti-influenza drug is needed. Summary of the Invention

[0005] In view of this, the object of the present invention is to provide an anti-influenza virus drug combination, a preparation and its use, which can synergistically inhibit influenza virus and the inflammation caused by it.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides an anti-influenza virus drug combination, which comprises andrographolide and tetrandrine.

[0008] Preferably, the active ingredients of the drug combination consist of andrographolide and tetrandrine.

[0009] Preferably, the pharmaceutical combination further comprises a pharmaceutically acceptable excipient.

[0010] Preferably, the mass ratio of the andrographolide to the tetrandrine is 15:5~25.

[0011] Preferably, the mass ratio of the andrographolide to the tetrandrine is 15:6, 15:12, 15:18 or 15:24.

[0012] The present invention provides a solid or liquid preparation for resisting influenza virus, comprising the above-mentioned drug combination.

[0013] Preferably, the solid or liquid preparation further comprises sodium carboxymethylcellulose solution.

[0014] Preferably, the mass volume percentage of the sodium carboxymethyl cellulose solution is 3-7%; the mass volume ratio of andrographolide, tetrandrine and sodium carboxymethyl cellulose solution is 15 mg: (5-25) mg: (18-22) mL.

[0015] The present invention provides a use of the above-mentioned drug combination or solid or liquid preparation in the preparation of anti-influenza virus drugs.

[0016] Preferably, the influenza virus comprises influenza A virus.

[0017] The present invention provides an application of the above-mentioned drug combination or solid or liquid preparation in preparing a drug for treating inflammation caused by influenza virus.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The present invention provides an anti-influenza virus drug combination, preparation, and use thereof. The drug combination has dual efficacy, can inhibit the replication of influenza viruses in vivo and in vitro, and can relieve inflammation caused by influenza viruses, with a synergistic effect. In addition, the andrographolide and tetrandrine in the drug combination of the present invention are highly safe, have abundant raw materials, are easy to obtain and prepare, and are suitable for industrial production. The present invention provides a new option for clinical anti-influenza virus drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The effects of different groups on the cytopathic effect of H1N1 influenza A virus infection, A is the result of combination drugs 1 to 6 inhibiting the cytopathic effect of H1N1 influenza A virus infection, B is the result of combination drugs 7 to 10 inhibiting the cytopathic effect of H1N1 influenza A virus infection, C is the result of combination drug 11 inhibiting the cytopathic effect of H1N1 influenza A virus infection;

[0021] Figure 2 This is the result of the combination of andrographolide and tetrandrine inhibiting influenza virus replication in vitro;

[0022] Figure 3 This is the result of the combination of andrographolide and tetrandrine inhibiting the expression of influenza virus proteins in vitro;

[0023] Figure 4 This is the result of tetrandrine combined with andrographolide inhibiting the changes in mRNA levels of inflammatory factors induced by influenza virus in vitro;

[0024] Figure 5 The results show the effects of tetrandrine, andrographolide and combined oral suspension 1 on the survival rate of mice infected with influenza virus.

[0025] Figure 6 The results show the effects of combined oral suspensions 1 to 5 on the survival rate of mice infected with influenza virus;

[0026] Figure 7 The results show the effects of combined oral suspensions 1-2, 6-7 on the survival rate of mice infected with influenza virus;

[0027] Figure 8 The results show the effects of different administration times of combined oral suspension 7 on the survival rate of mice infected with influenza virus;

[0028] Figure 9 The results of combined oral suspension 7 inhibiting influenza virus replication in vivo, A shows the effect of combined oral suspension 7 on M1 gene expression in lung tissue of a mouse model infected with influenza virus, and B shows the effect of combined oral suspension 7 on NS1 gene expression in lung tissue of a mouse model infected with influenza virus;

[0029] Figure 10 The results are as follows: Combination Oral Suspension 7 inhibits influenza virus protein expression in vivo;

[0030] Figure 11 The results show the effects of combined oral suspension 7 on lung indices in a mouse model of influenza virus infection;

[0031] Figure 12 The results show the effect of combined oral suspension 7 on lung tissue pathological damage in the influenza virus infected mouse model;

[0032] Figure 13 These are the results of the effect of combination oral suspension 7 on the release of inflammatory factors in the body caused by influenza virus. A is the result of the effect of combination oral suspension 7 on the release of IL-6 caused by influenza virus, B is the result of the effect of combination oral suspension 7 on the release of IL-1β caused by influenza virus, and C is the result of the effect of combination oral suspension 7 on the release of TNF-α caused by influenza virus. DETAILED DESCRIPTION

[0033] The present invention provides an anti-influenza virus drug combination, which comprises andrographolide and tetrandrine.

[0034] In the present invention, different combinations of tetrandrine, andrographolide, dihydroartemisinin and baicalin were made. The study found that the combination of tetrandrine and andrographolide was better than other drug combinations in treating influenza virus, and had a synergistic effect compared with the single tetrandrine and andrographolide drugs. In addition, the study found that the combination of tetrandrine and dihydroartemisinin, tetrandrine and baicalin, andrographolide and dihydroartemisinin, andrographolide and baicalin, dihydroartemisinin and baicalin, and tetrandrine, andrographolide, dihydroartemisinin and baicalin could not significantly inhibit the cytopathic effect caused by influenza virus. It can be seen that the selection of ingredients in the drug combination is crucial for inhibiting influenza virus, and not any combination of two or more drugs can inhibit influenza virus and have a synergistic effect. As a preferred embodiment, the active ingredients of the drug combination are composed of andrographolide and tetrandrine. In the present invention, the drug combination also includes pharmaceutically acceptable excipients. The excipients include one or more of solvents, adhesives, diluents, disintegrants, antioxidants, dispersants, wetting agents, cosolvents, buffers and surfactants. The dosage form of the drug preferably includes suspensions, tablets, pills, solutions, injections, granules, capsules or sprays. The drug described in the present invention can be administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously or nasally. The present invention has no special restrictions on the sources of andrographolide and tetrandrine, and a preparation method known in the art or a commercially available product can be used.

[0035] The structure of the andrographolide is as follows:

[0036] .

[0037] The structure of the tetrandrine is as follows:

[0038] .

[0039] In the present invention, the mass ratio of the andrographolide to the tetrandrine is preferably 15:5~25. Further preferably, the mass ratio of the andrographolide to the tetrandrine is preferably 15:6~24, such as the mass ratio of the andrographolide to the tetrandrine is 15:6, 15:12, 15:18 or 15:24. When the present invention prevents and / or treats influenza virus by using andrographolide and tetrandrine in different mass ratios, it is found that the combination of andrographolide and tetrandrine in the above mass ratio has a more significant anti-influenza virus effect and has a synergistic effect.

[0040] The present invention provides an anti-influenza virus solid or liquid preparation comprising the above-mentioned drug combination. The solid or liquid preparation further comprises a sodium carboxymethylcellulose solution, the mass volume percentage of which is preferably 3-7%, more preferably 4-6%, and even more preferably 5%. The solvent of the sodium carboxymethylcellulose solution is water. In one embodiment, the 5% sodium carboxymethylcellulose solution is prepared by weighing 1g of sodium carboxymethylcellulose and mixing it uniformly with 200mL of deionized water. The mass volume ratio of andrographolide, tetrandrine, and sodium carboxymethylcellulose solution is preferably 15mg:(5-25)mg:(18-22)mL, more preferably 15mg:(6-24)mg:(19-21)mL, and even more preferably 15mg:(6-24)mg:20mL, such as 15mg:6mg:20mL, 15mg:12mg:20mL, 15mg:18mg:20mL, or 15mg:24mg:20mL. The solid or liquid preparation includes a suspension, tablet, pill, solution, injection, granule, capsule or spray. The solid or liquid preparation is administered orally, rectally, intraperitoneally, subcutaneously, intramuscularly, intravenously or nasally. In the present invention, the sodium carboxymethyl cellulose can uniformly disperse andrographolide and tetrandrine in the liquid, ensuring uniformity of the dosage.

[0041] The present invention has found that after infection with H1N1 influenza A virus, after treatment with a combination of tetrandrine and andrographolide or a solid or liquid preparation, the expression levels of the viral NS1, M1 genes and NP, HA proteins were detected, and it was found that the combination of andrographolide and tetrandrine can significantly inhibit the expression levels of the above genes and proteins, inhibit the replication of influenza virus in vivo and in vitro, and at the same time significantly inhibit the increase in the levels of IL-6, TNF-α and IL-1β inflammatory factors caused by influenza virus. Therefore, the tetrandrine and andrographolide drug combination or solid or liquid preparation provided by the present invention can be used to treat influenza virus infection and inflammation caused by influenza virus infection.

[0042] The present invention provides a use of the above-mentioned drug combination or solid or liquid preparation in the preparation of anti-influenza virus drugs.

[0043] In the present invention, the influenza virus includes influenza A virus, such as H1N1 influenza A virus.

[0044] The present invention provides an application of the above-mentioned drug combination or solid or liquid preparation in preparing a drug for treating inflammation caused by influenza virus.

[0045] In the present invention, unless otherwise specified, all raw material components are commercially available products well known to those skilled in the art.

[0046] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0047] In the following examples, the preparation of tetrandrine, andrographolide, dihydroartemisinin, and baicalin stock solutions (cell experiments) was as follows: 20 mg of tetrandrine was accurately weighed using an electronic balance and dissolved in 6.4 mL of DMSO to prepare a 10 mM tetrandrine stock solution. 10 mg of andrographolide was accurately weighed using an electronic balance and dissolved in 2.8 mL of DMSO to prepare a 10 mM andrographolide stock solution. 10 mg of dihydroartemisinin was accurately weighed using an electronic balance and dissolved in 3.5 mL of DMSO to prepare a 10 mM dihydroartemisinin stock solution. 10 mg of baicalin was accurately weighed using an electronic balance and dissolved in 2.2 mL of DMSO to prepare a 10 mM baicalin stock solution.

[0048] Example 1

[0049] Combination of andrographolide, tetrandrine, dihydroartemisinin and baicalin inhibits cytopathic effects of influenza virus infection

[0050] (1) A549 cell culture

[0051] A549 cells were cultured in F12K medium containing 10% fetal bovine serum and 1% double antibody at 37°C in a 5% CO2 incubator. The cells were observed to be growing well. After culturing to the logarithmic growth phase, A549 cells were plated at 1×10 5 Cells / well were seeded into 96-well plates and cultured continuously.

[0052] (2) Influenza virus vaccination

[0053] The optimal concentrations of tetrandrine, andrographolide, dihydroartemisinin, and baicalin for individual administration were 6, 25, 10, and 25 μM. The concentrations of the two-way combination were halved, the concentration of the three-way combination was reduced to 0.33, and the concentration of the four-way combination was reduced to 0.25. Specifically:

[0054] Combination drug 1 was prepared by mixing 25 μM andrographolide solution and 25 μM baicalin solution at a volume ratio of 1:1 (denoted as And+Bai group);

[0055] Combination drug 2 was prepared by mixing 25 μM andrographolide solution and 6 μM tetrandrine solution at a volume ratio of 1:1 (denoted as And+Tet group);

[0056] Combination drug 3 was prepared by mixing 25 μM andrographolide solution and 10 μM dihydroartemisinin solution at a volume ratio of 1:1 (denoted as And+Dih group);

[0057] Combination drug 4 was prepared by mixing 25 μM baicalin solution and 6 μM tetrandrine solution at a volume ratio of 1:1 (denoted as Bai+Tet group);

[0058] Combination drug 5 was prepared by mixing 25 μM baicalin solution and 10 μM dihydroartemisinin solution at a volume ratio of 1:1 (denoted as Bai+Dih group);

[0059] Combination drug 6 was prepared by mixing 6 μM tetrandrine solution and 10 μM dihydroartemisinin solution at a volume ratio of 1:1 (denoted as Tet+Dih group);

[0060] Combination drug 7 was prepared by mixing 25 μM andrographolide solution, 25 μM baicalin solution, and 6 μM tetrandrine solution at a volume ratio of 1:1:1 (denoted as And+Bai+Tet group);

[0061] Combination drug 8 was prepared by mixing 25 μM andrographolide solution, 6 μM tetrandrine solution, and 10 μM dihydroartemisinin solution at a volume ratio of 1:1:1 (denoted as And+Tet+Dih group);

[0062] Combination drug 9 was prepared by mixing 25 μM baicalin solution, 6 μM tetrandrine solution, and 10 μM dihydroartemisinin solution at a volume ratio of 1:1:1 (denoted as Bai+Tet+Dih group);

[0063] Combination drug 10 was prepared by mixing 25 μM andrographolide solution, 25 μM baicalin solution, and 10 μM dihydroartemisinin solution at a volume ratio of 1:1:1 (denoted as And+Bai+Dih group);

[0064] Combination drug 11 was prepared by mixing 25 μM andrographolide solution, 25 μM baicalin solution, 6 μM tetrandrine solution and 10 μM dihydroartemisinin solution in a volume ratio of 1:1:1:1 (denoted as And+Bai+Tet+Dih group).

[0065] A549 cells were seeded into 96-well plates and cultured for 24 h. After the cells grew to 40-60% confluence, they were cultured with 200 TCID 50A549 cells were infected with H1N1 influenza A virus (H1N1, A / Puerto Rico / 8 / 34). After 1 hour, the virus solution was discarded, and cell culture medium containing combination drugs 1 to 11 (the cell culture medium was F12K medium containing 0.3% by volume BSA, 0.2 μg / mL trypsin-TPCK, and 1% by volume double antibody) was added to the influenza virus-infected cells. The control group consisted of A549 cells not infected with H1N1 influenza A virus and not treated with any drugs, and the H1N1 group consisted of A549 cells infected with H1N1 influenza A virus but not treated with any drugs. The cells were cultured at 37°C and 5% CO2.

[0066] (3) CCK8 detection of cell activity

[0067] After incubation at 37°C, 5% CO₂ for 48 h, the cells were washed twice with PBS. 100 μL of culture medium and 10 μL of CCK8 reagent were added to each well and incubated at 37°C for 10–30 min. The absorbance (OD) was recorded at 450 nm.

[0068] (4) Results

[0069] Figure 1 The results showed that the two-drug combination of andrographolide and tetrandrine (i.e., combination 2) significantly inhibited the cytopathic effect of influenza virus infection, surpassing the three-drug, four-drug, and other two-drug combinations (i.e., combination 1, and combinations 3 to 11). Furthermore, the Bai+Dih, Tet+Dih, And+Bai+Tet, And+Tet+Dih, Bai+Tet+Dih, And+Bai+Dih, and And+Bai+Tet+Dih groups (i.e., combinations 5 to 11) failed to inhibit the cytopathic effect of influenza virus infection, indicating that not every drug combination can achieve this cytopathic effect.

[0070] Example 2

[0071] Combination of andrographolide and tetrandrine inhibits influenza virus replication in vitro

[0072] (1) A549 cell culture

[0073] A549 cells were cultured in F12K medium containing 10% fetal bovine serum and 1% double antibody at 37°C in a 5% CO2 incubator. The cells were observed to be growing well. After culturing to the logarithmic growth phase, A549 cells were plated at 1×10 5 Cells / well were seeded into 12-well plates and cultured continuously.

[0074] (2) Influenza virus vaccination

[0075] A549 cells were seeded into 12-well plates and cultured for 24 h. After the cells grew to 40-60% confluence, they were cultured with 200 TCID 50 A549 cells were infected with H1N1 influenza A virus. After 1 hour, the virus solution was discarded, and a cell culture medium containing the combination drug 2 of Example 1 (the cell culture medium was F12K medium containing 0.3% by volume BSA, 0.2 μg / mL trypsin-TPCK, and 1% by volume double antibody) was added to the influenza virus-infected cells and cultured at 37°C and 5% CO2.

[0076] (3) Q-PCR detection of viral gene changes

[0077] RNA was extracted from cells infected with influenza virus after culturing at 37°C and 5% CO2 for 2 days in step (2) using a total RNA extraction kit according to the instructions. The extracted RNA was reverse transcribed using a cDNA first-strand synthesis premix kit according to the instructions. The M1 and HA target genes were amplified using the Talent fluorescence quantitative detection kit (SYBR Green).

[0078] (4) Western blot detection of viral protein changes

[0079] Cells were lysed using RIPA cell lysis buffer supplemented with phosphatase and protease inhibitors. Proteins from total cell lysates were separated by SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in TPBS for 2 hours and incubated overnight at 4°C with rabbit anti-HA (1:1000), mouse anti-NP (1:1000), and rabbit anti-β-actin primary antibodies. The membranes were washed four times for 5 minutes with TPBS and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000) for 1 hour at room temperature. After five washes in PBS-Tween buffer for 5 minutes, the chemiluminescence of the labeled proteins was revealed using HRP substrate and captured under an instrument.

[0080] (5) Results

[0081] The results are as follows Figure 2 As shown in Figure 2, the combination of andrographolide and tetrandrine (combination drug 2 in Example 1) inhibited the mRNA levels of viral M1 and HA genes. Figure 3 As shown, the combination of andrographolide and tetrandrine (combination drug 2 of Example 1) inhibited the expression of viral HA and NP proteins.

[0082] Example 3

[0083] Andrographolide and tetrandrine inhibit influenza virus inflammation in vitro

[0084] (1) A549 cell culture

[0085] A549 cells were cultured in F12K medium containing 10% fetal bovine serum and 1% double antibody at 37°C in a 5% CO2 incubator. The cells were observed to be growing well. After culturing to the logarithmic growth phase, A549 cells were plated at 1×10 5 Cells / well were seeded into 12-well plates and cultured continuously.

[0086] (2) Influenza virus vaccination

[0087] The cells seeded into 12-well plates were cultured for 24 h. After the cells grew to 40-60% confluence, they were cultured with 200 TCID 50 A549 cells were infected with influenza virus. After 1 hour, the virus solution was discarded and cell culture medium containing combination drug 2 of Example 1 (the cell culture medium was F12K medium containing 0.3% by volume BSA, 0.2 μg / mL trypsin-TPCK, and 1% by volume double antibody) was added to the influenza virus-infected cells and cultured at 37°C and 5% CO2.

[0088] (3) Q-PCR detection of viral gene changes

[0089] RNA was extracted from cells infected with influenza virus after culturing at 37°C and 5% CO2 for 2 days in step (2) using a total RNA extraction kit according to the instructions. The extracted RNA was reverse transcribed using a cDNA first-strand synthesis premix kit according to the instructions, and the target gene was amplified using the Talent fluorescence quantitative detection kit (SYBR Green).

[0090] (4) Results

[0091] The results are as follows Figure 4 As shown, andrographolide combined with tetrandrine (combination drug 2 of Example 1) can significantly inhibit the increase in mRNA levels of inflammatory factors such as IL-6 and TNF-α caused by influenza virus in vitro.

[0092] Example 4

[0093] At the animal level, the protective effect of andrographolide and tetrandrine combined oral suspension on influenza model mice, the inhibitory effect on influenza virus in the body, and the relief of inflammation caused by influenza virus were evaluated.

[0094] 1. Protective effects of andrographolide and tetrandrine alone and in combination on influenza virus-infected mice

[0095] (1) Preparation of andrographolide and tetrandrine combined oral suspension

[0096] 1g of sodium carboxymethylcellulose (CMCNa) was accurately weighed using an electronic balance and added to 200mL of deionized water. The mixture was shaken overnight to prepare a 5% CMCNa solution. Then, 150mg of andrographolide and 60mg of tetrandrine were accurately weighed using an electronic balance and suspended in 10mL of 5% CMCNa to prepare 6mg / mL tetrandrine and 15mg / mL andrographolide suspensions, respectively. Combined oral suspension 1 was prepared by suspending 150mg of andrographolide and 60mg of tetrandrine in 20mL of 5% CMCNa. For the positive drug group, 20mg of oseltamivir phosphate was weighed using an electronic balance and dissolved in 10mL of normal saline to prepare a 2mg / mL oseltamivir phosphate solution.

[0097] (2) Establishment of influenza virus infection mouse model: 6-8 week old Balb / c mice were randomly divided into groups and fed for 3 days. H1N1 influenza A virus was diluted with PBS to 4LD. 50 After anesthesia, mice were infected with 20 μL of influenza virus by intranasal drip, while the normal group was given intranasal saline.

[0098] (3) Grouping and administration: The mice were divided into normal control group (Control), positive drug group (H1N1+Ose), influenza virus infection group (H1N1), andrographolide group (H1N1+And), tetrandrine group (H1N1+Tet), andrographolide and tetrandrine combination group (H1N1+And+Tet), with 10 mice in each group. In the Control group, normal mice were given 5% CMCNa by gavage, 0.2 mL each time, once a day; in the H1N1+Ose group, influenza virus infected mice were given 2 mg / mL oseltamivir phosphate solution by gavage, 0.2 mL each time, once a day; in the H1N1 (model group), influenza virus infected mice were given 5% CMCNa, 0.2 mL each time by gavage, once a day; H1N1+And is to administer 15 mg / mL andrographolide suspension to influenza virus-infected mice by gavage, 0.2 mL each time by gavage, once a day, that is, 150 mg / kg / d; H1N1+Tet is to administer 6 mg / mL tetrandrine suspension to influenza virus-infected mice by gavage, 0.2 mL each time by gavage, once a day, that is, 60 mg / kg / d; H1N1+Tet+And is to administer combined oral suspension 1 to influenza virus-infected mice by gavage, 0.2 mL each time by gavage, once a day, that is, 75 mg / kg / dAnd+30 mg / kg / d Tet.

[0099] (4) Effects of tetrandrine, andrographolide, and their combined oral suspension 1 on the survival and body weight of model mice

[0100] The mice were given the drug once a day for 7 consecutive days after the challenge, and the survival status of the mice was recorded every day until the 15th day.

[0101] (5) Results

[0102] like Figure 5 Results showed that the combined oral suspension of andrographolide and tetrandrine delayed mortality in mice infected with influenza A virus (IAV). Tetrandrine (60 mg / kg) and andrographolide (150 mg / kg) alone also improved survival in influenza-infected mice. However, the combination of andrographolide and tetrandrine increased survival by 66.7% compared to the model group, even when the dose was reduced to half of the individual drugs. Using the half-dose additive method (see "Evaluation of Research Methods for Measuring the Effect of Combined Drugs," Guo Jianyou et al., Pharmacology and Clinical Practice of Traditional Chinese Medicine, 2005), q was calculated to be 1.8 > 1, indicating a synergistic effect. Tetrandrine and andrographolide alone increased survival by 37.5% and 37.5% compared to the model group, respectively. Therefore, the combination of andrographolide and tetrandrine can synergistically treat influenza A virus.

[0103] 2. Protective effects of different doses of andrographolide and tetrandrine oral suspension on influenza infection model mice

[0104] S1. Preparation of oral suspension containing andrographolide and tetrandrine in different ratios:

[0105] 1g of CMCNa was accurately weighed using an electronic balance, added to 200mL of deionized water, and shaken overnight to prepare a 5% CMCNa solution. Then, 60mg, 180mg, and 360mg of tetrandrine and 150mg, 450mg, and 900mg of andrographolide were accurately weighed using an electronic balance and suspended in 10mL of water. 5% CMCNa, respectively prepared into 6mg / mL, 18mg / mL, 36mg / mL of tetrandrine suspension, 15mg / mL, 45mg / mL, 90mg / mL of andrographolide suspension; andrographolide and tetrandrine were prepared in different ratios (1:3, 1:6, 1:1, 3:1, 6:1, the ratio value was calculated by comparing the amount of andrographolide and tetrandrine with the amount of andrographolide and tetrandrine in combination suspension 1), among which the 1:1 ratio of andrographolide and tetrandrine combination was the above-mentioned combination oral suspension 1; the 1:3 ratio of andrographolide and tetrandrine combination (denoted as combination oral suspension 2) was prepared by suspending 150mg of andrographolide and 180mg of tetrandrine in 20mL of 5% CMCNa. A 1:6 andrographolide and tetrandrine combination (designated as combined oral suspension 3) was prepared by suspending 150 mg of andrographolide and 360 mg of tetrandrine in 20 mL of 5% CMCNa. A 3:1 andrographolide and tetrandrine combination (designated as combined oral suspension 4) was prepared by suspending 450 mg of andrographolide and 60 mg of tetrandrine in 20 mL of 5% CMCNa. A 6:1 andrographolide and tetrandrine combination (designated as combined oral suspension 5) was prepared by suspending 900 mg of andrographolide and 60 mg of tetrandrine in 20 mL of 5% CMCNa. For the positive drug group, 20 mg of oseltamivir phosphate was weighed using an electronic balance and dissolved in 10 mL of normal saline to prepare a 2 mg / kg oseltamivir phosphate solution.

[0106] S2. The method for establishing the influenza virus infection mouse model is the same as step (2) of this example.

[0107] S3. Grouping and administration: The mice were divided into normal control group (Control), positive drug group (H1N1+Ose), influenza virus infection group (H1N1), 1:1 andrographolide and tetrandrine combination group (1:1 H1N1+And+Tet), 1:3 andrographolide and tetrandrine combination group (1:3 H1N1+And+Tet), 1:6 andrographolide and tetrandrine combination group (1:6 H1N1+And+Tet), 3:1 andrographolide and tetrandrine combination group (3:1H1N1+And+Tet), and 6:1 andrographolide and tetrandrine combination group (6:1 H1N1+And+Tet), with 10 mice in each group. Among them, the control group was gavage-administered 5% CMCNa to normal mice, 0.2 mL each time, once a day; H1N1+Ose was gavage-administered 2 mg / mL oseltamivir phosphate solution to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1 (model group) was gavage-administered 5% CMCNa to influenza virus-infected mice, 0.2 mL each time, once a day; 1:1 H1N1+Tet+And was gavage-administered combined oral suspension 1 to influenza virus-infected mice, 0.2 mL each time, once a day, i.e., 75 mg / kg / d And+30 mg / kg / d Tet; 1:3 H1N1+Tet+And was gavage-administered combined oral suspension 2 to influenza virus-infected mice, 0.2 mL each time, once a day, i.e., 75 mg / kg / d And+90 mg / kg / d Tet; 1:6 H1N1+Tet+And means that the influenza virus-infected mice were gavaged with the combination oral suspension 3, 0.2 mL each time, once a day, i.e., 75 mg / kg / d And + 180 mg / kg / d Tet; 3:1 H1N1+Tet+And means that the influenza virus-infected mice were gavaged with the combination oral suspension 4, 0.2 mL each time, once a day, i.e., 225 mg / kg / d And + 30 mg / kg / d Tet; 6:1 H1N1+Tet+And means that the influenza virus-infected mice were gavaged with the combination oral suspension 5, 0.2 mL each time, once a day, i.e., 450 mg / kg / d And + 30 mg / kg / d Tet.

[0108] S4. Effects of combined oral suspensions 1 to 5 on the survival of model mice.

[0109] The mice were given the drug once a day for 7 consecutive days after the challenge, and the survival status of the mice was recorded every day until the 15th day.

[0110] S5. Results

[0111] Figure 6The results showed that when using a 1:3 ratio of andrographolide and tetrandrine combined oral suspension, the death of mice infected with influenza virus can be significantly delayed, and the survival rate is increased by about 50%, which is the highest survival rate. The 3:1 ratio has the lowest improvement in the survival rate of mice, and the survival rate improvement rate of other different ratios is relatively low.

[0112] 3. Protective effects of different ratios of andrographolide and tetrandrine oral suspension on influenza model mice

[0113] A1. Preparation of oral suspension containing andrographolide and tetrandrine in different ratios:

[0114] Use an electronic balance to accurately weigh 1g of CMCNa, add it to 200mL of deionized water, shake overnight to prepare a 5% CMCNa solution. Then use an electronic balance to accurately weigh 60mg, 120mg, 180mg, 240mg of tetrandrine and 150mg of andrographolide, respectively, and suspend them in 10mL of 5% CMCNa, respectively prepared into 6mg / mL, 12mg / mL, 18mg / mL, 24mg / mL of tetrandrine suspension, and 15mg / mL of andrographolide suspension; andrographolide and tetrandrine were prepared in different ratios (1:1, 1:2, 1:3, 1:4, the ratio value was calculated by comparing the amount of andrographolide and tetrandrine with the amount of andrographolide and tetrandrine in combination suspension 1), among which the 1:1 ratio of andrographolide and tetrandrine combination was the above-mentioned combination oral suspension 1; the 1:2 ratio of andrographolide and tetrandrine combination (denoted as combination oral suspension 6) was prepared by suspending 150mg of andrographolide and 120mg of tetrandrine in 20mL of 5% CMCNa. A 1:3 combination of andrographolide and tetrandrine (designated as combined oral suspension 2) was prepared by suspending 150 mg of andrographolide and 180 mg of tetrandrine in 20 mL of 5% CMCNa. A 1:4 combination of andrographolide and tetrandrine (designated as combined oral suspension 7) was prepared by suspending 150 mg of andrographolide and 240 mg of tetrandrine in 20 mL of 5% CMCNa. For the positive drug group, 20 mg of oseltamivir phosphate was weighed using an electronic balance and dissolved in 10 mL of normal saline to prepare a 2 mg / kg oseltamivir phosphate solution.

[0115] A2. The method for establishing the influenza virus infection mouse model is the same as step (2) of this example.

[0116] A3. Grouping and administration: The mice were divided into normal control group (Control), positive drug group (H1N1+Ose), influenza virus infection group (H1N1), 1:1 andrographolide and tetrandrine combination group (1:1 H1N1+And+Tet), 1:2 andrographolide and tetrandrine combination group (1:2 H1N1+And+Tet), 1:3 andrographolide and tetrandrine combination group (1:3 H1N1+And+Tet), and 1:4 andrographolide and tetrandrine combination group (1:4H1N1+And+Tet), with 10 mice in each group. Among them, the control group was gavage-administered 5% CMCNa to normal mice, 0.2 mL each time, once a day; H1N1+Ose was gavage-administered 2 mg / mL oseltamivir phosphate solution to influenza virus-infected mice, 0.2 mL each time, once a day; H1N1 (model group) was gavage-administered 5% CMCNa to influenza virus-infected mice, 0.2 mL each time, once a day; 1:1 H1N1+Tet+And was gavage-administered combination oral suspension 1 to influenza virus-infected mice, 0.2 mL each time, once a day, i.e., 75 mg / kg / d And + 30 mg / kg / d Tet; 1:2 H1N1+Tet+And was gavage-administered combination oral suspension 6 to influenza virus-infected mice, 0.2 mL each time, once a day, i.e., 75 mg / kg / d And + 60 mg / kg / d Tet; 1:3 H1N1+Tet+And means that the influenza virus-infected mice were gavaged with the combination oral suspension 2, 0.2 mL each time, once a day, i.e., 75 mg / kg / d And + 90 mg / kg / d Tet; 1:4 H1N1+Tet+And means that the influenza virus-infected mice were gavaged with the combination oral suspension 7, 0.2 mL each time, once a day, i.e., 75 mg / kg / d And + 120 mg / kg / dTet.

[0117] A4. Effects of combined oral suspensions 1-2, 6-7 on survival and body weight in influenza virus infection model mice

[0118] The mice were given the drug once a day for 7 consecutive days after the challenge, and the survival status of the mice was recorded every day until the 15th day.

[0119] A5. Results

[0120] Figure 7 The results showed that when using a 1:4 ratio of andrographolide and tetrandrine combined oral suspension, the death time of mice infected with influenza virus can be delayed, and the survival rate is the highest, increasing by about 60%, while the survival rate improvement rate of other combinations with different ratios is relatively low.

[0121] 4. Protective effects of andrographolide and tetrandrine oral suspension administered at different times on influenza virus infection model mice

[0122] B1. Andrographolide and Tetrandrine Combined Oral Suspension Preparation:

[0123] 1g of CMCNa was accurately weighed using an electronic balance and added to 200mL of deionized water. The mixture was shaken overnight to prepare a 5% CMCNa solution. Then, 150mg of andrographolide and 240mg of tetrandrine were accurately weighed using an electronic balance and suspended in 10mL of 5% CMCNa to prepare a 24mg / mL tetrandrine suspension and a 15mg / mL andrographolide suspension, respectively. A 1:4 combination of andrographolide and tetrandrine (designated as combined oral suspension 7) was prepared by suspending 150mg of andrographolide and 240mg of tetrandrine in 20mL of 5% CMCNa. For the positive drug group, 2mg of oseltamivir phosphate was weighed using an electronic balance and dissolved in 10mL of normal saline to prepare a 2mg / kg oseltamivir phosphate solution.

[0124] B2. The method for establishing the influenza virus infection mouse model (administration on the first day after infection) is the same as step (2) of this example.

[0125] B3. Grouping and administration: The mice were divided into a normal control group (Control), a positive drug group (H1N1+Ose), an influenza virus infection group (H1N1), and a 1:4 combination of andrographolide and tetrandrine group (1:4 H1N1+And+Tet), with 10 mice in each group. Among them, the control group was gavage-administered 5% CMCNa to normal mice, 0.2 mL each time, once a day; the H1N1+Ose group was gavage-administered 2 mg / mL oseltamivir phosphate solution to influenza virus-infected mice, 0.2 mL each time, once a day; the H1N1 (model group) was gavage-administered 5% CMCNa to influenza virus-infected mice, 0.2 mL each time, once a day; the 1:4H1N1+Tet+And group was gavage-administered combined oral suspension 7 to influenza virus-infected mice, 0.2 mL each time, once a day, i.e. 75 mg / kg / d And + 120 mg / kg / d Tet.

[0126] B4. Effects of oral suspension 7 administered at different time combinations on the survival of model mice

[0127] The mice were given drugs on days 0 (control group), 1, 3, and 5 after infection, once a day for 6 consecutive days, and observed until day 15. The survival status of the mice was recorded every day.

[0128] B5. Results

[0129] Figure 8 Results showed that a 1:4 ratio of andrographolide and tetrandrine combined oral suspension administered one day after infection significantly delayed the death of mice infected with influenza virus, increasing survival by 80%, the highest survival rate. This rate was similar to that of oseltamivir, while the survival rate improvements at other administration times were relatively low. The later the combination drug intervention was administered after one day of infection, the less effective it was.

[0130] Example 5

[0131] Andrographolide and tetrandrine oral suspension inhibit influenza virus replication in vivo

[0132] (1) The preparation of andrographolide and tetrandrine oral suspension refers to step B1 of Example 4.

[0133] (2) Establishment of an influenza virus infection mouse model

[0134] The experiment used 6-8 week old Balb / c mice, which were randomly divided into groups. After 3 days of adaptive feeding, H1N1 influenza A virus was diluted with PBS to 4LD. 50 After anesthesia, mice were infected with 20 μL of influenza virus by intranasal drip, while the normal group was given intranasal saline.

[0135] (3) Grouping and administration: The mice were divided into a normal control group (Control), a positive drug group (H1N1+Ose), an influenza virus infection group (H1N1), and a 1:4 ratio of andrographolide and tetrandrine combination group (1:4 H1N1+And+Tet), with 10 mice in each group. Among them, the control group was gavage-administered 5% CMCNa to normal mice, 0.2 mL each time, once a day; the H1N1+Ose group was gavage-administered 2 mg / mL oseltamivir phosphate solution to influenza virus-infected mice, 0.2 mL each time, once a day; the H1N1 (model group) was gavage-administered 5% CMCNa to influenza virus-infected mice, 0.2 mL each time, once a day; the 1:4H1N1+Tet+And group was gavage-administered combined oral suspension 7 to influenza virus-infected mice, 0.2 mL each time, once a day, i.e. 75 mg / kg / d And + 120 mg / kg / d Tet.

[0136] (4) After the infection, the drug was administered on the first day and then once a day for 4 consecutive days.

[0137] (5) Q-PCR detection of viral gene changes in lung tissue of influenza mice

[0138] Lung tissues of mice in each group were collected 4 days after infection, and RNA was extracted from the lung tissues of mice in each group using an animal tissue total RNA extraction kit according to the instructions. The extracted RNA was reverse transcribed using a cDNA first-strand synthesis premix kit according to the instructions, and the M1 and NS1 target genes were amplified using the Talent fluorescence quantitative detection kit (SYBR Green).

[0139] (6) Western blot detection of changes in viral protein expression in lung tissue of influenza mice

[0140] Lung tissue was lysed using RIPA cell lysis buffer supplemented with phosphatase and protease inhibitors. Proteins from the total tissue lysate were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% skim milk in TPBS for 2 hours and incubated overnight at 4°C with rabbit anti-HA (1:1000), mouse anti-NP (1:1000), and rabbit anti-β-actin primary antibodies. The membrane was washed four times for 5 minutes with TPBS and incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (1:3000) for 1 hour at room temperature. After washing five times for 5 minutes in PBS-Tween buffer, the chemiluminescence of the labeled proteins was revealed using an HRP substrate and captured under an instrument.

[0141] (7) Results

[0142] Figure 9 The results showed that the combined oral suspension of andrographolide and tetrandrine (combination oral suspension 7) could significantly inhibit the expression of M1 and NS1 genes in the lung tissue of the influenza virus-infected mouse model on the 4th day.

[0143] also, Figure 10 The results showed that the combined oral suspension of andrographolide and tetrandrine (combination oral suspension 7) could also inhibit the expression of viral HA and NP proteins in the lung tissue of the influenza virus-infected mouse model on the 4th day.

[0144] Example 6

[0145] Andrographolide and tetrandrine oral suspension inhibits influenza virus-induced inflammation in vivo

[0146] (1) The preparation of andrographolide and tetrandrine oral suspension refers to step B1 of Example 4.

[0147] (2) Establishment of an influenza virus infection mouse model

[0148] The experiment used 6-8 week old Balb / c mice, which were randomly divided into groups. After 3 days of adaptive feeding, H1N1 influenza A virus was diluted with PBS to 4LD. 50After anesthesia, mice were infected with 20 μL of influenza virus by intranasal drip, while the normal group was given intranasal saline.

[0149] (3) Grouping and administration: The mice were divided into a normal control group (Control), a positive drug group (H1N1+Ose), an influenza virus infection group (H1N1), and a 1:4 ratio of andrographolide and tetrandrine combination group (1:4 H1N1+And+Tet), with 10 mice in each group. Among them, the control group was gavage-administered 5% CMCNa to normal mice, 0.2 mL each time, once a day; the H1N1+Ose group was gavage-administered 2 mg / mL oseltamivir phosphate solution to influenza virus-infected mice, 0.2 mL each time, once a day; the H1N1 (model group) was gavage-administered 5% CMCNa to influenza virus-infected mice, 0.2 mL each time, once a day; the 1:4H1N1+Tet+And group was gavage-administered combined oral suspension 7 to influenza virus-infected mice, 0.2 mL each time, once a day, i.e. 75 mg / kg / d And + 120 mg / kg / d Tet.

[0150] (4) After the infection, the drug was administered on the first day and then once a day for 4 consecutive days.

[0151] (5) Lung index measurement

[0152] Lung tissues of mice in each group were collected 4 days later, and the wet weight of lungs was measured using an electronic balance. Lung index was calculated (lung index = wet weight of lungs / body weight).

[0153] (6) HE pathological analysis

[0154] Lung tissues of mice in each group were collected on day 4, fixed with 4% paraformaldehyde, embedded in paraffin, and cut into 5 µm sections. The sections were stained with hematoxylin-eosin (HE), and electronic scanning was performed to analyze the pathological changes.

[0155] (7) ELISA detection of changes in inflammatory factors in lung tissue of influenza mice

[0156] Four days later, alveolar lavage fluid was collected from each group of mice and the ELISA instructions were used to detect inflammatory factors such as IL-6, TNF-α, and IL-1β.

[0157] (8) Results

[0158] Figure 11 The results showed that the combined oral suspension of andrographolide and tetrandrine (combination oral suspension 7) could significantly reduce the lung index of model mice on the 4th day. Figure 12The results showed that treatment with andrographolide and tetrandrine combined oral suspension (combination oral suspension 7) could reduce inflammatory cell infiltration and alveolar space thickness in mice. Figure 13 The results showed that andrographolide and tetrandrine oral suspension can significantly inhibit the increased levels of IL-6, TNF-α and IL-1β inflammatory factors caused by influenza virus.

[0159] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical combination, solid preparation or liquid preparation in the preparation of an anti-influenza virus drug, characterized in that: The drug combination consists of andrographolide and tetrandrine; The solid or liquid preparation comprises the pharmaceutical combination; The mass ratio of the andrographolide to the tetrandrine is 15:5~25.

2. The use according to claim 1, characterized in that The mass ratio of the andrographolide to the tetrandrine is 15:6, 15:12, 15:18 or 15:

24.

3. The use according to claim 1, characterized in that The solid or liquid preparations also include sodium carboxymethylcellulose solutions.

4. The use according to claim 3, characterized in that The mass volume percentage of the sodium carboxymethyl cellulose solution is 3-7%; the mass volume ratio of andrographolide, tetrandrine and sodium carboxymethyl cellulose solution is 15 mg: (5-25) mg: (18-22) mL.

5. The use according to claim 1, characterized in that The influenza virus includes influenza A virus.

6. Use of a pharmaceutical combination, solid preparation or liquid preparation in the preparation of a drug for treating inflammation caused by influenza virus, characterized in that: The drug combination is the drug combination according to claim 1 or 2; the solid or liquid preparation is the solid or liquid preparation according to any one of claims 1, 3 and 4.

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